52K Protein Research Articles

Expression of the spr1 gene in cultured tracheal epithelial cells and its regulation by retinoids before and after confluence.

The absence of vitamin A or vitamin A derivatives in culture media promotes squamous cell differentiation of tracheobronchial epithelial cells. This is especially true for the expression of a small proline-rich protein (20K; 98 amino acids) in pig trachea epithelial cells. Multigene families encode different small proline-rich proteins in different species, and these proteins are possible markers for squamous cell differentiation. 20K mRNA and 20K protein were detected in cells within 4 and 5 days in culture, respectively, when cells reached about 50% confluence, and expression increase 12-fold during cell proliferation until cells reached 100% confluence. Arotinoid (10(-9)M), a synthetic retinoid, essentially totally inhibited expression of 20K mRNA in proliferating tracheobronchial cells within 3 days of treatment while 20K protein levels were only decreased 4-fold after 5 days. However, if cells were exposed to arotinoid 3 days after reaching confluent growth, the levels of either 20K mRNA or 20K protein were unchanged. Cells exposed to arotinoid from the onset of culturing, and then removal of the retinoid from proliferating cells resulted in the expression of 20K mRNA and protein after 4 and 5 days as observed previously. 20K mRNA was not detected in cells that had been continuously exposed to arotinoid from the start of culture until 3 days post confluence, even 10 days following removal of arotinoid. Our results strongly suggest that the growth phase and state of cell differentiation greatly affect the response of these epithelial cells to vitamin A derivatives.

An intrinsic ATPase inhibitor binds near the active site of yeast mitochondrial F1-ATPase.

An ATPase inhibitor and its stabilizing factor, the 9K protein, are regulatory factors of F1F0-ATPase. The binding sites for these factors on F1 were examined using the zero length cross-linkers, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, and 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide. The cross-linked products were analyzed by immunoblotting after SDS-polyacrylamide gel electrophoresis. The inhibitor and the 9K protein cross-linked to the alpha and beta subunits of F1, indicating that they interacted with both subunits. Peptide mapping and amino acid sequence analysis of the cross-linked products after weak acid hydrolysis showed that the inhibitor cross-linked to the Pro334-Asp363 region of the beta subunit. Amino acid sequence analysis of the cross-linked peptide showed that the inhibitor binds to Asp363 of the beta subunit. As this region contains the amino acid residues, including Tyr359, that are modified by nucleotide analogs and form the active site, the inhibitor probably binds to the catalytic site of F1.

Juvenile hormone activation of gene transcription in locust fat body

Transcription of a vitellogenin gene ( VgB) was measured in nuclei isolated from fat body of adult female locusts, to study its relations to juvenile hormone (JH). During the first ovarian cycle, there was a correlation between the rise and fall in the hemolymph titer of JH and the rate of Vg gene transcription. At the oviposition stage, when the JH titer fell sharply, Vg transcription fell almost to zero; both JH titer and Vg transcription then rose again in the subsequent cycle. Administration of the JH analog, pyriproxyfen, to ethoxyprecocene-pretreated locusts was followed, after a lag of about 12 h, by initiation and rapid rise in Vg transcription. Data on transcription of the 21K and apoLpIII genes, although imprecise because of short probe sequences, are consistent with the induction of 21K protein and lack of effect on apoLpIII protein by JH. Total transcription in fat body nuclei was also elevated after pyriproxyfen administration to precocene-treated locusts but declined during the vitellogenic cycle, suggesting that rRNA synthesis is regulated by both JH and other cellular factors.

Characterization of a cytosolic activity that induces the formation of Golgi membrane tubules in a cell-free reconstitution system.

