bluuebunny/crossref_metadata_2025 huggingface dataset:
Myosin heads have a broad orientational distribution during isometric muscle contraction: time-resolved EPR studies using caged ATP.
Authors: Fajer P G , Fajer E A , Thomas D D
Published: May 2006

To study the orientation of spin-labeled myosin heads in the first few seconds after the production of saturating ATP, we have used a laser flash to photolyze caged ATP during EPR data acquisition. Rabbit psoas muscle fibers were labeled with maleimide spin label, modifying 60% of the myosin heads without impairing muscle fiber biochemical and physiological activity (ATPase and force). The muscle bundles were incubated for 30 min with 5 mM caged ATP prior to the UV flash. The flash, from an excimer laser, liberated 2-3 mM ATP, generating maximum force in the presence of Ca2+ and relaxing fully in the absence of Ca2+. Control experiments, using fibers decorated with labeled myosin subfragment, showed that the flash liberates sufficient ATP to saturate myosin active sites in all regions of the muscle bundles. To increase the time resolution, and to minimize the time of the contraction, we followed in time the intensity at a single spectral position (P2), which is associated with the high degree of orientational order in rigor. ATP liberation produced a rapid decrease of P2 with liberation of ATP, indicating a large decrease in orientational order in both relaxation and contraction. This transient was absent when caged AMP was used, ruling out nonspecific effects of the UV flash and subsequent photochemistry. The steady-state level of P2 during contraction was almost as low as that reached in relaxation, although the duration of the steady state was much more brief in contraction. Upon depletion of ATP in contraction, the P2 intensity reverted to the original rigor level, accompanied by development of rigor tension. The steady-state results obtained in the brief contractions induced by caged ATP are quantitatively consistent with those obtained in longer contractions by continuously perfusing fibers with ATP. In isometric contraction, most (88% +/- 4%) of the heads are in a population characterized by a high degree of axial disorder, comparable to that observed for all heads in relaxation. Since the stiffness of these fibers in contraction is 80% of the stiffness in rigor, it is likely that most of the heads in this highly disoriented population are attached to actin in contraction and that most actin-attached heads in contraction are in this disoriented population.
Vaccination with a live retrovirus: the nature of the protective immune response.
Authors: Ruprecht R M , Mullaney S , Bernard L D , Gama Sosa M A , Hom R C , Finberg R W
Published: May 2006

We tested 3'-azido-3'-deoxythymidine (zidovudine) combined with interferon alpha as chemoprophylaxis after exposing mice to Rauscher murine leukemia virus. Therapy started 4 hr after inoculation and administered for 20 days prevented viremia and disease in all 234 mice tested. When the animals were rechallenged with live virus after cessation of therapy, 96% were resistant. The nature of this protective immune response was analyzed: Passive serotherapy of naive mice challenged subsequently with Rauscher murine leukemia virus was only protective at a high dose of immune serum. Immune, but not naive, T cells alone were fully protective against virus challenge. We conclude that vaccination with a live retrovirus that cannot replicate because of pharmacological blockade induces a T-cell response capable of protecting against a lethal retrovirus-induced disease.
Ribosomes as sensors of heat and cold shock in Escherichia coli.
Authors: VanBogelen R A , Neidhardt F C
Published: May 2006

Nearly all cells respond to an increase in temperature by inducing a set of proteins, called heat shock proteins (HSPs). Because a large number of other stress conditions induce the HSPs (or at least the most abundant ones), this response is often termed the universal stress response. However, a careful study of conditions that truly mimic a temperature shift suggested that these proteins are induced in response to a change in the translational capacity of the cell. To test this directly, Escherichia coli cells were treated with antibiotics that target the prokaryotic ribosome. Two-dimensional gels were used to evaluate the ability of these drugs to alter the rate of synthesis of the HSPs. One group of antibiotics induced the HSPs, whereas a second group repressed the HSPs and induced another set of proteins normally induced in response to a cold shock. Depending on the concentration used, the induction of the heat or cold shock proteins mimicked a mild or severe temperature shift. In addition, antibiotics of the cold shock-inducing group were found to block high temperature induction of the HSPs. The results implicate the ribosome as a prokaryotic sensor for the heat and cold shock response networks, a role it may serve in eukaryotes as well.
Molecular cloning and characterization of human pyruvate dehydrogenase beta subunit gene.
Authors: Koike K , Urata Y , Koike M
Published: May 2006

A genomic clone encompassing the entire gene for the human pyruvate dehydrogenase beta subunit (PDH beta) has been isolated by screening a leukocyte genomic library with a nick-translated human foreskin fibroblast PDH beta cDNA probe. The 18-kilobase clone was characterized by restriction enzyme analysis, extensive DNA sequencing, and primer-extension analysis. The PDH beta structural gene is composed of 10 exons and 9 introns. All intron-exon splice junctions follow the GT/AG rule. The Alu family was found in introns 2 and 8. The 5' flanking region of the PDH beta gene contains a "CAAT" consensus promoter sequence but no "TATA" sequence. Primer-extension analysis indicated that the PDH beta gene transcription start site is an adenine residue located 132 bases upstream from the initiation codon in exon 1.
Residues in three conserved regions of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase are required for quaternary structure.
Authors: Fitchen J H , Knight S , Andersson I , Branden C I , McIntosh L
Published: May 2006

