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PubMed1991

Multiple roles of the pilus biogenesis protein pilD: involvement of pilD in excretion of enzymes from Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the genes pilB, pilC, and pilD encode proteins necessary for posttranslational modification and assembly of pilin monomers into pilus organelles (D. Nunn, S. Bergman, and S. Lory, J. Bacteriol. 172:2911-2919, 1990). We show that PilD, encoding a putative pilin-specific leader peptidase, also controls export of alkaline phosphatase, phospholipase C, elastase, and exotoxin A. pilD mutants accumulate these proteins in the periplasmic space, while secretion of periplasmic and outer membrane proteins appears to be normal. The periplasmic form of exotoxin A was fully mature in size, contained all cysteines in disulfide bonds, and was toxic in a tissue culture cytotoxicity assay, suggesting that in pilD mutants, exotoxin A was folded into its native conformation. The function of the other two accessory proteins, PilB and PilC, appears to be restricted to pilus biogenesis, and strains carrying mutations in their respective genes do not show an export defect. These studies show that in addition to cleaving the leader sequence from prepilin, PilD has an additional role in secretion of proteins that are released from P. aeruginosa into the surrounding media. PilD most likely functions as a protease that is involved in processing and assembly of one or more components of the membrane machinery necessary for the later stages of protein extracellular localization.

ADP Ribose Transferases↗
PubMed1985

Decreased amounts of core proteins I and II and the iron-sulfur protein in mitochondria from yeast lacking cytochrome b but containing cytochrome c1.

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in four mutants of yeast that lack a spectrally detectable cytochrome b and do not synthesize apocytochrome b. Quantitative analysis of intact mitochondria by immunoprecipitation or immunoblotting techniques with specific antisera revealed that the core proteins and the iron-sulfur protein were decreased 50% or more in the mitochondria from the mutants as compared to the wild type. Sonication of wild-type mitochondria did not result in any decrease in any of these proteins from the membrane; however, sonication of mitochondria from the four mutants resulted in a further decrease in the amount of these proteins suggesting that they are not as tightly bound to the mitochondrial membrane in the absence of cytochrome b. By contrast, the amounts of cytochrome c1 in the mitochondria, as determined both spectroscopically and immunologically, were not significantly affected by the absence of cytochrome b. In addition, no loss of cytochrome c1 was observed after sonication of the mitochondria suggesting that this protein is tightly bound to the membrane. These results suggest that the processing and/or assembly of these subunits of complex III into the mitochondrial membrane is affected by the absence of cytochrome b.

Cytochrome b Group↗
PubMed1985

An alternate pathway for the processing of the prolipoprotein signal peptide in Escherichia coli.

Previous studies showed that when the signal sequence plus 9 amino acid residues from the amino terminus of the major lipoprotein of Escherichia coli was fused to beta-lactamase, the resulting hybrid protein was modified, proteolytically processed, and assembled into the outer membrane as was the wild-type lipoprotein (Ghrayeb, J., and Inouye, M. (1983) J. Biol. Chem. 259, 463-467). We have constructed several hybrid proteins with mutations at the cleavage site of the prolipoprotein signal peptide. These mutations are known to block the lipid modification of the lipoprotein at the cysteine residue, resulting in the accumulation of unprocessed, unmodified prolipoprotein in the outer membrane. The mutations blocked the lipid modification of the hybrid protein. However, in contrast to the mutant lipoproteins, the cleavage of the signal peptides for the mutant hybrid proteins did occur, although less efficiently than the unaltered prolipo-beta-lactamase. The mutant prolipo-beta-lactamase proteins were cleaved at a site 5 amino acid residues downstream of the prolipoprotein signal peptide cleavage site. This new cleavage between alanine and lysine residues was resistant to globomycin, a specific inhibitor for signal peptidase II. This indicates that signal peptidase II, the signal peptidase which cleaves the unaltered prolipo-beta-lactamase, is not responsible for the new cleavage. The results demonstrate that the cleavage of the signal peptide is a flexible process that can occur by an alternative pathway when the normal processing pathway is blocked.

Amino Acid Sequence↗
PubMed1998

Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit--identification of a soluble precursor of the small subunit in a hypB mutant.