Using a cell-free reconstitution system, we have characterized a cytosol- and ATP-dependent process that leads to the formation of membrane tubules from isolated Golgi complexes. These membrane tubules are uniform in diameter (50-70 nm) and morphologically identical to ones normally seen in cells and to those which are enhanced following brefeldin A treatment. Tubulation was strictly dependent on an activity present in an organelle-free extract of bovine brain cytosol and hydrolyzable ATP. Tubule formation was saturable with respect to both cytosol and ATP with half-maximal induction occurring at approximately 0.5 mg/mL cytosol and 10-20 microM ATP. Mild proteolytic treatment of Golgi membranes significantly reduced the extent of tubulation to subsequently added cytosol, suggesting that the tubulation activity interacts with Golgi-associated membrane proteins. The cytosolic tubulation activity was heat-labile, nondialyzable, and precipitated in ammonium sulfate. This activity could be followed through various chromatographic steps to yield fractions enriched in a major 40 kDa protein and several other minor proteins of approximately 80, 60, and 30 kDa. Monospecific antibodies against the 40K protein inhibited the cytosol-dependent tubulation of Golgi membranes in the cell-free system. Gel filtration chromatography suggests that the tubulation activity has a native molecular weight of approximately 125,000-140,000. These results establish the existence of cytosolic protein factors that regulate the formation of Golgi membrane tubules, and will provide the means for a biochemical dissection of membrane tubulation.

The cowpea mosaic virus RNA 1-encoded 112 kDa protein may function as a VPg precursor in vivo.

Processing of the 112 kDa ('112K') protein encoded by cowpea mosaic virus RNA 1 was examined in cowpea mesophyll protoplasts using a transient expression system. Cleavage of the 112K protein occurred via two alternative pathways either into VPg and 110K (24K + 87K) or into 26K (VPg + 24K) and 87K proteins. The 26K protein can be further cleaved into VPg and 24K proteins. The results support a model in which the 112K protein functions as the precursor of VPg during initiation of replication.

Okadaic acid induces both augmentation and inhibition of opsonized zymosan-stimulated superoxide production by differentiated HL-60 cells. Possible involvement of dephosphorylation of a cytosolic 21K protein in respiratory burst

We found that okadaic acid (OA), a potent tumor promotor and a phosphatase inhibitor, has a unique opposing effect on opsonized zymosan (Op.-zym.)-elicited O 2 . − production by differentiated HL-60 cells in a narrow range of concentration but does not induce any O 2 . − production by itself. Okadaic acid magnified the O 2 . − production 2.5-fold at 1.0 μM, while it inhibited it at 2.0 μM or higher concentrations. This effect of OA did not correspond to the changes in the expression of surface receptors (CD11b/CD18, CR3) for Op.-zym., because they were weakly down-regulated by OA at any concentration. Two-dimensional gel electrophoresis revealed that in the absence of OA, Op.-zym. induced rapid dephosphorylation of a cytosolic 21K protein with a very slight increase in phosphorylation of membranous p47 phox , which is one of the cytosolic factors required for respiratory burst. In the presence of a stimulatory concentration (1.0 μM) of OA, the Op.-zym.-caused dephosphorylation of the 21K protein was still observed and the phosphorylation of p47 phox was enhanced. In the presence of an inhibitory concentration (2.0 or 5.0 μM) of OA, the Op.-zym.-induced dephosphorylation of the 21K protein was strongly inhibited while p47 phox was heavily phosphorylated. Acid hydrolysis of the 21K phosphoprotein yielded only phosphoserine as a phosphoamino acid. Furthermore, at least part of the 21K protein seemed to be associated with p67 phox and p47 phox , because it was co-immunoprecipitated with those cytosolic factors. These results suggest that a cytosolic 21K protein plays an important role in respiratory burst through dephosphorylation by a phosphoserine phosphatase, and that the dephosphorylated 21K protein may work synergistically with the phosphorylated p47 phox on the pathway for activation of the respiratory burst oxidase.

