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R E Cohen

Publications and source records attributed to R E Cohen.

At least 73 records · Page 4Linked to original sources
PubMed1991

Uncoupling ubiquitin-protein conjugation from ubiquitin-dependent proteolysis by use of beta, gamma-nonhydrolyzable ATP analogues.

Pathways of ubiquitin-dependent protein degradation have in common two requirements for ATP. Ubiquitin activation by the enzyme E1 is accompanied by ATP hydrolysis to yield AMP and PPi, and during conjugate breakdown, the ubiquitin-dependent protease hydrolyzes ATP to ADP and Pi. We show here that either of two beta, gamma-nonhydrolyzable ATP analogues, 5'-adenylyl imidodiphosphate or 5'-adenylyl methylenediphosphate, can support ubiquitin-protein conjugation. With the ubiquitin-dependent protease, however, neither analogue could substitute for ATP. Thus, the substitution of a beta, gamma-nonhydrolyzable analogue for ATP offers a simple method to uncouple ubiquitin conjugation from proteolysis in crude systems. On the basis of pyrophosphate exchange kinetics, E1 has apparent Km and Vmax values that are similar for ATP and the analogues, but substrate inhibition by 5'-adenylyl methylenediphosphate made use of the beta, gamma-imido analogue preferable. In one application, beta, gamma-imido-ATP was used in combination with ubiquitin aldehyde (an inhibitor of ubiquitin-protein isopeptidases) to establish that several unfolded RNase A derivatives are recognized equally as ubiquitination substrates. This result extends an earlier study [Dunten, R. L., & Cohen, R. E. (1989) J. Biol. Chem. 264, 16739-16747] to show that conjugate yields, upon which relative ubiquitination rates were based, were not influenced by differential ubiquitin-dependent proteolysis. In a second application, ATP and beta, gamma-imido-ATP were compared in a pulse-chase experiment to investigate the contributions of ATP-dependent proteolysis and isopeptidase activities to conjugate stability.

Adenosine Triphosphate↗
PubMed1991

The structures of ubiquitin conjugates of yeast Iso-2-cytochrome c.

Ubiquitin (Ub) conjugates to Saccharomyces cerevisiae iso-2-cytochrome c were formed in vitro in a rabbit reticulocyte extract (Fraction II). In the presence of ubiquitin-aldehyde, used to inhibit ubiquitin-protein isopeptidases in Fraction II, mono-, di-, and triubiquitinated cytochrome c conjugates accumulated in a 1.2:1.0:0.2 molar ratio. CNBr digestions showed that, in all three conjugates, Ub attachment was within the first 73 amino acids of the cytochrome c. For the two most abundant conjugates, this region was further narrowed to the first 30 residues by peptide mapping with Staphylococcus aureus V8 protease. N-terminal protein sequencing identified Lys-13 as the major ubiquitination site in each conjugate. For di- and triubiquitinated iso-2-cytochrome c, this suggested that Ub2 and Ub3 multiubiquitin chains extend from Lys-13. This conclusion was supported by a variation of protein sequencing in which polypeptides recovered after Edman degradation were analyzed to determine at which cycle(s) radiolabeled Ub or Ubn was cleaved from the conjugate. Because of the sensitivity afforded by the use of 125I-Ub in this "stutter-step" sequencing method, minor ubiquitination at Lys-8 also was detected. Thus, Ub2-iso-2-cytochrome c conjugates contain mostly Ub2 at Lys-13 with a small fraction of conjugates having single Ubs on 2 residues, Lys-8 and Lys-13. Similarly, Ub3-iso-2-cytochrome c predominantly has a Ub3 chain on Lys-13, although minor species with combinations of Ub1 and Ub2 distributed on Lys-8 and Lys-13 also may be present. This specificity is discussed in the context of iso-2-cytochrome c structure.

Adenosine Triphosphate↗
PubMed1991

Specific disulfide cleavage is required for ubiquitin conjugation and degradation of lysozyme.