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal tryptic-fragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416-4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.

Amino Acid Sequence↗
PubMed1984

Subcellular localization of the synthesis and glycosylation of chondroitin sulfate proteoglycan core protein.

In order to study the temporal and topological events involved in the processing and assembly of chondroitin sulfate proteoglycan, a fractionation scheme involving differential centrifugation and discontinuous sucrose density gradient centrifugation was developed for homogenates of chick embryo sternal chondrocytes. The precursors in these subcellular fractions were examined by a series of pulse, pulse-chase, and continuous labeling experiments. When chondrocytes were pulsed for 20 min with [35S]methionine, an immunoprecipitable core protein precursor with an approximate molecular size of 376,000 Da was localized to the rough endoplasmic reticulum fractions. Further incubation under chase conditions showed the presence of the 376,000-Da species as well as two additional polypeptides of higher molecular masses in the smooth membrane-enriched fractions within the next 2 h. This translocation did not occur in the presence of the energy transfer inhibitor carbonyl cyanide-m-chlorophenylhydrazone. The labeling pattern of the newly synthesized core protein precursor with either [3H] mannose or [3H]glucosamine showed that N-linked oligosaccharide addition was found on the earliest synthesized product in the rough endoplasmic reticulum, and the addition of this oligosaccharide was inhibited by co-incubation with tunicamycin. Furthermore, the high mannose oligosaccharide was susceptible to cleavage by endo-beta-N-acetylglucosaminidase H, while upon chase approximately 56 and 31% of the glucosamine- and mannose-labeled oligosaccharides, respectively, were processed to resistant forms, presumably in the Golgi complex. Both direct assay of glycosyl- and sulfotransferases requisite for addition of chondroitin sulfate chains and sensitivity of intracellular precursors to chondroitinase, keratanase , and endoglycosidase H suggest that only the N-linked oligosaccharides are added in the rough endoplasmic reticulum and glycosaminoglycan chain addition occurs predominantly in smooth membranes.

Animals↗
PubMed1993

VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae.

vma12 mutants of the yeast Saccharomyces cerevisiae, which were originally identified as calcium-sensitive (cls) mutants that were also respiratory deficient (Pet-), have a defect in vacuolar membrane H(+)-ATPase activity (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The VMA12 gene was cloned by complementation of the growth defects of vma12 mutants. The nucleotide sequence of the gene predicts a polypeptide of 215 amino acids (25.2 kDa) with two putative membrane-spanning domains. A null vma12 mutant, constructed by chromosomal deletion of the gene, is viable but has completely lost the vacuolar membrane H(+)-ATPase activity and exhibits the same growth defects as observed for the original vma12 mutants. Synthesis and targeting of the subunits of the H(+)-ATPase in the delta vma12 mutant cells were examined by Western blotting analyses of whole cell and vacuolar membrane protein extracts. None of the peripheral membrane subunits that we analyzed (the 69-, 60-, 42-, and 27-kDa subunits) was detected in the vacuolar membrane fractions, although the cellular levels of these polypeptides appeared to be normal. The 100- and 17-kDa integral membrane subunits of the enzyme were absent or present at a substantially reduced level in mutant vacuolar membrane fractions. Anti-Vma12p antibodies recognized a vacuolar protein with the expected molecular mass of 25 kDa. However, the Vma12 protein was not detected in the vacuolar membrane ATPase complex that had been solubilized with a zwitterionic detergent, ZW3-14, and purified by glycerol gradient centrifugation (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). These results indicate that the VMA12 gene product is not a component of the active vacuolar ATPase complex and instead suggest that this protein is required during the process of assembly and/or targeting of the enzyme complex to the vacuolar membrane.

Amino Acid Sequence↗
PubMed2002

Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed.

Antigens↗
PubMed1999

The role of the L4 33K gene in adenovirus infection.