Proteins in fluid from the proximal vas deferens of normal fertile and vasectomized men

Fluids were collected from the proximal vas deferens of 18 normal fertile men and 32 vasectomized men during vasectomy or vasovasostomy, respectively, and the protein concentration and pattern of proteins were then analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). In normal fertile men and vasectomized men there were no significant differences in total protein concentrations between fluids from the left and right vas deferens. The total protein concentration of proximal vas fluid from vasectomized men (82.8 +/- 7.5 mg/ml; mean +/- SEM) was significantly higher than in normal fertile men (36.8 +/- 4.5 mg/ml). In vasectomized men there was no significant correlation between the total protein concentration in vas fluid and the duration of vasectomy. The patterns of protein bands in proximal vas fluid differed from those in seminal plasma. There was no relationship between the pattern of protein bands in vas fluid and the duration of vasectomy. Five major groups of proteins from proximal vas fluid were identified and no new major proteins were found in proximal vas fluid were identified and no new major proteins were found in proximal vas fluid after vasectomy. The percentage of 80K and 67K protein bands in vasectomized men was higher than that in normal fertile men. It is suggested that, after vasectomy, the physiological environment of the proximal vas deferens may be changed. One of these changes may be the higher total protein concentration (especially the 80K and 67K proteins) in vas fluid.

Cloning and characterization of an autonomous replication sequence from Coxiella burnetii.

A Coxiella burnetii chromosomal fragment capable of functioning as an origin for the replication of a kanamycin resistance (Kanr) plasmid was isolated by use of origin search methods utilizing an Escherichia coli host. The 5.8-kb fragment was subcloned into phagemid vectors and was deleted progressively by an exonuclease III-S1 technique. Plasmids containing progressively shorter DNA fragments were then tested for their capability to support replication by transformation of an E. coli polA strain. A minimal autonomous replication sequence (ARS) was delimited to 403 bp. Sequencing of the entire 5.8-kb region revealed that the minimal ARS contained two consensus DnaA boxes, three A + T-rich 21-mers, a transcriptional promoter leading rightwards, and potential integration host factor and factor of inversion stimulation binding sites. Database comparisons of deduced amino acid sequences revealed that open reading frames located around the ARS were homologous to genes often, but not always, found near bacterial chromosomal origins; these included identities with rpmH and rnpA in E. coli and identities with the 9K protein and 60K membrane protein in E. coli and Pseudomonas species. These and direct hybridization data suggested that the ARS was chromosomal and not associated with the resident plasmid QpH1. Two-dimensional agarose gel electrophoresis did not reveal the presence of initiating intermediates, indicating that the ARS did not initiate chromosome replication during laboratory growth of C. burnetii.

Induction of calbindin-D 28K gene and protein expression by physiological stimuli but not in calcium-mediated degeneration in rat PC12 pheochromocytoma cells

To understand the role of calbindin-D 28K in neuronal degeneration, we examined its expression in differentiated PC12 cells in response to calcium intoxication, using the ionophore A23187 treatment, that results in cell degeneration and death. We first established that calbindin-D 28K is expressed in PC12 cells. The amounts of calbindin-D 28K mRNA and protein were increased by the differentiation factors, NGF and retinoic acid, but not by vitamin D 3. Calbindin-D 28K expression was also significantly up-regulated by stimuli (depolarization, low concentrations of Ca 2+ ionophore A23187) which increase intracellular calcium levels within the physiological range. In contrast, the calbindin-D 28K mRNA and protein concentrations were not modulated by high concentrations of A23187, which resulted in cell degeneration and death. Experiments with the antisense oligonucleotides showed that, although the calbindin-D 28K protein levels were decreased significantly, the progression of degenerative changes induced by calcium via A23187, was not altered.

Cotton-top tamarins with spontaneous colitis contain circulating antibodies reactive to human colonic Mr 40k protein, an autoantigen associated with human ulcerative colitis (UC)

Cotton-top tamarins with spontaneous colitis contain circulating antibodies reactive to human colonic Mr 40k protein, an autoantigen associated with human ulcerative colitis (UC)

Estrogen, not testosterone, creates male predominance of a P4501-related cytochrome in adult guinea pig adrenals.

