scholarly journals Asparagine-linked glycoprotein biosynthesis in rat epididymis. Presence of a mannosidase II-like enzyme

1991 ◽  
Vol 277 (1) ◽  
pp. 213-221 ◽  
Author(s):  
M D Skudlarek ◽  
M C Orgebin-Crist ◽  
D R P Tulsiani
Keyword(s):  
Antigenic Site ◽  
Indirect Evidence ◽  
Similar Function ◽  
Membrane Component ◽  
Complex Type ◽  
Present Evidence ◽  
Processing Enzymes ◽  
Golgi Membranes ◽  
Sds Page ◽  
Rat Epididymis

Previous studies from this laboratory using p-nitrophenyl alpha-D-mannoside (p-NPM) as substrate provided no evidence for the presence of mannosidase II in the rat epididymis [Skudlarek & Orgebin-Crist (1988) J. Reprod. Fertil. 84, 611-617]. However, rat epididymal epithelial cells cultured in the presence of swainsonine, an inhibitor of mannosidase II, produce abnormally processed N-linked glycoproteins containing hybrid-type oligosaccharides instead of complex-type [Tulsiani, Skudlarek & Orgebin-Crist (1990) Biol. Reprod. 43, 130-138], a result providing indirect evidence for the presence of mannosidase II-like enzyme in rat epididymis. In the studies described here, we present evidence for the occurrence of this processing enzyme in rat epididymal Golgi membranes. This enzyme is an integral Golgi membrane component. Like liver mannosidase II, the epididymal enzyme cleaves alpha 1,3- and alpha 1,6-linked mannosyl residues from GlcNAcMan5GlcNAc. However, unlike liver mannosidase II, the epididymal enzyme shows no activity towards the synthetic substrate, p-NPM. The epididymal mannosidase cross-reacts with liver anti-(mannosidase II) antibody, a result suggesting that the two enzymes share a common antigenic site(s). Immunoblotting studies following resolution of liver and epididymal Golgi membranes on SDS/PAGE show that, whereas the liver mannosidase II was resolved as a doublet of Mr 120,000 and 122,000, only the Mr 120,000 band was observed in the epididymal Golgi membranes. Immunoblotting of the Golgi-rich fractions, resolved under non-denaturing conditions, showed different patterns of charge and/or size isomers from the two tissues. These studies demonstrate tissue-specific differences in processing enzymes with similar function.

Response of aggregating chick corneal cells to modifiers of N-linked oligosaccharides, endoglycosidase H and deoxymannojirimycin

1988 ◽  
Vol 89 (3) ◽  
pp. 405-413
Author(s):  
J. Overton
Keyword(s):  
Fine Structure ◽  
Present Report ◽  
Single Cells ◽  
Cellular Responses ◽  
Complex Type ◽  
Normal Complement ◽  
Sds Page ◽  
Moderate Rate ◽  
High Mannose ◽  
Endoglycosidase H

Chick corneal epithelium takes on its mature conformation between 11 and 16 days of incubation. Earlier work has shown that desmosome frequency increases during this period, reaching its highest rate at 15 1/2 days. In the present report aggregation rates of cells from embryos of 11 days and those of 15 1/2 days are compared. Younger cells, which form fewer desmosomes, aggregate at a more moderate rate than older cells. In addition, younger cells bind less concanavalin A (ConA) than older cells. To determine if increase in ConA binding could be related to these cellular responses, aggregating cells were exposed to endoglycosidase H (EndoH) and to deoxymannojirimycin. This treatment should permit comparison of the response of cells that have a normal complement of N-linked oligosaccharides with those that have reduced high-mannose or complex type sugars. The effectiveness of EndoH under the conditions used was confirmed by failure of treated glycoprotein after separation by SDS-PAGE and electroblotting to bind ConA. Aggregation rates of both older and younger cells were unaffected, as measured by disapperance of single cells, though older cells formed somewhat smaller aggregates at the highest dosage used. Desmosome formation was markedly reduced in the presence of the enzyme, even in the absence of other changes in the fine structure. At the highest dose of the enzyme the fine structure of older but not younger cells showed indications of blockage of transport. Deoxymannojirimycin appears to cause a build-up of high-mannose groups, since treated cells showed increased incorporation of [3H]mannose.(ABSTRACT TRUNCATED AT 250 WORDS)

A perichromosomal region contains proteins phosphorylated during mitosis in Xenopus laevis cells