The late phase of adenovirus infection is characterized not only by the synthesis of late proteins and the assembly of new virions, but also by the inhibition of early gene expression and host cell translation. Previous work has demonstrated that both of these inhibitory effects depend upon expression from the major late transcription unit (MLTU), controlled by the major late promoter (MLP). Furthermore, the repression of early gene expression has been shown to be mediated in trans, suggesting a role for one or more MLTU-encoded soluble factor(s). A possible candidate for such a factor is the L4-encoded 33K gene product, a protein conserved throughout the Mastadenoviridae, but of no known function. To test the role of this protein in viral infection, a stop codon was placed at the 20th position of the 33K ORF. Viable virus with genomes containing the mutation were recovered in an overlap recombination assay. Phenotypic analysis revealed that the mutant virus had a significant deficiency in both kinetics of replication and final yield, as compared to the wild-type virus. Detailed analysis of infected cells showed that there was no detectable change in the regulation of expression of several early genes and the pIX gene. This suggests either that 33K is not involved in this late phase phenomenon or that this function is replaceable by another late protein(s). Late protein synthesis and accumulation were similar to those in wild-type-infected cells. However, the reduced yield of infectious mutant virus could be accounted for by a marked deficiency in the accumulation of intermediate particles and completed capsids, suggesting a role for 33K in the process of assembly. In addition there was a small but reproducible deficiency in the shutoff of host cell translation. These results show that the 33K protein plays an important, although apparently not essential, function in the late phase of virus infection.

Capsid↗
PubMed2006

Micropatterned avidin arrays on silicon substrates via photolithography, self-assembly and bioconjugation.

Processes for micropatterning protein arrays on inorganic substrates have gained attention in the development of biosensors and clinical diagnostics. This study demonstrates a chemically selective method based on photolithographic deposition of gold patterns with the subsequent attachment of functionalized alkanethiols via molecular self-assembly. Selective capping of carboxy groups on alkanethiols by N-hydroxysuccinimide esters allowed the deposition of methoxypoly(ethylene glycol) silane as a blocking agent on unpatterned regions. Carboxylates were used to couple a form of avidin to create a microarray of protein. This microarray was successfully probed with biotinylated quantum dots. In-process characterization methods included grazing-angle Fourier-transform IR spectroscopy, ellipsometry, contact-angle goniometry, atomic-force microscopy and fluorescence microscopy.

Avidin↗
PubMed1991

Secretion of VLDL, but not HDL, by rat hepatocytes is inhibited by the ethanolamine analogue N-monomethylethanolamine.

The role of phospholipids in the assembly and secretion of very low density lipoproteins (VLDL) has been investigated by incubation of monolayer cultures of rat hepatocytes with monomethylethanolamine, an analogue of ethanolamine and choline. The cellular concentration of phosphatidylmonomethylethanolamine was increased 17-fold in response to treatment of hepatocytes with monomethylethanolamine. The secretion of phosphatidylcholine, triacylglycerol, and the apolipoproteins BH, BL, and E into VLDL was inhibited by approximately 50% in hepatocytes incubated with monomethylethanolamine, compared to untreated cells. Cell viability was unaffected by treatment with the ethanolamine analogue, as was cellular protein synthesis. The mechanism by which monomethylethanolamine reduced VLDL secretion was examined. Since monomethylethanolamine is a structural analogue of ethanolamine and choline, an obvious hypothesis for explanation of the effect on VLDL secretion was that phosphatidylcholine biosynthesis, which is required for VLDL secretion (Z. Yao and D. E. Vance. 1988. J. Biol. Chem. 263: 2998-3004) was inhibited. However, the biosynthesis of phosphatidylcholine from [3H]choline or from [3H]glycerol was not significantly reduced in the analogue-treated, compared with the untreated, hepatocytes. Nor was the incorporation of [3H]glycerol into cellular triacylglycerol altered in the monomethylethanolamine-treated cells. Furthermore, addition of monomethylethanolamine to hepatocytes did not reduce the rate of biosynthesis of phosphatidylethanolamine either from CDP-ethanolamine or from phosphatidylserine, nor was phosphatidylserine biosynthesis from [3-3H]serine affected. The 50% inhibition of VLDL secretion elicited by monomethylethanolamine was apparently specific for VLDL because there was no difference in secretion of HDL (lipid or apoprotein moieties) or albumin by cells incubated with or without the ethanolamine analogue. The experiments showed that inhibition of VLDL secretion by monomethylethanolamine was not the result of decreased biosynthesis of phospholipids, triacylglycerols, or cholesteryl esters. More subtle effects of the ethanolamine/choline analogue, for example interference by the increased amount of phosphatidylmonomethylethanolamine, in the process of assembly of lipids with apoB remain a possibility.