To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), small subunits with single amino acid substitutions in three regions of relative sequence conservation were produced by directed mutagenesis of the rbcS gene from Anabaena 7120. These altered small subunits were cosynthesized with large subunits (from an expressed Anabaena rbcL gene) in Escherichia coli. Mutants were analyzed for effects on quaternary structure and catalytic activity. Changing Glu-13S (numbering used is that of the spinach enzyme) to Val, Trp-67S to Arg, Pro-73S to His, or Tyr-98S to Asn prevented accumulation of stable holoenzyme. Interpretation of these results using a model for the three-dimensional structure of the spinach enzyme based on x-ray crystallographic data suggests that our small subunit mutants containing substitutions at positions 13S and 67S probably do not assemble because of mispairing or nonpairing of charged residues on the interfacing surfaces of the large and small subunits. The failure of small subunits substituted at positions 73S or 98S to assemble correctly may result from disruption of intersubunit or intrasubunit hydrophobic pockets, respectively.
Intracellular-pH dependence of Na-H exchange and acid loading in quiescent and arginine vasopressin-activated mesangial cells.
Authors: Boyarsky G , Ganz M B , Cragoe E J , Boron W F
Published: May 2006

We studied intracellular pH (pHi) regulation in the absence of HCO3- in single mesangial cells (MCs) with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(and -6)carboxyfluorescein. Our approach was to acid load the cells by an NH+4 prepulse and to monitor the subsequent pHi recovery. Previous work on MCs and other cells has shown that the recovery is prevented by adding ethylisopropyl amiloride (EIPA) or removing Na+ before the recovery begins, suggesting that at low pHi only Na-H exchange contributes to the recovery. This conclusion is often extrapolated to the entire pHi range. To test this, we interrupted the recovery with EIPA at various pHi values, finding that EIPA unmasked a background acidification that was negligible at pHi less than approximately 6.7 but increased steeply at higher pHi values. Correcting the total recovery rate for this EIPA-insensitive component, we found that the EIPA-sensitive (Na-H exchange) rate fell steeply with increasing pHi between 6.3 and 6.7 but was relatively pHi insensitive between 6.7 and 7.2. Thus, the recovery halts as pHi approaches approximately 7.2 not so much because Na-H exchange slows, but because acid loading accelerates. Applying the mitogen arginine vasopressin (AVP; 100 nM) caused a rapid pHi decrease of approximately 0.4, followed by a slower increase to a level approximately 0.15 higher than the initial pHi. Coincident with this biphasic change in pHi was a biphasic change in Na-H exchange kinetics. In the early phase (i.e., pHi recovery commencing approximately 8 min after AVP addition), AVP linearized the pHi dependence of the exchanger; its rate was unaffected by AVP at pHi less than approximately 6.7 but was progressively inhibited at higher pHi values. In the later phase (i.e., pHi recovery commencing approximately 14 min after AVP addition), AVP shifted this linear pHi dependence in the alkaline direction; the exchanger was stimulated at pHi less than 6.9 but was modestly inhibited at higher pHi values (i.e., in the physiological range). At all times, AVP greatly inhibited background acid loading. Thus, AVP raises steady-state pHi not because Na-H exchange is stimulated but because, although the exchanger is inhibited, acid loading is inhibited even more.
Treatment of Pneumocystis carinii pneumonia with 1,3-beta-glucan synthesis inhibitors.
Authors: Schmatz D M , Romancheck M A , Pittarelli L A , Schwartz R E , Fromtling R A , Nollstadt K H , Vanmiddlesworth F L , Wilson K E , Turner M J
Published: May 2006

Pneumocystis carinii pneumonia is a major cause of death in AIDS patients in the United States. The presently available treatments have limited use due to a high incidence of adverse reactions. Therefore, there is an urgent need for a safer method for treatment and prevention of this disease. Recent evidence has suggested that P. carinii is related to fungi and that the wall of the cyst form contains 1,3-beta-glucan as a major constituent. Based on this, several proposed 1,3-beta-glucan synthesis inhibitors were evaluated for their ability to control P. carinii pneumonia in vivo. Compounds from two classes of 1,3-beta-glucan synthesis inhibitors, the echinocandins and papulacandins, were found to be effective against P. carinii.
Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells.
Authors: Anagnostou A , Lee E S , Kessimian N , Levinson R , Steiner M
Published: May 2006

Erythropoietin is known to be a hematopoietic growth factor with a singularly specific action on the proliferation and differentiation of erythroid progenitor cells. We have observed a dose-dependent proliferative action of human recombinant erythropoietin on human umbilical vein endothelial cells and bovine adrenal capillary endothelial cells. Binding studies with radioiodinated recombinant human erythropoietin revealed a large number (approximately 27,000) of an apparent single class of receptors with an affinity in the 10(-9) M range. Linkage of the radiolabeled ligand to its receptor via a bifunctional crosslinking agent allowed us to identify an endothelial cell protein of 45 kDa as the principal receptor associated with this mitogenic effect of erythropoietin. Recombinant human erythropoietin also enhanced the migration of endothelial cells.
25-Hydroxyvitamin D3-1 alpha-hydroxylase in porcine hepatic tissue: subcellular localization to both mitochondria and microsomes.
Authors: Hollis B W
Published: May 2006