Both ubiquitin conjugation and ubiquitin-dependent degradation of chicken egg white lysozyme in a reticulocyte lysate depend on the presence of a reducing agent. We present evidence that the reduction of a specific disulfide bond, namely that at Cys6-Cys127, facilitates ubiquitination and is a prerequisite to the formation of a multiubiquitin chain on one of at least four chain initiation sites on lysozyme. The Cys6-Cys127 disulfide bond in lysozyme can be specifically reduced, and the modified protein can be isolated after carboxymethylation of the 2 resulting cysteines. This modified lysozyme no longer requires the presence of a reducing agent for ubiquitin conjugation and degradation. Inhibition of ubiquitination by the dipeptide Lys-Ala revealed that this modified lysozyme, like the unmodified protein, is recognized via the binding of the ubiquitin protein ligase, E3, to the substrate's N-terminal lysyl residue. Both the rate and the extent of ubiquitin-lysozyme conjugation, however, are significantly higher with this modified substrate. Likewise, ubiquitin-dependent degradation of 6,127-reduced/carboxymethylated lysozyme was 2-4-fold faster than degradation of the unmodified counterpart. These results are consistent with an interpretation that the modified lysozyme mimics an intermediate formed at the rate-limiting step of the degradation of lysozyme in the reticulocyte lysate. Reduction of the Cys6-Cys127 disulfide bond is expected to unhinge the N-terminal region of lysozyme, and we propose that the recognition of this otherwise stable protein by the ubiquitin pathway is due to facilitated binding of E3 that results from such a conformational transition.

Chromatography, Ion Exchange↗
PubMed1991

Immunochemistry and immunogenicity of low molecular weight human salivary mucin.

The purposes of this study were to examine the immunogenicity of the low molecular weight human salivary mucin (MG2) and determine its distribution within major and minor human salivary glands. Anti-MG2 sera were produced in Balb/c mice by a variety of immunization schedules. Chromatographically or electrophoretically purified MG2 and partially purified mucin chromatographic fractions exposed to mild denaturing conditions were not immunogenic. Only MG2 without prior exposure to urea or guanidine was able to elicit an immune response. A murine anti-MG2 monoclonal antibody (clone 1/F9) was produced and its monospecificity confirmed by immuno-dot blotting and SDS-PAGE Western transfer. Clone 1/F9 (IgG1; kappa) was of moderate affinity and was directed to a Pronase- and TPCK trypsin-sensitive but periodate-resistant epitope which was not blood group- or sialic acid-specific. Immunocytochemical studies of frozen tissue sections with clone 1/F9 using both indirect and direct methods revealed that MG2 was more heterogeneously distributed within submandibular than labial glands and was not found in parotid or palatine glands. The use of a polyclonal rabbit anti-MG2 reagent in either frozen or paraffin-embedded tissues gave the same immunocytochemical results as those obtained with the monoclonal antibody.

Adult↗
PubMed1990

Immunochemistry of high molecular-weight human salivary mucin.

The purpose of this study was to determine the distribution of mucin glycoprotein 1 (MG1) within submandibular, parotid, labial and palatine salivary tissues. Formalin-fixed and frozen tissue sections were examined histochemically with PAS, Alcian blue and Meyer's mucicarmine, and immunocytochemically with an anti-mucin glycoprotein 1 monoclonal antibody (clone 3/E8). Clone 3/E8 was produced in Balb/c mice using mucin-enriched chromatographic fractions from submandibular-sublingual saliva. The monospecificity of 3/E8 was confirmed by immuno-dot blotting and SDS-PAGE/electrophoretic transfer. Clone 3/E8 (IgG1; kappa) was of moderate affinity, and was directed to a carbohydrate-containing, TPCK-trypsin-insensitive and pronase-insensitive epitope on this mucin, which was not blood-group specific. The location of mucin glycoprotein 1 was determined by both indirect (peroxidase-antiperoxidase) and direct methods. Mucin glycoprotein 1 was localized within all labial acini examined, but was not found within parotid tissues. Histochemical methods stained all submandibular, palatine and labial acini, but immunocytochemistry with monoclonal antibody revealed heterogeneous staining with clone 3/E8 in submandibular and palatine tissues. These findings suggest the presence of mucin glycoprotein 1-specific acinar cell subpopulations within human submandibular and palatine salivary tissues.

