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Within the context of an international collaborative study of the evolution of hypertension in the black diaspora, we determined the allelic distribution of hypertension candidate genes for the renin-angiotensin system in three populations of African origin. The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) and the M235T and T174M variants of the angiotensinogen (AGT) gene were examined in individuals from Nigeria, Jamaica, and the United States. Large differences in the prevalence of hypertension were recorded in door-to-door surveys, ranging from 16% in Nigeria to 33% in the United States. The frequency of the D allele was similar in all groups (54%, 59%, and 63% in Nigeria, Jamaica, and the United States, respectively). The 235T allele of the AGT gene was found in 81% of US and Jamaican blacks and 91% of Nigerians; very little variation was seen for the T174M marker. Despite large differences in hypertension rates, genetic variation at the index loci among these groups was modest. Overall, the frequency of the ACE*D allele was only slightly higher than that reported for European and Japanese populations, whereas the AGT 235T allele was twice as common. Compared with blacks in the western hemisphere, Nigerians had a higher frequency of the 235T allele, which is consistent with 25% European admixture in Jamaica and the United States. The results indicate the potential for etiologic heterogeneity in genetic factors related to hypertension across ethnic groups while suggesting that environmental exposures most likely explain the gradient in risk in the comparison among black populations. |
Heat shock/stress proteins (HSP) act as molecular chaperones, protect cells from injury, and are involved in the immune response. We investigated the effects of the immunomodulating bacterial extracts OM-85 on the stress response in normal human peripheral blood monocytes. While OM-85 did not induce the classical HSP, we show here, using 2D gel electrophoresis combined with immunoblotting, the induction of the glucose regulated protein grp78 (the immunoglobulin heavy chain binding protein BiP) along with the described accumulation of pro-interleukin-1 beta. The increased Ca2+ mobilization observed with OM-85 is the likely second messenger for grp78 induction. Recent studies are in favor of a protective role of grp78 against cytokine-mediated cytotoxicity and apoptosis. We suggest that grp78 induction following exposure to OM-85 explains, at least in part, the immunodulatory and protective effects of the bacterial extracts. |
Recently, a cDNA was isolated from hydra with extensive homology to a mammalian and invertebrate gene which codes for a protein called laminin binding protein (LBP). In this paper we describe the protein expression of the hydra LBP in Escherichia coli. On SDS gels the recombinant hydra LBP displayed an apparent molecular mass of 43 kDa, although the calculated mass, including six additional histidines, is 33.7 kDa. Polyclonal antibodies were produced against the hydra recombinant LBP. The antiserum reacted with a 42-kDa and a 43-kDa protein from Hydra vulgaris and from a multiheaded mutant of Chlorohydra viridissima, respectively. In hydra, LBP RNA and protein were highly expressed in cells with short cell cycles, such as all cells of the interstitial cell lineage, less in slowly cycling epithelial cells, and at very reduced levels or not at all in differentiated cells. Higher expression in the multiheaded mutant of C. viridissima than in H. vulgaris, the cells of which differ in doubling time, hint at a function in cell proliferation. This is supported by the finding that in vitro hydra LBP is a substrate for the cell-cycle-specific kinase CDC2. |
Bone resorption by mononucleated cells was studied in the acellular bone of a teleost fish (Oreochromis niloticus) by histological and enzyme histochemical observations and by transmission electron microscopy. Bone resorbing cells (osteoclasts) were identified by their location at the sites of bone resorption, their frequent association with a band of concentrated activity of tartrate-resistant acid phosphatase at the bone surface and by the presence or lack of certain enzymes. Tartrate-resistant acid phosphatase was used as a marker for osteoclasts, and alkaline phosphatase as a marker for osteoblasts. Osteoclasts in O. niloticus are not multinucleated; however, during intense bone resorption, they form cell aggregations that resemble multinucleated giant cells in mammals. Conversely, during less intense bone degradation, osteoclasts are flat, have long narrow cytoplasmic processes and resemble the bone-lining cells of mammals. All bone-resorbing cells in O. niloticus are mononucleated and lack a ruffled border. Similarities to and differences from bone resorption by mononucleated cells in mammals are discussed. |
tRNA splicing is essential in yeast and humans and presumably all eukaryotes. The first two steps of yeast tRNA splicing, excision of the intron by endonuclease and joining of the exons by tRNA ligase, leave a splice junction bearing a 2'-phosphate. Biochemical analysis suggests that removal of this phosphate in yeast is catalyzed by a highly specific 2'-phosphotransferase that transfers the phosphate to NAD to form ADP-ribose 1"-2" cyclic phosphate. 2'-Phosphotransferase catalytic activity is encoded by a single essential gene, TPT1, in the yeast Saccharomyces cerevisiae. We show here that Tpt1 protein is responsible for the dephosphorylation step of tRNA splicing in vivo because, during nonpermissive growth, conditional lethal tpt1 mutants accumulate 2'-phosphorylated tRNAs from eight different tRNA species that are known to be spliced. We show also that several of these tRNAs are undermodified at the splice junction residue, which is always located at the hypermodified position one base 3' of the anticodon. This result is consistent with previous results indicating that modification of the hypermodified position occurs after intron excision in the tRNA processing pathway, and implies that modification normally follows the dephosphorylation step of tRNA splicing in vivo. |
Recently, the cell-volume-regulated serine-threonine protein kinase h-sgk was cloned from a human hepatoma cell line. The sgk gene was shown to be induced by cell shrinkage in many different mammalian cell lines. In this study, two highly conserved serine-threonine protein kinases, sgk-1 and sgk-2, were cloned from rectal gland tissue of the spiny dogfish (Squalus acanthias). Both kinases showed a distinct pattern of tissue specificity, with high expression levels in kidney, intestine, liver and heart. In rectal gland slices sgk-1 transcription was induced by exposure to hypertonic solution, reduction of the extracellular urea concentration, and addition of the secretagogues vasoactive intestinal polypeptide (VIP) and carbachol. The shark sgk-1 serine-threonine protein kinase may therefore provide a link between cell volume, Cl–secretion and protein phosphorylation state in shark rectal gland cells. |
The type II Na/Pi-cotransporter is expressed preferentially in renal proximal tubular epithelial cells. Comparison of the 5′ flanking region of the human NPT-2 gene with the opossum cell line (OK cell) and the murine Npt2 promoters revealed two conserved regions, one representing a putative C/EBP alpha site, the other a consensus TATA-box. In contrast to the OK cell and murine Npt-2 gene, the human exon 1 is flanked by two Alu-repeats, a short 90-bp inverted Alu element which is located within the promoter region and a full-length forward repeat present in intron 1. A 497-bp human promoter fragment including the inverted Alu-repeat was cloned in front of a luciferase reporter gene. The construct was active in OK and HeLa-S3 cells but no activity could be detected in the human monocyte cell line U937, the murine renal cortex cell line MCT and the dog kidney cell line MDCK. A twofold increase in promoter activity was observed in HeLa-S3 cells for a 5′ truncated fragment of 253 bp missing the inverted Alu-repeat. In the OK cell system the absence of the Alu-repeat was unable to modify promoter activity. In electrophoretic mobility shift assays (EMSAs) with a 31-bp oligonucleotide representing the conserved region with homology to C/EBP alpha we could provide evidence for specific DNA/protein interactions with nuclear extracts derived from kidney and liver cell lines but not for HeLa-S3 and U937 nuclear extracts. Specific interactions could also be observed with nuclear extracts from renal cortex, medulla and rat liver but not from rat spleen, intestine and heart. Southern-Western blotting techniques suggest that a 31-kDa nuclear protein from kidney-derived cells binds to the C/EBP-like region of the NPT2 promoter. |
Aquaporins are transmembrane proteins that contain six bilayer-spanning domains, connected by loops A to E. The hourglass model predicts that the conserved loops B and E are essential for the formation of the water pore. To test the importance of loops B and E in the determination of the transport characteristics, we exchanged loops B and/or E between AQP0, AQP2, and AQP3. Detailed functional, immunoblot and immunocytochemical analyses of expression in Xenopus oocytes revealed that six out of the nine chimeric aquaporin proteins were not functional, because of misrouting. AQP0 with loop E of AQP2 was not impaired in its routing and revealed a low water permeability equal to that of wild-type AQP0. AQP2 with loop B of AQP0 was also routed normally and gave a high water permeability, similar to that of wild-type AQP2. AQP0 with loops B and E of AQP2 (AQP0–2BE) did not result in an increase in water permeability and was partly misrouted. However, the plasma membrane expression was high enough to expect an increase in water permeability, as loops B and E of AQP2 confer AQP2’s water permeability to AQP0. Although it is unclear for the dual chimera (AQP0–2BE), the parental water permeabilities obtained in oocytes expressing AQP0 with loop E of AQP2 or AQP2 with loop B of AQP0 indicate that, besides loops B and E, other parts of the AQP protein are important in the determination of the characteristics of the channel. |
Hemichannels of rat connexin 46 (rCx46) were expressed in Xenopus laevis oocytes and analysed by two-electrode voltage-clamp experiments. It is established that rCx46 hemichannels can be activated at low external Ca2+ and positive membrane potentials. Upon larger depolarizations, the hemichannels of oocytes activate in a time-dependent manner, occasionally followed by a spontaneous inactivation. We found that, in the absence of inactivation, treatment of oocytes with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an activator of protein kinase C (PKC), reversibly reduced the amplitude of the rCx46-mediated current and, after an incubation time of about 30 min, induced inactivation of the voltage-dependent current. After wash-out of OAG the corresponding membrane conductance increased and the inactivation behaviour disappeared. The OAG-induced inactivation, as well as the spontaneous inactivation, could be removed by application of the specific PKC inhibitor calphostin C and also by phloretin. The data provide evidence that the activation and inhibition of PKC affect the rCx46-mediated membrane conductance as well as the voltage-dependent current inactivation in an inverse manner. |
Recent studies have implicated a variant of the angiotensin-converting enzyme gene (ACE), associated with increased activity of this enzyme, in the development and progression of diabetic nephropathy. This study provides a systematic review of all cross-sectional, case-control, and cohort studies in patients with insulin-dependent (IDDM) or non-insulin-dependent (NIDDM) diabetes mellitus of any race, examining the relationship between the ACE-insertion/deletion polymorphism and nephropathy. Nineteen studies in 21 populations published between 1994 and 1997 presenting data on 5336 patients were reviewed. Two investigators independently assessed the studies on methodologic quality, performance of study, and association between the ACE-insertion/deletion polymorphism and nephropathy. Separate analyses of the relationship between genotype and allele frequencies were performed for patients with IDDM and NIDDM by race, using Peto's odds ratio. In Caucasians with IDDM, pooling was not performed due to heterogeneity of the studies, but among the homogeneous studies, no association was detected. Likewise, no association was observed in Caucasian patients with NIDDM (odds ratio [OR], 1.10; 95% confidence interval [95% CI], 0.83 to 1.45). In Asian patients with NIDDM, the risk of nephropathy was increased in the presence of the DD or ID genotype (OR, 1.88; 95% CI, 1.42 to 2.85). Although this analysis fails to confirm an association between the ACE-insertion/deletion genotype and nephropathy in Caucasians with NIDDM or IDDM, a role for this genetic marker in Asian patients cannot be ruled out. However, due to methodologic limitations of individual studies, no definite conclusions can be drawn from this analysis. Clearly, more rigorous methodology needs to be applied in future studies. |
The last step of tRNA splicing in yeast is catalyzed by Tpt1 protein, which transfers the 2'-phosphate from ligated tRNA to NAD to produce ADP-ribose 1"-2"-cyclic phosphate (Appr>p). Structural and functional TPT1 homologs are found widely in eukaryotes and, surprisingly, also in Escherichia coli, which does not have this class of tRNA splicing. To understand the possible roles of the Tpt1 enzymes as well as the unusual use of NAD, the reaction mechanism of the E. coli homolog KptA was investigated. We show here that KptA protein removes the 2'-phosphate from RNA via an intermediate in which the phosphate is ADP-ribosylated followed by a presumed transesterification to release the RNA and generate Appr>p. The intermediate was characterized by analysis of its components and their linkages, using various labeled substrates and cofactors. Because the yeast and mouse Tpt1 proteins, like KptA protein, can catalyze the conversion of the KptA-generated intermediate to both product and the original substrate, these enzymes likely use the same reaction mechanism. Step 1 of this reaction is strikingly similar to the ADP-ribosylation of proteins catalyzed by a number of bacterial toxins. |