Animals↗
PubMed1994

Selective assembly of laminin variants by human carcinoma cells.

BACKGROUND: The laminins are heterotrimeric basement membrane glycoproteins. Eight subunits that can be assembled into laminins have been characterized and are known as: A, B1, B2, S, M, K, B2t, B1k laminin chains. Although many neoplastic cells secrete laminins and some of them even assemble basement membranes, the pattern of production of various laminin subunits remains to be explored. EXPERIMENTAL DESIGN: The expression of laminin was examined in several human carcinoma cells using a panel of specific cDNA probes as well as polyclonal and chain specific monoclonal antibodies. For this purpose a human laminin S chain 2 kb cDNA was isolated and characterized and used together with existing probes for laminin chains. RESULTS: All carcinoma cell lines had a high level of expression of three light chains (B1, S and B2) mRNA. In contrast, the heavy chains of laminin, A and M, were expressed in negligible amounts as detected by Northern blotting and PCR. The only exception was the HU-1 lung adenocarcinoma cell line which expressed significant quantities of laminin M chain mRNA and lower levels of laminin A chain mRNA. The presence in the HU-1 cells of translated polypeptides was demonstrated by immunofluorescence staining. The cells contained both B1 and S chain laminin in the cell layer, but preferentially secreted the B1 chain into the culture supernatant as shown by Western blotting. The 300 to 400 kDa M chain immunoreactive band was found in laminin secreted into the culture medium of HU-1 cells. Immunoprecipitation of biosynthetically labeled proteins showed that the M chain was synthesized as a complex with B chains. Little or no A chain laminin was detected in the culture medium supernatant. HU-1 cells also synthesized the newly described laminin variant, epiligrin which was secreted into the medium. Thus, the HU-1 cells secreted two laminin variants: M-B1-B12 laminin and epiligrin into the culture medium. Immunostaining of HU-1 nude mice tumors showed that tumor basement membranes contained M, B1, and B2 laminin and epiligrin immunoreactivity but apparently no S chain. CONCLUSIONS: All human carcinoma cell lines produced laminin chains B1, B2 and S, but no or little A or M. The only exception was the lung carcinoma cell line HU-1. Human HU-1 carcinoma cells in culture synthesize several homologous laminin chains and regulate the process of assembly, secretion and deposition of laminin variants into tumor basement membranes. These data indicate that the tumor cells vary among themselves with regards to laminin production and that some of them, like HU-1 may produce essentially all laminin chains simultaneously.

Amino Acid Sequence↗
PubMed1983

Expression and disposition of the murine mammary tumor virus (MuMTV) envelope gene products by murine mammary tumor cells.

Three murine mammary tumor virus (MuMTV)-producing epithelial cell lines derived from murine mammary tumors were examined in order to identify the MuMTV-specific cell surface antigens and their distribution on the cell surface, to study the kinetics of the MuMTV envelope precursor processing, virus assembly, and release, and to characterize the soluble MuMTV antigens that are shed into culture medium. Cell surface labeling experiments showed that only the mature MuMTV envelope glycoproteins gp52 and gp36 were exposed on the cell surface, and that gp52 was more abundant than gp36. In cells producing large quantities of MuMTV, expression of gp52 on the cell surface was shown by immunoelectron microscopy to be localized predominantly on the surface of budding virions and not on smooth areas of the cell surface where virus was not budding. The cell surface associated gp36 was found not to be incorporated into budding virions. A few cells in all three cell lines were found to produce only a few or no MuMTV particles and in these cells, unlike in the high virus-producing cells, considerable quantities of gp52 were expressed on the surface membrane. All three cell lines were found to shed large amounts of the MuMTV env precursor polyprotein as well as the mature non-virion-associated glycoprotein, gp52, into the culture medium. The envelope precursor protein (P75env) that was shed into the culture medium was found to differ from the predominant form of the cellular env precursor (Pr70env) in that (1) P75env migrated with an apparent higher molecular weight than Pr70env in SDS gels; (2) Pr70env contained only the core oligosaccharide, whereas P75env contained fucose in addition to the core sugars; (3) two-dimensional gel electrophoretic analysis showed that Pr70env could be resolved into three to four components migrating in the basic region of the isoelectric focussing gel (pH 7-8), whereas P75env was resolved into 9-13 components migrating in a more acidic region of the gel (pH 5-7). The molecular structure of the exfoliated gp52 was found to be similar to that of the gp52 that was incorporated into the virions although the virion-associated gp52 was not the source of the gp52 in the medium. Our quantitative pulse-chase studies suggest that of the two populations of MuMTV env precursors that are present in MuMTV-producing cells, only Pr70env is processed intracellularly to give rise to the mature MuMTV envelope proteins gp52 and gp36.