Aged↗
PubMed1990

Atypical stromal cells of lower female genital tract.

Atypical stromal cells were identified in either the cervix or vagina in 27 (13%) of 205 autopsy specimens. They were more commonly observed in the vulva, occurring in 29 (78.4%) of 37 vulvar surgical specimens. In only seven patients were the cells present as a continuous band; five in cervix or vagina and two in vulva. Electronmicroscopic examination confirmed the fibroblastic nature of these cells. Although hormonal influences may be responsible for this stromal alteration, their occurrence in elderly patients and in males at other sites is not consistent with a specific hormonal profile. It is important not to misdiagnose these cells as malignant.

Adolescent↗
PubMed1990

Ectopic gingival sebaceous glands presenting as localized periodontitis.

The development of ectopic gingival sebaceous glands is a very unusual condition which is histologically similar to Fordyce's granules when they occur within the buccal or labial mucosa. In this report, we present a rare case of ordinarily innocuous, ectopic gingival sebaceous glands, which presented clinically and radiographically as localized advanced periodontitis. Histological and immunocytochemical evaluation of tissue removed from the periodontal lesion following excisional biopsy supported the diagnosis of an ectopic gingival sebaceous defect. This case illustrates the necessity of considering less frequently occurring entities in the differential diagnosis of localized lesions which appear to be periodontal in origin.

Adult↗
PubMed1990

Gingival manifestations of Wegener's granulomatosis.

Localized Wegener's granulomatosis is characterized by necrotizing granulomatous disease affecting the upper and lower respiratory tracts, mucosa, and skin. Without treatment, disseminated necrotizing vasculitis and focal necrotizing glomerulonephritis usually follow. Although oral lesions are relatively common, they have been infrequently described and have rarely been reported as the sole manifestation of this disease. In this report, we present two cases of early Wegener's granulomatosis which featured hyperplastic gingival lesions as the initial presenting lesion. Clinical findings and histological evaluation of tissue obtained from the gingival lesion following incisional and excisional biopsies supported the diagnosis of Wegener's granulomatosis. These cases illustrate that less-frequently occurring entities should be considered in the differential diagnosis of localized gingival lesions which fail to respond to conventional therapy.

Aged↗
PubMed1990

Pulmonary blastoma with malignant melanoma component.

Pulmonary blastomas are rare primary tumors that consist of tubular or glandular structures embedded in an undifferentiated mesenchymal stroma. Focal cartilage, bone, and skeletal muscle as well as squamous differentiation have been described in these tumors. We report a unique case of a pulmonary blastoma showing a malignant melanoma component. Immunohistochemical stains for S100 protein and HMB-45 were positive in the areas of melanocytic differentiation.

Carcinoma, Squamous Cell↗
PubMed1990

Tissue distribution of an inducible cystatin in isoproterenol-treated rats.

A well-characterized, monospecific rabbit antiserum directed to an isoproterenol-inducible type 2 salivary cystatin was used for immunocytochemical localization of this cystatin in rat salivary glands, as well as in other organs of normal and isoproterenol-treated rats. Immunocytochemical analysis revealed a moderate staining of secretory granules within the acinar cells of submandibular glands, which was more pronounced in tissues obtained from female rats. In addition, the inducible cystatin was readily detected within granular convoluted tubule cells and striated duct cells of submandibular glands of both male and female rats, although not all such structures were stained. Cystatin was also localized in the proximal convoluted tubule cells of the kidney in isoproterenol-treated female rats. Western blotting and Ouchterlony double diffusion analysis showed that the cystatin from submandibular gland and kidney extracts was immunologically identical.

Animals↗
PubMed1989

Recognition of modified forms of ribonuclease A by the ubiquitin system.