Microtubules are involved in the positioning and movement of organelles and vesicles and therefore play fundamental roles in cell polarization and differentiation. Their organization and properties are cell-type specific and are controlled by microtubule-associated proteins (MAP). E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) has been identified as a microtubule-stabilizing protein predominantly expressed in epithelial cells. We have used human skin and primary keratinocytes as a model to assess a putative function of E-MAP-115 in stabilizing and reorganizing the microtubule network during epithelial cell differentiation. Immunolabeling of skin sections indicated that E-MAP-115 is predominantly expressed in the suprabasal layers of the normal epidermis and, in agreement with this observation, is relatively abundant in squamous cell carcinomas but barely detectable in basal cell carcinomas. In primary keratinocytes whose terminal differentiation was induced by increasing the Ca2+ concentration of the medium, E-MAP-115 expression significantly increased during the first day, as observed by northern and western blot analysis. Parallel immunofluorescence studies showed an early redistribution of E-MAP-115 from microtubules with a paranuclear localization to cortical microtubules organized in spike-like bundles facing intercellular contacts. This phenomenon is transient and can be reversed by Ca2+ depletion. Treatment of cells with cytoskeleton-active drugs after raising the Ca2+ concentration indicated that E-MAP-115 is associated with a subset of stable microtubules and that the cortical localization of these microtubules is dependent on other microtubules but not on strong interactions with the actin cytoskeleton or the plasma membrane. The mechanism whereby E-MAP-115 would redistribute to and stabilize cortical microtubules used for the polarized transport of vesicles towards the plasma membrane, where important reorganizations take place upon stratification, is discussed. |
The systematics of Botrychium subgenus Botrychium has been controversial, primarily because reduction in frond size and complexity has limited the number of characters available for discrimination of species. The recognition of many polyploid species has magnified the difficulty of classification because allopolyploids are often morphologically intermediate between their progenitor diploids. In order to evaluate species limits and sectional boundaries, we surveyed and compared 16 of the 24 currently recognized species for isozymic variation. Little or no intrapopulational variation was detected, but the variation present was consistent with the hypothesis that Botrychium species are primarily inbreeding. Interspecific comparisons distinguished six diploid species and provided evidence of molecular differentiation between the cryptic sister species B. lunaria and B. crenulatum. Evidence of possible progenitor/descendant relationships was found for certain diploid/polyploid relationships. Using enzyme bands shared between species, realignment of the sectional assignment of several species is proposed. Anomalous banding patterns in certain individuals suggested that gene silencing or homoeologous chromosome pairing might be operating in B. minganense, B. hesperium, and B. matricariifolium. Isozyme data also showed that some populations of species presumed to be uniformly diploid possessed isozyme patterns typical of polyploids. All available molecular data indicate that members of Botrychium subgenus Botrychium are actively evolving at diploid and polyploid levels. |
Molecular dynamics calculations were carried out on models of two synthetic leucine-serine ion channels: a tetrameric bundle with sequence (LSLLLSL)(3)NH(2) and a hexameric bundle with sequence (LSSLLSL)(3)NH(2). Each protein bundle is inserted in a palmitoyloleoylphosphatidylcholine bilayer membrane and solvated by simple point charge water molecules inside the pore and at both mouths. Both systems appear to be stable in the absence of an electric field during the 4 ns of molecular dynamics simulation. The water motion in the narrow pore of the four-helix bundle is highly restricted and may provide suitable conditions for proton transfer via a water wire mechanism. In the wider hexameric pore, the water diffuses much more slowly than in bulk but is still mobile. This, along with the dimensions of the pore, supports the observation that this peptide is selective for monovalent cations. Reasonable agreement of predicted conductances with experimentally determined values lends support to the validity of the simulations. |
The structural, dynamical, and thermodynamic properties of a model potassium channel are studied using molecular dynamics simulations. We use the recently unveiled protein structure for the KcsA potassium channel from Streptomyces lividans. Total and free energy profiles of potassium and sodium ions reveal a considerable preference for the larger potassium ions. The selectivity of the channel arises from its ability to completely solvate the potassium ions, but not the smaller sodium ions. Self-diffusion of water within the narrow selectivity filter is found to be reduced by an order of magnitude from bulk levels, whereas the wider hydrophobic section of the pore maintains near-bulk self-diffusion. Simulations examining multiple ion configurations suggest a two-ion channel. Ion diffusion is found to be reduced to approximately 1/3 of bulk diffusion within the selectivity filter. The reduced ion mobility does not hinder the passage of ions, as permeation appears to be driven by Coulomb repulsion within this multiple ion channel. |
Gamma-aminobutyric acid type A (GABA(A)) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the alpha(1)M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from alpha(1)Ala(291) to alpha(1)Val(307). The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS(-) was assayed electrophysiologically. Cysteines substituted for alpha(1)Ala(291) and alpha(1)Tyr(294) reacted with pCMBS(-) applied both in the presence and in the absence of GABA. Cysteines substituted for alpha(1)Phe(298), alpha(1)Ala(300), alpha(1)Leu(301), and alpha(1)Glu(303) only reacted with pCMBS(-) applied in the presence of GABA. We infer that the pCMBS(-) reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of alpha(1)Ala(291), a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents. |
The activity of many membrane proteins depends on a conformational transition that is often strongly influenced by small membrane-soluble solutes. This allosteric modulation may be direct, involving binding to the protein at localized sites of varying specificity, or may be indirect, resulting from altered membrane properties. In the present paper, a general expression for solute-protein titration curves is predicted, using an indirect mechanism that couples solute-induced changes in the lateral pressure profile of the bilayer to a shift in protein conformational equilibrium. When the common practice of fitting dose-response data to the Hill equation is applied to these curves, the fits are found to be reasonably good, with large Hill coefficients. Because this would commonly be interpreted as evidence of the existence of multiple sites with strong positive cooperativity, it is argued that caution must therefore be exercised in the interpretation of titration data in the absence of direct evidence of the existence of binding sites. The form of the titration curve predicted from this lateral pressure mechanism is shown to be quite general for indirect mechanisms. It is also shown that this form is the same as would be obtained from classical models of binding cooperativity, such as that of Monod, Wyman, and Changeux, in the limit of an infinite number of sites with vanishingly small site affinity. |
Weak anharmonic coupling of two soft molecular vibrations is shown to cause pronounced temperature dependence of the corresponding resonance Raman bands. The developed theory is used to interpret the temperature dependence of the iron-histidine band of deoxyheme proteins and model compounds. It is shown that anharmonic coupling of the iron-histidine and heme doming vibrations must cause pronounced broadening of the band, its asymmetry, and shift of its maximum to the red upon heating. It also can lead to a structured shape of this band at room temperature. Proper consideration of the anharmonic coupling allows simulation of the temperature dependence of the iron-histidine band shape of horse heart myoglobin in the temperature interval of 10-300 K, using the minimum number of necessary parameters. Analysis of this temperature dependence clearly shows that the iron-histidine band of deoxyheme proteins is sensitive to the glass-liquid phase transition in the protein hydration shell, which takes place at 160-190 K. |
The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules. |
The on-line high-pressure cell NMR technique was used to study pressure-induced changes in the tertiary structure and dynamics of a globular protein, basic pancreatic trypsin inhibitor (BPTI). Practically all the proton signals of BPTI were observed with (1)H two-dimensional NMR spectroscopy at 750 MHz at variable pressure between 1 and 2000 bar. Chemical shifts, nuclear Overhauser effect (NOE), and line shapes were used to analyze conformational and dynamic changes of the protein as functions of pressure. Linear, reversible, but nonuniform pressure-induced chemical shift changes of practically all the C(alpha) protons and side chain protons showed that the entire secondary and tertiary structures are altered by pressure within the folded ensemble of BPTI. The high field shift tendency of most side chain proton signals and the increase in NOE intensities of some specific side chain protons indicated a site-specific compaction of the tertiary structure. Pressure dependence of ring flip rates was deduced from resonance line shapes of the slices of the two-dimensional NMR spectrum for ring proton signals of Tyr-35 and Phe-45. The rates of the flip-flop motions were considerably reduced at high pressure, from which activation volumes were determined to be 85 +/- 20 A(3) (or 51.2 ml/mol) and 46 +/- 9 A(3) (or 27.7 ml/mol) for Tyr-35 and Phe-45, respectively, at 57 degrees C. The present experiments confirm that pressure affects the entire secondary and tertiary structures of a globular protein with specific compaction of a core, leading to quite significant changes in slow internal dynamics of a globular protein. |
Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer. |
Microtubule-associated proteins (MAPs) have been hypothesized to regulate microtubule dynamics and/or functions. To test hypotheses concerning E-MAP-115 (ensconsin) function, we prepared stable cell lines expressing conjugates in which the full-length MAP (Ensc) or its microtubule-binding domain (EMTB) was conjugated to one or more green fluorescent protein (GFP) molecules. Because both distribution and microtubule-binding properties of GFP-Ensc, GFP-EMTB, and 2x, 3x, or 4xGFP-EMTB chimeras all appeared to be identical to those of endogenous E-MAP-115 (ensconsin), we used the 2xGFP-EMTB molecule as a reporter for the behavior and microtubule-binding function of endogenous MAP. Dual wavelength time-lapse fluorescence imaging of 2xGFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimera associated with the lattice of all microtubules immediately upon polymerization and dissociated concomitant with depolymerization, suggesting that dynamics of MAP:microtubule interactions were at least as rapid as tubulin:microtubule dynamics in the polymerization reaction. Presence of both GFP-EMTB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellular microtubules at all cell cycle stages suggested that the MAP might function in modulating stability or dynamics of microtubules, a capability shown previously in transiently transfected cells. Although cells with extremely high expression levels of GFP-EMTB chimera exhibited stabilized microtubules, cells expressing four to ten times the physiological level of endogenous MAP exhibited microtubule dynamics indistinguishable from those of untransfected cells. This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likely serves to modulate microtubule functions or interactions with other cytoskeletal elements. |
The higher level relationships within Apiaceae (Umbelliferae) subfamily Apioideae are controversial, with no widely acceptable modern classification available. Comparative sequencing of the intron in chloroplast ribosomal protein gene rpl16 was carried out in order to examine evolutionary relationships among 119 species (99 genera) of subfamily Apioideae and 28 species from Apiaceae subfamilies Saniculoideae and Hydrocotyloideae, and putatively allied families Araliaceae and Pittosporaceae. Phylogenetic analyses of these intron sequences alone, or in conjunction with plastid rpoC1 intron sequences for a subset of the taxa, using maximum parsimony and neighbor-joining methods, reveal a pattern of relationships within Apioideae consistent with previously published chloroplast DNA and nuclear ribosomal DNA ITS based phylogenies. Based on consensus of relationship, seven major lineages within the subfamily are recognized at the tribal level. These are referred to as tribes Heteromorpheae M. F. Watson & S. R. Downie Trib. Nov., Bupleureae Spreng. (1820), Oenantheae Dumort. (1827), Pleurospermeae M. F. Watson & S. R. Downie Trib. Nov., Smyrnieae Spreng. (1820), Aciphylleae M. F. Watson & S. R. Downie Trib. Nov., and Scandiceae Spreng. (1820). Scandiceae comprises subtribes Daucinae Dumort. (1827), Scandicinae Tausch (1834), and Torilidinae Dumort. (1827). Rpl16 intron sequences provide valuable characters for inferring high-level relationships within Apiaceae but, like the rpoC1 intron, are insufficient to resolve relationships among closely related taxa. |
Begonia dregei and B. homonyma (Begoniaceae), rare plants endemic to coastal forests of eastern South Africa, are two closely related species with high levels of variation among populations in the shape of leaves. Distribution of genetic variation and genetic relatedness were investigated in 12 populations of B. dregei and seven of B. homonyma using polyacrylamide gel electrophoresis. Twelve of the 15 enzyme loci examined were polymorphic, but only seven loci were polymorphic within at least one population. Genetic diversity measures indicated that the among-population gene differentiation represents >90% of the total genetic component in both species considered individually or combined. This indicated restricted gene flow, consistent with the limited dispersal abilities of Begonia generally and the ancient separation of isolated forest patches. Genetic distances among populations are much higher than usually found within species. Allozyme data provide no support for the recognition of B. dregei and B. homonyma as distinct species. |