Animals↗
PubMed2003

Molecular imaging of protein-protein interactions: controlled expression of p53 and large T-antigen fusion proteins in vivo.

Protein-protein interactions control essential steps in signal transduction pathways and other intracellular processes, and assembly of protein complexes modulates and responds to the regulatory events that exist in living animals. We have used microPET and fluorescence imaging to detect interactions between p53 tumor suppressor and large T antigen (TAg) of SV40 virus in a tetracycline-inducible two-hybrid system. To additionally validate this molecular imaging technique, we investigated whether expression of the reporter gene, comprised of a mutant thymidine kinase from herpes simplex virus 1 fused to green fluorescent protein could quantify relative differences in amounts of interacting hybrid proteins. In HeLa cells stably transfected with the reporter gene and interacting (p53-TAg) or noninteracting (p53 and polyoma virus coat protein) pairs of proteins, treatment with doxycycline produced time- and dose-dependent increases in expression of hybrid proteins. Proportional increases in amounts of reporter gene were produced only in cells expressing p53 and TAg. In mice bearing xenografts of these stably transfected HeLa cells, amounts of hybrid proteins were regulated with doxycycline. Both microPET imaging and biodistribution studies showed time- and dose-dependent increases in accumulation of the reporter substrate 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine only in p53-TAg tumors. Fluorescence microscopy of excised tumors also showed corresponding changes in expression of the fusion reporter gene in response to binding of p53 and TAg. These data demonstrate that the imaging two-hybrid system responds in a proportional fashion to increasing amounts of interacting proteins in vivo.

Acyclovir↗
PubMed2003

Actin-based endosome and phagosome rocketing in macrophages: activation by the secretagogue antagonists lanthanum and zinc.

Although motile endocytic vesicles form actin-rich rocket tails [Merrifield et al., 1999: Nature Cell Biol 1:72-74], the mechanism of intracellular organelle locomotion remains poorly understood. We now demonstrate that bone marrow macrophages treated with lanthanum and zinc ions, well-known secretagogue antagonists, reliably exhibit vesicle motility. This treatment results in accentuated membrane ruffling and the formation of phagosomes and early endosomes that move rapidly through the cytoplasm by assembling actin filament rocket tails. Protein-specific immunolocalization demonstrated the presence of Arp2/3 complex in the polymerization zone and throughout the actin-rich tail, whereas N-WASP was most abundant in the polymerization zone. Although Arp2/3 and N-WASP play essential roles in nucleating filament assembly, other processes (i.e., elongation and filament cross-linking) are required to produce forces needed for motility. Efficient elongation was found to require zyxin, VASP, and profilin, proteins that interact by means of their ABM-1 and ABM-2 proline-rich motifs. The functional significance of these motifs was demonstrated by inhibition of vesicle motility by the motif-specific ABM-1 and ABM-2 analogues. Furthermore, lanthanum/zinc treatment also facilitated the early onset of actin-based vaccinia motility, a process that also utilizes Arp2/3 and N-WASP for nucleation and the zyxin-VASP-profilin complex for efficient elongation. Although earlier studies using cell extracts clouded the role of oligoproline sequences in activating the polymerization zone, our studies emphasize the importance of evaluating motility in living cells.

Actin-Related Protein 2↗
PubMed2004

Septal columns in rodent barrel cortex: functional circuits for modulating whisking behavior.