The substrate specificity of the ubiquitin (Ub) conjugation system was explored with regard to recognition of unfolded conformation and/or oxidized methionine residues in six derivatives of bovine RNase A. Based on the following observations, ubiquitination of RNase A substrates by the enzymes in a rabbit reticulocyte extract appears to correlate with unfolded conformation rather than with methionine oxidation. 1) Methionine oxidation in already unfolded forms of RNase A does not enhance ubiquitination. 2) Fluorescence measurements and iodoacetate trapping of free sulfhydryls show that the disulfide bonds of MetSO-RNase A, in which the 4 methionine residues are oxidized to the sulfoxide, are reduced by 2 mM dithiothreitol (DTT) in standard Ub conjugation assays so that this derivative also is unfolded. 3) Although MetSO-RNase A is ubiquitinated in the absence of DTT, its intrinsic fluorescence, cation-exchange properties, and susceptibility to reduction indicate a non-native conformation. 4) Methionine sulfoxide-containing peptides that mimic regions of RNase A fail to inhibit conjugation of 125I-Ub to MetSO-RNase A. Ub adducts to two of the six derivatives (MetSO- and reduced/carboxamidomethylated MetSO-RNase A) increase when DTT is omitted from the reactions. Ubaldehyde, an inhibitor of isopeptidases that disassemble Ub-protein conjugates, increased product yields and reduced or abolished the DTT effect, suggesting that an isopeptidase specific for these two RNase A derivatives may be inactivated by oxidation. Ub conjugates of the other RNase A derivatives also increase with Ub-aldehyde but are unaffected by DTT.

Amino Acid Sequence↗
PubMed1988

Signet-ring clear-cell basal cell carcinoma.

Basal cell carcinoma displays a myriad of histopathologic variants, some of which are related to the different lines of differentiation, vis-a-vis, squamous, pilar, eccrine, or sebaceous. We herein report an example of a rare signet-ring, clear-cell variant. Our diagnosis is primarily based on the histopathologic features of the tumor, namely, the dermal nests of tumor cells with peripheral palisading and focally retracted fibroblastic stroma. Several nests are folliculocentric. The tumor cells are glycogen-rich, mucin-negative, pankeratin-positive, cytokeratin-negative, S100 protein-negative, and carcinoembryonic antigen-negative. Based on the histopathology and the results of the special stains we propose that the signet-ring clear-cell variant of basal cell carcinoma is differentiating in the direction of the outer root sheath cells of the pilar structure.

Aged↗
PubMed1987

Immunochemical quantitation of alpha-amylase and secretory IgA in parotid saliva from people of various ages.

The alpha-amylase (128 subjects) and secretory IgA (118 subjects) concentration in stimulated parotid saliva of healthy individuals aged 23-84 years, was determined. They were divided into three age groups: I, 23-39; II, 40-59; and III, 60-84 years old. The concentrations (microgram/ml) of alpha-amylase for group I = 803.6; II = 648.0; and III = 652.4; the concentrations for secretory IgA for group I = 96.2; II = 101.8; and III = 97.7. The Kruskal-Wallis test revealed no significant differences between groups for alpha-amylase or secretory IgA.

Adult↗
PubMed1987

The Armero tragedy: lessons for mental health professionals.

A U.S. mental health consultant worked closely with medical personnel soon after a volcanic eruption and mud avalanche killed about 22,000 persons and devastated the area around Armero, Colombia. The consultant conducted workshops and courses on crisis intervention for health personnel operating disaster relief units and for mental health professionals, pediatric nurses, and family workers; she also provided consultations to clinic and shelter directors and case consultation with hospitalized victims. Observations of early postdisaster responses of hospitalized victims showed recurring themes such as victims' ambivalence about learning the full extent of the disaster and their own losses, delayed mourning because many bodies could not be recovered, somatic expressions of anxiety and fear, and the use of primitive defenses, such as magical thinking.

Colombia↗
PubMed1987

Structural aspects of salivary glycoproteins.