Cell type-specific microtubules, such as the Sertoli cell microtubules and the manchette and flagellum microtubules of the spermatids, play essential roles in spermatogenesis. We identified the gene encoding E-MAP-115 (epithelial microtubule-associated protein of 115 kD) as a retinoic acid-inducible gene using gene trap mutagenesis in mouse embryonic stem cells. The gene trap insertion led to a null allele of the E-MAP-115 gene and, in agreement with its high expression in the testis, male mice homozygous for the mutation were sterile because of deformation of spermatid nuclei and subsequent gradual loss of germ cells. Consistent with a possible role for E-MAP-115 in stabilizing microtubules, microtubule associations in the mutant were morphologically abnormal in the manchette of spermatids and in Sertoli cells. We hypothesize that the abnormal microtubules in these two cell types are responsible for deformation of spermatid nuclei and germ cell loss, respectively, and indicate an essential role for E-MAP-115 in microtubule functions required for spermatogenesis. |
We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-μm microcarriers and 1 μg of pCMVL plasmid DNA at a delivery pressure of 200 psi. Time course studies revealed luciferase activity peaking at 18 hours and decreasing to 30% of the maximum at day 8 after DNA transfer. Onset of reporter gene (GFP) expression was detected at 13 minutes after DNA delivery, and by 65 minutes approximately 100% of the cells in the target area exhibited GFP expression. No germline association or integration events were detected in a screen of approximately 250,000 zebrafish sperm cells by fluorescence in situ hybridization at 15 or 30 days after delivery of 1 μg of pCMVL DNA, suggesting incidental male germline integration should not be considered as a risk factor when using the biolistic DNA delivery parameters and target tissues described. |
We used enzyme electrophoresis to evaluate genetic diversity in 32 populations of Calystegia collina, a clonal plant species endemic to serpentine outcrops in northern California (USA). Of 34 loci examined 56% were polymorphic, but on average only 17% were polymorphic within local populations. Neither the total number of alleles nor the number of multilocus genotypes differed significantly between populations in small vs. large serpentine outcrops. Genetic and geographic distances between populations were positively correlated, but this relationship was not significantly affected by the isolation of serpentine outcrops. Populations were highly differentiated (F(st) = 0.417) and little genetic variation was explained by geographic region or serpentine outcrop.Observed heterozygosity within populations almost always exceeded Hardy-Weinberg expectations. In many populations, all 30 sample ramets were uniformly heterozygous at one or more loci yet were genetically variable at other loci. These results imply that many C. collina populations originate from one or a few genetic founders, with little recruitment from seeds. Genetic variation within uniformly heterozygous populations must be the product of multiple, closely related founders or somatic mutations within the population. We conclude that vegetative reproduction, perhaps coupled with somatic mutation, helps maintain genetic diversity in these isolated but long-lived populations. |
I compared the C(4) grass flora and climatic records for 32 sites in the United States. Consistent with previous studies, I found that the proportion of the grass flora that uses the NADP malic enzyme (NADP-ME) variant of C(4) photosynthesis greatly increases with increasing annual precipitation, while the proportion using the NAD malic enzyme (NAD-ME) variant (and also the less common phosphoenolpyruvate carboxykinase [PCK] variant) decreases. However the association of grass subfamilies with annual precipitation was even stronger than for the C(4) decarboxylation variants. Analysis of the patterns of distribution by partial correlation analysis showed that the correlations between the frequency of various C(4) types and rainfall were solely due to the association of the C(4) types with particular grass subfamilies. In contrast, there was a strong correlation of the frequency of the different subfamilies with annual precipitation that was independent of the influence of the different C(4) variants. It therefore appears that other, as yet unidentified, characteristics that differ among grass subfamilies may be responsible for their differences in distribution across natural precipitation gradients. |
The ability of microorganisms to form biofilms has been well documented. Bacterial cells make a transition from a planktonic state to a sessile state, replicate, and subsequently populate a surface. In this study, organisms that initially colonize a ``clean'' surface are referred to as ``primary'' biofilm cells. The progeny of the first generation of sessile cells are known as ``secondary'' biofilm cells. This study examined the growth of planktonic, primary, and secondary biofilm cells of a green fluorescent protein producing (GFP+) Pseudomonas aeruginosa PA01. Biofilm experiments were performed in a parallel plate flow cell reactor with a glass substratum. Individual cells were tracked over time using a confocal scanning laser microscope (CSLM). Primary cells experience a lag in their growth that may be attributed to adapting to a sessile environment or undergoing a phenotypic change. This is referred to as a surface associated lag time. Planktonic and secondary biofilm cells both grew at a faster rate than the primary biofilm cells under the same nutrient conditions. |
The influence of substrate concentration on plasmid transfer frequency in the rhizosphere was investigated using a physical model employing a hollow fiber membrane instead of a real root. The absolute number of transconjugants produced increased with increasing substrate (glucose) concentration, but the plasmid transfer frequency decreased exponentially with increasing substrate concentration from 4.4 × 10(-3) at 90 μg ml(-1) glucose to 1.35 × 10(-5) at 3600 μg ml(-1) glucose. These results were found to be heavily dependant on donor to recipient ratio and distribution of strains, but independent of initial donor and recipient inoculum density and regime. These observations also show that plasmid transfer frequency is reduced at high substrate concentrations, which suggests that plasmid transfer is either stimulated when growth limiting nutrient is scarce or inhibited when it is abundant. |
Reproduction of polyploid Rubus species is described as facultatively apomictic. Pollination is needed for seed set, but most seedlings are produced asexually by pseudogamy. Although sexual processes may occur, clonal diversity can be extremely low. We performed a pollination experiment to investigate the breeding system and used allozyme and AFLP markers to analyze genetic variation among and within seed families in R. armeniacus and R. bifrons. Pollination either with self or outcross pollen was necessary to trigger seed set. Outbreeding marginally increased the number and quality of seeds compared with selfing. The enzyme PGI revealed some genetic variation within seed families. Seven other enzyme systems were monomorphic. The more detailed AFLP analyses with five primer pairs detected the same rate of genetic variation (14-17% of seedlings were genetically distinct) and confirmed the allozyme results for the same individuals. No genetic variation was found between the seed families from within a species collected in widely separated populations, but clear species-specific differences were observed. The results support the view that polyploid Rubus species are pseudogamous apomicts with low genetic diversity among and within seed families. However, sexual reproduction occasionally occurs and contributes to the maintenance of genetic variation within natural populations. |
The ability of the rodent brain to support plasticity-related phenomena declines with increasing age. A decreased coordination of genes implicated in brain plasticity may be one factor contributing to this decline. Synaptic rearrangement that occurs after seizure activity is regarded as a model of brain plasticity. In a rat model of seizure-related brain plasticity, we found that the induction of immediate-early genes, as exemplified by c-fos and tissue plasminogen activator ( tPA), is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure, and the levels of c-fos and tPA mRNAs induction are decreased in the cortex and in the hippocampus of 30 month old rats, as compared to the levels expressed by 3 month old rats. In addition, at the peak induction, the TPA transcripts were restricted to certain cortical layers of the older rats. Surprisingly, in applying the same experimental paradigm to late genes, we found that there was a shift toward earlier times in the maximum expression of growth-related molecules, the microtubule-associated protein 1B (MAP1B) mRNA, which was very evident in 18 month old rats. Aberrant immunolabeling of MAP1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that (1) one consequence of aging, besides decreases in the levels of mRNA, is a progressive loss of coordination in gene activity following the administration of a stimulus; (2) since c-fos, TPA and MAP1B have been implicated in neuronal plasticity, these findings could explain, in part, the limited plasticity of the aging brain. |
Oxidative phosphorylation is the main endogenous source for the generation of reactive oxygen species (ROS). In order to investigate the influence of enhanced ROS production on the in vitro senescence of Wi-38 fibroblasts, cells were cultivated in medium with elevated (hypertonic) NaCl concentrations. The number of active Na(+)/K(+)-ATPase molecules per cell was found to be increased. A rise in both respiration and glycolysis as evidenced by the increases in oxygen and glucose consumption and lactate production was revealed. Cells stayed alive in medium with NaCl concentrations of up to 0.30 M and could be adapted to growth under these hypertonic conditions (high-NaCl tolerant cells). These cells exhibited an increased cell size and protein content. A growing number of cells showed stress fibers and granulation. The proliferation rate and the maximum number of cumulative population doublings of these high-NaCl tolerant cultures were reduced and saturation density was decreased. Thus, these cells under energetic stress due to increased energy requirements for active ion transport expressed features typical for aging in vitro. We conclude therefore that energetic stress induces premature aging in human diploid fibroblasts. |
Four variants of Kranz anatomy occur in the Cyperaceae. Three of these anatomical types (fimbristyloid, chlorocyperoid, and eleocharoid) are unique among taxa with C(4) photosynthesis in that the photosynthetic carbon reduction tissue (PCR, functional equivalent of bundle sheath) is located within the vascular strand and is separated from the primary carbon assimilation tissue (PCA, positional equivalent of mesophyll) by the mestome sheath layer. In the fourth anatomical type, rhynchosporoid, PCR tissue is located in the position of the mestome sheath. In this study, we compared two aspects of development of PCR and PCA tissues in representatives of the C(3) and C(4) types: (1) ontogenetic derivation and (2) cellular differentiation. Analysis of the planes of cell division associated with procambial strand formation indicated that PCR tissue is always derived from the procambium, while PCA tissue is derived from the ground meristem. These cell lineages remain distinct after the initial organization of vascular strands. Analysis of cell differentiation using accumulation of cell-type-specific photosynthetic enzymes as markers of differentiation indicated that, with one exception, a low level of non-cell-specific enzyme accumulation preceded abundant and cell-specific accumulation of photosynthetic enzymes at the distal end of the leaf elongation zone. Enzyme accumulation coincided spatially (and temporally) with structural aspects of cell differentiation. Previous cladistic analyses have indicated that these anatomical types represent separate evolutionary origins of the C(4) pathway, and the differences in developmental pathways observed here reflect these independent origins from C(3) ancestors. |
Cost of antihypertensive drugs in Spain raises 100.000 millions of pesetas. The utilization of new drugs more expensive than classics diuretics and beta blockers is the main reason of this cost increase.The Joint National Commission on Hypertension 6th Report supports the utilization of diuretics and beta blockers as the first choice in patients without any special condition, based in their best efficience. Other professional group don't point out any therapeutic drugs because each of them have their indication. Health authorities have implemented measures intended more to achieve savings than to improve prescriptions. There are not any studies which demonstrate that the second type of measures are more efficient than first one. |
Numerous epidemiological studies have shown an inverse correlation between moderate consumption of alcoholic drinks and the risk of coronary heart disease. Wine, especially red wine, may be more favorable in this respect than beer or spirits because of its high content of flavonoids. These polyphenols originate from the skins, seeds, and vine stems of the grapes while some are formed during the process of vinification. In nature they exhibit a wide range of biological effects as antioxidants, antimicrobials, and modulators of various enzyme systems. Potential beneficial effects for humans have been demonstrated in experimental studies and include influences on the oxidation of LDL-cholesterol, on platelet aggregation, and on prostaglandin and nitric oxide metabolism. However, most of these studies concern semi in vivo experiments and research in animal models; data from human intervention trials are scarce. Furthermore, little is known about the absorption, bioavailability, and bioactivity of most of these compounds because of difficulties in reliable quantification in biological fluids. Until these issues are well addressed, and despite the enthusiasm and faith of many believers, evidence-based medicine justifies a critical attitude towards the application of these compounds outside the context of scientific research. Yet, there is no need to deny their potential, nor should we close our eyes to the blessings of the grape. |
Elevated serum total sialic acid (TSA) has been shown to be associated with increased cardiovascular mortality. It has been postulated that atherogenesis is a postprandial phenomenon. We tested the hypothesis that serum TSA and other acute phase proteins, namely C-reactive protein (CRP) and fibrinogen, may be related to the postprandial state. |