In vitro studies were performed to assess the ability of hepatic homogenates, mitochondria, and microsomes to 1 alpha-hydroxylate 25-hydroxyvitamin D3 [25(OH)D3]. Addition of 25(OH)D3 to either hepatic mitochondria or microsomes caused a concentration-dependent increase in the production of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Hepatic homogenates also produced purported 1,25(OH)2D3, although at a much reduced efficiency as compared with hepatic mitochondria or microsomes. Purported 1,25(OH)2D3 synthesized by hepatic mitochondria or microsomes was identified by its mobility on several high-performance liquid chromatographic systems and, ultimately, by its ability to interact with the bovine thymus 1,25(OH)2D3 receptor protein. Production of 1,25(OH)2D3 by hepatic mitochondria and microsomes was dependent on time of incubation, protein content, and pH of incubation medium, and it required an adequate source of reducing equivalents. Generation of 1,25(OH)2D3 by these organelles could be totally blocked by the cytochrome P-450 inhibitor ketoconazole. The microsomal 1 alpha-hydroxylase could not be saturated even at the highest concentration (240 microM) of 25(OH)D3 used. The mitochondrial 1 alpha-hydroxylase, however, displayed saturation at approximately 40 microM 25(OH)D3. Eadie-Hofstee reciprocal plot analysis of the hepatic mitochondrial 1 alpha-hydroxylase gave a Km of 17 microM 25(OH)D3 and a Vmax of 481 pg of 1,25(OH)2D3 per min per mg of protein. Because of its inability to achieve substrate saturation, meaningful kinetic parameters could not be calculated for the hepatic microsomal 1 alpha-hydroxylase. These data demonstrate the liver to be an even more dynamic organ than was previously believed with respect to vitamin D metabolism in that the liver has the potential to produce 1,25(OH)2D3 in situ by at least two separate mechanisms.
Purification and characterization of cytolytic and noncytolytic human natural killer cell subsets.
Authors: Lebow L T , Bonavida B
Published: May 2006

Natural killer (NK) cells form three functionally distinct populations of effectors: competent cytolytic effectors able to bind and kill target cells and two subsets of nonlytic effectors, one able and the other unable to bind target cells. A flow cytometric method was developed, based on size and two-color fluorescence of NK cell-target conjugates, for the characterization and sorting of highly purified subpopulations--killer cells, nonkiller binder cells, and free cells. Ultrastructural examination revealed that granule content was reduced in the killer cells and absent in most of the binder cells. Quantitative differences in the expression level of HLA class I, CD11b (C3bi receptor), and CD16 (receptor for the Fc portion of IgG) antigens could differentiate the subsets. The killer phenotype was HLAlo, CD11bvery hi, and CD16very lo; the binder phenotype was CD11bhi and CD16lo; and the free-cell phenotype was CD11blo and CD16hi. Cell activation was not requisite for lytic function because no difference in either expression of activation markers or cell cycle could be established among the sorted subpopulations. Although recycling function was inhibited, retention of lytic activity was enriched 4-fold in the sorted killer cell population. These results represent characterization of a successful bulk isolation of competent killer, nonkiller binder, and free cells in human NK-cell populations and should aid our understanding of NK-cell development, lineage, and function.
Molecular cloning and protein structure of a human blood group Rh polypeptide.
Authors: Chérif-Zahar B , Bloy C , Le Van Kim C , Blanchard D , Bailly P , Hermand P , Salmon C , Cartron J P , Colin Y
Published: May 2006

cDNA clones encoding a human blood group Rh polypeptide were isolated from a human bone marrow cDNA library by using a polymerase chain reaction-amplified DNA fragment encoding the known common N-terminal region of the Rh proteins. The entire primary structure of the Rh polypeptide has been deduced from the nucleotide sequence of a 1384-base-pair-long cDNA clone. Translation of the open reading frame indicates that the Rh protein is composed of 417 amino acids, including the initiator methionine, which is removed in the mature protein, lacks a cleavable N-terminal sequence, and has no consensus site for potential N-glycosylation. The predicted molecular mass of the protein is 45,500, while that estimated for the Rh protein analyzed in NaDodSO4/polyacrylamide gels is in the range of 30,000-32,000. These findings suggest either that the hydrophobic Rh protein behaves abnormally on NaDodSO4 gels or that the Rh mRNA may encode a precursor protein, which is further matured by a proteolytic cleavage of the C-terminal region of the polypeptide. Hydropathy analysis and secondary structure predictions suggest the presence of 13 membrane-spanning domains, indicating that the Rh polypeptide is highly hydrophobic and deeply buried within the phospholipid bilayer. In RNA blot-hybridization (Northern) analysis, the Rh cDNA probe detects a major 1.7-kilobase and a minor 3.5-kilobase mRNA species in adult erythroblasts, fetal liver, and erythroid (K562, HEL) and megakaryocytic (MEG01) leukemic cell lines, but not in adult liver and kidney tissues or lymphoid (Jurkat) and promyelocytic (HL60) cell lines. These results suggest that the expression of the Rh gene(s) might be restricted to tissues or cell lines expressing erythroid characters.
Genes for tryptophan biosynthesis in the archaebacterium Haloferax volcanii.
Authors: Lam W L , Cohen A , Tsouluhas D , Doolittle W F
Published: May 2006

