bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of rat α-lactalbumin and casein mRNA between polyadenylated and nonpolyadenylated RNA
Authors: Nadin-Davis Susan , Mezl Vasek A.
Published: December 2009

mRNAs for the two types of milk protein were determined in the polyadenylated (poly(A)+) and nonpolyadenylated (poly(A)−) RNA fractions prepared from lactating rat mammary gland, to assess if a high fraction of poly(A)− mRNA is a general characteristic of milk protein mRNAs. Use of the [3H]poly(U) assay to evaluate the effectiveness of oligo(dT)-cellulose and poly(U)-Sepharose for the preparation of these samples showed that similar, essentially poly(A)-free, RNA samples could be prepared with either resin when the appropriate sample to resin ratio was used. The poly (A) tracts in rat mammary poly(A)+ RNA displayed bimodal distribution with peaks at 40 and 80 nucleotides. Translation of these samples confirmed previous observations that mammary poly(A)− RNA contains high amounts of translational activity; however, it is shown that translation gives an overestimate of the mRNA activity in this fraction. Analyses with complementary DNAs indicated that only 24–35% of the casein mRNA and 14–33% of the α-lactalbumin mRNA are present in poly(A)− RNA. Milk protein mRNAs are, therefore, comparable with other abundant mRNAs. Evidence is presented indicating that partial degradation can greatly reduce the extent of polyadenylation with little effect on the amount and on the translational activity of the mRNA.
Comparison of the effects of heat shock and metal-ion stress on gene expression in cells undergoing myogenesis
Authors: Atkinson Burr G. , Cunningham Tanja , Dean Rob L. , Somerville Martin
Published: December 2009

Subjecting 9-day-old quail embryos to an elevated temperature in ovo causes limb, breast, and brain tissues to shift their patterns of protein synthesis from the production of a broad spectrum of different proteins to the new and (or) enhanced synthesis of a small number of heat-shock proteins (HSPs). The HSPs synthesized by undifferentiated breast tissue in ovo (relative masses (Mrs) 88 000, 82 000, 64 000, and 25 000) are similar to those synthesized by explanted breast tissue or by primary cultures of breast myoblasts heat-shocked in culture. Heat-shocked, 120-hour-old myotube cultures synthesize HSPs similar to those detected in heat-shocked myoblasts except that myotubes also exhibit enhanced synthesis of a 55 000 dalton polypeptide and little or no synthesis of a 25 000 dalton HSP; the failure to thermally induce a 25 000 dalton polypeptide in myotubes is related to the fused nature of these cells rather than to their state of differentiation. Myoblasts, as well as myotubes, cultured in the presence of elevated amounts of arsenite, copper, or zinc also synthesize new and (or) enhanced amounts of polypeptides with isoelectric points and immunochemical properties similar to the 25 000 and 64 000 dalton HSPs. However, elevated levels of these metal ions fail to stimulate new and (or) enhanced synthesis of other HSP-like proteins. These results demonstrate that, although the protein synthetic response of myogenic cells to chemical and thermal stress may be similar in some respects, a number of the synthetic responses are clearly different.
Relationship between hyperthermia-induced heat-shock proteins and thermotolerance in Morris hepatoma cells
Authors: Landry J. , Chrétien P.
Published: December 2009

We recently demonstrated that heat treatments, capable of triggering thermotolerance development in MH-7777 cells, also induce a transiently increased synthesis of a small set of polypeptides, a phenomenon similar to the heat-shock-protein (HSP) induction demonstrated in various organisms. A close temporal relationship was also found between HSP synthesis and thermotolerance development, and the degradation kinetics of the newly synthesized HSPs very closely paralleled the decay of the thermotolerant state. In the present study, we extended our analysis to determine whether various experimental conditions known to affect thermotolerance development (duration and temperature of the conditioning treatment, step-down heating, ethanol exposure) will also influence HSP synthesis and, conversely, whether agents known to induce (sodium arsenite) or to impair (cycloheximide) HSP production will similarly affect thermotolerance. The results suggested that the expression of HSP and thermotolerance in MH-7777 cells are most likely regulated by some interrelated mechanisms, but indicated that HSP synthesis is neither a sufficient or necessary condition for thermotolerance development.
Proteases and posttranslational processing of prohormones: a review
Authors: Lazure Claude , Seidah Nabil G. , Pélaprat Didier , Chrétien Michel
Published: December 2009

Pioneering work on insulin and on lipotropin, published in 1967, led to the formulation of the hypothesis that biologically active peptides such as peptide hormones are derived from larger precursor molecules by enzymatic cleavage at basic amino acid pairs. Since then, an ever-increasing number of hormones and active peptides including neuropeptides have been found to be contained within the sequence of a larger precursor protein and are released by limited proteolytic cleavage. The hypothesis concerning the posttranslational maturation of precursor molecules into smaller active entities is now firmly established in many tissues (e.g., pituitary gland, pancreas) and for all kinds of organisms (e.g. mammals, fish), though the nature of the enzyme(s) responsible for this maturation process still remains unknown. This article presents current knowledge of the intracellular processing of precursor molecules, especially the conversion of prohormone to hormone. The discussion deals principally with the evidences concerning the localization of the maturation process mainly in the secretory granules, the role of basic amino acid pairs at the cleavage site, and the possible involvement of serine and (or) thiol endopeptidases in that process.
Somatostatin radioreceptor assay: development and application to the measurement of somatostatinlike activity in the rat central nervous system
Authors: Rorstad O. P. , Schonbrunn A. , Martin J. B.
Published: December 2009