An extremely rare case of malignant schwannoma of the esophagus with lymph node metastasis is reported. A 49-year-old woman was found to have an abnormal shadow on a chest X-ray film taken during an annual checkup. Upper gastrointestinal series showed extrinsic pressure on the middle thoracic esophagus, without a mucosal lesion. An exploratory operation was performed, with a tentative diagnosis of esophageal leiomyoma. The tumor was enucleated with part of the esophageal mucosa, and a few enlarged lymph nodes around the tumor were dissected. The resected tumor was an elastic firm mass, measuring 8.2 x 5.8 x 3.7 cm, and had a smooth surface. Histological examination of the tumor revealed the proliferation of spindle-shaped cells with chromatin-rich nuclei. The nuclei were variable in size and showed remarkable atypia. A paraesophageal lymph node had same findings as the main tumor. Immunohistochemically, the tumor cells were diffusely positive for S-100 protein and neuron-specific enolase. The pathological diagnosis of this tumor was malignant esophageal schwannoma with lymph node metastasis. Esophageal schwannoma is extremely rare. We reviewed the literature on 19 cases of esophageal schwannoma, including that in our patient. The majority of the tumors were benign. Only three cases of schwannoma were malignant, and this is the first reported case of malignant schwannoma with lymph node metastasis. |
To study apoptosis induced by cadmium chloride and the alteration in activity of stress activated protein kinase (SAPK) in adrenocortical cells. |
Previously, studies have demonstrated that the effects of both a laboratory-produced vancomycin and a clinically available vancomycin were mediated, in part, by activation of both H(1) and H(2) receptors; however, other mechanisms may play a role in the vascular changes associated with vancomycin, since neither H(1) and H(2) receptor blockade has completely abolished the vasodilator responses to vancomycin in any model system. To study the mechanisms of vancomycin interactions in the hindlimb vascular bed of the rat, responses of two types of vancomycin preparations were studied. Vancomycin prepared for either clinical or laboratory use produced an initial short-lived period of vasoconstriction followed by a prolonged period of vasodilation in the hindlimb vascular bed. Responses to both the vancomycins and histamine on systemic arterial vasodilation were significantly decreased after administration of both the H(1)-receptor antagonist diphenhydramine and the H(2)-receptor antagonist famotidine. Verapamil, an L-type calcium channel blocker, significantly reduced the vasopressor responses to clinical vancomycin but not the vasopressor responses to laboratory vancomycin. Enalaprilat, and angiotensin-converting enzyme blocker, significantly reduced the vasodilator responses but not the vasoconstrictor responses of clinical vancomycin and significantly reduced the vasoconstrictor responses but not the vasodilator responses to laboratory vancomycin. Meclofenamate, a cyclo-oxygenase inhibitor, and N(omega)-L-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase inhibitor, had no significant effect on the biphasic responses with either vancomycin preparations. Atropine, an anticholinergic-antimuscarinic receptor antagonist, and propranolol, a beta adrenergic blocker, had no significant effect on vancomycin responses. Finally, ondansetron, a serotonin receptor blocker, and HOE 140, a bradykinin receptor blocker, also had no significant effect on vancomycin responses. These data suggest that both vancomycin preparations (clinically available and laboratory prepared) caused biphasic responses that differed from the dose-dependent vasodilation elicited by histamine. Both vancomycin preparations' vasodilator responses appear to be modulated, in part, by a histamine receptor--sensitive mechanism, while vancomycin-induced vasoconstrictor responses appear to be modulated, in part, by angiotensin-converting enzyme and L-type calcium channel--sensitive mechanisms in the rat hindlimb vascular bed. These data also suggest that the vascular responses of vancomycin are preparation dependent. |
To explore the effect of different vector structure on hF IX protein expression in murine myoblasts in order to construct more efficient expression vector for my oblast-mediated gene therapy for hemophilia B. |
The effects of sodium butyrate (SB) and trichostatin A (TSA) on cell proliferation andapoptosis against human glioma T98G, U251MG, and U877MG cells were investigated. Upon exposure to either SB or TSA, cell proliferation was reduced, and apoptosis detected by DNA fragmentation analysis and the cleavage of CPP32 was induced. Previously, we reported that SB increased the expression levels of p21 (WAF-1) and inhibited G1-S transition of the cell cycle. In this study, we showed that TSA also increased p21 expression, suggesting that histone deacetylase (HDAC) inhibitors may up-regulate p21 protein in common and thus arrest proliferation in the G1 phase of the cell cycle. To further determine the underlying molecular mechanisms of apoptosis with either SB or TSA treatment, we studied the expression levels of apoptosis-related proteins in human glioma cells. SB increased the expression of the Bad protein, although the expression of Bcl-2, Bcl-xL, Bax, and Fas was not changed by theaddition of SB. TSA treatment also up-regulated the expression of Bad protein. The results suggest that HDAC inhibitors such as SB and TSA induce apoptosis through an increase in Bad protein in human glioma cells in vitro. |
With the use of RT-PCR (reverse transcriptase-polymerase chain reaction), Northern blot analysis, and Western blot analysis, seven primary brain lymphomas were examined for the state of the MMACI tumor suppressor gene. Nucleotide analysis of RT-PCR clones revealed no abnormality in the MMAC1 coding sequence in each case. Although Northern blot revealed variation among cases in the signal intensities for MMAC1 mRNA, Western blot revealed a distinct MMAC1 protein band in all cases, suggesting that the actual MMAC1 expressions were similar. In Western blot analysis of phosphorylated Akt (p-Akt), which is regulated positively by PI3K (phosphoinositide-3 kinase) and negatively by MMAC1, all the lymphomas revealed an Akt band but not a p-Akt band, suggesting that the MMAC1 phosphatase activity was maintained in each case. These findings suggest that the MMAC1 gene is normal in its coding sequence, gene expression, and phosphatase activity in the lymphomas. Thus, unlike the p16 and p15 tumor suppressor genes, which are frequently deleted and inactivated in brain lymphoma and represent a striking contrast to systemic lymphoma, MMAC1 may not play an important role in carcinogenesis in this tumor, as in the systemic counterpart. |
The tumor suppressor p16/CDKN2A/INK4a gene is frequently mutated, mostly by homozygous deletions in high-grade gliomas. Although the p16 protein suppresses cell proliferation primarily through inhibition of cell-cycle progression at the G1 phase, other phenotypic changes in glioma cells associated with p16INK4a alterations have not been fully described. To determine the roles of p16 alterations in glioma formation, we have established ecdysone-driven inducible p16 expression in the human glioblastoma cell line CL-4, which were derived from p16-null U87MG cells. Here we show that exogenous p16 expression in CL-4 cells results in morphological changes, with large and flattened cytoplasm, which are associated with increased formation of cytoplasmic actin-stress fibers and vinculin accumulation in the focal adhesion contacts. Adhesion of CL-4 cells to extracellular matrix proteins, such as laminin, fibronectin, and type IV collagen, significantly increased upon exogenous p16 expression, which correlated with increased expression of integrin alpha5 and alphav. Expression of a small GTP-binding protein, Rac, also decreased. Following epidermal growth factor stimulation, phosphorylation of MAP kinases ERK1 and 2 and induction of an early immediate gene product, c-Fos, were significantly reduced in CL-4 cells with p16 expression. These results suggest that the tumor suppressor p16 may exert its antitumor effects through modulation of multiple aspects of glioblastoma phenotypes, including proliferation, invasiveness, and responsiveness to extracellular growth stimuli. |
Nearly fifty years after their introduction, corticoids continue to be one of the main pillars of dermatological therapy in general and in atopic dermatitis in particular. However, their use is limited by local and systemic adverse effects.There is enormous demand for anti-inflammatory agents not belonging to the corticoid group that would be useful in the different inflammatory dermatoses.In the last few years the therapeutic arsenal for atopic dermatitis has increased with two distinct groups of drugs: topical immunomodulators and leukotriene inhibitors. Both groups of drugs are new and new compounds belonging to these types of drugs will probably appear in the next few years.Among the alternatives to corticoids are the immunomodulators and the most promising of these are macrolide antibiotics. Tacrolimus (FK 506) and pimecrolimus (ASM 981) belong to this group of substances with a high capacity to inhibit T lymphocyte activation. Although they also act on other cells playing a role in atopic dermatitis (mastocytes, Langerhans' cells, B lymphocytes) their action on T lymphocytes seems to be the most important. In T lymphocytes, these drugs act by inhibiting the action of calmodulin, a vital enzyme in the activation chain of these cells that ends in the production of interleukin 2 and other proinflammatory cytokines.The accumulated evidence of the various publications seems to indicate that tacrolimus is a safe and effective treatment of atopic dermatitis in patients aged 2 years or more. The drug was approved for clinical use 3 years ago in Japan and more than 1 year ago in the United States. The number of randomized clinical trials comparing this drug with placebo or various corticoids demonstrate that its action is overwhelming. Its anti-inflammatory action is similar to that of high-potency topical corticoids. Two doses daily are required and the drug has been tested in patients with moderate-to-severe atopic dermatitis. Published data from more than 10,000 patients guarantee its safety. The most frequent short-term adverse effect is a burning sensation in the site of application lasting a few minutes.Pimecrolimus has not yet been commercialized in any country and is currently undergoing phase III clinical investigations. It has been tested in three clinical trials in patients aged more than 3 months old with mild-moderate atopic dermatitis. Its safety profile is good and similar to that of tacrolimus.The number and quality of the data provided by studies of treatment with leukotriene inhibitors (zafirlukast, montelukast and zileuton) is much lower but these substances seem to warrant further investigation. Only five small series undergoing treatment with these products have been published. |
Large-scale, high precision drug discovery calculations, such as predicting protein folding or small-molecule protein inhibitors, have frustrated computational chemists because the supercomputers currently available are insufficiently powerful. The increasing power of PCs offers an alternative approach by harnessing 'idle time' from corporate and home computers that are connected to the Internet or an intranet. However, although the approach has the potential of offering hundreds or thousands of years of computer time per elapsed day, the architecture constraints require computational chemists to choose their methods and applications with care. Some algorithms, such as those for molecular simulations, are generally not appropriate, whereas virtual screening of molecules for protein inhibition works well. |
K-252b, a potent inhibitor of protein kinases blocked a late phase of long-term potentiation (LTP) in area CA1 of rat hippocampal slices, resulting in decremental LTP. It also prevented the slowly developing increase in sensitivity of CA1 neurons to iontophoretically administered alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) which was seen in control slices that exhibit nondecremental LTP. However, K-252b applied 60 - 180 min after the induction of LTP had no effect on the potentiated synaptic and AMPA-induced responses. A K-252b-sensitive protein kinase may therefore be involved in a slowly developing postsynaptic component of LTP. |
Calcium channel currents have been recorded from cultured rat sensory neurons at clamp potentials of between -30 and +120 mV. At large depolarizing potentials between +50 and +120 mV, the current was outward. This outward current was shown to be largely due to ions passing through calcium channels, because it was substantially although generally incompletely blocked by Cd2+ (1 mM) and omega-conotoxin (1 microM). Internal GTP-gamma-S (100 microM) and to a lesser extent GTP (1 mM) reduced the amplitude and slowed the activation of the outward, as well as the inward calcium channel current. Baclofen (100 microM) reversibly inhibited both the inward and outward currents. These results suggest that the effect of baclofen and G protein activation on calcium channel currents is not due to a shift in the voltage-dependence of channel availability. |
Acetylcholinesterase is secreted in the central nervous system (independently of cholinergic transmission) in a non-classic, non-enzymatic capacity. A light-emitting reaction has recently been established that demonstrates release of this protein from the substantia nigra of a guinea pig with a temporal resolution corresponding to real time, i.e. 'on-line'. In this study the technique has been applied to investigate the significance of this novel phenomenon in the generation of specific types of movement. During locomotion a 'pulsatile' release of acetylcholinesterase occurs much more frequently than in other situations. However, these pulses of released acetylcholinesterase are of shorter duration than the respective periods of locomotion that caused them. Furthermore, as episodes of movement are repeated, the release of acetylcholinesterase becomes less likely. These observations suggest that the phenomenon does not simply reflect ongoing movement. Indeed, chewing behaviour is frequently initiated when acetylcholinesterase release occurs during locomotor activity. Hence, acetylcholinesterase released in association with locomotion may favour the onset of further types of movement. |
