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PubMed2005

Expression of dominant negative Rho-binding domain of Rho-kinase in organ cultured human eye anterior segments increases aqueous humor outflow.

PURPOSE: Based on pharmacological inhibition of activity, a role has been proposed for Rho-kinase in the modulation of aqueous outflow and intraocular pressure (IOP). This study employed a molecular approach to specifically address the role of Rho-kinase in the modulation of aqueous humor outflow. METHODS: Adenoviral vectors expressing green fluorescent protein alone (Ad-GFP) or the dominant negative Rho-binding domain of Rho-kinase and GFP (Ad-DNRK-GFP) were utilized in these experiments. Human and porcine primary trabecular meshwork (TM) cells were infected with 30 MOI (multiplicity of infection) of Ad-GFP alone or with Ad-DNRK-GFP. Changes in cell shape, actomyosin cytoskeletal integrity, and the status of myosin light chain (MLC) phosphorylation were evaluated. The aqueous outflow facility was determined in organ cultured anterior segments of human cadaver eyes infected with 10(7) pfu adenoviral vectors (Ad-GFP or Ad-DNRK-GFP) using a constant flow perfusion system. RESULTS: Expression of DNRK resulted in changes in cell shape (cell rounding, cell-cell detachment) and decreased actin stress fiber and focal adhesion staining in TM cells. These cellular changes were associated with substantially reduced myosin light chain phosphorylation. Additionally, organ cultured human eye anterior segments infected with Ad-DNRK-GFP exhibited a significant increase in the outflow facility (80%, n=9) compared to control anterior segments infected with Ad-GFP (5%). CONCLUSIONS: This study demonstrated that specific inhibition of Rho-kinase activity in trabecular meshwork cells led to alterations in cell shape and presumed contractile properties, and we hypothesize that these morphological and contractile events underlie the observed effects of dominant negative Rho-kinase on the aqueous humor outflow facility. These data provide molecular evidence for the hypothesis of Rho-kinase being a potential cellular target involved in the regulation of aqueous humor outflow resistance, with implications for novel glaucoma therapy.

Actins↗

PubMed1992

The primary structure of the flavoprotein D-aspartate oxidase from beef kidney.

The complete primary structure of the peroxisomal flavoenzyme D-aspartate oxidase from beef kidney has been determined by analyses of the peptides obtained through fragmentation of the carboxymethylated protein with trypsin, CNBr, heptafluorobutyric acid/CNBr and Staphylococcus aureus V8 protease. The protein consists of a single polypeptide of 338 residues, accounting for a M(r) of 37,305 for the apoprotein. A form of the enzyme lacking Lys-338 and therefore ending with Pro-337 has been detected. The N-terminal residue is blocked. Seven cysteines and no disulfide bridges are present. Residue 228 can be either Ile or Val. Thus, D-aspartate oxidase presents two types of heterogeneity in the polypeptide chain in addition to the one already described concerning the possible content of FAD or 6-hydroxyflavin adenine dinucleotide. Comparison of the primary structure of D-aspartate oxidase with other known sequences reveals that D-aspartate oxidase is homologous with D-amino acid oxidase (another flavo-oxidase) and does not present significant sequence similarities with any other protein, including flavoenzymes.

Amino Acid Oxidoreductases↗

PubMedDate not supplied

[Biochemical mechanisms of temperature adaptation in the (+) and (-) strains of Blakeslea trispora].

Evidence obtained with industrial beta-carotene-superproducing (+)T and (-)T strains, which fail to form zygotes, suggests that the lipids in the mycelium of the (-) strain of Blakeslea trispora lack linolenic acid. This circumstance apparently accounts for the fact that the (+) and (-) strains of B. trispora use different adaptive mechanisms to cope with an increase or decrease in cultivation temperature. In the (+) strain, temperature adaptation is based on changes in the ratio between linoleic and linolenic acyls and, also, involves shortening of acyl chains. In addition, the (+) strain contains a larger amount of protective carbohydrates, such as arabitol and trehalose. This strain is characterized by the presence of glycerol, a cryothermoprotector that protects fungal cells at low temperatures. The (-) strain lacks these biochemical mechanisms, but its neutral lipids contain a comparatively high amount of sterols and their esters. These facts enable us to interpret the enhanced thermotolerance of the (-) strain and its capacity to grow at high temperatures in terms of biochemical adaptation. In the light of the data obtained with wild-type and industrial strains, it is suggested that the lack of linolenic acid in the lipids should be considered an essential sex-specific property of the heterothallic strains of Blakeslea trispora.