Functional receptors for the neuropeptide somatostatin have previously been characterized in a clonal line (GH4C1) of rat pituitary tumour cells. We have utilized GH4C1 cell receptors to develop a radioreceptor assay for the measurement of somatostatinlike activity in biological samples. The concentration of somatostatinlike activity was determined in various regions of the rat central nervous system by both radioreceptor assay and two radioimmunoassays. For all tissue samples tested, less somatostatinlike activity was measured by radioreceptor assay than by radioimmunoassay. Furthermore, the ratio of the measured activity in the two assays varied from 0.31 to 0.76. It is likely that these differences are at least partially due to the different affinities of GH4C1 cell receptors and the two antibodies for somatostatin-14 and somatostatin-28 which are both present in the rat central nervous system.
Biosynthesis of neurohypophysial hormones: historical and current events
Authors: Moore Graham J. , Rosenior Joseph C.
Published: December 2009

Properties of the neurohypophysial hormones and neurophysins are presented and historical events contributing to our present understanding of the biosynthesis of these molecules are reviewed. Recent findings, including cloning of mRNA and identification of putative giant precursors, are discussed in the context of precursor processing systems and possible biochemical and physiological mechanisms.
Regulation of oxytocin receptor concentration in rat uterine explants by estrogen and progesterone
Authors: Soloff Melvyn S. , Fernstrom Mats Axel , Periyasamy Sumudra , Soloff Solweig , Baldwin Sam , Wieder Michael
Published: December 2009

Incubation of uterine explants from immature rats with 0.01–100 ng of 17β-estradiol/mL resulted in approximately a fivefold increase in the number of oxytocin receptors per milligram of protein in 48 h. This increase was maintained for at least an additional 48 h in the presence of estrogen. When the explants were incubated with 1 μg progesterone/mL from the outset, the concentration of oxytocin receptors was the same as initial (0 time) levels. The estrogen-induced increase in oxytocin receptor concentration was blocked by incubation with cycloheximide, an inhibitor of protein synthesis. Once increased, however, the concentration of oxytocin receptors exhibited no turnover for at least a 48-h period in the presence of estrogen. The addition of progesterone and estrogen to explants with elevated receptor levels resulted in almost a 60% reduction in oxytocin receptor concentration by 24 h, with no change in affinity of the receptor for oxytocin. The reduction in receptor concentration by progesterone was not prevented by cycloheximide. The progesterone effect may involve inactivation or degradation of oxytocin receptors or activation of substances that are inhibitory to oxytocin binding. The effects of estradiol and progesterone on oxytocin receptor concentration in uterine explants are similar to those seen when the steroids are administered in vivo. The explant system, therefore, should prove useful in clarifying factors and processes that are involved in regulation of oxytocin receptor concentration in the uterus and in the initiation of parturition in the rat.
Oxytocin receptors in rat involuting mammary gland
Authors: Soloff Melvyn S. , Wieder Michael H.
Published: December 2009

Oxytocin-receptor concentrations in the rat mammary gland were determined by Scatchard analyses with [3H]oxytocin. There was about a 100-fold increase in the number of receptors per mammary gland between the 1st day of pregnancy and late lactation. The number of receptors then fell markedly during postweaning mammary regression, but rose again during a second pregnancy and lactation cycle. The changes in oxytocin-receptor number corresponded to changes in alkaline phosphatase activity per mammary gland. These results strongly support data suggesting that alkaline phosphatase, like oxytocin receptors, is a specific marker for mammary myoepithelial cells. Despite the fall in oxytocin-receptor number per mammary gland during postweaning regression, the concentration of receptors, expressed per milligram of protein, increased 10-fold over lactating levels on the 6th day of regression. Thereafter, receptor concentrations declined, but were still elevated about fivefold over lactating levels on the 15th day of regression. It is likely that the increased concentration of receptors was due to a decrease in the relative amount of nontarget secretory cells. The factors that regulate the concentration of oxytocin receptors on mammary myoepithelial cells are presently unknown; however, the involuting mammary system may be practical for obtaining enriched populations of oxytocin-sensitive myoepithelial cells.
Insulin binding to differentiating muscle cells in culture
Authors: Klip Amira , Li Grace , Walker Denise
Published: December 2009

Saturable binding of 125I-labelled insulin was detected on L6 myoblasts grown in monolayers. Binding was proportional to the number of cells and was pH sensitive, decreasing above pH 7.0. Binding also decreased with increasing temperature in the range 22–37 °C. Binding of insulin over a range of hormone concentrations (10−10–10−6 M) was analyzed by Scatchard plots. The data are compatible with the coexistence of two components with Kd of 3 and 190 nM. The number of both types of binding sites increased with cell differentiation (upon alignment and cell fusion), when expressed either per unit protein, DNA, or surface membrane cholesterol. On the other hand, their association constants did not vary. Binding of insulin to a nonfusing mutant of L6 cells resembled binding to the parent myoblasts before alignment.
Receptors for insulin and basic somatomedin: immunological and affinity-chromatographic cross-reactivity
Authors: Armstrong Glen D. , Hollenberg Morley D. , Bhaumick Banani , Bala R. Marvin , Maturo III Joseph M.
Published: December 2009