The expression of growth-associated protein GAP-43 mRNA in spinal cord and dorsal root ganglion (DRG) neurons has been studied using an enzyme linked in situ hybridization technique in neonatal and adult rats. High levels of GAP-43 mRNA are present at birth in the majority of spinal cord neurons and in all dorsal root ganglion cells. This persists until postnatal day 7 and then declines progressively to near adult levels (with low levels of mRNA in spinal cord motor neurons and 2000 - 3000 DRG cells expressing high levels) at postnatal day 21. A re-expression of GAP-43 mRNA in adult rats is apparent, both in sciatic motor neurons and the majority of L4 and L5 dorsal root ganglion cells, 1 day after sciatic nerve section. High levels of the GAP-43 mRNA in the axotomized spinal motor neurons persist for at least 2 weeks but decline 5 weeks after sciatic nerve section, with the mRNA virtually undetectable after 10 weeks. The initial changes after sciatic nerve crush are similar, but by 5 weeks GAP-43 mRNA in the sciatic motor neurons has declined to control levels. In DRG cells, after both sciatic nerve section or crush, GAP-43 mRNA re-expression persists much longer than in motor neurons. There was no re-expression of GAP-43 mRNA in the dorsal horn of the spinal cord after peripheral nerve lesions. Our study demonstrates a similar developmental regulation in spinal cord and DRG neurons of GAP-43 mRNA. We show moreover that failure of re-innervation does not result in a maintenance of GAP-43 mRNA in axotomized motor neurons. |
The discovery of molecular markers which are selectively expressed during the development of specific classes of rat central nervous system macroglia has greatly advanced our understanding of how these cells are related. In particular, it has been shown in tissue culture that oligodendrocytes and some astrocytes (type-2) may be derived from a common progenitor cell (O-2A progenitor). However, the existence of type-2 astrocytes in vivo has yet to be unequivocally established. Recently, it has been reported that the neural-specific growth-associated protein-43 (GAP-43, otherwise known as B-50, F1, pp46 and neuromodulin) may be expressed by cells of the O-2A lineage in vitro. We set out to examine the cellular specificity of GAP-43 in O-2A progenitors and their descendants in vitro and in vivo. Using a polyclonal antiserum against a GAP-43 fusion protein we have shown the presence of immunoreactive GAP-43 in the membranes of bipotential O-2A glial progenitor cells and type-2 astrocytes by Western blotting and immunocytochemistry of cells in culture. In contrast to previous studies, double labelling with mature oligodendrocyte markers showed that GAP-43 is down-regulated during oligodendrocyte differentiation in vitro. Immunohistochemical staining of sections of developing rat brain demonstrated the same developmental regulation of GAP-43, suggesting that oligodendrocytes only express GAP-43 at immature stages. In addition, normal and reactive astrocytes in tissue sections were not labelled with GAP-43. |
Nerve growth factor (NGF) induces transient Fos-immunoreactivity (Fos-IR) independently of any other factor, both in newly isolated rat sympathetic neurons and in established cultures after NGF deprivation. The same proportion of neurons that express Fos-IR in response to NGF also survive. In addition to direct stimulation of Fos-IR expression, the presence or recent exposure to NGF is required to obtain Fos-IR expression by other stimuli. In newly isolated neurons no Fos-IR is detected in response to stimulation by serum alone and a response to depolarization or cyclic AMP is obtained only if neurons are stimulated within a short period after ganglion excision. In established cultures none of these stimuli, nor the trauma of cutting neurites or spiking cell bodies with a microinjection needle induce Fos-IR unless NGF is present or had been removed for <8 - 16 h. The lack of response is not due to a general decrease in the rate of protein or RNA synthesis. These findings show that in regenerating sympathetic neurons NGF induces c-Fos and suggest that NGF may activate a master trigger that is required for c-Fos expression to be induced by other stimuli. |
4-hydroxyphenylpyruvate dioxygenase (HPD) (EC 1.13.11.27) is a key enzyme involved in tyrosine catabolism. Congenital HPD deficiency is a rare, relatively benign condition known as hereditary type III tyrosinemia. The severe type I tyrosinemia, caused by a deficiency of fumarylacetoacetate hydrolase which functions downstream of HPD in the tyrosine degradation pathway, is often associated with decreased expression of HPD, and interestingly, inhibition of HPD activity seems to ameliorate the clinical symptoms of type I tyrosinemia. The HPD gene was previously mapped to the chromosomal region 12q24-->qter. In the present study high-resolution chromosome mapping localized the HPD gene to 12q24.31. DNase I footprinting, revealed that four regions of the HPD promoter were protected by rat liver nuclear proteins. Computer-assisted analyses suggested that these elements might bind Sp1/AP2, HNF4, HNF3/CREB, and C/EBP, respectively. In transient transfection experiments, the proximal 271bp of the promoter conferred basal transcriptional activation in human Chang cells. Sequences in intron 1 were able to enhance the activity of this basal promoter. Finally, vaccinia virus-based expression provided evidence that HPD is subject to phosphorylation, and furthermore, allowed mapping of the HPD protein in the human keratinocyte 2D database. |
The 90kDa heat shock protein (Hsp90) is one of the most abundant protein and essential for all eukaryotic cells. Many proteins require the interaction with Hsp90 for proper function. Upon heat stress the expression level of Hsp90 is even enhanced. It is assumed, that under these conditions Hsp90 is required to protect other proteins from aggregation. One property of Hsp90 is its ability to undergo autophosphorylation. The N-terminal domain of Hsp90 has been shown to contain an unusual ATP-binding site. A well-known inhibitor of Hsp90 function is geldanamycin binding to the N-terminal ATP-binding site with high affinity. Recently it was shown that Hsp90 possesses a second ATP-binding site in the C-terminal region, which can be competed with novobiocin. Autophosphorylation of Hsp90 was analysed by incubation with gamma(32)P-ATP. Addition of geldanamycin did not interfere with the capability for autophosphorylation, while novobiocin indeed did. These results suggest that the C-terminal ATP-binding site is required for autophosphorylation of Hsp90. |
To investigate the regulating role of protein kinase C(PKC) and the effects of losartan intervention on the expression of pulmonary arterial collagen in chronic hypoxic rat models. |
To study the relationship between tau hyperphosphorylation and the function of glutamate transporter okadaic acid (OA), a protein phosphatase inhibitor, 20 ng in a 0.5 microl volume, was injected into the frontal cortex of rat brain and immunostaining was used to observe the phosphorylation of tau protein and the expression of excitatory amino acid transporter 1 (EAAT1) in the brain following the injection. The results showed that (1) the neurons in the center of the injection region displayed cytoplasmic shrinkage, swelling, nuclear pyknosis, and dislocation at the early stage, and necrosis appeared 3 d after the injection. However, most neurons in the peri-injected areas showed normal morphological characters with immuno positive reaction for AT8, a tau phosphorylated marker; (2) morphological analysis showed that tau hyperphosphorylation caused by OA treatment was mainly observed in the axons and dendrites of neuronal cells at 6 h in the cell body at 1 d, which brought about dystrophic neurites and neurofibrillary tangle (NFT)-like pathological changes; (3) the induction of glutamate transporter EAAT1 was observed in the involved areas corresponding to that with AT8 immunopositive staining, and the number of EAAT1-positive staining cells markedly increased at 12 h (P<0.01), peaked at 1 d (P<0.001), then decreased at 3 d following the injection. Combined with a confocal laser scanning microscopic analysis, double fluorescent immunostaining showed that EAAT1 positive staining appeared in neurons as well as astrocytes in the peri-injected areas of the frontal cortex. These results demonstrate that OA increases glutamate transporter EAAT1 expression in neurons while it induces tau hyperphosphorylation. However, the mechanism and significance of the induction of glutamate transporter EAAT1 expression remain to be further elucidated. |
To explore the changes in adrenomedullin (ADM) and receptor activity-modifying protein 2 (RAMP2) mRNA in myocardium and vessels in hypertension, a hypertensive rat model was prepared by administering L-NNA. Contents of ADM in plasma, myocardium and vessels were measured by radioimmunoassay (RIA). The levels of pro-ADM mRNA of myocardium and vessels were determined by competitive quantitative RT-PCR. The results showed that L-NNA induced hypertension and cardiomegaly. The ratio of heart to body weight increased by 35.5% (P<0.01). In hypertensive rats the ir-ADM in plasma, myocardium and vessels was increased by 80%, 72% and 57% (P<0.01), respectively compared with the control. The amounts of ADM mRNA in myocardium and vessels were increased by 50% and 109.2% (P<0.05), respectively, and the amounts of RAMP2 mRNA was increased by 132% and 87% (P<0.01), respectively, compared with control. The levels of ADM in myocardium and vessels were positively correlated with RAMP2 mRNA, the correlation coefficients were 0.741 and 0.885 (P<0.01), respectively. The results obtained indicate that in hypertensive rats, ADM is elevated in plasma, myocardium and ves-myocardium and vessel, and ADM and RAMP2 mRNA are up-regulated in myocardium and vessel. The ADM/RAMP2 system may play an important role in the pathogenesis of hypertension. |
The activity of NADH-cytochrome b(5) reductase (b(5)R) and the levels of hydrogen peroxide in the thyroid of patients with Graves' disease (GD) and normal controls were measured with potassium ferricyanide as substrate and with the homovanillic acid fluorescence assay. The activity of b(5)R and the level of H2O2 in GD thyroid were definitely higher than those in normal control, but the activity of catalase in GD thyroid was not significantly different from that in normal thyroid. After addition of p-chloromercuribenzoate, a b(5)R inhibitor, the activity of b(5)R in GD and normal thyroid decreased by 85%, the level of H2O2 decreased by 50%, and protein-bound iodine (PBI) formation by 52%. There is a positive correlation between the level of H2O2 and the activity of b(5)R. Our data indicate that b(5)R participates in thyroid hydrogen peroxide biosynthesis, and is an important enzyme in the production of H2O2. |
The development of new solid catalysts for use in industrial chemistry has hitherto been based to a large extent upon the empirical testing of a wide range of different materials. In only a few exceptional cases has success been achieved in understanding the overall, usually very complex mechanism of the chemical reaction through the elucidation of individual intermediate aspects of a heterogeneously catalyzed reaction. With the modern approach of combinatorial catalysis it is now possible to prepare and test much more rapidly a wide range of different materials within a short time and thus find suitable catalysts or optimize their chemical composition. Our understanding of the mechanisms of reactions catalyzed by these materials must be developed, however, by spectroscopic investigations on working catalysts under conditions that are as close as possible to practice (temperature, partial pressures of the reactants, space velocity). This demands the development and the application of new techniques of in situ spectroscopy. This review will show how this objective is being achieved. By the term in situ (Lat.: in the original position) is meant the investigation of the chemical reactions which are taking place as well as the changes in the working catalysts directly in the spectrometer. |
The visual pigment rhodopsin constitutes the interface between the physical event of light absorption and the biochemical process of visual transduction within the photoreceptor cells. The signal transduction is initiated by an 11-cis→all-trans photoisomerization of the retinal chromophore of rhodopsin which causes a series of thermally driven conformational changes of the chromophore and the protein moiety. A rhodopsin conformation is generated which allows interaction with a heterotrimeric G-protein. Two recent publications follow the chromophore motions after light absorption by cross-linking experiments and by solid-state NMR spectroscopy. |
Semicrystalline microparticles of high molecular mass polyethylene can be prepared as aqueous dispersions by catalytic emulsion polymerization of ethylene with a mini-emulsified catalyst precursor (D$\rm{\frown}$D=salicylaldimine, L=pyridine). Stable polymer latexes are formed. |
The covalent linkage of a hairpin polyamide, which binds in the minor groove, to camptothecin provides an efficient system to direct topoisomerase I mediated DNA cleavage to specific sites. These conjugates are equally as potent at targeting the enzyme to a single site in a DNA fragment as camptothecin conjugates of ligands that bind in the major groove (triplex-forming oligonucleotides). |