Adaptation, Physiological↗

PubMed2006

[Coercion and active interference in psychiatry].

It is becoming increasingly clear that the autonomy and legal position of psychiatric patients are impediments to their adequate care. There is a strong emphasis on the voluntary cooperation of the patient involved. The patient as a person appears to be more important than his or her health status. There will always be tension between the right to self-determination and the dangers to the patient's health. There is a need for a law addressing the necessity to treat. In addition, a chain of care should be developed encompassing the whole spectrum of care institutions and centred around the individual's needs for food, shelter, care, financial support and medical attention.

Coercion↗

PubMed1991

Characterization of heparan sulfate proteoglycan from calf lens capsule and proteoglycans synthesized by cultured lens epithelial cells. Comparison with other basement membrane proteoglycans.

After extraction with 4 M guanidinium chloride and purification by DEAE-cellulose chromatography, the heparan sulfate proteoglycan (HSPG) of calf anterior lens capsule was found to consist of two immunologically related components (Mr = 340,000 and 250,000) which upon deglycosylation with trifluoromethanesulfonic acid yielded core proteins with Mr values of 170,000 and 145,000. The heparan sulfate chains were uniform in size (Mr = 14,000) and manifested a clustering of sulfate groups in a peripheral domain. From the decrease in Mr observed after heparitinase digestion, it could be estimated that 6 and 11 glycosaminoglycan chains were present in the Mr = 250,000 and 340,000 components respectively. The occurrence of N-linked oligosaccharides was evident from the size difference of the heparitinase- and trifluoromethane-sulfonic acid-treated proteoglycans (approximately 20 kDa), as well as from the presence of a substantial number of mannose residues; furthermore, interaction of the capsule proteoglycan with Bandeiraea simplicifolia I suggested that these carbohydrate units contains terminal alpha-D-Gal groups. Cultured lens epithelial cells deposited a single [35S]sulfate-labeled proteoglycan into their matrix (Mr = 400,000) which was immunologically related to the lens capsule proteoglycan and contained only heparan sulfate chains. In addition to this component, the medium from these cells contained an immunologically unrelated HSPG (Mr = 150,000) as well as a chondroitin sulfate proteoglycan (Mr = 240,000). Examination of bovine glomeruli indicated that, in addition to the previously described 200-kDa HSPG, an immunologically related 350-kDa component was also present. This size heterogeneity, which is comparable to that seen in the lens capsule, is most readily attributable to proteolytic processing of a precursor molecule. Studies with polyclonal antibodies demonstrated only limited cross-reactivities between the Engelbreth-Holms-Swarm proteoglycan and the components from lens capsule and glomerular basement membrane; since even the latter two differed somewhat in their antigenic sites, it would appear that cell- and species-dictated genetic differences as well as post-translational events contribute to the diversity observed in basement membrane HSPGs.

Amino Acids↗

PubMed1990

Reaction of insulin with reduced glutathione.

An aggregate of insulin, molecular weight about 70,000, was formed when it was incubated with GSH. The aggregate product converted to a low molecular weight product, presumably the insulin A- and B-chains, on addition of urea to mixtures. This conversion requires sulfhydryl-disulfide interchange as a sulfhydryl reagent inhibited it, in part. The aggregate does not form with EDTA in mixtures, but as before, a product of lower molecular weight than insulin was formed. These results support the previously held view that the disulfide bonds formed by insulin are influenced by its structure.

Animals↗

PubMed1990

The effects of Medicaid reimbursement method and ownership on nursing home costs, case mix, and staffing.

In this study we analyze the effects of ownership and Medicaid cost containment initiatives on nursing home costs, payer mix, case mix, and staffing, using 1981 Medicare cost reports and Medicare/Medicaid Automated Certification files. As Medicaid cost containment incentives become stronger, nursing homes respond by decreasing case mix and commensurately decreasing staffing. When these incentives are especially strong, staffing is reduced beyond the apparently appropriate level, given the case mix. In addition, while chain facilities have lower costs than other types of facilities, these lower costs do not appear to come from either increased cream-skimming or reduced staffing levels.

Aged↗

PubMed1990

Characterization of human anti-cytomegalovirus monoclonal antibody as biologics.

