PubMed Health⌕ Search

Biomedical subjects

Y Milner

Publications and source records attributed to Y Milner.

At least 19 recordsLinked to original sources
PubMed2000

Changes in protein kinase C during vitellogenesis in the crayfish Cherax quadricarinatus--possible activation by methyl farnesoate.

During ovarian maturation in the crayfish Cherax quadricarinatus, changes in ovarian protein kinase C (PKC) isoenzymes take place in parallel to yolk accumulation (as shown by immunoblot analysis). Significant changes were recorded in the amounts of specific isoenzymes and in their distribution between the cytosol and the membranes. Ovarian maturation was accompanied by the appearance of high- and low-molecular-weight immunoreactive PKC isoenzyme species. Among the isoenzymes tested, PKC alpha was the most clearly activated during ovarian maturation, as shown by significant translocation from the cytosol to the particulate fraction and the appearance of high-molecular-weight species. Moreover, a similar picture was obtained in the ovaries of intersex individuals upon induction of secondary vitellogenesis by androgenic gland ablation. Immunohistological staining showed PKC alpha to be localized mainly in the cytosol of premature oocytes, whereas in later maturation stages, it was concentrated around the nucleus in a vesicular structure and in the oocyte membrane. In secondary vitellogenic stages, PKC was localized in the plasma membrane and apparently in follicular cells. In addition, its activity was demonstrated by in vitro phosphorylation assays of a crayfish ovarian homogenate. Activation of total PKC phosphorylation of histone, an external substrate, was induced by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate (TPA) or methyl farnesoate. Both TPA and methyl farnesoate stimulated activation of PKC alpha in organ culture, causing its translocation from the cytosol to the membranes and inducing autophosphorylation of threonine residues. The changes in PKC isoenzymes during ovarian maturation in the crayfish suggest their involvement in this process as well as a possible regulatory role for methyl farnesoate through a direct effect on some PKC isoenzymes.

Animals↗
PubMed2000

The distribution of pemphigus vulgaris-IgG subclasses and their reactivity with desmoglein 3 and 1 in pemphigus patients and their first-degree relatives.

BACKGROUND: Pemphigus vulgaris (PV) autoantibodies (PV-IgG) have been found in 40-70% of sera of first-degree relatives of pemphigus patients. OBJECTIVES: To determine the possible role of PV-IgG subclasses in the pathogenesis of the disease. PATIENTS AND METHODS: Study groups comprised 25 PV patients, 55 unaffected family members and 56 sera of healthy individuals. Indirect immunofluorescence (IIF) staining and Western immunoblotting (WB) techniques were used to determine total PV-IgG and PV-IgG subclasses and their reactivity to desmoglein (Dsg) 1 and 3. RESULTS: By IIF staining, circulating PV-IgG were found in 64% of the patients, in 15% of the relatives and in none of the controls (P < or = 0.001); by WB the results were 91%, 49% and 12%, respectively (P < or = 0.001). The distribution of PV-IgG subclasses 1-3 was similar among patients and their relatives. PV-IgG4 was found in 62% of the patients but in only one relative and was absent in the controls (P < or = 0.001). PV-IgG1, 2 and 4 were found to react mainly with Dsg3 and PV-IgG3 mainly with Dsg1 and 3. CONCLUSIONS: These results support the concept of a genetic predisposition in pemphigus. The non-complement-fixing PV-IgG4 and at least one complement-fixing PV-IgG subclass appear to be involved in the pathogenesis of the disease. The absence of PV-IgG4 among relatives who were PV-IgG carriers seems to be linked to the fact that they do not develop pemphigus. The exact nature of this linkage is still unclear.

Adult↗
PubMed2000

Increase of oxidatively modified protein is associated with a decrease of proteasome activity and content in aging epidermal cells.

For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker 1-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins.

Adolescent↗
PubMed1999

Identification of the gregarization-associated dark-pigmentotropin in locusts through an albino mutant.

In response to crowding, locusts develop characteristic black patterns that are well discernible in the gregarious phase at outbreaks. We report here a dark-color-inducing neuropeptide (dark-pigmentotropin) from the corpora cardiaca of two plague locusts, Schistocerca gregaria and Locusta migratoria. The chromatographic isolation of this neuropeptide was monitored by using a bioassay with an albino mutant of L. migratoria. The neurohormone, consisting of 11 amino acids, is identical to [His7] corazonin, previously isolated from corpora cardiaca of another acridid without known function. The present results show that even in isolated (solitary) nymphs, [His7] corazonin induces gregarious black patterns. Its primary structure shows some similarity with the vertebrate melanophore stimulating hormone.

Animals↗
PubMed1998

Vitamin A inhibits cytokines produced by type 1 lymphocytes in vitro.