Antimony potassium tartrate (APT) is a complex salt that until recently was used worldwide as an anti-schistosomal drug. APT was efficacious in humans only if administered intravenously at a near-lethal total dose of 36 mg/kg. Because unconfirmed epidemiologic studies suggested a possible association between APT treatment and bladder cancer, prechronic toxicity studies were initiated with APT to select a route of administration and appropriate doses in the event chronic studies were needed. To determine the most appropriate route for longer-term studies, toxicity and concentrations of tissue antimony were compared in F344/N rats and B6C3F1 mice that were administered APT in drinking water or by i.p. injection for 14 or 16 days. The animals were assigned to dose groups, 5/sex/species. Drinking water doses, estimated by water consumption, were 0, 16, 28, 59, 94, or 168 mg/kg in rats and 0, 59, 98, 174, 273, or 407 mg/kg in mice; i.p. doses were 0, 1.5, 3, 6, 11, or 22 mg/kg in rats and 0, 6, 13, 25, 50, or 100 mg/kg in mice. APT was poorly absorbed and relatively nontoxic when given orally. There was no mortality or histopathological lesions in rats or mice receiving doses of APT as high as 168 or 273 mg/kg, respectively. One mouse in the highest dose group (407 mg/kg) died, and there were treatment-related lesions in the liver and forestomach of most mice in this dose group. In contrast, i.p. administration of the drug was much more toxic, resulting in the deaths of rats administered 22 mg/kg; kidney and liver lesions were found in these rats. In mice, i.p. administration of APT caused deaths and liver lesions at dose levels one-fourth of those that caused similar effects by oral administration. All male and female mice injected with 100 mg/kg APT died; half of the female mice given 50 mg/kg APT died; additional deaths occurred with doses as low as 6 mg/kg. Hepatocellular necrosis and inflammation of the liver capsule were present in both sexes of mice in the 50 mg/kg dose groups. As a result of these findings, an i.p. dose regimen was selected for subsequent studies. Groups of ten male and female F344/N rats and B6C3F1 mice were given 0, 1.5, 3, 6, 12, or 24 mg/kg doses of APT 3 times per week for 13 weeks by i.p. injection. Rats were more sensitive than mice to the toxic effects of APT, exhibiting dose-related mortality and reduction in body weight. Four male rats in the 24 mg/kg dose died; body weights in both sexes of rats from this dose group and in male rats from the 12 mg/kg dose group were 10-20% below controls. No clinical signs of toxicity in the mice, nor gross or microscopic lesions, could be attributed to APT. Increased concentrations of antimony, considered to be dose-related, were detected in the blood, liver, kidney, spleen, and heart of rats, and in the liver and spleen of mice. In rats, hepatocellular degeneration and necrosis were associated with dose-related elevations in activities of the liver-specific serum enzymes, sorbitol dehydrogenase and alanine aminotransferase. By alternating the site of abdominal injection and the days of treatment, mesenteric inflammation at the site of administration was minimized in the rats and mice, indicating that the i.p. route would be suitable for chronic studies. Hepatotoxicity in rats occurred in dose groups where there was little evidence of renal toxicity and no cardiac toxicity; thus, serial measurement of liver-specific serum enzyme activities may be useful to monitor the presence and progression of hepatocellular degeneration in longer-term exposures. Synonyms: APT; Tartar emetic; tartrated antimony; tartarized antimony; potassium antimonyltartrate; Bis [μ-[2,3-dihydroxy- butanedioato(4-)-O1, O2:O3, O4]]-diantimonate dipotassium trihydrate (stereoisomer). |
A spontaneous high expression of heat shock protein 70 (HSP 70) was found to arise in zebrafish (Danio rerio) at the larval stage (84 hr after fertilization). The level of HSP 70 in 84-hr-old larvae was estimated to be six- to eightfold that of 12-hr-old embryos. As heat-induced HSP 70 synthesis in many eukaryotic organisms is known to be mediated by a transcriptional-dependent pathway activated by heat shock factor 1 (HSF-1), we then examined if the spontaneous and heat-induced HSP 70 synthesis in zebrafish were controlled by the same mechanism. Although the transient increase of a 62-kDa HSF-1-like polypeptide in 72- to 96-hr-old larvae seemed to correlate with the onset of the spontaneous HSP 70 production, an anti-HSF-1 antibody cocktail supershifted the heat shock element (HSE) binding complex induced by stressed but not by unstressed zebrafish extracts. Northern blot and quantitative RT-PCR analysis demonstrated the predominant presence of the cognate form of hsp 70 mRNA (hsc 70 mRNA) in developing zebrafish. The extent of heat-induced HSP 70 production in 84-hr-old larvae matched well with a dramatic increase in hsp 70 mRNA accumulation, while no apparent increase in total hsp 70 mRNA could be detected in 72- to 84-hr-old unstressed larvae by northern blot analysis. The stable expression of hsc 70 mRNA specific to beta-actin mRNA in normal zebrafish was confirmed by RT-PCR analysis. Hence, the spontaneous high expression of HSP 70 in zebrafish is believed to be controlled by a mechanism different from the HSF-1-dependent transcriptional activation of hsp 70 under heat stress. J. Exp. Zool. 293:349-359, 2002. |
We previously produced four monoclonal antibodies to testicular proteins of a teleost, the Nile tilapia. One of the monoclonal antibodies, TAT(Testicular Antigen of Tilapia)-10, recognizes a Mr=27,000 protein (27 kD protein), which is present in A and early B type spermatogonia, spermatids, and spermatozoa in testis. In order to clarify the function of this protein, molecular cloning was conducted. The cDNA for the 27 kD protein contains a complete open reading frame encoding 220 amino acid residues. The predicted amino acid sequence of the 27 kD protein was homologous to those of the ubiquitin carboxy-terminal hydrolases (UCH) reported in mammals. The measurement of the ubiquitin-releasing activity of the recombinant 27 kD protein revealed that the protein is the active form of UCH. Northern blot analysis showed that the UCH mRNA was expressed in ovary and brain in addition to the testis. Immunohistochemical study showed that, in brain, UCH was localized especially on the olfactory organ including the olfactory bulb and olfactory epithelium in olfactory rosetta, suggesting the involvement of the protein in chemoreceptive function. In the Tilapia ovary, UCH localized especially in pre-vitellogenic oocytes, suggesting that the enzyme activity could be important in oocyte growth. This is the first report for the cDNA cloning and cellular localization of UCH in fish. J. Exp. Zool. 293:368-383, 2002. |
Previous studies have revealed that although the brains of cypriniform teleosts (iberian barb, Barbus comiza; carp, Cyprinus carpio; goldfish, Carassius auratus) are rich in glial fibrillary acidic protein (GFAP), they have, however, areas devoid of GFAP immunoreactivity. The largest ones of these are in the rhombencephalon, e.g., the zones of the sensory and motor neurons in the vagal lobe. Our studies in amniotes suggested that the GFAP immunonegative areas could be characteristic of the more advanced brains (avian and mammalian), whereas no similar areas were found in reptiles. A similar tendency was found in the Chondrichthyes, i.e., GFAP immunonegative areas appeared as brain complexity progressed. The question arose whether the GFAP immunonegative brain areas in the Teleostei were also the result of such a tendency. Within the radiation of ray-finned fishes (Actinopterygii), Chondrostei represent a less advanced level as compared to the Teleostei. Therefore, the distribution of GFAP immunoreactivity was investigated in the rhombencephalon of the sterlet (Acipenser ruthenus) as a representative of Chondrostei, and in the carp. Serial vibratome sections were processed according to the avidin-biotinylated horseradish peroxidase method.Several comparable GFAP immunoreactive structures were found in the two species: the dense periventricular ependymoglial plexus, the midsagittal glial septum, the small glial septa separating the nerve fiber bundles, and the wide glial endfeet lining the meningeal surface. In the vagal lobe in the zones adjacent to the meningeal and ventricular surfaces, the glial structures also proved to be similar. In contrast to the carp, however, no areas were found devoid of GFAP immunoreactivity in the sterlet.The results suggest that this trend of glial evolution, i.e., GFAP immunonegative areas appearing as brain complexity progressed, is a common feature shared by Actinopterygii, Amniota, and Chondrichthyes, despite their separate evolutionary histories. J. Exp. Zool. 293:395-406, 2002. |
Morphological and chemical studies on the cuticle during the molt cycle of the crab Scylla serrata were performed in order to understand the layer formation. Cuticle ultrastructure was studied by scanning electron microscopy (SEM). Energy-dispersive, X-ray diffraction, and X-ray fluorescence analysis were used for identification of the elements and phases in the inner surface of the cuticle. In the first stage (A) of cuticle formation, a thin pellicle organized as an irregular fragmented structure is built. It is composed mainly of alpha-chitin/protein beta-keratin-like complexes where heterogeneous mineral nucleation occur. It is impregnated by ferric concretions, responsible for the brown colour of the carapace. At the beginning of the mineralization process, a spheroidal inorganic phase appears consisting of dicalcium phosphate dihydrate (DCPD) Ca/P=1.00, octacalcium phosphate (OCP) Ca/P=1.33 associated with hydromagnesite and bromapatite traces. During further cuticle development in the remaining A stage and in the beginning of the B stage, calcite and magnesian calcite are formed from the precursor calcium phosphate phase. The next development in the C stages is characterized by intense calcareous thickening consisting mainly of calcite and of magnesian calcite, which become the major mineral fraction of the cuticle. Organic-inorganic complex precipitations exhibit different aspects as spongiform, filamentary helicoidal, and concentric radial arrangements during C1, C2, and C3, respectively. During different stages of the cuticle formation in Scylla serrata, these mineral deposits may partially result from the balance among different organic contents, mainly between alpha-chitin and protein beta-keratin-like compounds. On the other hand, the calcium crystallization on apatite and calcite polymorphic structures may be influenced by variations of physico-chemical factors in the cuticle compartment. J. Exp. Zool. 293:414-426, 2002. |
This study was intended to establish a method of purification of HPV16 L1 protein expressed in a prokaryotic system and to obtain the purified protein. The prokaryotic expression vector pGEX-4T-1-HPV16 L1 was constructed and transformed into E. coli BL21 cell, and induced by 1 mM IPTG to express HPV16L1 protein. The inclusion bodies were isolated and solubilized with 8 M urea. After the urea was removed by gradual dialysis, the denatured L1 protein were renatured and then were purified by affinity chromatography. The results showed that HPV16L1 protein formed inclusion bodies in bacterial expression system, suggesting that this assay can be used to purify HPV16L1 protein and hence provide a basis for studying the applications of HPV16 L1 protein. |
Cellular mechanics is a branch of tissue engineering and cellular engineering. As one of the important method, loading different mechanical stimuli to culturing cells in vitro so as to study the influence that the stress has on the cells is one of the important fields of cellular mechanics. This paper reviews the experimental methods for mechanically stimulating the cells in vitro, according to the different loading modalities, the methods can be categorized into micropipette aspiration, compression loading, substrate distention, fluid shear, etc. And it also points out their advantages and disadvantages. |
The A mating-type locus of the mushroom Coprinus cinereus contains three or more paralogous pairs of genes encoding two families of homeodomain proteins (HD1 and HD2). A successful mating brings together different allelic forms of at least one gene, and this is sufficient to trigger initial steps in sexual development. Previous studies have suggested that development is regulated by heterodimerization between HD1 and HD2 proteins. In this report, we describe 5[prime] gene deletions and 5[prime] end exchanges showing that the N-terminal regions of the proteins are essential for choosing a compatible partner but not for regulating gene transcription. Using an in vitro glutathione S-transferase association assay, we demonstrated heterodimerization between HD1 and HD2 proteins and found that heterodimerization only occurs between compatible protein combinations. The N-terminal regions of the proteins were sufficient to mediate dimerization, and N-terminal swaps resulted in a predicted change in dimerization specificity. By analyzing the N-terminal amino acid sequences of HD1 proteins, we identified two potential coiled-coil motifs whose relative positions vary in paralogous proteins but are both required for in vivo function. |
By means of in situ hybridization and immunohistochemistry, the protein localization and gene expression of cyclin B1 in spermatogenic cells were characterized during the spermatogenesis of rabbits. The results showed that the cyclin B1 mRNA in rabbit spermatogenic epithelium was expressed dominantly in primary spermatocytes. The expression was observed in round spermatids with a gradual decline in the process of metamorphosis of the spermatids, but not in elongated spermatids and sperms. Cyclin B1 protein was expressed in mitotic spermatogonia and meiotic spermatocytes and was observed predominantly in round and elongated spermatids. These results indicate that the expression of cyclin B1 mRNA and localization of cyclin B1 protein are dependent on the developmental stages of spermatogenic cells. |
Neomycin is one of the aminoglycoside antibiotics, and on the cellular level it inhibits phospholipase C. The effects of neomycin on the acetylcholine (ACh)-induced current (I(ACh)) were studied in pheochromocytoma (PC12) cells by using the whole-cell clamp technique. The I(ACh) on PC12 cells proved to be generated through activation of the neuronal nicotinic receptor. ACh (30 micromol/L) induced an inward current at a holding potential of -80 mV. When the cells were applied with neomycin (0.01~1 mmol/L) and ACh (30 micromol/L) simultaneously, an inhibitory effect of neomycin on the peak of I(ACh) was observed. This effect was fast, reversible and concentration-dependent. Pretreatment with neomycin for 3~8 min had no influence on its inhibitory effect. Activation of protein kinase C by using an exogenous activator exerted an inhibitory action on I(ACh). However, intracellular dialysis with a PKC inhibitor (PKCI 19-31, 0.1~5 micromol/L) did not affect the inhibitory effect of neomycin. The results obtained suggest that neomycin exerts an inhibitory effect on I(ACh) without involvement of the blockage of phospholipase C. |
To observe the expression of a protein inducible by transforming growth factor-beta (betaig-h3) in normal cornea and keratoconus and discuss the effects of extracellular matrix on keratoconus. |
To explore the effects of As(2)O(3) on BCR-ABL protein level and signal transduction in chronic myeloid leukemia (CML) cells. |
To study the nature of extracellular matirx (ECM) remodeling and its role in airflow obstruction in a rat model of chronic obstructive pulmonary disease (COPD), and to observe the role of nacetylcystein (NAC), protein kinase C (H(7)) and TGF-beta monocolonal antibody in the regulation of extracellular matrix remodeling in the airway wall. |
Reducing the loss of energy by a patient during laparoscopic surgery supports better quality of life. Homo sapiens are homeothermic and are thus only able to effectively tolerate temperature changes ranging from 37+/ -O.5°C. Maintenance of body temperature both intra- and post-operatively is essential. Variations in temperature may bring about changes in enzyme reactions and metabolic processes in the body. Maintenance of body temperature in the central core, including the brain, thoracic and abdominal organs, is most vital. |