Human placental receptors for insulin and for human basic somatomedin (BSM, analogous to human insulinlike growth factor I (IGF-I)) were selectively cross-link labelled with 125I-labelled insulin and BSM using disuccinimidyl suberate under conditions whereby cross-linking of 125I-labelled insulin to the BSM receptor and of 125I-labelled BSM to the insulin receptor was avoided. We have found that the cross-link-labelled receptors for insulin and BSM, present in equivalent amounts in human placenta membrane preparations, both cross-react with antibodies directed against purified rat liver insulin receptor. Compared with the human insulin receptor, the BSM receptor cross-reacted with the antireceptor antibody to a level of roughly 9%, indicating a limited sequence homology between the insulin and BSM receptors. Since both receptors are present in comparable amounts in solubilized placenta membrane preparations, we sought methods for the selective purification of both receptors from such extracts, so as to provide a basis for further comparative structural studies of the two receptors. We have observed that both receptors were adsorbed by affinity columns of insulin-agarose, in a manner that did not yield insulin receptor entirely free from the BSM receptor. As an alternative for receptor purification, we have found that immunoaffinity columns using antiligand antibody should provide a means for the selective isolation of cross-link labelled receptor from tissues in which both are present in equivalent amounts.
Posttranslational modification of prothyroid hormone
Authors: Eggo M. C. , Drucker D. , Cheifetz R. , Burrow G. N.
Published: December 2009

Posttranslational processing of thyroglobulin may influence its role as the prothyroid hormone. We have examined the role of glycosylation in the subsequent export and iodination of thyroglobulin. We find that glycosylation is necessary both for the movement of thyroglobulin through the cell and for iodination. During posttranslational processing, thyroglobulin is also phosphorylated and carboxymethylated. The implications of these postsynthetic modifications are discussed.
Presence of α-glucosidases in the male reproductive system of the rat and hormonal influences
Authors: Grandmont Anne-Marie , Chapdelaine Pierre , Tremblay Roland R.
Published: December 2009

The use of a synthetic substrate (p-nitrophenyl-α-D-glucoside) to measure α-glucosidase activity has allowed us to demonstrate the presence of acid and neutral α-glucosidases in the reproductive organs of the male rat. Both enzymes increased in the epididymis, particularly in the caput segment, along with initiation of spermatogenesis at puberty; it then started decreasing after 12 weeks of life. Similar variations were not recorded in testis, prostate, and seminal vesicles. Castration led to a significant decrease of acid and neutral α-glucosidases in all accessory reproductive organs, but administration of testosterone proprionate (50 μg/day for 10 days) restored the enzyme activity to its original level. When estradiol-17β (5 mg) was administered simultaneously with testosterone (500 μg), the antagonistic effect of estradiol on testosterone was particularly evident on the levels of neutral α-glucosidases which reached the castration range, while the acid α-glucosidase remained unchanged in epididymis, prostate, and seminal vesicles. These results show that both acid and neutral α-glucosidases may be influenced by gonadal hormones in the male rat.
Regulation of the androgen receptor in human genital skin fibroblasts, with a review of sex steroid receptor regulation by homologous and heterologous steroids
Authors: Pinsky Leonard , Kaufman Morris , Gil-Esteban Carmen , Sumbulian Datevik
Published: December 2009

When normal human genital skin fibroblasts are cultured for 3 days with 2–3 nM methyltrienolone (R1881, a synthetic nonmetabolizable androgen), they augment their specific androgen-receptor activity (two- to four-fold) with a time pattern that is always most rapid in the first 24 h, usually peaks by 48 h, and often declines, sometimes substantially, in the third 24-h interval. The time pattern is highly reproducible within experiments on confluent monolayers and is not influenced by the presence or absence of 10% fetal calf serum in the medium, the temperature (37 vs. 40 °C) at which the incubation is conducted, or the basal activity (up to 40 fmol/mg protein) that is saturated by incubation for 30–45 min at 37 °C. It does appear to be influenced by an unusually rapid increase in the first 24 h and by nonconfluent density of the monolayers, but these factors do not explain the considerable interexperimental variation, within and among cell lines, under nominally identical conditions. The time pattern is interpreted to represent an initial phase of "up-regulation" that is followed by one of compensatory adjustment, despite a constant level of unmetabolized ligand. A review of sex steroid receptor regulation by homologous and heterologous sex steroids reveals numerous examples of apparently homologous regulatory behavior.
Analysis of triacylglycerols by reversed-phase high pressure liquid chromatography with direct liquid inlet mass spectrometry
Authors: Marai L. , Myher J. J. , Kuksis A.
Published: December 2009

Natural triacylglycerols were resolved by high pressure liquid chromatography on Supelcosil LC-18 columns using a gradient of 30–90% propionitrile in acetonitrile as eluting solvent. The effluent was admitted to a quadrupole mass spectrometer via a direct liquid inlet interface. The mass spectra of the solutes were recorded in the chemical ionization mode. The triacylglycerol elution profile was obtained from the total ion current. Individual molecular species of the triacylglycerols were identified from the (MH)+ and the (MH—RCOOH)+ ions. The reversed-phase system allowed the separation of triacylglycerols on the basis of both carbon number and double-bond number, as well as of certain critical pairs of triplets of triacylglycerols with the same partition number. The method is applicable to the determination of triacylglycerol composition of both plant and animal fats. The minimum amount of sample which is required is about 50 ng in the scanning mode. However, 100 times more material must be injected in the liquid chromatograph because only 1/100th of the effluent is being injected into the mass spectrometer.
Metabolism of NG-monomethyl-L-arginine
Authors: Paik Woon Ki , Abou-Gharbia Magid , Swern Daniel , Lotlikar Prabhakar , Kim Sangduk
Published: December 2009