The protective functions of saliva are attributed, in part, to its serous and mucous glycoproteins. We have studied, as representative molecules, the proline-rich glycoprotein (PRG) from human parotid saliva and the high (MG1) and low (MG2) molecular weight mucins from submandibular-sublingual saliva. PRG (38.9 kDa) contains 40% carbohydrate consisting of 6 triantennary N-linked units and a single peptide chain of 231 amino acids, 75% of which = PRO + GLY + GLN. PRG's secondary structure is comprised of 70% random coil (naked regions) and 30% beta-turns (glycosylated domains). MG1 (greater than 10(3) kDa) contains 15% protein (several disulfide linked subunits), 78% carbohydrate (290 units of 4-16 residues), 7% sulfate, and small amounts of covalently linked fatty acids. MG2 (200-250 kDa) contains 30% protein (single peptide chain), 68% carbohydrate (170 units of 2-7 residues), and 2% sulfate. The major carbohydrate units of MG2 are: NeuAc alpha 2,3Gal beta 1,3GalNAc,Gal beta 1,3GalNAc, and Fuc alpha 1,2Gal beta 1,3GalNAc. MG1 contains hydrophobic domains, as evidenced by its ability to bind fluorescent hydrophobic probes; MG2 does not. Collectively, the biochemical and biophysical comparisons between MG1 and MG2 indicate that these two mucins are structurally different. Several functional properties of MG1, MG2, and PRG have been examined, including their presence in two-hour in vivo enamel pellicle, binding to synthetic hydroxyapatite, lubricating properties, and interactions with oral streptococci. The data presented suggest that these glycoproteins may have multiple functions which are predicated, in part on their carbohydrate units. The potential significance of the structure-function relationships of these glycoproteins to the oral ecology is discussed.

Dental Pellicle↗
PubMed1986

Kinetics of the interaction of N-(phosphonacetyl)-L-aspartate with the catalytic subunit of aspartate transcarbamoylase. A slow conformational change subsequent to binding.

The binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) to the active sites of both the free catalytic subunit of aspartate transcarbamoylase and the intact holoenzyme causes conformational changes which have been studied extensively. However, no kinetic information has been available about the sequence of events occurring during the formation or dissociation of the complexes. Stopped flow kinetics, 31P saturation transfer NMR spectroscopy, and presteady-state kinetics were used to monitor the interaction of PALA with the catalytic subunit (or a derivative containing nitrotyrosyl chromophores which served as spectral probes). The various experimental approaches lead to a mechanism that includes a rapid binding of PALA with an "on" rate of about 10(8)M-1s-1 and an "off" rate of 28 s-1, followed by a much slower isomerization of the complex with a forward rate constant of 0.18 s-1. Analysis of the presteady-state bursts of enzyme activity when the protein is added to a mixture of substrates and PALA and of the lag in activity when the PALA complex with catalytic subunit is added to substrates yielded a rate constant for the reverse isomerization of 0.018s-1. Thus, the conformational change subsequent to PALA binding leads to a 10-fold increase in the equilibrium constant for complex formation. Stopped flow kinetic measurements of the spectral change resulting from mixing the complex of PALA and nitrated protein with native enzyme showed a slow process with a t1/2 of about 11 s, whereas 31P saturation transfer NMR experiments yielded at t1/2 of about 260 ms for the dissociation of PALA from the complex. This apparent disparity is understood in terms of the two-step binding scheme where rapid dissociation of the initial ligand X enzyme complex is measured by the NMR technique and the slow isomerization of the complex is responsible for the bulk of the stopped flow signal.

Aspartate Carbamoyltransferase↗
PubMed1986

Botulism: emotional impact on patient and family.

During the third largest outbreak of botulism reported in the United States, affective responses of patients and their family members were assessed to monitor and compare the emotional distress experienced by the two groups during the initial, acute phase of the life-threatening illness. Ratings of 12 patients and 16 family members indicated that family members were significantly more fearful and depressed than patients during the first week and as fearful and depressed as patients during the second week of hospitalization/treatment. Anxiety and helplessness decreased significantly in both groups by week 2. Results illustrate the impact of catastrophic illness on the entire family system and provide support for the utility of family-oriented, crisis interventions. In future outbreaks of catastrophic illness, the early identification of emotional distress and informational needs and provision of appropriate counselling to family members as well as patients may be clinically indicated and valuable in facilitating the coping process.

Adult↗