To determine whether Gilbert's syndrome increases the risk of gallstone formation in children with chronic hemolytic disease, we studied 44 children with this diagnosis. Gallstones were detected by abdominal ultrasonography. This took place annually in scheduled examinations or in the context of acute abdominal pain. In all patients, the mean values of hemoglobin, reticulocyte and serum bilirubin in the chronic phase were recorded. In addition, TA insertion in the A(TA)nTATAA motif within the promoter region of the enzyme uridine-diphosphate-glucuronyl transferase (UGT1A1) was screened, since this is typically associated with GS.We found 10 (22.7 %) homozygotes for the mutated allele TA*7/TA*7, 12 (27.3 %) TA*6/TA*6 heterozygotes and 22 (50 %) homozygotes for the wild-type allele TA*6/TA*6. No statistically significant differences were found in the values of hemoglobin (Kruskal-Wallis test 2.496; p > 0.05) or in reticulocyte count (Kruskal-Wallis test 1.696; p > 0,05) between the three groups of patients, suggesting a similar degree of hemolysis. Patients with the UGT1A1 TA*7/TA*7 genotype showed higher mean serum bilirubin levels than did patients who were homozygous for the wild-type allele (Mann-Whitney test 35.5; p < 0.05). None of the patients with the TA*6/TA*6 genotype developed gallstones, whereas this complication was found in 2 of 12 (16.6 %) heterozygotes and 6 of 10 (60 %) homozygotes for the allele with TA insertion. In this latter group, 4 patients presented acute pancreatitis as a consequence of gallstone formation.The association between increased bilirubin load due to chronic hemolytic disease and diminished hepatic conjugation leads to raised serum bilirubin levels and consequently to an increased risk of gallstone formation. Therefore, we recommend screening for Gilbert's syndrome in children in the initial phases of chronic hemolytic diseases. |
Although plasma cholesterol levels are not generally associated with Alzheimer disease (AD) incidence, in vitro studies have found that increased cellular cholesterol levels are associated with increases in -amyloid (A ) production, with a concomitant decrease in sAPPa, the secreted non amyloidogenic fragment of the amyloid precursor protein (APP). In two previous studies using a mouse model for AD-like pathology, non-physiological high-cholesterol diet has been shown to increase plasma and cerebral cholesterol levels, but have resulted in conflicting results on cerebral A levels. In the present study APPSWE male transgenic mice were fed either a chow diet or a physiological high-fat high-cholesterol Western-type diet until the mice reached 1 year of age. Mice fed the Western type diet, compared to the low-fat chow diet, had increased body weight, plasma and cerebral cholesterol levels, as well as a 50% increase in cerebral A levels. Cerebral levels of total APP were not altered while cerebral apoE levels were increased in the mice fed the Western-type diet, versus the chow-fed mice. These data demonstrate that chronic intake of a non-toxic high-cholesterol diet, which is similar to a human diet in fat and cholesterol content, was effective in increasing A levels and further suggests that dietary cholesterol and/or fat may be a risk factor for AD. |
Increased circulating cholesterol has been long linked to an increased risk of coronary artery disease (CAD), and is now linked to an increased risk of developing Alzheimer s disease (AD). We first showed the neuropathologic link between CAD and AD as increased incidence of cerebral senile plaques in both disorders. We then showed that AD-like neuropathology occurred in the brains of cholesterol-fed rabbits; including increased -amyloid (Ab). Currently there are a number of transgenic mouse models of AD that exhibit enhanced Ab pathology if cholesterol diet is administered. Culture studies clearly show that excess cholesterol enhances beta-metabolism of amyloid precursor protein (APP) and production of -amyloidogenic peptides, and that sufficiently reducing cholesterol levels by inhibition of synthesis completely inhibits all beta-metabolism of APP. Our finding that the elevated levels of Ab in rabbits fed cholesterol diet could be cleared from the brain by resuming a control diet prompted the hypothesis that lowering cholesterol levels in the blood of AD patients may be of some clinical benefit. Pilot data suggests that therapeutically lowering circulating cholesterol may attenuate Ab production in the cholesterol-fed rabbit brain, may stabilize cognitive performance in mildly impaired AD patients, and may reduce the risk of developing AD. Accordingly, we have initiated a double-blind treatment trial evaluating Atorvastatin Na+ among 120 mild-to-moderately impaired AD subjects randomized to one of two groups receiving placebo or active drug once a day. Atorvastatin is one of a general class of HMG-CoA reductase inhibitor drugs called statins that lower cholesterol by inhibition of synthesis. We chose to use Atorvastatin in this AD Treatment Trial because it does not cross the blood-brain-barrier, and believe it would be ill-advised to use a statin that does. This position stems from the observations that excess cholesterol inhibits cholesterol synthesis and increases Ab production, that Ab kills cells in part by inhibiting cholesterol synthesis, and that statins acting at the neuronal level could further exacerbate degeneration in AD by further inhibition of necessary cholesterol synthesis. |
This review summarizes the recent studies to understand the mechanisms of sarcopenia of aging. A decrease in mitochondrial and nuclear gene transcriptions in skeletal muscle is likely to be responsible for reduced synthesis rates of muscle mitochondrial protein, myosin heavy chain (MHC) and actin. A decrease in muscle mitochondrial protein synthesis could contribute to reduced mitochondrial function. A decrease in synthesis rate of MHC and actin, the key contractile proteins could be responsible for reduced muscle strength. The MHC synthesis rate seems to result from a selective decrease in transcription of MHC isoforms (MHCIIa and IIx) responsible for fast-twitch fibers. Resistance training increases MHC-I isoform mRNA levels with an overall increase in MHC synthesis rate. Aerobic training increases muscle oxidative enzymes equally in young and old but its impact on overall mitochondrial function remains to be clearly defined. Long-term studies are needed to determine the potential benefits and undesirable effects of replacements of various hormones that decline with aging. An individualized exercise prescription involving both aerobic and resistance training is definitely helpful to overcome many aging-related muscle dysfunctions. |
Myostatin is a recently discovered member of the TGF-b superfamily of genes. It is expressed in skeletal muscle and believed to suppress muscle growth. Myostatin-null mice develop skeletal muscles that are 2-3x larger than wild type mice. Serum and intramuscular concentrations of myostatin-immunoreactive protein are increased in AIDS-muscle wasting and are inversely related with fat-free mass (FFM). |
Advancing age appears to alter the chemical and physical properties of skeletal muscle proteins. Alterations include: reduced contractile, mitochondrial, and enzyme protein synthesis rates, altered expression and post-translational modifications to muscle proteins, reduced maximum voluntary muscle strength, reduced muscle strength per unit muscle mass and muscle power. These age-associated impairments in muscle protein quantity and quality contribute to physical disability and frailty, a loss of independent function, the risk of falling and fractures, and contribute to escalating health care costs. Progressive resistance exercise training is a potent, non-pharmacologic, effective therapy that opposes the impairments in muscle protein quantity and quality in middle age and physically frail adults. In the absence of contraindications to exercise, muscle proteins adapt to an exercise training stimulus despite the depredation of age. The proposed pathogenesis for some of these impairments is briefly reviewed. Evidence that supports the use of progressive resistance exercise training to restore muscle quality and quantity in elderly adults is reviewed. |
To make a thorough study on the clinicopathologic significance of the three-dimensional structural alteration of the p53 protein in papillary thyroid carcinomas and to provide an objective criterion for the evaluation of PTC prognosis. |
To study the effect of selenium (Se) and iodine (I) and the compound of both on the proto-oncogenes c-fos and c-jun mRNA and their protein expression in the cultured rat hippocampus neurons. |
This paper was designed to observe the colocalization of 11beta-HSD1 and GR, and its significance in the rat hippocampus. Immunocytochemical dual-staining showed that not only 11beta-HSD1 but also GR immunoreactive substances were present in the cultured rat hippocampal neurons. Moreover, they were colocalized in the same hippocampal neuron. Synthetic glucocorticoid dexamethasone (DEX) up-regulated the protein expression and activity of 11beta-HSD1 in the cultured hippocampal neurons, as determined by Western blot and thin layer chromatography (TLC) respectively. The transfection of PC12 cells with the plasmid containing promoter sequence of 11beta-HSD1 gene and the reporter gene of CAT enzyme was conducted. DEX up-regulated the reporter gene expression in the system described above. The up-regulation of 11beta-HSD1 and reporter gene expression induced by DEX were both blocked by GR antagonist RU38486. Our study suggests that the colocalization of 11beta-HSD1 and GR in the hippocampus may be implicated in the up-regulation of 11beta-HSD1 expression by glucocorticoids combining to its promoter region, which in turn produces more biologically active glucocorticoids necessary for the binding of low affinity of GR. |
The effects of adrenomedullin (ADM) on the L-type calcium currents (I(Ca,L)) and the mechanism of the signal transduction process were studied. Enzymatically isolated guinea-pig ventricular myocytes were used to measure ICa,L with whole-cell patch-clamp techniques. ADM at the concentrations of 1-100 nmol/L decreased ICa,L in a dose-dependent manner (P<0.05). ADM22-52) (100 nmol/L), a specific ADM-receptor antagonist, completely abolished the ADM-induced inhibition of ICa,L. Pretreatment of the cells with H-89 (10 micromol/L), a specific PKA inhibitor, did not attenuate the effects of ADM. Intracellular application of 10 micromol/L PKC19-36), a specific PKC inhibitor, prevented the ADM-induced inhibition of the ICa,L, while the specific PKC activator PMA could mimic the effects of ADM on the ICa,L. PMA (1 micromol/L) decreased the ICa,L by 32.26+/-4.20%(P<0.05). These findings indicate that ADM can inhibit the ICa,L in guinea-pig ventricular myocytes, and the inhibition is mediated by the specific ADM-receptor and an activation of protein kinase C. |
To explore the effects of ATP concentration in the medium and hypoxia exposure on mitochondrial DNA expression at transcriptional and translational level, rats were exposed to hypoxia in a hypobaric chamber simulating 4000 m above sea level for 3 d (acute hypoxia) or 40 d (chronic hypoxia). Cerebral cortex mitochondria were isolated from control and hypoxia-exposed rats by centrifugation program. The activities of intramitochondrial RNA and protein synthesis were measured respectively by the methods of incorporation of (3)H-UTP or (3)H-Leucine in a cell-free system in vitro in isolated organelle. The effect of different ATP concentrations in medium on incorporation activity of mitochondria from control rat brains was observed. The results showed that there was a 40% reduction in RNA synthesis and a 60% inhibition in protein synthesis in isolated mitochondria in vitro in acute hypoxia exposure compared to control. But in chronic hypoxic exposure, the inhibition of both RNA synthesis and protein synthesis was alleviated, being 72% and 76% of the normoxic control, respectively. Furthermore, the effect of ATP concentration in medium on mitochondrial RNA and protein synthesis in vitro showed two phases. The mitochondrial RNA and protein synthesis were inhibited when ATP concentration was either above or below 1 mmol/L in the incubation medium. These results indicate that hypoxia exposure affects the expression of mtDNA at both transcription and translation levels. It also suggests that the improvement of mitochondrial semi-automation during chronic hypoxic exposure may be at least one of the cellular mechanisms of body adaptation to hypoxia. The regulation of ATP in mitochondrial RNA and protein synthesis is therefore an economic and effective mode of regulation. |
This study was intended to evaluate the effects of hypoxic exposure on gene expression and coordination of cytochrome oxidase (COX) subunits I (COX I) and IV (COX IV) encoded by mtDNA and nDNA respectively in rat cerebral cortex. Male Wistar rats were exposed to hypoxia in a hypobaric chamber simulating high altitude at 5000 m for 2, 5, 15 and 30 d. Control rats were fed outside the hypobaric chamber (the height was 300 m above sea level). Rats were sacrificed and mitochondria from cerebral cortex were isolated by differential centrifugation at each time point. COX I and COX IV proteins in isolated rat cerebral cortex mitochondria were detected by Western blot analysis and mRNA in the cerebral cortex by RT-PCR. The ratios of protein and mRNA were used to estimate the coordinative expression of two subunits. The results showed that COX I mRNA increased significantly at 2 and 5 d, and decreased to the control level at 15 and 30 d; COX IV mRNA remarkably increased at 2, 5 and 15 d, and dropped below the control level at 30 d. The mRNA ratio of COX IV to COX I reached a peak at 15 d, but showed no differences between other time points. The Western blot analysis of COX I and COX IV in isolated rat cerebral cortex mitochondria showed no obvious changes during hypoxic exposure. Our findings demonstrate that hypoxia can affect mRNA expression of COX I and COX IV and their coordination, while protein expression of both subunits are stable and coordinative. This study suggests that the expression of COX I and COX IV proteins during hypoxic exposure is coordinately regulated by post-transcriptional mechanisms. |