Rat kidney contains an enzyme which hydrolyzes NG-monomethyl-L-arginine to give rise to the formation of ornithine and N-methylurea. Confirmation of formation of these reaction products is carried out colorimetrically as well as radiochemically employing NG-monomethyl-L-[ornithine-14C(U)] arginine. This pattern of reaction products suggests that the enzyme responsible is an arginase type. However, the kidney enzyme is quite distinct from the hepatic arginase; commercial bovine hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is completely inactive toward NG-monomethyl-L-arginine. Among various rat tissues examined, the hydrolytic activity is the highest in kidney, followed by the activity in liver and pancreas.
Theoretical simulation of ionic transport through a transmembrane channel
Authors: Fraga Serafin , Nilar S. H. M.
Published: December 2009

The two-site model of Urry for the ionic conduction through a gramicidin A channel has been confirmed by theoretical calculations, using a method developed at this laboratory, which yields the potential energy profile of the ion along the channel. Preliminary results show that the pore closure could start with the spatial rearrangement of the —HN—CH3 terminal chain under the influence of the ion already in the channel.
An optical stopped-flow and 1H and 113Cd nuclear magnetic resonance study of the kinetics and stoichiometry of the interaction of the lanthanide Yb3+ with carp parvalbumin
Authors: Corson David C. , Lee Lana , McQuaid Gerard A. , Sykes Brian D.
Published: December 2009

The rate constants for the dissociation of the lanthanide Yb3+ from the CD and EF calcium-binding sites of carp parvalbumin (isoelectric point, 4.25) have been measured using optical stopped-flow and 1H nuclear magnetic resonance (NMR) methods. The off-rate constants for Yb3+ are 1.5 × 10−1 and 1.3 × 10−3 s−1 respectively, at pH 6.6 and 23 °C. The relative displacement of Ca2+ from the two sites by Yb3+ was determined from the observed amplitude of the fast and slow kinetic phases. Yb3+-shifted 1H-NMR spectra of parvalbumin are presented as a function of pH, concentration, and H2O:D2O ratio to relate the NMR results to the kinetic and optical results. The displacement of Cd2+ from parvalbumin by Yb3+ was studied using 113Cd NMR. All of the results show the sequential displacement of Ca2+ from the CD and EF sites of parvalbumin by Yb3+. Some results are also presented for Tb3+ and Gd3+.
Characterization of lecithin–cholesterol acyltransferase from human plasma. 3. Chemical properties of the enzyme
Authors: Chong Kui Song , Jahani Mehrnoosh , Hara Shinichi , Lacko Andras G.
Published: December 2009

The polypeptide molecular weight of lecithin–cholesterol acyltransferase (LCAT) (45 000) was obtained by deducting the weight of carbohydrate moiety (25%, w/w) from the total molecular weight of 60 000. LCAT was found to have a relatively high content of glutamic acid, aspartic acid, glycine, and leucine residues and four half-cystines. The carbohydrate content was found to be about 25% (w/w): hexoses, 13%; hexosamines, 6.2%; and sialic acid, 5.4%. The total number of 408 amino acid residues per mole and the mean residue weight of 110.3 were found. From fluorescence spectroscopy analysis, 6–7 mol of tryptophan were found per mole of LCAT in 10 mM phosphate (pH 7.4). However, when LCAT was digested by the mixture of chymotrypsin and pronase the tryptophan residues increased to 10–11 mol/mol of LCAT, which agrees well with data obtained previously by ultraviolet absorption spectroscopy. A partial specific volume of 0.707 mL/g was determined by compositional analysis. Human LCAT was found to have a relatively high extinction coefficient [Formula: see text] of 21 at 280 nm and neutral pH. Two residues of cysteine per mole of LCAT were estimated both in the presence or absence of sodium dodecyl sulfate by titration with 5,5′-dithiobis-2-nitrobenzoic acid. The enzyme showed a lower tendency to staining with Coomassie blue R-250 than bovine serum albumin. The enzyme was rapidly inactivated by diisopropyl fluorophosphate (DFP), regardless of whether the free sulfhydryl were blocked or not. The enzyme was also irreversibly inhibited by cysteine above concentrations of 1 mM. Neither the high density lipoprotein nor liposome substrate was able to protect against the inhibition by cysteine or DFP.
Calmodulin: an introduction
Authors: Stevens Frits C.
Published: December 2009

Calmodulin is the most widely distributed and most versatile member of a family of calcium-binding proteins which probably serve as receptors for the Ca2+ signal. Through the regulation of a wide variety of intracellular enzymes, calmodulin plays a key role in many physiological processes. An understanding of the different mechanisms by which calmodulin exerts its influence is beginning to emerge and an interesting hypothesis regarding the interrelationship between the cAMP and Ca2+ second messenger systems has recently been put forward.
On the mechanism of interaction between calmodulin and calmodulin-dependent proteins
Authors: Wang Kuo Chieh , Mutus Bulent , Sharma Rajendra K. , Lam Hing-Yat Peter , Wang Jerry H.
Published: December 2009