1991 ◽  
Vol 98 (3) ◽  
pp. 309-315
Author(s):  
S.M. Dilworth
Keyword(s):  
Xenopus Laevis ◽  
Molecular Mass ◽  
Nuclear Protein ◽  
Antigenic Site ◽  
Relative Molecular Mass ◽  
Specific Expression ◽  
Storage Mechanism ◽  
Phosphorylated Form ◽  
Sds Page ◽  
Specific Manner

An antibody that recognizes the phosphorylated form of nucleoplasmin has identified another nuclear protein whose antigenic form is regulated in a mitosis-specific manner, with a dramatic increase in binding occurring in all mitotic cells. The protein is localised around the periphery of condensed chromosomes during mitosis in a manner analogous to another nucleoplasmin-related polypeptide NO38. Mitosis-specific expression of the antigenic site is dependent on phosphorylation of the polypeptide; binding of the antibody is dramatically reduced by prior incubation of the polypeptide with phosphatases. Migration on SDS-PAGE suggests that the protein has an exceptionally large relative molecular mass, in excess of 400,000. The probable mitosis-specific phosphorylation and location of this antigen suggests a subcellular storage mechanism for proteins during mitosis.

Applicability of Baumrind's parent typology to collective cultures: Analysis of cultural explanations of parent socialization effects

2005 ◽  
Vol 29 (6) ◽  
pp. 552-563 ◽  
Author(s):  
Nadia Sorkhabi
Keyword(s):  
Parenting Styles ◽  
Child Outcomes ◽  
Child Rearing ◽  
Authoritative Parenting ◽  
Similar Function ◽  
Future Directions ◽  
Present Evidence ◽  
Authoritarian Parenting ◽  
Parent Socialization ◽  
Rearing Effects

This article reviews studies that have examined whether Baumrind's parenting styles are related to child outcomes similarly in cultures where independence is said to be emphasized versus cultures where interdependence is said to be emphasized. I present evidence showing that Baumrind's parenting styles have similar function in both collectivist and individualist cultures. Based on these studies, I argue against the claim of some researchers that authoritarian parenting is not detrimental or authoritative parenting beneficial to the development of young people in cultures that are said to emphasize interdependence. However, more research is needed before conclusions can be reached about the extent to which the culture construct explains child-rearing effects on child development. Future directions for research, which include the importance of identifying diverse forms of parenting within interdependent cultures so as to distinguish the influence of functional and dysfunctional forms of parenting on child outcomes, are suggested.

Monoclonal Antibodies Directed to the Calcium-Free Conformation of Human Protein S

1989 ◽  
Vol 62 (02) ◽  
pp. 708-714 ◽  
Author(s):  
Santica Marcovina ◽  
Raffaella Coppola ◽  
Carla Valsecchi ◽  
Adele Zoppo ◽  
Cecilia Gelfi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Protein ◽  
Antigenic Site ◽  
Fluid Phase ◽  
Protein S ◽  
Human Protein ◽  
Sephadex Column ◽  
Sds Page ◽  
Igg1 Subclass ◽  
Sepharose 4B

SummaryFour mouse hybridomas secreting monoclonal antibodies specific for human protein S (PS) have been generated. The antibodies, all of the IgG1 subclass, were designated S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient on warfarin therapy, and plasma from a patient with no free PS and only PS bound to C4b-binding protein. The slopes of the curves show that the monoclonal antibodies reacted equally with all the tested forms of PS indicating that the antigenic site(s) to which the monoclonal antibodies are directed are present and exposed in free and bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed, ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent with a buffer containing 4 mmol/1 CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained by chromatography of the barium citrate absorbate on a DEAE-Sephadex column. The protein peak containing the bulk of PS was subsequently applied to the immunoadsorbent and eluted with 4 mmol/1 CaCl2. These studies demonstrate that EDTA-dependent monoclonal antibodies can simplify the isolation of PS from human plasma.

scholarly journals Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery

Proteomes ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 36 ◽  
Author(s):  
Raj Luxmi ◽  
Crysten Blaby-Haas ◽  
Dhivya Kumar ◽  
Navin Rauniyar ◽  
Stephen M. King ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Sexual Reproduction ◽  
Growth Factor Signaling ◽  
Carboxypeptidase B ◽  
Peptide Processing ◽  
Processing Enzymes ◽  
Unicellular Green Alga ◽  
Sds Page ◽  
Catalytically Active ◽  
Cysteine Endopeptidases

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

scholarly journals Reconstitution of vesiculated Golgi membranes into stacks of cisternae: requirement of NSF in stack formation.