Recent technical advances permit direct genetic approaches for isolating genes and mapping auxotrophic mutations in the halophilic archaebacterium (Archaea) Haloferax volcanii. Twenty-nine mutations in tryptophan biosynthesis mapped to two separate chromosomal locations. DNA sequencing of one gene cluster shows a unique gene order (trpCBA) and unusual potential secondary structures in the 5'-flanking region.
Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms.
Authors: Kranz J E , Holm C
Published: May 2006

Studies of cell physiology and structure have identified many intriguing proteins that could be analyzed for function by using the power of yeast genetics. Unfortunately, identifying the homologous yeast gene with the two most commonly used approaches--DNA hybridization and antibody cross-reaction--is often difficult. We describe a strategy to identify yeast homologs based on protein function itself. This cloning-by-function strategy involves first identifying a yeast mutant that depends on a plasmid expressing a cloned foreign gene. The corresponding yeast gene is then cloned by complementation of the mutant defect. To detect plasmid dependence, the colony color assay of Koshland et al. [Koshland, D., Kent, J. C. & Hartwell, L. H. (1985) Cell 40, 393-403] is used. In this paper, we test the feasibility of this approach using the well-characterized system of DNA topoisomerase II in yeast. We show that (i) plasmid dependence is easily recognized; (ii) the screen efficiently isolates mutations in the desired gene; and (iii) the wild-type yeast homolog of the gene can be cloned by screening for reversal of the plasmid-dependent phenotype. We conclude that cloning by function can be used to isolate the yeast homologs of essential genes identified in other organisms.
Characterization of a fibrillar collagen gene in sponges reveals the early evolutionary appearance of two collagen gene families.
Authors: Exposito J Y , Garrone R
Published: May 2006

We have characterized cDNA and genomic clones coding for a sponge collagen. The partial cDNA has an open reading frame encoding 547 amino acid residues. The conceptual translation product contains a probably incomplete triple-helical domain (307 amino acids) with one Gly-Xaa-Yaa-Zaa imperfection in the otherwise perfect Gly-Xaa-Yaa repeats and a carboxyl propeptide (240 amino acids) that includes 7 cysteine residues. Amino acid sequence comparisons indicate that this sponge collagen is homologous to vertebrate and sea urchin fibrillar collagens. Partial characterization of the corresponding gene reveals an intron-exon organization clearly related to the fibrillar collagen gene family. The exons coding for the triple-helical domain are 54 base pairs (bp) or multiples thereof, except for a 57-bp exon containing the Gly-Xaa-Yaa-Zaa coding sequence and for two unusual exons of 126 and 18 bp, respectively. This latter 18-bp exon marks the end of the triple-helical domain, contrary to the other known fibrillar collagen genes that contain exons coding for the junction between the triple-helical domain and the carboxyl propeptide. Compared to other fibrillar collagen genes, the introns are remarkably small. Hybridization to blotted RNAs established that the gene transcript is 4.9 kilobases. Together with previous results that showed the existence of a nonfibrillar collagen in the same species, these data demonstrate that at least two collagen gene families are represented in the most primitive metazoa.
Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae.
Authors: Barnes D E , Johnston L H , Kodama K , Tomkinson A E , Lasko D D , Lindahl T
Published: May 2006

Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.
Expression of the maize Cat3 catalase gene is under the influence of a circadian rhythm.
Authors: Redinbaugh M G , Sabre M , Scandalios J G
Published: May 2006

The accumulation of Cat3 catalase mRNA in the green leaves of maize (Zea mays) exhibits an unusual circadian rhythm. The steady-state level of the Cat3 transcript varies dramatically over the day in seedlings grown on a 12-hr photoperiod. Low or undetectable levels of the Cat3 mRNA are found late in the dark and early in the light period, while the transcript accumulates to high levels late in the light and early in the dark period. This dramatic diurnal variation in mRNA level does not occur with the maize Cat1 or Cat2 catalase transcripts. The diurnal fluctuation in Cat3 mRNA persists when the seedlings are transferred to continuous light or darkness, which indicates the influence of a circadian rhythm. The lack of influence of red and/or far-red light on this diurnal variation in transcript level indicates that the circadian rhythm is not regulated by phytochrome. Run-on transcription assays show that changes in the rate of Cat3 gene transcription closely parallel the observed changes in steady-state mRNA levels. This circadian rhythm in Cat3 gene expression and the resulting high levels of mRNA which accumulate late in the light period suggest that the catalase 3 (CAT-3) isozyme is being synthesized for accumulation in the dark period. This, together with the high level of Cat3 mRNA and CAT-3 protein accumulation in dark-grown maize shoots, suggests that the activity of the CAT-3 isozyme might be associated with a metabolic process important in shoot cells in the dark.
Lipid domains in the ram sperm plasma membrane demonstrated by differential scanning calorimetry.
Authors: Wolf D E , Maynard V M , McKinnon C A , Melchior D L
Published: May 2006