Molecular and kinetic studies of the interaction between calmodulin and calmodulin-dependent proteins have been reviewed. Several calmodulin-dependent proteins have been purified to homogeneity and characterized in terms of subunit structure in recent years. The results indicate that these proteins do not contain a common subunit as the basis of calmodulin binding. A monoclonal antibody capable of interacting with several calmodulin-dependent proteins has been obtained, suggesting that these proteins contain common structure. It seems that hybridoma technology may be used for probing calmodulin-binding domains in the calmodulin-dependent proteins. Using a fluorescent-labelled cyclic nucleotide phosphodiesterase, the interaction between calmodulin and the enzyme in the absence of Ca2+ can be demonstrated, and the equilibrium constant of the reaction can be determined. The study further defines the multiple interactions in the activation of the cyclic nucleotide phosphodiesterase by Ca2+ and calmodulin. Previous kinetic results along with the present results are summarized and used to elucidate the regulatory significance of the multiple Ca2+-binding of calmodulin.
Recent progress towards understanding the roles of the basement membrane in development
Authors: Sanders E. J.
Published: December 2009

A brief description of the structure of basement membranes is presented including results of immunocytochemical investigations. A number of developmental events are considered in which the basement membrane has been implicated in the regulation of morphogenesis. One of the clearest developmental functions appears to be the maintenance of the lobular morphology of glandular epithelia, where the basement membrane supplies a supportive scaffold. The contributions of the glycoproteins laminin and fibronectin to basement membrane function are discussed with reference to potential roles for this structure as a substratum for morphogenetic cell migrations and as a mediator of inductive interactions in embryogenesis. Examples used to illustrate these proposals include the mesodermal migrations of gastrulation, kidney tubule morphogenesis, and limb bud development. Evidence from these and other sources lend support to current view for an active and changing role for the basement membrane in the epithelial–mesenchymal interactions of embryonic development.
Epithelial induction of osteogenesis in embryonic chick mandibular mesenchyme: a possible role for basal lamina
Authors: Exan R. J. Van , Hall B. K.
Published: December 2009

The initiation of osteogenesis at 7 days in the embryonic chick mandibular mesenchyme depends on an epithelial induction in the mandible to day 4. This article reviews a series of experiments conducted to study the nature of this inductive mechanism. Transfilter tissue recombinations were used to determine whether direct tissue apposition was required for induction. Ultrastructural studies of the epithelial–mesenchymal interface were conducted to see if direct epithelial–mesenchymal cell–cell contacts occurred during the inductive stage in vivo. Epithelial cells were cultured on Millipore filters for 28 days and allowed to deposit extracellular products. These products were tested for inductive activity. Findings from these three sets of experiments were discussed with respect to the inductive mechanism. Our results indicate that the induction is not mediated by a diffusible substance and that direct apposition of the two tissues is required. The mechanism of induction, however, does not require direct epithelial–mesenchymal cell to cell contacts. This suggests that a nondiffusible component of the extracellular matrix may be involved. Epithelial extracellular products are inductively active and have the appearance of basal lamina. The active component of the extracellular product is proteinaceous, perhaps collagen, and appears to be situated in the epithelial basal lamina. The role of basal lamina in epithelial–mesenchymal interactions is discussed.
Acrosin: immunochemical demonstration of multiple forms generated from bovine and human proacrosin
Authors: Elce John S. , McIntyre Elise J.
Published: December 2009

A rabbit antiserum was prepared against purified bovine spermatozoal acrosin (EC 3.4.21.10), and the specific immunoglobulin G (IgG) was isolated by immunoaffinity chromatography. This IgG was shown to be monospecific for acrosin by rocket immunoelectrophoresis and by Western blotting of sodium dodecyl sulfate – polyacrylamide gels onto nitrocellulose sheets, followed by indirect immunodetection. In extracts of bovine spermatozoa prepared in the presence of 50 mM benzamidine, a single form of acrosin was detected, having a relative mass (Mr) of 48 000, which is presumed to be proacrosin. At least four further intermediate forms of acrosin were detectable in extracts prepared in the absence of benzamidine and in the various column eluates, having Mr values of 47 000, 44 000, 42 000, and 40 000, while the final product of the purification had a Mr of 37 500. The rabbit antibovine acrosin antiserum reacted also with human acrosin on Western blots. In this way, human proacrosin was found to have a Mr of 50 000 and to be convertible into intermediate forms of Mr 48 500, 44 500, 40 500, and 37 500, while the final product had a Mr of 35 500.
Stripping of proteins from submitochondrial particles of rat skeletal muscle or bovine heart by chemical uncouplers
Authors: Yamada Esther W. , Huzel Norman J.
Published: December 2009