Human anti-cytomegalovirus (CMV) monoclonal antibody designated C23 was purified from the culture fluid of hybridoma cells which were generated by cell fusion of human lymphocytes and mouse myeloma cells. The purified C23 was found to be identical to human gammaglobulin (HGG) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and non-reducing conditions and in gel filtration chromatography. C23 was not contaminated with either aggregated IgG molecules or mouse immunoglobulin chains. In addition, the formulated C23 preparation showed an anti-complement activity low enough to permit its use as a biologic. A virus neutralization titer of C23 was about 1,000 times higher than the titers of HGG preparations. All the tested CMV strains were susceptible to neutralization by C23 and this neutralization was not affected by addition of either beta 2 microglobulin or fresh human serum. These results suggest that human monoclonal antibody C23 is as safe as conventional HGG preparations which have been used in humans, and much more effective in providing host protection against CMV infection.

Animals↗

PubMed1990

Two dissociable phases in the contractile response of the guinea pig isolated vas deferens to adenosine triphosphate.

The effects of the ATP affinity label periodate-oxidized ATP (ATP-2',3'-dialdehyde; P-ATP) on contractile responses of the guinea pig vas deferens to ATP was characterized and compared to the effects of the specific P2x-purinoceptor photoaffinity label antagonist, arylazido aminopropionyl ATP (ANAPP3). After incubation of vas deferens with 10(-2) M P-ATP for 5 min, the second phase of biphasic contractions to ATP was inhibited selectively. The inhibitory effect of P-ATP was specific for ATP, and resulted from affinity labeling in that it was long-lasting, was not reversed by washing, was related in magnitude to exposure period and was attenuated by ATP present during the incubation. In contrast to P-ATP, ANAPP3 inhibited selectively the first phase of ATP-induced responses. P-ATP and ANAPP3 together inhibited both phases of response. P-ATP inhibited selectively the second phase of responses to 5'-substituted ATP analogs which develop a prolonged second phase [e.g., adenosine 5'-O-(3-thiotriphosphate) and adenosine tetraphosphate] or an abbreviated second phase (e.g., beta,gamma-methylene ATP, beta,gamma-imido ATP and alpha,beta-methylene ATP). The greater the duration of the second phase, the more pronounced was the inhibitory effect. Two distinct and dissociable mechanisms mediate the biphasic response to ATP: the initial phase involves ANAPP3-sensitive P2x-purinoceptors, whereas the second is blocked by P-ATP and appears to involve cleavage of the phosphate chain. An additional effect observed as a result of P-ATP-treatment was potentiation of the first phase of contraction to ATP. This novel and irreversible effect may arise from the inhibition of degradative ecto-phosphohydrolase activity.

Adenosine Triphosphate↗

PubMed1987

Three different erythrocyte surface molecules are required for spontaneous T cell rosette formation.

We have obtained three anti-sheep erythrocyte (E) monoclonal antibodies (MAb) which block rosettes with human T cells. They also block rosettes with E from all the species we have tested, including rosettes with autologous E. They define three different minor components on the E surface: MAb N217 precipitates a single 42-kilodalton (kDa) chain and MAb N4 a single 14-kDa chain, and MAb N23 immunoprecipitates under nonreducing conditions a single band at approximately 220 kDa, which is resolved under reducing and alkylating conditions, in two bands migrating at approximately 110 kDa and approximately 220 kDa. Thus MAb N23 is likely to react with a complex made of covalently linked 100-kDa chains associated in a noncovalent fashion with approximately 220 kDa chains. In addition, we have observed a puzzling phenomenon, i.e., that binding the MAb N23 first increases, to a large extent, the amount of N4 epitopes which can be subsequently detected on E. This effect was not observed when N217 MAb or antiglycophorin (either monoclonal or polyclonal) antisera were first bound on E. Therefore the N23 and N4 molecules must interact on the E surface. This finding discloses the complex interactions between the T cell and E surface component that must occur in the process of rosette formation, including that with autologous E. These observations are of interest in view of the recent evidence that the CD2 molecule is involved both in adhesion and activation aspects of T cell functioning.

Antibodies, Monoclonal↗

PubMed1989

Are laminin binding sites on tumor cell surface involved in the indomethacin-induced sensitivity to natural cytotoxic cells?