The effect of vitamin A (retinol) on cell-mediated immune responses was studied. As an experimental model, Leishmania major infection in mice was used. In this model, resistant mouse strains develop a type 1 response, while susceptible strains develop a type 2 response. Using lymph node cells and T-cell lines developed from infected susceptible and resistant mice, it was found that vitamin A inhibited lymphocyte proliferation in a dose-dependent manner. By separately incubating antigen-presenting cells and T cells with vitamin A, it was shown that the inhibitory effect was on the T cells. Type 1 cytokine (IFN-gamma, GM-CSF, IL-2) secretion in vitro in response to stimulation with specific antigen was also inhibited in a dose-dependent manner, whereas secretion of type 2 cytokines (IL-4 and IL-10) was not affected by vitamin A. The inhibitory effect was also observed in PMA-stimulated (but not Con A-stimulated) lymphocytes and was noticeable even if the vitamin was added as late as 24 h after initiation of the incubation period. Since PMA does not operate via a receptor-coupled signaling pathway but rather directly affects the protein kinase C (PKC) pathway, we have measured the effect of vitamin A on PKC in situ activation. Incubation of lymphocytes and antigen in the presence of vitamin A caused inhibition of PKC isoenzymes translocation to the particulate cell fraction, as measured by immunoblotting. The results presented indicate that, when added to cell cultures in vitro, vitamin A inhibits only secretion of type 1 but not type 2 cytokines, possibly through an inhibitory effect on protein kinase C activity.

Animals↗
PubMed1997

Circulating pemphigus IgG in families of patients with pemphigus: comparison of indirect immunofluorescence, direct immunofluorescence, and immunoblotting.

BACKGROUND: Patients with pemphigus vulgaris (PV) are genetically linked to two alleles of the HLA subgroup, and circulating antibodies were found in first-degree relatives of these patients, thus showing genetic predisposition. OBJECTIVE: Our purpose was to determine the occurrence of circulating true PV-IgG in patients' relatives. METHODS: Circulating PV-IgG was determined in 21 first-degree relatives of 12 patients with PV by indirect immunofluorescence on monkey esophagus, carcinoma A431 cultures, and Western immunoblotting. Direct immunofluorescence was performed on skin biopsy specimens of 20 relatives. RESULTS: Circulating PV-IgG was detected in 15 relatives (71%) by all methods tested. Good correlation was found between immunoblot reactivity and immunofluorescence. Of the 15 "positive" relatives, only five showed fixation of IgG to epidermal cells in vivo. CONCLUSION: The permeability of the epidermis or epidermal cell reactivity in vivo probably controls the expression of disease in patients' relatives.

Adult↗
PubMed1991

The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae.

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.

Animals↗
PubMed1991

The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

Animals↗
PubMed1991

The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗
PubMed1990

Effect of androgenic gland ablation on morphotypic differentiation and sexual characteristics of male freshwater prawns, Macrobrachium rosenbergii.

Mature males of the freshwater prawn, Macrobrachium rosenbergii (de Man), may change from one to another morphotype, according to a set sequence. Small males may develop into orange-claw males and orange-claw males into dominant blue-claw males. Each of the three morphotypes demonstrates distinctive reproductive behavior and secondary sexual characteristics. The role of the androgenic gland in this morphotypic transformation was examined experimentally by bilateral androgenic gland ablation (andrectomy) of small males and orange-claw males. For andrectomy initiated in the small male morphotype, transformation to the next morphotype was permitted (orange-claw), but subsequent transformation to the blue-claw morphotype was blocked. Andrectomy of orange-claw males did not prevent transformation into the blue-claw. Andrectomy on both small and orange-claw males caused disappearance of the genital papillae and atrophy of the sperm ducts and testes. The growth rates of the andrectomized small and orange-claw males were significantly lower than those of the unoperated and sham-operated controls. We conclude that androgenic gland factors control not only the differentiation of male secondary sexual characteristics but also morphotypic differentiation. Bioassays based on the results of this study will be instrumental in the characterization of such a factor(s).

Animals↗
PubMed1990

Modulation of the binding and endocytosis of concanavalin A by guinea pig keratinocytes: reversible antagonistic effects of cholesterol and phospholipid-liposomes.

Alteration of guinea pig keratinocyte membrane microviscosities (eta) by liposomes of varying composition was determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Measurements performed either with whole cell suspensions or Percoll-separated cell subpopulations, indicate a similar membrane microviscosity (eta = 3.37 poise +/- 10%) compared to those microviscosities reported for other cell types. Our findings show that treatment of guinea pig keratinocytes with liposomes composed of phospholipids results in a decreased membrane microviscosity (1.95 poise), whereas treatment of the cells with an emulsion of cholesterol hemisuccinate, or liposomes composed of cerebrosides, causes an increase in membrane microviscosity (3.85 poise and 5.55 poise +/- 10%, respectively). Changes in membrane fluidity had no significant effect on cell viability. A reduced membrane microviscosity resulted in a decrease in the binding of Concanavalin A to keratinocytes, whereas an increased microviscosity resulted in an increased binding of Concanavalin A. Furthermore, endocytosis of Concanavalin A bound to keratinocytes plasma membranes was not significantly affected by a reduced membrane microviscosity, whereas an increased membrane microviscosity completely blocked the endocytosis of Concanavalin A. Another novel observation was that membranes "fluidified" by phospholipid liposomes could be "rigidified" by treatment with cholesterol hemisuccinate and vice versa. Moreover, these alternate changes in membrane microviscosity resulted in simultaneous alternate changes in the binding of Concanavalin A to the keratinocyte surface.