Proteins of similar molecular weights were stripped from submitochondrial particles (A particles) of rat skeletal muscle or bovine heart by treatment with classical chemical uncouplers at 0 °C as with Ca2+. Proteins released included two of high molecular weight (about 43 000 and 30 000), an ATPase inhibitor protein (IF1)as well as the Ca2+-binding lipoprotein that has previously been shown to protect the mitochondrial ATPase complex against inhibition by N,N′-dicyclohexylcarbodiimide (DCCD). The latter two proteins were purified to a high degree. The crude fraction obtained by stripping with chemical uncouplers also contained traces of an additional protein (relative mass (Mr) ~ 13 000) which was also found upon aging of the crude fraction stripped by Ca2+. It was not found in aged preparations of either purified IF1 or the lipoprotein, but appeared when IF1 and the lipoprotein were mixed and aged together. Pretreatment of the mixture with 2-mercaptoethanol prior to electrophoresis did not remove the hybrid. More phospholipid was stripped from A particles by chemical uncouplers than by Ca2+, but less protein was stripped. Phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, and cardiolipin were identified in the phospholipid fractions.
The circular dichroism of ovoverdin and other carotenoproteins from the lobster Homarus americanus
Authors: Young N. Martin , Williams Ross E.
Published: December 2009

The circular dichroic (CD) spectra of the α-, β′-, and γ-crustacyanins, ovoverdin, and the yellow lobster-shell protein were measured in the region 200–750 nm, for comparison with the CD spectrum of the free carotenoid astaxanthin. The two carotenoid chromophores of ovoverdin gave a CD spectrum with a series of bands of alternating sign and ellipticities up to 1.9 × 105 degree∙cm2∙dmol−1, comparable to the low temperature CD spectrum of astaxanthin in the UV region. The visible region of ovoverdin also contained strong CD bands where astaxanthin itself has very weak ones. The blue (640 nm) chromophore of ovoverdin gave a broad negative CD feature quite different from the blue chromophores of the three crustacyanins. The crustacyanins have a broad positive feature between 400 and 610 nm with ellipticities up to 2 × 105 degree∙cm2∙dmol−1, followed at higher wavelengths by a negative band with an ellipticity up to 1 × 105 degree∙cm2∙dmol−1. Gaussian curve-fitting procedures showed the positive features to consist of a minimum of two or three CD bands. In addition to the exciton bands at the main visible absorption, the yellow lobster-shell protein had a pair of CD bands of equal but opposite ellipticity associated with an absorption band between 250 and 340 nm. The UV regions of the CD spectra of the five carotenoproteins have bands from both carotenoid and protein chromophores, and possible assignments of these bands to one or the other of the two types of chromophore are proposed.
Measurement of the absolute synthesis of soluble and insoluble elastin by chick aortic tissue
Authors: Keeley Fred W. , Johnson Dorothy J.
Published: December 2009

The rate of in vitro synthesis of soluble and insoluble elastin by thoracic aorta of 2-day-old chicks has been measured in absolute terms. In the absence of β-aminopropionitrile (βAPN), the steady state level of soluble elastin was 120 pmol/100 mg of aortic tissue or 3.7 μg/whole aorta segment. The rate of synthesis of elastin in vitro was approximately 130 μg/day per whole aorta segment. This is three- to four-fold lower than the estimated rate of in vivo synthesis for a comparable segment of aortic tissue at the same stage of development. Pulse-chase experiments suggested that this difference was not due to in vitro proteolysis of a significant proportion of the newly synthesized soluble elastin, but rather that the conversion of soluble to insoluble elastin was both rapid and efficient. These experiments also indicated the presence in aortic tissue of a substantial pool of elastin of intermediate solubility. Although inclusion of βAPN in the incubation medium resulted in an increase in the amount of soluble elastin in aortic tissue, the rate of accumulation of newly synthesized soluble elastin in the presence of this inhibitor of cross-linking was not linear, but decreased with incubation time. Furthermore, although βAPN effectively suppressed the appearance of insoluble elastin for at least 2 h, some escape from the effect of this inhibitor was seen with further incubation. In general, βAPN significantly depressed elastin synthesis.
Structural elucidation of the capsular polysaccharide of Streptococcus pneumoniae type 9N
Authors: Rosell Karl-Gunnar , Jennings Harold J.
Published: December 2009

The specific capsular polysaccharide of Streptococcus pneumoniae type 9N (American type 9) contains D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-mannose in the molar ratio of 2:1:1:1. Accumulated data from spectroscopic (13C and 1H nuclear magnetic resonance) and methylation analyses of the native and specifically degraded polysaccharide indicated that it was linear and composed of the following pentasaccharide repeating unit; —4)-α-D-GlcpA-(1 → 3)-α-D-Glcp-(1 → 3)-β-D-ManpNAc-(1 → 4)-α-D-Glcp-(1 → 4)-β-D-GlcpNAc(1 →. Structural regions in the type 9N polysaccharide common to those of types 9A, 9L, and 9V have been identified which account for the cross-reactivity of this groups of polysaccharides.
In vitro binding of propranolol and progesterone to native and desialylated human orosomucoid
Authors: Wong Allan K. L. , Hsia J. Carleton
Published: December 2009

A comparison of propranolol and progesterone binding to native and desialylated human orosomucoid was studied by means of equilibrium dialysis. The association constants of propranolol and progesterone binding to native human orosomucoid under physiological conditions were 8.4 × 105 and 3.2 × 105 M−1, respectively. Enzymatic desialylation of human orosomucoid removed 95% of the sialic acid content and reduced the binding affinity of propranolol from 8.4 × 105 to 6.0 × 105 M−1, but the affinity of progesterone was not affected. In addition, desialylation reduced the percent binding for propranolol, indicating that electrostatic attraction of the positive charge on propranolol by sialic acid residues on human orosomucoid had some effect on the binding ability of purified orosomucoid for propranolol. The present data suggest that the electrostatic attraction between sialic acid and propranolol is partially responsible for the preferential binding of basic drugs to orosomucoid in plasma.
Metabolic labelling of oligosaccharide chains of glycoproteins in a rat myoblast cell line and its lectin resistant mutants
Authors: Gilfix B. M. , Sanwal B. D.
Published: December 2009