1995 ◽  
Vol 129 (3) ◽  
pp. 577-589 ◽  
Author(s):  
U Acharya ◽  
J M McCaffery ◽  
R Jacobs ◽  
V Malhotra
Keyword(s):  
High Speed ◽  
Protein Transport ◽  
Rat Kidney ◽  
Average Diameter ◽  
Cell Extract ◽  
Processing Enzymes ◽  
Golgi Membranes ◽  
Transport Vesicles ◽  
Target Membrane

We have developed an in vitro system to study the biochemical events in the fusion of ilimaquinone (IQ) induced vesiculated Golgi membranes (VGMs) into stacks of cisternae. The Golgi complex in intact normal rat kidney cells (NRK) is vesiculated by treatment with IQ. The cells are washed to remove the drug and then permeabilized by a rapid freeze-thaw procedure. VGMs of 60 nm average diameter assemble into stacks of Golgi cisternae by a process that is temperature dependent, requires ATP and a high speed supernatant from cell extract (cytosol), as revealed by immunofluorescence and electron microscopy. The newly assembled stacks are functionally active in vesicular protein transport and contain processing enzymes that carry out Golgi specific modifications of glycoproteins. The fusion of VGMs requires NSF, a protein known to promote fusion of transport vesicles with the target membrane in the exocytic and endocytic pathways. Immunoelectron microscopy using Golgi specific anti-mannosidase II antibody reveals that VGMs undergo sequential changes in their morphology, whereby they first fuse to form larger vesicles of 200-300-nm average diameter which subsequently extend into tubular elements and finally assemble into stacks of cisternae.

scholarly journals Postnatal changes in N-linked oligosaccharides of glycoproteins in rat liver

1988 ◽  
Vol 253 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S Kato ◽  
S Oda-Tamai ◽  
N Akamatsu
Keyword(s):  
Rat Liver ◽  
Concanavalin A ◽  
Post Partum ◽  
Deae Cellulose ◽  
Complex Type ◽  
Processing Enzymes ◽  
Significant Difference ◽  
Neonatal Liver ◽  
Old Rats ◽  
Rat Livers

[3H]Mannose-labelled glycopeptides in the slices of livers from neonatal and 1-, 2-, 3- and 5-week-old rats were characterized by column chromatographies on Sephadex G-50 and concanavalin A-Sepharose and by endo-beta-N-acetylglucosaminidase H digestion. The proportion of complex-type glycopeptides was increased with time until 2 weeks post partum and then returned to the neonatal level. This was mainly due to the increased proportion of concanavalin A-bound (biantennary) species. These changes were accompanied by consistent changes in the activities of processing enzymes in liver microsomal fraction, especially of N-acetylglucosaminyltransferase I. Complex-type glycopeptides from neonatal and 2- and 5-week-old rat livers were further characterized by column chromatographies on Bio-Gel P-6 and DE 52 DEAE-cellulose in combination with neuraminidase digestion. No significant difference was found between concanavalin A-bound species from neonatal liver and those from liver 5 weeks post partum, most of which were sialylated. Concanavalin A-bound species 2 weeks post partum were comparatively smaller in size and less sialylated. On the other hand, there was no significant difference among concanavalin A-unbound species from the three different sources, most of which were sialylated. Since glycoproteins from regenerating rat liver also contain a higher proportion of complex-type oligosaccharides, as previously reported, such changes in N-linked oligosaccharides of glycoproteins may be related to control of the growth of liver cells.

scholarly journals A multisubunit particle implicated in membrane fusion.

1992 ◽  
Vol 117 (3) ◽  
pp. 531-538 ◽  
Author(s):  
D W Wilson ◽  
S W Whiteheart ◽  
M Wiedmann ◽  
M Brunner ◽  
J E Rothman
Keyword(s):  
Lipid Bilayers ◽  
Sedimentation Coefficient ◽  
Direct Interaction ◽  
Eukaryotic Cells ◽  
Membrane Component ◽  
Particle Assembly ◽  
Golgi Membranes ◽  
Alpha Beta ◽  
Attachment Proteins ◽  
Hydrolysis Of