Prostaglandins are secreted by a variety of tumor cell lines. The prostaglandin synthesis inhibitor indomethacin (IND) inhibits 3LL tumor growth after both intramuscular or intrasplenic transplantation (45 and 72%, respectively). Moreover, when tumor cells were cultured with IND, the sensitivity of 3LL cells to natural cytotoxic (NC) effector cells was increased (30%) and a higher cytotoxicity was reached when both target and effector cells were treated. This effect was reversed partially or totally when the assay was performed in the presence of laminin or an octapeptide from the laminin B1 chain. In addition, we correlate the increased cytotoxicity mediated by IND with an enhanced ability of 3LL tumor cells to bind labeled laminin (55%). In summary, our results show that the blockage or modulation of cell surface laminin binding components could be directly correlated with the sensitivity of tumor target cells to be eliminated by way of natural cytotoxicity.

Amino Acid Sequence↗

PubMed1989

Type XII collagen. A large multidomain molecule with partial homology to type IX collagen.

Three overlapping cDNAs encoding alpha 1 (XII) collagen have been isolated and sequenced. The DNAs define five sequence domains within the chain. Three domains are nontriple-helical; two are relatively short triple-helical regions. The amino acid sequences of tryptic peptides derived from 16- and 10-kDa pepsin-resistant fragments isolated from tendon extracts are in full agreement with the deduced sequences of the triple-helical regions. Two of the five sequence domains in alpha 1 (XII), one triple-helical and one nontriple-helical, show a high degree of similarity to regions in type IX collagen chains. In addition, examination of seven exons in the alpha 1 (XII) gene shows that the gene is, in part, similar to the structure of type IX collagen genes. Therefore, collagen types IX and XII are partially homologous. The alpha 1 (XII) sequence data predict an asymmetric structure for type XII collagen molecules, fully consistent with the rotary shadowing images. These images show a triple-helical 75-nm tail attached through a central globule to three finger-like structures, each 60 nm long (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156).

Amino Acid Sequence↗

PubMed1989

Ca2+-dependent binding of a synthetic Arg-Gly-Asp (RGD) peptide to a single site on the purified platelet glycoprotein IIb-IIIa complex.

The platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a member of the integrin receptor family that recognizes adhesive proteins containing the Arg-Gly-Asp (RGD) sequence. In the present study the binding characteristics of the synthetic hexapeptide Tyr-Asn-Arg-Gly-Asp-Ser (YNRGDS, a sequence present in the fibrinogen alpha-chain at position 570-575) to purified GP IIb-IIIa were determined by equilibrium dialysis. The binding of 125I-YNRGDS to GP IIb-IIIa was specific, saturable, and reversible. The apparent dissociation constant was 1.0 +/- 0.2 microM, and the maximal binding capacity was 0.92 +/- 0.02 mol of 125I-YNRGDS/mol of GP IIb-IIIa, indicating that GP IIb-IIIa contains a single binding site for RGD peptides. The binding of 125I-YNRGDS to purified GP IIb-IIIa showed many of the characteristics of fibrinogen binding to activated platelets: the binding was inhibited by fibrinogen, by the monoclonal antibody A2A9, and by the dodecapeptide from the C terminus of the fibrinogen gamma-chain. In addition, the binding of 125I-YNRGDS to GP IIb-IIIa was divalent cation-dependent. Our data suggest that two divalent cation binding sites must be occupied for YNRGDS to bind: one site is specific for calcium and is saturated at 1 microM free Ca2+, whereas the other site is less specific and reaches saturation at millimolar concentrations of either Ca2+ or Mg2+. The results of the present study support the hypothesis that the RGD domains within the adhesive proteins are responsible for their binding to GP IIb-IIIa.

Amino Acid Sequence↗

PubMedDate not supplied

Identification of ibopamine metabolites in rat and dog urine.

Metabolism of ibopamine (N-methyldopamine-O,O'-diisobutyryl ester) was studied in rats and dogs. The compound was well absorbed in both species when given orally. Most of the administered radiolabel (74-94%) was excreted within 24 hr in urine of both species. The major metabolite in rat urine was 4-glucuronylepinine (63% of the total administered dose). Minor metabolites identified were 4-O-glucuronyl-3-O-methylepinine, 3,4-dihydroxyphenylacetic acid (DOPAC), DOPAC-glucuronide, homovanillic acid (HVA), and HVA-glucuronide. Free epinine and epinine sulfate were detected in the range of less than 1% of the total administered dose. Metabolite patterns in dog urine were different from those of rat urine. The major metabolite was epinine-3-O-sulfate (62% of the total administered dose). Minor metabolites identified in dog urine were DOPAC-sulfate, HVA-sulfate, and free HVA. Free epinine was detected but in the range of less than 1% of the total administered dose. These results showed that ibopamine underwent extensive hydrolysis in vivo to epinine, which was subsequently conjugated and excreted as major metabolites in urine. In addition, side chain degradation of epinine led to minor metabolites, which were excreted in urine as free and conjugated forms. The route of conjugation of ibopamine metabolites is species dependent.