Mammalian sperm plasma membranes, in contrast to those of mammalian somatic cells, exhibit a significant fraction of lipid that does not diffuse laterally in the plane of the membrane. This nondiffusing fraction results from lipid-lipid interactions. Similar nondiffusing fractions are found in mixed-lipid model systems that contain coexistent gel and fluid domains. These results suggest that the sperm plasma membrane may also exhibit lateral phase segregations of lipids and may contain significant amounts of gel-phase lipid. In this paper we use differential scanning calorimetry to show that, in contrast to the plasma membranes of mammalian somatic cells, the plasma membrane from the anterior region of the head of ram sperm exhibits at least two major endothermic transitions, one centered at approximately 26 degrees C and one centered at approximately 60 degrees C. The heats of these transitions are consistent with gel-to-fluid transitions in model membranes. These transitions are observed both in plasma membrane vesicles and in rehydrated lipid extracts made from these vesicles. These results demonstrate that at physiological temperatures the lipids of the ram sperm plasma membrane are segregated into coexistent fluid and gel domains. Since sperm encounter a wide range of temperatures during their development, these phase transitions may be important in establishing dynamic domains of lipid requisite for epididymal storage and fertilization.
Casein expression in cytotoxic T lymphocytes.
Authors: Grusby M J , Mitchell S C , Nabavi N , Glimcher L H
Published: May 2006

A cDNA that expresses a mRNA restricted to cytotoxic T lymphocytes (CTL) and mammary tissue has been isolated and characterized. The deduced amino acid sequence from this cDNA shows extensive homology with the previously reported amino acid sequence for rat alpha-casein. Indeed, the presence of a six-residue-repeated motif that is specific for rodent alpha-caseins strongly supports the identification of this cDNA as mouse alpha-casein. Northern (RNA) blot analysis of many hematopoietic cell types revealed that this gene is restricted to CTL, being expressed in four of six CTL lines examined. Furthermore, CTL that express this gene were also found to express other members of the casein gene family, such as beta- and kappa-casein. These results suggest that caseins may be important in CTL function, and their potential role in CTL-mediated lysis is discussed.
Synporins--synthetic proteins that emulate the pore structure of biological ionic channels.
Authors: Montal M , Montal M S , Tomich J M
Published: May 2006

A class of proteins that mimic the fundamental pore structure of authentic ionic channels has been designed, synthesized, and characterized. The design is based on our earlier result that a 23-mer peptide with the sequence of the M2 segment of the Torpedo californica acetylcholine receptor delta subunit--Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe -Leu- Leu-Leu-Thr-Ser-Gln-Arg--forms cation-selective channels in lipid bilayers, presumably by self-assembly of conductive oligomers. Accordingly, a tethered parallel tetramer was synthesized with four M2 delta peptides attached to a carrier template--a 9-amino acid backbone with four attachment sites. As expected, the complete 101-residue protein does form channels in lipid bilayers reproducing several features that are characteristic of authentic acetylcholine receptor channels, such as single-channel conductance, cation selectivity, transitions between closed and open states in the millisecond time range, and sensitivity to local anesthetic channel blockers. An analogue protein, in which the serine residue in position 8 is replaced with alanine in each of the four M2 delta 23-mer peptides ([Ala8]M2 delta), also forms channels that, however, exhibit lower single-channel conductance. By contrast, a similar tethered tetramer with M1 delta peptides does not form channels, in accord with expectations. The general validity of this strategy to other channel sequences and oligomer numbers is anticipated. Thus, synporins--a term coined to identify this class of synthetic pore proteins--enrich our armamentarium directed toward the elucidation of structure-function relationships.
Plasminogen-activator inhibitor type 1 is a potent natural inhibitor of extracellular matrix degradation by fibrosarcoma and colon carcinoma cells.
Authors: Cajot J F , Bamat J , Bergonzelli G E , Kruithof E K , Medcalf R L , Testuz J , Sordat B
Published: May 2006

We have analyzed the role of plasminogen-activator inhibitor type 1 (PAI-1) in the regulation of tumor cell-mediated extracellular matrix degradation. Immunocytochemical analysis revealed PAI-1 associated with microgranular and fibrillar material of the extracellular matrix and demonstrated the presence of PAI-1 as a cell surface-associated antigen. Transforming growth factor beta significantly reduced matrix degradation mediated by HT-1080 human fibrosarcoma cells. This inhibition was correlated with an increase in PAI-1 antigen expression, whereas urinary-type plasminogen activator (u-PA) secretion was unaffected. In this experimental system, PAI-1 regulated extracellular matrix breakdown, as added PAI-1 inhibited matrix solubilization, whereas monoclonal antibodies to PAI-1 increased it. A cell line (LPAI) producing high levels of biologically active PAI-1 was established by transfection of a human PAI-1 cDNA clone into mouse L cells. Coculture experiments demonstrated that LPAI cells prevented matrix degradation by Lu-PA cells (L cells expressing high levels of u-PA) or Co-115 human colon carcinoma cells (expressing tissue-type plasminogen activator). These results indicate that PAI-1 may play a critical role in the regulation of extracellular matrix degradation during tumor cell invasion.
T-cell receptor-negative natural killer cells display antigen-specific cytotoxicity for microvascular endothelial cells.
Authors: Bender J R , Pardi R , Engleman E
Published: May 2006