The N-ethylmaleimide sensitive fusion protein (NSF) is required for fusion of lipid bilayers at many locations within eukaryotic cells. Binding of NSF to Golgi membranes is known to require an integral membrane receptor and one or more members of a family of related soluble NSF attachment proteins (alpha-, beta-, and gamma-SNAPs). Here we demonstrate the direct interaction of NSF, SNAPs and an integral membrane component in a detergent solubilized system. We show that NSF only binds to SNAPs in the presence of the integral receptor, resulting in the formation of a multisubunit protein complex with a sedimentation coefficient of 20S. Particle assembly reveals striking differences between members of the SNAP protein family; gamma-SNAP associates with the complex via a binding site distinct from that used by alpha- and beta-SNAPs, which are themselves equivalent, alternative subunits of the particle. Once formed, the 20S particle is subsequently able to disassemble in a process coupled to the hydrolysis of ATP. We suggest how cycles of complex assembly and disassembly could help confer specificity to the generalized NSF-dependent fusion apparatus.

Two site-specific radioimmunoassays which demonstrate the presence of proteolytically modified insulin-like growth factor-binding protein-3 in the circulation

1993 ◽  
Vol 137 (2) ◽  
pp. 321-328 ◽  
Author(s):  
S. C. Cwyfan Hughes ◽  
J. A. H. Wass ◽  
J. M. P. Holly
Keyword(s):  
Growth Factor ◽  
Binding Protein ◽  
Antigenic Site ◽  
Binding Domain ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Sds Page ◽  
Igf Binding ◽  
Harsh Conditions ◽  
Igfbp 3

ABSTRACT The presence of proteolytic activity in the circulation directed against insulin-like growth factor binding protein-3 (IGFBP-3) was originally described in pregnancy but has subsequently been described in a number of catabolic and other pathological conditions. However, detection of this proteolytically modified IGFBP-3 has been demonstrated, in most cases, following separation using SDS-PAGE and this has led to speculation that its occurrence may be an artefact of the harsh conditions employed. Using two sitespecific antisera, one of which recognizes as its antigenic site a region of IGFBP-3 which is close to that of the IGF-binding domain, we have developed two radioimmunoassays which, when compared, can reveal alterations in the IGF-binding domain of IGFBP-3. The presence of IGFBP-3 modified by the circulating protease is denoted in this system by a divergence in the IGFBP-3 levels observed; this divergence has been shown to coincide with protease activity as determined by Western ligand blotting. The use of these assays has confirmed that the IGF-binding site of IGFBP-3 undergoes proteolytic modification in the circulation during a number of pathologies. Journal of Endocrinology (1993) 137, 321–328

Evidence for initial calcite-aragonite composition of Lower Algal Chert Member ooids and stromatolites, Paleoproterozoic Gunflint Formation, Ontario, Canada

2000 ◽  
Vol 37 (9) ◽  
pp. 1229-1243 ◽  
Author(s):  
M G Sommers ◽  
S M Awramik ◽  
K S Woo
Keyword(s):  
Aqueous Solution ◽  
Indirect Evidence ◽  
Mineral Species ◽  
High Fidelity ◽  
Compelling Evidence ◽  
Present Evidence ◽  
Primary Mineral ◽  
Two Stages ◽  
Initial Mineral ◽  
Chemical Sediments

The chert members of the Paleoproterozoic Gunflint Formation, Ontario, Canada, are commonly regarded as examples of primary silica chemical sediments. This interpretation is founded upon the ubiquitous nature of silica, high fidelity of preservation of microfossils found within, and lack of observed primary carbonate. Previous arguments for silica replacement of carbonate are based on indirect evidence and are largely dismissed. Thus, Gunflint microfossils are regarded as having lived in a silica-rich environment, which makes them unusual compared to most other pre-Phanerozoic microfossils that are known from silicified carbonates. We present evidence that carbonate was a primary mineral species of the Lower Algal Chert Member (where most of the microbiota are found). Most compelling are iron-stained, low-Mg calcite ooids containing (1) well-preserved growth laminae, (2) textures indicating a disrupted tangential fabric of growth laminae, and (3) quartz crosscutting the growth laminae. These indicate the initial mineral was a carbonate, possibly aragonite. Less-compelling evidence is found in stromatolite columns. Specularite crystals in low-Mg calcite display crosscutting relationships with calcite and quartz indicating that calcite was primary, specularite secondary, and quartz tertiary. The crosscutting relations show that silicification took place in at least two stages. One of the silicification events took place very early in diagenesis. Thus the Gunflint microbiota may not have existed in a radically different environment (silica precipitating) than those of most other known Proterozoic microbiota. High fidelity of preservation of microfossils does not always indicate that the mineral entombing them was the primary mineral precipitated from aqueous solution.

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