The hypothalamic decapeptide gonadotrophin-releasing hormone (GnRH) binds to specific receptors on pituitary gonadotrophs. These receptors belong to the family of G protein-coupled receptors. Their activation leads to phosphoinositide breakdown with generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. These second messengers initiate Ca2+ release from intracellular stores and activation of protein kinase C, both of which are important for gonadotrophin secretion and synthesis. Prolonged activation of GnRH receptors by GnRH leads to desensitization and consequently to suppressed gonadotrophin secretion. This is the primary mechanism of action of agonistic GnRH analogues. By contrast, GnRH antagonists compete with GnRH for receptors on gonadotroph cell membranes, inhibit GnRH-induced signal transduction and consequently gonadotrophin secretion. These compounds are free of agonistic actions, which might be beneficial in certain clinical applications. |
Expression microarray was employed in this study to investigate whether the ion channels and their regulatory elements encoding genes participate in the immune response to Mycobacterium tuberculosis infection. The results of a virulent strain were compared with those of the clinically isolated strains. The data demonstrate that K(+), Na(+), Ca(2+) and Cl(-) channels and their regulatory elements, such as the G protein, receptor and second messenger, protein kinase and protein phosphatase were involved in the immune reaction. The clinical strain affected more types of ion channels and respective regulatory elements. The data provides clues for further scrutiny into the role of ion channels and related elements in the interaction between Mycobacterium tuberculosis and host macrophage. |
MTT analysis and intracellular calcium measurement by using confocal laser scanning microscopy were used to study the possible mechanism of protective effect of pituitary adenylate cyclase activating polypeptide 27 (PACAP27) from beta amyloid protein (Abeta)-induced neurotoxicity. The results showed that treatment with PACAP (less than 0.1 micromol/L) increased the survival and reproductive ability of neuro-2a cells and protected the neuro-2a cells from being injured by Abeta. The protective effect of PACAP27 was reversed by the competitive PACAP receptor antagonist PACAP6-27. An increase in intracellular calcium was observed when the cells were challenged with Abeta and PACAP. But the calcium increase induced by Abeta kept stable for a long time while PACAP caused a transient rise in intracellular calcium. The intracellular calcium increase induced by Abeta was blocked by pretreatment with PACAP for 10 min. It is suggested that the neuroprotective effect of PACAP against neuronal damage induced by Abeta may result from its role in inhibiting the sustained rise in intracellular calcium. |
We examined the effect of endogenous and exogenous nitric oxide (NO) on protein kinase C (PKC) activity induced by angiotensin II (Ang II) in cultured neonatal rat cardiomyocytes. The results are as follows. The activity of PKC was increased by Ang II (0.01-10 micromol/L) in a dose-dependent manner, but decreased by NO precursor L-arginine (L-Arg) (10 micromol/L-10 mmol/L) in a dose-dependent manner in cultured neonatal rat cardiomyocytes. Pretreatment with L-Arg (100 micromol/L) decreased significantly Ang II -activated PKC activity and PKC activity induced by phorbol 12-myristate 13-acetate (PMA) ( 10 micromol/L), a PKC activator. Pretreatment with N(G)-nitro-L-argingie methyl ester (L-NAME), a nitric oxide synthase (NOS) blocker, may inhibit significantly the role of L-Arg on Ang II - and PMA-activated PKC activity. The activity of PKC was also decreased by NO donor sodium nitroprusside (SNP) (10 micromol/L-1 mmol/L) in a dose-dependent manner in cultured neonatal rat cardiomyocytes. Pretreatment with SNP (10 micromol/L) decreased significantly Ang II - and PMA-activated PKC activity. These results indicate that PKC was controlled by both NO and Ang II. PKC may be a cross talk between Ang II and NO in cardiomyocytes. NO abolished the activity of PKC and impaired PKC downstream signaling transduction pathway cascades. |
To investigate the role of activated nuclear factor-kappaB (NF-kappaB) in experimental allergic encephalomyelitis (EAE), the activity and protein expression of NF-kappaB p65 in rat brain tissues, which were extracted from EAE rats at 1, 7, 14 and 21 d respectively after EAE was induced by CFA-GPSCH, were measured with electrophoretic mobility shift assay and immunohistochemistry. The relationship between activated NF-kappaB and symptoms of EAE was also investigated. The results showed that protein expression level and the activity of NF-kappaB were very low in the brain of the control group. After EAE was induced, the activity of NF-kappaB and the level of the protein expression in the brains increased gradually with the development of symptoms and brain pathology of EAE. On d 14, both the activity and the level of protein expression in the brains reached a peak, the positive cells of NF-kappaB were mainly located at the choroid plexuses and subfornical organ, as well as around the regions of sleeve-like lesion foci, which were coincident with the locations of lesions of EAE. The incidence, symptoms, reduction of the body weight and pathology of EAE rats brains at the above locations were most significant. On d 21 the activity of NF-kappaB and level of the protein expression reduced gradually, which was in parallel with a gradual alleviation of the symptoms of EAE rats. After a specific inhibitor of NF-kappaB, PDTC was applied, the symptoms and pathological lesions of EAE rat brain were mitigated markedly. The above results indicate that the dynamic changes in the activity and protein expression of NF-kappaB were in parallel with the changes in symptoms and pathological lesion of EAE rat brains. In conclusion, the activated NF-kappaB in the brain may play a critical role in the pathogenesis of EAE, and application of some inhibitors of NF-kappaB, such as PDTC, may be one of the effective therapeutic methods for prevention and treatment of EAE. |
For studying the expression and distribution of angiotensinogen (AGT), the C-teminus of rat AGT gene was expressed in E.coli. Rabbits were immunized with expressed AGT protein and sera from different rabbits were raised. ELISA showed a high titre (1:25600) of the antiserum. With the antiserum, Western blotting recognized not only the prokaryotic expressed AGT, but also the endogenous AGT protein in liver tissue of both rats and humans. Using this antiserum, immunohistochemistry showed the expression of AGT protein in islet cells of human pancreas as well as in epithelium of human bile duct. These results suggest that the prokaryotic expressed AGT protein is an effective immunogen for the preparation of anti-AGT antiserum. Our present work provides an important tool for study of the pathophysiological role of AGT as well as local renin-angiotensin system. |
The effect of zinc deficiency on proliferation and differentiation of rat osteoblast was studied in vitro. The osteoblasts were isolated from the rat calvaria, and were subcultured in DMEM medium. Zinc deficiency model in cell culture was made by chelating zinc from the medium with a special chelator TPEN. Incorporation of 3H-thymidine was measured to show the DNA synthesis at different time point, and cell cycle was analyzed by flow cytometry. A histochemistry method was used to detect changes of osteoblast cytoskeleton. Incorporation of 3H-proline was measured to show the synthesis of collagen. Alkaline phosphatase activity (AKP) and osteocalcin content were measured by enzyme dynamics method and radioimmunoassay respectively. The results showed that the incorporation of 3H-thymidine in the zinc deficient group was significantly less than that in the control group at different time points. The cells were blocked at G2/M phase of cell cycle, which is related to the impairment of cytoskeleton. Collagen synthesis, osteocalcin content and AKP activity in the zinc deficient group were reduced significantly as compared with those in the control group. But no differences in all the measurements were observed between the control group and the group of zinc deficiency supplemented with zinc. Therefore, it was concluded that zinc deficiency inhibited the proliferation and differentiation of rat osteoblast in vitro. |
In this article, the influence of factors related to different levels (seg, provincial, county and household) on the proportion of protein intake from animal food was evaluated by using multi-level modeling. It was found that after being adjusted by individual factors, the factors related to the household, such as household income, dietary habit and knowledge of nutrition were the most important factors affecting the individual's showed animal protein intake. In addition, the results showed that the difference of animal protein intake in various places depended not only on the increase of household income, but also dietary behavior and agriculture crops in different areas. Comparison of different models indicates that in analyzing the data from large-scale health survey with hierchical structure, multi-level modeling is strongly recommended. |
There is evidence that the myocytes produce dynorphin and dynorphin-like peptides, which are kappa opioid receptor (kappa-OR) agonists. Activation of kappa-OR, a dominant opioid receptor in the heart, alters the cardiac function in vivo and in vitro. The observations suggest that the endogenous kappa-opioid peptides may act as autocrines or paracrine in regulation of cardiac functions. Myocardial ischemia is a common cause of heart disorders, which is manifested in decreased myocardial performance, arrhythmia and infarct. When myocardial ischemia occurs, the sympathetic discharge increases, which in turn increases the work-load and oxygen consumption. This exacerbates the situation induced by ischemia. One of the mechanisms with which the body protects against ischemia-induced injury/arrhythmia is inhibition of stimulation of beta-adrenoceptor (beta-AR), the receptor mediating the actions of sympathetic stimulation. kappa-Opioids inhibit the beta-AR activation. The inhibition of the beta-AR activation is due to inhibition of Gs-protein and to a lesser extent the adenylyl cyclase of the signaling pathway mediating beta-AR stimulation by a pertussis sensitive G-protein that mediates kappa-OR activation. Another mechanism against ischemia-induced injury is preconditioning, which is defined as prior exposures to ischemia or other insults make the heart more tolerant to subsequent and more severe insults. Protection occurs immediately or 1-3 days after preconditioning. kappa-OR mediates protection of preconditioning with ischemia or metabolic inhibition, one of the consequences of ischemia, in the heart. Activation of kappa-OR by U50488H, a selective kappa-OR agonist (pharmacological preconditioning with U50488H, UP), activates protein kinase C (PKC), opens K(ATP) channels and increases the production of heat shock proteins. Blockade of PKC, or closing of the K(ATP) channels or inhibition of the synthesis of the heat shock protein abolishes the cardioprotection of UP. The findings indicate the important roles of PKC, the K(ATP) channels and the heat shock protein in cardioprotection of UP. In addition, UP also attenuates the Ca(2+) overload, a precipitating cause of cardiac injury, induced by ischemic insults, indicating that UP may confer cardioprotection via at least partly attenuating the Ca(2+) overload. Most interestingly, blockade of the K(ATP) channels with channel blockers, that abolishes the delayed cardioprotection of UP, also attenuates the inhibitory effect of UP on Ca(2+) overload, suggesting that the cardioprotective effect of opening of the K(ATP) channels may be due at least partly to the prevention/attenuation of Ca(2+) overload. |
The effect of protein kinase C (PKC) signaling pathway on the activity of voltage-dependent delayed rectifier potassium channel (K(V)) and the expression of K(V) isoform K(V)1.5 in rat bronchial smooth cells (BSMCs) were investigated with whole-cell patch clamp, Western-blot and RT-PCR techniques. The results showed: (1) phorbol 12-myristate 13-acetate (PMA), a PKC activator, caused a significant inhibition of K(V) channel currents in rat BSMCs. The inhibition was partly abolished by Ro31-8220, a PKC inhibitor. (2) PMA caused a significant suppression of the expression of K(V)1.5 mRNA and protein in rat BSMCs. These effects were attenuated by Ro31-8220. The results suggest that in rat BSMCs PKC activation inhibits K(V) currents and down-regulates the expression of K(V)1.5. |
To define the molecular basis of ethanol dependence, changes in the phosphorylation of cAMP response element binding protein (CREB) in the nucleus accumbens of rats after acute and chronic ethanol administration were detected using immunohistochemistry. The results demonstrate that the expression of phospho-CREB (p-CREB) protein in the rat nucleus accumbens significantly increased after 15 min of acute ethanol exposure, reaching a peak at 30 min after ethanol administration. The increment remained after 1 or 6 h of ethanol exposure compared to the control rats. In contrast, chronic intake of ethanol solution obviously decreased the expression of p-CREB protein compared to the control rats. The decrement remained 24 h or 72 h after ethanol withdrawal, and returned to the control levels after 7 d of ethanol withdrawal. The results suggest that an acute ethanol administration led to an increase in the phosphorylation of CREB in the nucleus accumbens, but chronic ethanol administration produced a decrement, which is possibly one of the molecular mechanisms of alcohol dependence. |
Recent evidence indicates that the aberrant neuronal expression of mitotic proteins in Alzheimer's disease (AD) brain may be related to AD pathological changes. To investigate whether the toxicity of beta-amyloid protein (Abeta) induces mitotic proteins expression in adult rat brain, we used immunohistochemical and integral optical density analytic method to analyze the adult rat brains, which had been injected with Abeta(25-35) into unilateral amygdala. Results showed that the levels of neurofibrillary tangle (NFT) related phosphorylated tau protein and apoptosis related protein Bax were increased in Abeta(25-35) injected rat brains, meanwhile the aberrantly expression of mitotic protein cyclin A and cyclin B1 was also detected at 7 d after operation, but the level of cyclin A decreased and cyclin B1 disappeared at 21 d. Immunofluorescence double labeling presented that cyclin B1 was partially co-localized with Bax or phosphorylated tau protein, whereas Bax and phosphorylated tau protein seldom co-localized. These results suggest that Abeta causes mitotic protein expression in adult brain neurons, which may die through apoptosis or may be affected by AD NFT-related tau phosphorylation. |
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