Based upon prior demonstrations that human microvascular endothelial cells (ECs) could serve as natural killer (NK) cell targets, we established NK cell lines and clones by repeated stimulation of highly purified CD16-positive, CD3/T-cell receptor (Ti)-negative cells with allogeneic ECs. After 3 weeks in culture these lymphoid cells, which neither expressed surface CD3/Ti molecules nor rearranged Ti beta- or gamma-chain genes and which lysed K562 human erythroleukemia cells, displayed specific cytotoxicity for the stimulating ECs. Furthermore, freshly isolated NK cells bound and then removed from each of several allogeneic EC lines displayed selective cytotoxicity for the adsorbing EC line. These results provide evidence for alloantigen-specific recognition of microvascular ECs by NK cells that appears to be determined, at least in part, at the level of adherence. We discuss the implications of these findings with respect to the rejection of vascularized organ allografts.
Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions.
Authors: Ylä-Herttuala S , Rosenfeld M E , Parthasarathy S , Glass C K , Sigal E , Witztum J L , Steinberg D
Published: May 2006

Oxidation of low density lipoprotein (LDL) enhances its atherogenicity, and inhibition of such oxidation decreases the rate of progression of atherosclerotic lesions. The mechanism of LDL oxidation in vivo remains uncertain, but in vitro studies have suggested that cellular lipoxygenases may play a role by initiating lipid peroxidation in LDL. In situ hybridization studies using a 15-lipoxygenase riboprobe and immunostaining using antibodies against 15-lipoxygenase showed strongly positive reactivity largely confined to macrophage-rich areas of atherosclerotic lesions. Polymerase chain reaction with 15-lipoxygenase-specific oligonucleotides and restriction enzyme digestions of the amplified fragment were used to confirm the presence of 15-lipoxygenase message in the reverse-transcribed lesion mRNA. Immunostaining with antibodies reactive with oxidized LDL (but not with native LDL) indicated that the lipoxygenase colocalizes with epitopes of oxidized LDL, compatible with a role for macrophage lipoxygenase in the oxidation of LDL in vivo. Since oxidized LDL is chemotactic for blood monocytes, early lesions might progress at a markedly accelerated rate because of further recruitment of more monocytes which, in turn, would increase further the rate of oxidation of LDL. These data suggest that therapy targeted to block macrophage lipoxygenase activity might decrease the rate of development of atherosclerotic lesions.
Inhibition of eicosanoid biosynthesis by glucocorticoids in humans.
Authors: Sebaldt R J , Sheller J R , Oates J A , Roberts L J , FitzGerald G A
Published: May 2006

Therapeutic doses of glucocorticoids are thought to inhibit prostaglandin and leukotriene formation in humans. Several studies in animals, however, have failed to demonstrate modulation of eicosanoid biosynthesis by steroids in vivo. We administered prednisone (60 mg/day) to eight healthy volunteers and measured eicosanoid formation by a variety of cell types in vivo and ex vivo, using sensitive and specific physicochemical assays. We found that the in vivo course of prednisone failed to inhibit the synthesis of thromboxane A2, prostaglandin I2 (prostacyclin), prostaglandin E2, and leukotriene E4 in vivo and of leukotriene B4 ex vivo. Biosynthesis of leukotriene B4, thromboxane B2, and prostaglandins F2 and E2 by macrophage-rich bronchoalveolar lavage cells was strongly suppressed. These findings indicate that therapeutic regimens of glucocorticoids suppress eicosanoid biosynthesis in human macrophages but not in a number of other cell types with steroid receptors, the capacity for eicosanoid formation, and lipocortin-like material.
Differential regulation of skeletal alpha-actin transcription in cardiac muscle by two fibroblast growth factors.
Authors: Parker T G , Chow K L , Schwartz R J , Schneider M D
Published: May 2006

In cardiac muscle, acidic and basic fibroblast growth factors (aFGF and bFGF) regulate at least five genes in common (including alpha and beta myosin heavy chains, atrial natriuretic factor, and the sarcoplasmic reticulum calcium ATPase), provoking a generalized "fetal" phenotype similar to events in pressure-overload hypertrophy; however, aFGF and bFGF differentially control the striated alpha-actins. bFGF stimulates and aFGF inhibits skeletal alpha-actin transcripts associated with the embryonic heart, whereas cardiac alpha-actin mRNA is inhibited by aFGF but not bFGF. To elucidate mechanisms for these selective and discordant actions of aFGF and bFGF on cardiac muscle, chicken skeletal and cardiac alpha-actin promoter-driven reporter genes were introduced into neonatal rat cardiac myocytes by electroporation. Skeletal alpha-actin transcription was selectively stimulated by bFGF, whereas the cardiac alpha-actin promoter was unaffected. In contrast, aFGF suppressed both transfected alpha-actin genes. The differential regulation of skeletal alpha-actin transcription was equivalent with either purified or recombinant FGFs and was observed with 5' flanking sequences from either nucleotide -202 or -2000 to nucleotide -11. Positive and negative modulation of alpha-actin transcription by growth factors corresponded accurately to the endogenous genes in all permutations studied. These investigations provide a model for reciprocal control of gene transcription by aFGF vs. bFGF.
Abnormally high expression of proteasomes in human leukemic cells.
Authors: Kumatori A , Tanaka K , Inamura N , Sone S , Ogura T , Matsumoto T , Tachikawa T , Shin S , Ichihara A
Published: May 2006