We have determined the experimental conditions under which optimum hydrolysis of the various oligosaccharides of membrane glycopeptides can be achieved in a permanent line of rat skeletal myoblasts and its wheat germ agglutinin resistant mutants. The labelled glycopeptides were isolated by extensive pronase digestion of whole cells. Labelling studies demonstrated that label from fucose and galactose remained largely in these two sugars, while glucosamine labelled sialic acid and N-acetylglucosamine. Mannose label was found in both mannose and fucose.
Some properties of aminopeptidase associated with rat brain cortical synaptosomes
Authors: Chan William W.-C. , Demmer Wolfgang , Brand Karl
Published: December 2009

To understand the breakdown of peptides in the brain, we studied the aminopeptidase associated with synaptosome particles. A continuous spectrophotometric assay in stirred cuvettes was used to follow the kinetics of inactivation by EDTA and by diethyl pyrocarbonate. The sensitivity of the enzyme towards puromycin and leucine hydroxamate was also determined. The results are consistent with the presence of a single species of aminopeptidase in freshly prepared synaptosome. This enzyme is capable of degrading Met-enkephalin in vitro and is distinct from microsomal leucine aminopeptidase. Storage of synaptosomes by freezing and subsequent thawing changed some properties of the enzyme and partially solubilized the enzyme. These studies suggest that there are advantages in studying the enzyme in its native particle-bound state.
Histone Hl° expression during developmental growth of rat liver
Authors: Larue Hélène , Bissonnette Elyse , Bélanger Luc
Published: December 2009

Histone Hl° appears in rat liver nuclei around the time of birth; it increases to near-adult levels by 30 days of age, in waves which correlate with distinct phases of liver developmental growth. Hl° emerges as the liver terminates the bulk of its proliferative activity, but it accumulates mainly during the terminal cytodifferentiation phases leading to polyploidization. Premature interruption of liver DNA synthesis in neonatal rats, with cytosine arabinoside or dexamethasone, induces some accumulation of Hl° in the liver. Our data suggest that DNA synthesis arrest conditions in part, but is not the main developmental event underlying Hl° expression during differentiation of rat liver.
Studies on TI-2 trypsin inhibitor of Streptomyces griseus
Authors: Mukherjee Mina
Published: December 2009

The trypsin inhibitor TI-2 produced by Streptomyces griseus Cal in a medium, containing beef extract and peptone as the nitrogen source, was purified by ammonium sulfate precipitation and gel filtration. Amino acid analysis showed that it contained hydroxyproline and carbohydrate whereas tyrosine, tryptophan, and cystine were absent. The composition of the inhibitor protein thus showed a similarity to collagen. Growth of the organism in a medium with gelatin as the sole nitrogen source resulted in the production of the same TI-2 inhibitor. Incubation of gelatin with either concentrated culture filtrate, trypsin, or chymotrypsin also yielded trypsin inhibitors. The product isolated from the in vitro incubation of gelatin with trypsin appeared to be the same as TI-2. To the author's knowledge, this is the first report that peptides obtained by enzymic hydrolyses of gelatin are trypsin inhibitors.
Effects of glucocorticoid hormones on lipid-synthetic enzymes from different adipose tissue regions and from liver
Authors: Lau David C. W. , Roncari Daniel A. K.
Published: December 2009

The regional diversity of adipose tissue is dramatically accentuated in states of glucocorticoid excess, in which certain fat depots expand, while others contract. We have studied the molecular basis of this redistribution by determining the activity of fatty acid synthetase and enzymes catalyzing di- and tri-acylglycerol synthesis, in subcellular fractions from four adipose depots of rats injected with dexamethasone and from interscapular and epididymal adipocyte precursors after addition of either dexamethasone or corticosterone to confluent monolayers in secondary culture. Subcellular fractions from cervical and interscapular adipose tissue, as well as from cultured interscapular precursors, revealed a general increase in specific enzyme activity. The opposite trend was observed for retroperitoneal and epididymal fat tissue, as well as cultured epididymal precursors. Fatty acid synthetase and cytosolic phosphatidate phosphohydrolase appeared to be most responsive. The findings in culture indicate that the regional effects of glucocorticoids are partly independent of other circulating or neural factors. Injections of dexamethasone led to significantly enhanced specific activity of all the lipid-synthetic enzymes assayed in subcellular fractions from liver. Differences in hormonal influences between liver and certain fat tissue regions represent tissue specificity. In addition, the diverse effects of glucocorticoids on various adipose tissue depots indicate regional or "intratissue" specificity.
In vivo detection of cryoprotectants and lipids in overwintering larvae using carbon-13 nuclear magnetic resonance spectroscopy
Authors: Buchanan G. W. , Storey K. B.
Published: December 2009

Natural abundance carbon-13 nuclear magnetic resonance (NMR) spectra of intact, acclimated Eurosta solidaginis larvae have been obtained. The spectra show peaks assignable to lipid components and the presence of one major type of monounsaturated fatty acid. Chloroform-soluble extracts of whole larvae support this. The major nonlipid carbon components were the two cryoprotectant polyols glycerol and sorbitol. Carbon-13 NMR is a useful tool for analysis of cryoprotectant molecules in whole, living cold acclimated freezing tolerant insects.
A comparison of the rates of incorporation of fatty acids during the rapid synthesis in vitro of endogenous triacylglycerols by neuronal nuclei
Authors: Baker R. Roy , Chang Huu-yi
Published: December 2009