Guinea pig adrenal microsomes possess a distinctive cytochrome P450 that is immunochemically related to P4501 and correlates with microsomal capacity for xenobiotic metabolism. This 52K protein and the capacity for metabolizing compounds such as ethylmorphine are located in the zona reticularis, are suppressed by ACTH, and are predominant in adult males. The protein is undetectable and the enzyme activity is low in young prepubertal animals. In males, both increase with age. However, in females, the protein remains undetectable, and ethylmorphine demethylase activity remains low into adulthood. Despite this clear sex difference through puberty and into sexual maturity, we recently observed that in female retired breeders, both the 52K cytochrome P450 and the capacity for metabolism of ethylmorphine appear at levels equal to those in males of comparable age. As estrogen levels are low in female retired breeders, we decided to investigate whether estrogen plays a role in maintaining the low levels of this protein and of xenobiotic metabolism seen in younger females. In a series of gonadectomy and hormone replacement experiments, we demonstrated that estrogen suppressed the levels of both protein and enzyme activity in adult guinea pigs. Ovariectomy resulted in the appearance of the 52K cytochrome P450 and of ethylmorphine demethylase in female adrenal microsomes at levels comparable to those seen in adult males. Estrogen replacement suppressed the increase in both protein concentration and enzyme activity. In hemiovariectomized cycling females, compensatory hypertrophy of the remaining ovary occurred, and the characteristic low levels of the 52K P450 and enzyme activity were maintained. Furthermore, estrogen treatment of male guinea pigs suppressed levels of both the 52K P450 and ethylmorphine demethylase activity in male adrenals. These experiments demonstrate that estrogen plays a significant role in the regulation of this protein. Testosterone, on the other hand, was not required to maintain the higher levels of 52K P450 and correlated enzyme activity in adult males. The levels of both were the same in normal, castrated, and sham-operated males, treated with testosterone or vehicle alone or left untreated. In fact, castration of prepubertal males resulted in a rapid rise to adult male levels of both immunodetectable protein and enzyme activity, implying that some suppressive agent of testicular origin effects the gradual increase that normally occurs with age in males.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationships between Grain Hardness and the Contents of Prolamin and Three Anti-fungal Proteins in Sorghum

Abstract The contents of three anti-fungal proteins of Mr 18k, 26k and 30k were determined by enzyme-linked immunosorbent assay (ELISA) for 40 cultivars of sorghum that varied in grain hardness and prolamin content. Statistically significant relationships were observed between the contents of the anti-fungal proteins and prolamin content, particularly for the Mr 18k (r=0·64) and 30k (r=0·73) proteins. Analysis of hard and soft portions of two cultivars shared that the Mr 18, 26 and 30k proteins were present at a higher level in the corneous endosperm than in the floury endosperm. Immunofluorescence microscopy, using antisera raised in rabbits against these proteins, revealed that the anti-Mr 18k antiserum reacted primarily with the protein bodies and along the cell walls, where the protein bodies are concentrated. Labelling was also more intense in the outer than in the inner endosperm. The Mr 26k protein was associated primarily with the cell walls and not with the protein bodies. The peripheral endosperm was labelled to the greatest extent as detached by immunofluorescence. No fluorescence was observed for the Mr 30k protein. The results indicate a locational and quantitative link between the sorghum prolamins and anti-fungal proteins. This may be due to common genetic modulation or linkage.

An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis.

The tyrosine kinase activity of c-Src is stimulated during mitosis by dephosphorylation of its regulatory tyrosine residue. This is associated with increased accessibility of its Src homology-2 (SH2) domain for binding a phosphotyrosine-containing peptide. But physiological targets of activated c-Src in mitosis have not yet been identified. Here we report that a 68K protein (p68) becomes tyrosine-phosphorylated and physically associates with Src during mitosis in mouse fibroblasts. p68 independently binds the Src SH2 and SH3 domains in vitro and both domains are required for p68 phosphorylation and binding in vivo. p68 is closely related to the p62 protein that is associated with the Ras GTPase-activating protein (GAP) and selectively binds, directly or indirectly, polyribonucleotides. Because the Src SH3 domain also binds heterogeneous nuclear ribonucleoprotein K, these results raise the intriguing possibility that c-Src may regulate the processing, trafficking or translation of RNA in a cell-cycle-dependent manner.