In rodents, each mystacial whisker is represented in the granular layer of primary somatosensory (SI) cortex by a compact cluster of cells known as a barrel, and barrels are separated from each other by domains that are called septa. Vertical columns of neurons aligned with each barrel act as a functional assembly to process information from a "principal" whisker, but a functional role has not been identified for vertical columns of neurons that are aligned with the septa. To determine whether these septal columns provide the main source of projections to primary motor (MI) cortex, we placed retrograde tracers in MI cortex and analyzed the location of the retrogradely labeled neurons with respect to the septal and barrel compartments of SI barrel cortex. In cases in which SI barrel cortex was sectioned tangentially, retrogradely labeled neurons in the extragranular layers of SI were plotted and superimposed onto reconstructions of the layer IV barrel field. In each of these cases, most labeled neurons were located above or below the septal regions of layer IV. When SI barrel cortex was sectioned coronally, we observed multiple columns of labeled SI neurons that were vertically aligned with the septal zones of layer IV. These results indicate that columns of neurons that are vertically aligned with the septa, or septal columns, are functionally linked by virtue of their projections to MI cortex. We hypothesize that these septal columns represent an interconnected and functionally distinct circuit that transmits information to MI and other brain regions involved in motor control.

Animals↗
PubMed1988

Defective intercellular cohesion in glycosylation mutants of Dictyostelium discoideum.

In order to identify the biological roles of protein-linked oligosaccharides, we have isolated mutants by a selection for amoebae with temperature-sensitive defects in glycan assembly and processing. Of these, 75% were also temperature sensitive for development [Boose and Henderson, 1986]. Two such mutants with distinct developmental phenotypes and glycosylation patterns are described. Mutant HT7 cannot complete aggregation at the restrictive temperature and is defective in expression of EDTA-resistant cohesion. The biochemical defect appears to be early in glycan processing. A revertant of HT7 has recovered aggregation capability, EDTA-resistant cohesion, and reverted almost totally to wild-type glycosylation. Mutant HT15 aggregates at the restrictive temperature but then disperses into a cell lawn. It is less deficient in EDTA-resistant cohesion than HT7 and has a different glycosylation profile. These results provide strong support for a role of protein N-linked oligosaccharides in aggregation-stage intercellular cohesion.

Agglutination↗
PubMed1995

Cassette baculovirus vectors for the production of chimeric, humanized, or human antibodies in insect cells.

Plasmid cassette-transfer vectors pBHuC chi and pBHuC gamma l have been designed which enable the construction of recombinant baculoviruses directing the co-expression of complete immunoglobulin in insect cells. We describe the application of these vectors for the expression of a human/mouse chimeric monoclonal antibody of potential immunosuppressive clinical value derived from a mouse anti-human CD29 monoclonal antibody (Mu-K20). The chimeric K20 light and heavy chains produced in sf9 insect cells were correctly processed and assembled into a normal immunoglobulin which is secreted into the culture medium of infected cells. The chimeric mAb Ch-K20-sf9 reproduces in vitro the functional properties of the parental mouse K20, including affinity and inhibition of lymphocyte proliferation. These results demonstrate that the baculovirus/insect cell expression system is suitable for the expression of fully active monoclonal antibodies of therapeutic value. Our generic cassette approach makes this system a very flexible and convenient one for the rapid production of either chimeric, humanized or human mAb with heavy and light chains of any isotype.

Animals↗
PubMed1988

Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain.

The in vitro phosphorylation of chicken desmin by the catalytic subunit of cAMP-dependent protein kinase was analysed. Phosphorylated desmin loses the ability to form intermediate filaments (IFs). Fragmentation at the sole cysteine and mild chymotryptic treatment show a differential phosphorylation of the three structural domains. Only the amino-terminal head domain is the target of the kinase. Peptide analysis shows that serine 29 is fully phosphorylated, while serine 35 and 50 are phosphorylated at least at 22 and 50% respectively. All three sites show the sequence arginine-X-serine with X being a small residue. These results strengthen the view that the nonhelical head domain has a strong influence on filament integrity most likely via a direct influence of some of its arginine residues. Taken together with previous results (Inagaki et al., 1987) on the phosphorylation of vimentin by kinase A, a new view on IFs emerges. Phosphorylation could allow for regulatory processes in assembly and turnover.

Amino Acid Sequence↗