The incorporation of radioactive palmitate, oleate, linoleate, and arachidonate into endogenous triacylglycerols was followed in vitro using neuronal nuclei (N1) isolated from cerebral cortices of 15-day-old rabbits. Specific rates of incorporation of fatty acids into N1 triacylglycerols were 33–42 times and more than 100 times the corresponding values for cerebral cortex homogenates and microsomal fractions (P3), respectively. Acyl-CoA synthetase specific activities in N1 were 2.2 to 3.2 times the specific rates for fatty acid incorporation into N1 triacylglycerols. Using single fatty acids, N1 acyl-CoA synthetase showed a preference for linoleate which was more highly marked in linoleate–palmitate and linoleate–arachidonate competitions. In fatty acid incorporation into N1 triacylglycerols a preference for linoleate in competition with palmitate was noted; however, there was also a relatively higher utilization of arachidonate shown competitively than was noted in acyl-CoA synthesis. The data suggested that N1 diacylglycerol acyl transferase shows a selectivity for arachidonoyl-CoA in comparison with CoA esters of palmitate or oleate. Molecular class analyses of radioactive triacylglycerol products indicated that native endogenous N1 diacylglycerols bearing arachidonate or fatty acids of equal or higher unsaturation were used preferentially in N1 triacylglycerol synthesis. This preference was significantly decreased when higher levels of endogenous diacylglycerols were produced in N1 following a phospholipase C preincubation.
Inhibition of fusion of skeletal myoblasts by tunicamycin and its reversal by N-acetylglucosamine
Authors: Gates G. A. , Kaur H. , Sanwal B. D.
Published: December 2009

The differentiation of a permanent line of rat skeletal myoblasts is inhibited by a low concentration of tunicamycin in the growth medium. At a level of 0.9 μg/mL, mannose incorporation in trichloroacetic-acid-insoluble material is inhibited to the extent of about 50% by the antibiotic. Blotting of glycoproteins of the cytoplasmic membrane of myoblasts separated by gel electrophoresis by radioiodinated concanavalin A revealed that four major glycoproteins of 230 000, 145 000, 119 000, and 46 000 daltons were present in lower relative amounts in the plasma membrane following tunicamycin treatment. The 119 000 dalton glycoprotein was a major radioiodinated protein in intact cells and was presumably localized on the periphery of the membrane. The effect of tunicamycin on both fusion and glycosylation of membrane proteins could be reversed by N-acetylglucosamine, but not by the protease inhibitors leupeptin and pepstatin.
On the interaction between synthetic detergents and enzymatic proteins
Authors: Treves Cristina , Vincenzini Maria Teresa , Favilli Fabio , Vanni Paolo , Baccari Vincenzo
Published: December 2009

Eight highly purified and crystalline enzymes representative of fundamental metabolic pathways and of cellular structure were tested with seven detergents. The enzyme–detergent interaction was studied as a time-independent (inhibition) or time-dependent (inactivation) phenomenon. Our results imply a specificity of detergent action on the enzymes studied.
Permeability of bilayers composed of mixtures of saturated phospholipids: evidence for compositional segregation of lipids at phase boundaries
Authors: Tse Man Yat , Singer Michael
Published: December 2009

Lipid bilayers composed of a binary mixture of saturated phospholipids display broad permeability maxima in the temperature region of phase separation. We have proposed that this enhanced diffusion occurs through defects which develop at boundaries between liquid crystal and gel state domains and that one of the lipid components of the mixture might specifically congregate at these defects. To explore this postulate we have performed the following type of experiment. Liposomes were formed from different mole ratios of the lipid pairs 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dimyristoyl GPC) – 1,2-distearoyl-sn-glycero-3-phosphocholine (distearoyl GPC) and dimyristoyl GPC – 1,2-dimyristoyl-sn-glycero-3-phosphoethanolarnine (dimyristoyl GPE) and 22Na effluxes measured over an extensive temperature span (15 to 60 °C). For a given mixture we plotted 22Na movement through the boundary regions which develop during phase separation against the fraction of dimyristoyl GPC in the coexisting solid and liquid phases at that temperature. As dimyristoyl GPC is added to either distearoyl GPC or dimyristoyl GPE, boundary efflux remains low until the mole fraction of dimyristoyl GPC in the coexisting solid and liquid phases reaches 0.1 and 0.4 (for dimyristoyl GPC – distearoyl GPC) and 0.3 and 0.7 (for dimyristoyl GPC – dimyristoyl GPE), respectively. As the fraction of dimyristoyl GPC in the coexisting phases exceeds these values, boundary permeability rises and with further increments in this phospholipid, the mixtures begin to assume permeability characteristics similar to that of dimyristoyl GPC alone. These observations suggest that at low mole fractions of dimyristoyl GPC, the boundary regions are populated by the distearoyl GPC or dimyristoyl GPE molecules and hence efflux rates are small. As the fraction of dimyristoyl GPC in the coexisting phases exceeds the values noted above, this phospholipid congregates at the boundary regions, thus altering their barrier properties.