Immunohistochemical localization of calcium-binding proteins in the human cutaneous sensory corpuscles

The localization of the calcium-binding proteins (CaBP) calbindin-D 28k (CB), parvalbumin (PV) and S-100 protein (S100P) in the human cutaneous sensory corpuscles was studied by immunohistochemical procedure using monoclonal antibodies. Occurrence of CB, PV and S100P immunoreactivity (IR) was observed in the lamellar cells of Meissner's corpuscles. In the pacinian corpuscles, S100P IR was restricted to the inner-core cells whereas CB and PV IR were found labelling the axon, inner core, outer core and capsule. At the light-microscope level of resolution, the presence of IR in the axon of Meissner's corpuscles cannot be ensured. Since calcium ions (Ca 2+) seem to participate in the mechanoreceptor electrogenesis, present results suggest that CaBP could be involved in buffering and/or transport of Ca 2+ within the specialized cells surrounding the axon tips of sensory corpuscles, thus, maintaining the periaxonal microenvironment.

Nucleotide sequence and genome characterization of rice yellow mottle virus RNA

The genome of rice yellow mottle virus (RYMV) is a single-stranded positive-sense RNA that is not polyadenylated, and has an M(r) of 1.4 x 10(6). We present here the 4550 nucleotide (nt) sequence of RYMV RNA, and its predicted genomic organization. The RYMV genomic RNA contains four open reading frames (ORFs). The first (nt 80 to 553) encodes a protein containing 157 amino acids with a predicted M(r) of 17.8K. No function has yet been attributed to this product. ORF2 (nt 608 to 3607) encodes a polyprotein of 999 amino acids, with a predicted M(r) of 110.7K. The first 134 amino acids of ORF2 are predicted to be the genome-linked protein, VPg, followed by the viral protease, the helicase and the RNA-dependent RNA polymerase. ORF3 is within the boundaries of ORF2 and is predicted to encode a polypeptide with 126 amino acids and an M(r) of 13.7K. No function has yet been attributed to this protein. ORF4 (nt 3447 to 4166), which overlaps the 3' terminus of ORF2, encodes a 26K protein. This polypeptide has been identified as the RYMV coat protein. The data presented here confirm that RYMV belongs to the sobemovirus group and thus is a member of the picorna-like family of plant viruses.

Comparison of Phorbol Ester-induced Responses in Rabbit and Human Platelets and Difference in Shape Change

Phorbol 12,13-dibutyrate (PDBu) induced aggregation and ATP release of washed human and rabbit platelets in a concentration-dependent manner. The EC(50) for PDBu-induced aggregation of washed human and rabbit platelets were 67.6 ± 3.3 and 32.7 ± 5.9 nM, respectively. PDBu and l-oleoyl-2-acetylglycerol (OAG) also caused a shape change in rabbit platelets detected by microscopic and turbidimetric techniques. Both 20K and 47K proteins of rabbit platelets were phosphorylated by PDBu treatment. All the PDBu-induced responses of human and rabbit platelets were blocked by a protein kinase C inhibitor, staurosporine, in a concentration-dependent manner. In both human and rabbit platelets, PDBu induced no phosphoinositide breakdown, caused no increase of [Ca(2+)](f) and induced no formation of TxB(2). Indomethacin, BN52021, nitroprusside and cytochalasin B did not inhibit, whereas verapamil, prostaglandin E, and EGTA inhibited PDBu-induced aggregation of washed human and rabbit platelets. Apyrase and creatine phosphate/creatine phosphokinase inhibited PDBu-induced aggregation, but did not affect the shape change or 20K myosin light chain phosphorylation of rabbit platelets. In Ca(2+)-free Tyrode's solution containing 1 mM EGTA, PDBu induced a shape change in rabbit platelets and this effect was inhibited by staurosporine and cytochalasin B. It is concluded that PDBu and OAG induce a shape change in rabbit platelets probably through phosphorylation of 20K myosin light chain.

Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin.