Animals↗

PubMed1987

Expression of T cell receptor-alpha and -beta subunits in human thymocytes. An immunocytologic study.

The expression of the T cell receptor (Ti)-alpha and -beta subunits in human thymocytes was studied with the use of two rabbit antisera directed at constant regions of human Ti-alpha- and Ti-beta-chains (H36 and H38, respectively). Immunoperoxidase techniques were employed to count by light and electron microscopy the cells, in the various thymocyte subsets, bearing Ti-alpha and Ti-beta subunits. Of the unfractionated thymocytes, 88% +/- 5 SD and 56% +/- 8 SD were labeled by H38 and H36, respectively. More than 90% of cells in cluster of differentiation (CD)-1+ (mainly cortical) and CD1-CD3+ (mainly medullary) subsets were stained with H38. When tested by H36, 51% +/- 6 SD of CD1+ and 81% +/- 8 SD of CD1-CD3+ thymocytes were positive. In the immature CD3-CD1- subpopulation, less than 3% of cells reacted with H36, whereas 15% +/- 3 SD were stained by H38. Flow cytometry revealed that CD1+ (mainly cortical) thymocytes expressed CD3 surface antigen in a percentage similar to that of CD1+ cells positive for Ti-alpha subunits. Indirect double labeling procedures with immunogold- and peroxidase-conjugated second antibodies demonstrated that almost all CD1+/Ti-alpha + cells expressed the surface CD3 antigen, whereas a large proportion of CD1+/Ti-beta + cells did not. These results indicate a sequential expression of Ti-beta and Ti-alpha subunits during intrathymic T cell differentiation. They also suggest that assembly and surface expression of the CD3-Ti complex are linked to the production of Ti-alpha-chains in addition to Ti-beta subunits. Last, the expression of Ti-alpha and Ti-beta subunits was studied in peanut agglutinin (PNA)+, CD1+ blasts representing the main, spontaneously proliferating intrathymic pool. The lack of Ti-alpha and Ti-beta subunits and the absence of surface CD3 antigen on most of these blasts suggest that immature T cells are compelled to proliferate in the thymus in a CD3-Ti complex independent manner.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1986

Periplasmic cytochrome C552 of Escherichia coli K12. Oligomeric structure and its role in nitrite reduction.

Perisplasmic cytochrome C552 was purified from a cold shock fluid. We demonstrated that this protein could reduce nitrite into ammonium. The reducing equivalents could be donated by formate or NADH through some segment of the membrane respiratory chain. FAD addition was necessary when pure cytochrome was used to reduce nitrite. The predominant form was found to have a molecular weight of about 65 to 70 K daltons, composed of three 21,000 d. subunits. Hemes may or may not be present on the subunit.

Chromatography, Gel↗

PubMed1979

Transplantation in miniature swine. V. Characterization of Ia antigens.

The complexity of the I region analog associated with the MHC of miniature swine has been probed by sequential antibody precipitation studies of Ia antigens. Treatment of solubilized lymphocyte preparations from MSLA homozygotes of the DD haplotype with excess AA anti-DD alloantiserum led to precipitation of only a portion of the total Ia antigens, as determined by secondary precipitation of the remaining material with CC anti-DD serum. The presumed I region of miniature swine must therefore code for more than one Ia antigen-bearing polypeptide chain. In addition, certain mouse alloantisera that had previously been shown to react with rat Ia antigens were tested for reactivity with swine Ia antigens. Anti-Iak mouse alloantisera precipitated Ia molecules from every swine extract tested, regardless of MHC type, precluding genetic mapping studies. However, sequential precipitation studies demonstrated that the cross-reactive mouse alloantisera reacted only with a subclass of swine Ia antigens, again suggesting genetic complexity of the pig Ia locus.

Animals↗

PubMed1985

Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells.

The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. A cellular proteoglycan associated with the peritubular cells is described which has properties similar to those of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. SC III has properties similar to those of a major Sertoli cell-secreted protein previously defined as a dimeric acidic glycoprotein. The stimulation by follicle-stimulating hormone of the incorporation of [35S]SO2(-4) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III (i.e. dimeric acidic glycoprotein), and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.

Animals↗