Animals↗
PubMed1989

Binding modes of IgG from pemphigus autoimmune sera onto guinea pig keratinocytes and the fate of bound IgGs.

Pemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P-IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P-IgG onto living keratinocytes only. This was shown with several Pemphigus sera or purified P-IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and "Km" values for P-IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]-P-IgG onto Percoll-separated (living) keratinocytes showed the existence of two classes of sites: 2 x 10(6) sites/cell high-affinity sites (Kd = 1.5 x 10(-6) M total IgG) and 25 x 10(6) sites/cell low-affinity sites (Kd = 6 x 10(-5) M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light-scattering signal, the RNA content, the size and morphology, and the P-IgG binding to the cells. The results indicated that P-IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell-sorter analysis of cells with membrane-bound P-IgG, coupled to direct determination of P-IgG released in the medium, revealed the fate of bound P-IgG: 40-60% of the P-IgGs were released in the medium within 30 minutes at 37 degrees C. This was accompanied and followed by a much slower, metabolic energy-dependent, internalization process of the membrane-bound P-IgG. The internalization has been confirmed by electron microscopy of bound P-IgG labeled with protein A-gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit "antisurface" antibodies.

Animals↗
PubMed1989

IL-1 production by T6 (CD1a) positive cord blood mononuclear cells (Langerhan's cell precursors?).

Human cord blood (CB) mononuclear cells were fractionated into peanut agglutinin positive (PNA+) and PNA negative (PNA-) subsets. The PNA+ subset was enriched for T6+(CD1a)Ia+ cells, which we have previously shown to resemble the Langerhans cells (LCs) of the skin, and therefore described as circulating LCs precursors. Supernatants of PNA+ and PNA- cells, and of FACS purified populations of T6+ CB cells, cultured with and without LPS, were tested for IL-1 activity. It was found that cord blood PNA+ mononuclear cells as well as purified populations of T6+ CB cells produce significant amounts of, both extracellular and cell associated, IL-1 as compared to PNA- and T6- cells, and comparable to those produced by macrophages. LPS stimulation mainly affected T6+ cells. It can be concluded that cord blood T6+ cells, presumably LCs precursors, are capable of IL-1 production.

Antigens, CD1↗
PubMed1988

Protein phosphorylation in Mycoplasma gallisepticum.

Incubation of the soluble fraction derived from Mycoplasma gallisepticum cells with [gamma-32P]ATP results in the phosphorylation of several endogenous proteins. One protein with an apparent molecular mass of 55 kDa was the acceptor of more than 95% of the radioactive phosphate. This protein was also found to be radiolabeled in intact cells grown in the presence of [32P]orthophosphate. Acid hydrolysis of the phosphorylated 55-kDa protein followed by two-dimensional electrophoresis revealed that the 32P-labeled material co-migrated with phosphoserine. The in vitro phosphorylation of the 55-kDa protein has an optimum pH of 5.5-6.0 and is not affected by various metabolites of glycolysis, by cAMP or by calmodulin with or without Ca2+. The phosphorylation is dependent upon divalent cations, a dependency that is best fulfilled by the simultaneous addition of Ca2+ and Zn2+ that act in a specific and cooperative manner. Of a variety of possible exogenous protein acceptors tested, the endogenous protein kinase was capable to phosphorylate only phosvitin. The phosphorylation of the 55-kDa protein is reversible through the activity of a phosphoprotein phosphatase present in the soluble fraction of M. gallisepticum. The phosphoprotein phosphatase has an optimum pH of 7.5-8.0, is inhibited by NaF and stimulated to a large extent by inorganic phosphate and arsenate and to a lesser extent by pyrophosphate ATP and ADP. The possible association of the reversible protein phosphorylation to cell shape and gliding motility of M. gallisepticum are discussed.

Adenosine Triphosphate↗
PubMed1988

Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide.

A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-D-arginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (D-BAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. cereus T was inhibited by p-chloromercuribenzene sulfonic acid. The D-BAPAases of B. subtilis and B. cereus T had a Michaelis constant for D-BAPA of 2.9 x 10(-5) M and 1.4 x 10(-4) M, respectively. D-BAPAase is an intracellular enzyme localized in the protoplast (80 to 90% in soluble form in the cytoplasm). The ability to cleave D-BAPA is suggested as an additional chemotaxonomic characteristic of sporeforming bacteria of the genera Bacillus and Clostridium.

Bacillus↗
PubMed1987

Inhibition of malaria parasite invasion of human erythrocytes by a lymphocyte membrane polypeptide.

Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level.

Antimalarials↗