Three synaptic proteins, syntaxin, SNAP-25 and synaptobrevin, were recently identified as targets of clostridial neurotoxins that irreversibly inhibit synaptic vesicle fusion. Experiments searching for membrane receptors for N-ethylmaleimide-sensitive fusion protein (NSF), which has an important role in membrane fusion, revealed an ATP-dependent interaction of the same three synaptic proteins with NSF and its soluble attachment proteins. Thus, two independent approaches identify syntaxin, synaptobrevin and SNAP-25 as components of the synaptic vesicle fusion machinery, but their mode of action is unclear. We have now discovered a brain protein of relative molecular mass 67,000 (67K) which binds stably to syntaxin. Amino-acid sequencing and complementary DNA cloning revealed that the 67K protein is encoded by the mammalian homologue of the Caenorhabditis elegans gene unc-18. In C. elegans, unc-18 belongs to a group of genes defined by mutations with a paralytic phenotype and accumulations of acetylcholine, suggesting a defect in neurotransmitter release. The binding of the mammalian homologue of unc-18 (Munc-18) to syntaxin requires the N terminus of syntaxin whereas that of SNAP-25 involves a more C-terminal sequence. Our data suggest that Munc-18 is a previously unidentified essential component of the synaptic vesicle fusion protein complex.

Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit.

The major events of the cell division cycle are triggered by periodic changes in the activity of cyclin-dependent protein kinases (CDKs). In mammals, the members of the CDK family include CDK2 and CDC2, which are thought to be involved in the control of DNA replication and mitosis, respectively. The protein kinase activity of these enzymes is controlled by a complex array of mechanisms. Activation of the CDK catalytic subunit requires association with a positive regulatory subunit (cyclin) and phosphorylation (at Thr 160 in CDK2). This activated complex can be inhibited by additional phosphorylation at Thr 14 and Tyr 15. Here we report the identification of a new mechanism for the regulation of CDK2 activity. We find that CDK2/cyclin complexes in mouse fibroblasts associate tightly with a 20K protein (CAP20). Complexes containing CAP20 were isolated from cell lysates and found to have negligible kinase activity, indicating that CAP20 association in vivo may inhibit CDK2 activity. We purified CAP20 from 3T3 cells and found that low concentrations of the protein completely inhibit the kinase activity of CDK2 in vitro. Thus CAP20 represents a new negative regulatory subunit that inhibits the activity of CDK2/cyclin complexes in mammalian cells.

45K actin filament-severing protein from sea urchin eggs: interaction with phosphatidylinositol-4,5-bisphosphate.

An actin filament-severing activity of 45K protein isolated from sea urchin eggs was abolished when this protein was incubated with phosphatidylinositol-4,5-bisphosphate (PIP2). This effect was specific to PIP2 since phosphatidylinositol, phosphatidylinositol-4-monophosphate, inositol-1,4,5-trisphosphate, and phosphatidylserine did not show such an effect at the same concentration. Digestion of PIP2 with phospholipase C eliminated the effect. On the other hand, PIP2 did not affect either the formation of 45K protein-actin complex or actin filament-capping activity of the complex. Possible implication of the binding of PIP2 to 45K protein in cytoskeleton formation after fertilization of sea urchin eggs is discussed.

A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies.

A panel of six neutralizing murine monoclonal antibodies (MAbs) to equine arteritis virus (EAV) was produced. The MAbs were characterized by Western immunoblotting assay and competitive ELISA. The six MAbs identify a single neutralization site on a 29K envelope glycoprotein. Deglycosylation of viral proteins prior to immunoblotting showed that the 29K protein is the glycosylated form of a 20K protein. Equine anti-EAV serum also strongly bound the 29K glycoprotein, as well as an unglycosylated protein of 17K. The equine antisera to EAV blocked the binding of a selected MAb to EAV, whereas normal equine serum did not. Two neutralization-resistant escape mutant (EM) variants of the EAV prototype were produced using MAb 6D10. The phenotypic properties of the EM viruses were characterized by neutralization and immunoblotting assays with two MAbs (6D10 and 5G11). The two MAbs failed to neutralize either EM virus, and they did not react in an immunoblot assay with any proteins of the EM viruses. In contrast, binding of the equine antiserum to viral proteins was equivalent with prototype and EM virus strains. These data clearly indicate that a 29K envelope glycoprotein expresses at least one neutralization determinant of EAV.