Proteasomes are eukaryotic ring-shaped or cylindrical particles with multicatalytic protease activities. To clarify the involvement of proteasomes in tumorigenesis of human blood cells, we compared their expression in human hematopoietic malignant tumor cells with that in normal peripheral blood mononuclear cells. Immunohistochemical staining showed considerably increased concentrations of proteasomes in leukemic cells from the bone marrow of patients with various types of leukemia and the predominant localization of these proteasomes in the nuclei. Moreover, enzyme immunoassay and Northern blot analysis indicated that the concentrations of proteasomes and their mRNA levels were consistently much higher in a variety of malignant human hematopoietic cell lines than in resting peripheral lymphocytes and monocytes from healthy adults. Proteasome expression was also greatly increased in normal blood mononuclear cells during blastogenic transformation induced by phytohemagglutinin; their expression increased in parallel with induction of DNA synthesis and returned to the basal level with progress of the cell cycle. Thus, abnormally high expression of proteasomes may play an important role in transformation and proliferation of blood cells and in specific functions of hematopoietic tumor cells.
Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis.
Authors: Battista J R , Ohta T , Nohmi T , Sun W , Walker G C
Published: May 2006

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. The UmuD protein shares homology with a family of proteins that includes LexA and several bacteriophage repressors. UmuD is posttranslationally activated for its role in mutagenesis by a RecA-mediated proteolytic cleavage that yields UmuD'. A set of missense mutants of umuD was isolated and shown to encode mutant UmuD proteins that are deficient in RecA-mediated cleavage in vivo. Most of these mutations are dominant to umuD+ with respect to UV mutagenesis yet do not interfere with SOS induction. Although both UmuD and UmuD' form homodimers, we provide evidence that they preferentially form heterodimers. The relationship of UmuD to LexA, lambda repressor, and other members of the family of proteins is discussed and possible roles of intact UmuD in modulating SOS mutagenesis are discussed.
Structure of the T-cell antigen receptor: evidence for two CD3 epsilon subunits in the T-cell receptor-CD3 complex.
Authors: Blumberg R S , Ley S , Sancho J , Lonberg N , Lacy E , McDermott F , Schad V , Greenstein J L , Terhorst C
Published: May 2006

The T-cell antigen receptor (TCR) consists of heterodimeric glycoproteins (TCR alpha beta or gamma delta) that demonstrate homology with immunoglobulins. Noncovalently associated with the alpha beta (or gamma delta) heterodimer are at least five nonvariant proteins (CD3-gamma, -delta, -epsilon, -zeta, and -eta), which together comprise the TCR-CD3 complex. The stoichiometry of the antigen receptor has been assumed to be either alpha beta gamma delta epsilon zeta zeta or alpha beta gamma delta epsilon zeta eta. In this paper we provide several lines of evidence that support the notion that the mature TCR-CD3 complex on the cell surface contains two CD3-epsilon polypeptide chains. Transfection of two murine T cell-T cell hybridomas with the human DNA encoding CD3-epsilon protein demonstrated that both murine and human CD3-epsilon chains were present within the same TCR-CD3 complex. Analysis of thymocytes isolated from transgenic mice that expressed high copy numbers of the human CD3-epsilon gene showed that the heterologous human CD3-epsilon subunits were coexpressed with murine CD3-epsilon in the same TCR-CD3 complex. Since CD3-epsilon was shown to form disulfide-linked homodimers both in human and murine T cells, the two CD3-epsilon subunits present in the TCR-CD3 complex were in direct contact with one another. The presence of two CD3-epsilon polypeptide chains in close proximity to one another in the TCR-CD3 complex may have important implications for its assembly and its signal transduction mechanisms.
Fatty acids inhibit apical membrane chloride channels in airway epithelia.
Authors: Anderson M P , Welsh M J
Published: May 2006

Apical membrane Cl- channels control the rate of transepithelial Cl- secretion in airway epithelia. cAMP-dependent protein kinase and protein kinase C regulate Cl- channels by phosphorylation; in cystic fibrosis cells, phosphorylation-dependent activation of Cl- channels is defective. Another important signaling system involves arachidonic acid, which is released from cell membranes during receptor-mediated stimulation. Here we report that arachidonic acid reversibly inhibited apical membrane Cl- channels in cell-free patches of membrane. Arachidonic acid itself inhibited the channel and not a cyclooxygenase or lipoxygenase metabolite because (i) inhibitors of these enzymes did not block the response, (ii) fatty acids that are not substrates for the enzymes had the same effect as arachidonic acid, and (iii) metabolites of arachidonic acid did not inhibit the channel. Inhibition occurred only when fatty acids were added to the cytosolic surface of the membrane patch. Unsaturated fatty acids were more potent than saturated fatty acids. Arachidonic acid inhibited Cl- channels from both normal and cystic fibrosis cells. These results suggest that fatty acids directly inhibit apical membrane Cl- channels in airway epithelial cells.
Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells.
Authors: Yaswen P , Smoll A , Peehl D M , Trask D K , Sager R , Stampfer M R
Published: May 2006

A human cDNA library obtained from cultured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease in expression might be relevant to epithelial transformation. One clone identified by this procedure corresponded to a 1.4-kilobase mRNA, designated NB-1, whose expression was decreased greater than 50-fold in HMECs tumorigenically transformed in vitro after exposure to benzo[a]pyrene and Kirsten sarcoma virus. Sequence analysis of NB-1 cDNA revealed an open reading frame with a high degree of homology to calmodulin. NB-1 expression could be demonstrated by polymerase chain reaction amplification in normal breast, prostate, cervix, and epidermal tissues. The presence of NB-1 transcripts was variable in primary breast carcinoma tissues and undetectable in tumor-derived cell lines of breast, prostate, or other origins. NB-1 mRNA expression could be down-regulated in cultured HMECs by exposure to reconstituted extracellular matrix material, while exposure to transforming growth factor type beta increased its relative abundance. The protein encoded by NB-1 may have Ca2+ binding properties and perform functions similar to those of authentic calmodulin. Its possible roles in differentiation and/or suppression of tumorigenicity in epithelial tissues remain to be examined.
Electron transfer to the binuclear center in cytochrome oxidase: catalytic significance and evidence for an additional intermediate.
Authors: Malatesta F , Sarti P , Antonini G , Vallone B , Brunori M
Published: May 2006

We have followed, by transient kinetics, the reduction of cytochrome a3 in the presence of carbon monoxide under different experimental conditions. We have observed that the internal electron transfer rate accounts for the turnover number, and both display the same pH and temperature dependence [pKa = 7.4 and activation energy (Ea) = 14.7 +/- 0.1 kcal/mol]. Moreover, comparison of the time course of cytochrome c oxidation and cytochrome a3 reduction indicates that two electrons are transferred internally and with different rates to the oxygen-binding site. A kinetic model based on sequential internal electron transfer pathways, describing quantitatively the experimental data, is presented and discussed.
p53 mutations in colorectal cancer.
Authors: Rodrigues N R , Rowan A , Smith M E , Kerr I B , Bodmer W F , Gannon J V , Lane D P
Published: May 2006

Immunohistological staining of primary colorectal carcinomas with antibodies specific to p53 demonstrated gross overexpression of the protein in approximately 50% of the malignant tumors examined. Benign adenomas were all negative for p53 overexpression. To determine the molecular basis for this overexpression we examined p53 protein expression in 10 colorectal cancer cell lines. Six of the cell lines expressed high levels of p53 in ELISA, cell-staining, and immunoprecipitation studies. Direct sequencing and chemical-mismatch-cleavage analysis of p53 cDNA by using the polymerase chain reaction in these cell lines showed that all cell lines that expressed high levels of p53 were synthesizing mRNAs that encoded mutant p53 proteins. In two of those four cell lines where p53 expression was lower, point mutations were still detected. Thus, we conclude that overexpression of p53 is synonymous with mutation, but some mutations would not be detected by a simple immunohistochemical analysis. Mutation of the p53 gene is one of the commonest genetic changes in the development of human colorectal cancer.
Direct determination of phospholipid lamellar structure at 0.34-nm resolution.
Authors: Dorset D L , Beckmann E , Zemlin F
Published: May 2006

Low-dose, high-resolution electron microscopy combined with conventional direct-phasing methods based on the estimates of triplet-structure invariants are used to determine phase values for all observed electron-diffraction-structure factor magnitudes from epitaxially oriented multilamellar paracrystals of the phosphospholipid 1,2-dihexadecyl-sn-glycerophosphoethanolamine. The reverse Fourier transform of these phase-structure factors is a one-dimensional electrostatic potential map that strongly resembles the electron-density maps calculated from similar x-ray-diffraction data. Determination of the phase values for the electron-diffraction data with structure invariants alone is nearly as successful as the combined use of two separate methods, assigning values to 13 of the 16 reflections--i.e., the electrostatic potential map closely resembles the one calculated with all data.
Characterization of a complete immunoglobulin heavy-chain variable region germ-line gene of rainbow trout.
Authors: Matsunaga T , Chen T , Törmänen V
Published: May 2006

A germ-line heavy-chain variable region (VH) gene (RTVH431) has been isolated from a rainbow trout (Salmo gairdneri) and characterized by complete nucleotide sequencing. It is characteristic of VH, as shown by the conserved octamer and TATA motif in the 5' region, the heptamernonamer recombination signal sequence in the 3' region, and the 18-amino-acid-long hydrophobic leader interrupted by an intron. The 98-amino-acid-long VH coding region has 50-70% nucleotide sequence homology and 40-60% amino acid sequence homology with VHS of various vertebrate species. We have also found unique or species-specific amino acid residues in the VHS of rainbow trout, amphibia (Xenopus), reptile (Caiman), and shark (Heterodontus) in our sequence analyses. The RTVH431 has an unusual amino acid in the conserved 34th position in complementarity-determining region 1 of VH. Southern hybridization results suggest the presence of a large gene family related to RTVH431 in the trout genome. The complex evolution of antibody V genes is discussed.