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Biomedical subjects

J Horwitz

Publications and source records attributed to J Horwitz.

At least 109 records · Page 6Linked to original sources
PubMed1989

Muscarinic receptor stimulation increases inositol-phospholipid metabolism and inhibits cyclic AMP accumulation in PC12 cells.

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A covalent change in alpha crystallin during opacification of the Emory mouse lens.

Polyclonal antisera made against synthetic peptides corresponding to the N-terminus (anti-alpha NT), the C-terminus (anti-alpha CT), and to an internal sequence (anti-alpha 147-161) of bovine alpha-A2 crystallin have been used to quantitatively probe Western blots of proteins from the Emory mouse cataractous lens. Relative to proteins from transparent lenses of the control Carworth Farm Webster (CFW) mouse, there is no significant difference in binding of the anti-alpha NT and anti-alpha CT sera to alpha crystallin from lenses of cataractous Emory mice versus transparent lenses of CFW mice at any of the ages studied. In contrast, antiserum to an internal sequence (anti-alpha 147-161) binds significantly better to alpha crystallin from cataractous Emory mice lenses. Together, these results demonstrate covalent changes in the alpha crystallin molecule during opacification of the Emory mouse lens, which like those occurring in the human senile cataractous lens, result in increased binding of the anti-alpha 147-161 serum.

Aging↗
PubMed1988

Tau-crystallin/alpha-enolase: one gene encodes both an enzyme and a lens structural protein.

tau-Crystallin has been a major component of the cellular lenses of species throughout vertebrate evolution, from lamprey to birds. Immunofluorescence analysis of the embryonic turtle lens, using antiserum to lamprey tau-crystallin showed that the protein is expressed throughout embryogenesis and is present at high concentrations in all parts of the lens. Partial peptide sequence for the isolated turtle protein and deduced sequences for several lamprey peptides all revealed a close similarity to the glycolytic enzyme enolase (E.C. 4.2.1.11). A full-sized cDNA for putative duck tau-crystallin was obtained and sequenced, confirming the close relationship with alpha-enolase. Southern blot analysis showed that the duck genome contains a single alpha-enolase gene, while Northern blot analysis showed that the message for tau-crystallin/alpha-enolase is present in embryonic duck lens at 25 times the abundance found in liver. tau-Crystallin possesses enolase activity, but the activity is greatly reduced, probably because of age-related posttranslational modification. It thus appears that a highly conserved, important glycolytic enzyme has been used as a structural component of lens since the start of vertebrate evolution. Apparently the enzyme has not been recruited for its catalytic activity but for some distinct structural property. tau-Crystallin/alpha-enolase is an example of a multifunctional protein playing two very different roles in evolution but encoded by a single gene.

Amino Acid Sequence↗
PubMed1988

Relationship between psychosocial variables and school absenteeism in kindergarten children.

Experimental and clinical studies have demonstrated that psychosocial factors may influence various immune responses. Recently, the relationship between psychologically stressful events and immunomodulation in humans has been reported. Few studies, however, have demonstrated clinically significant increases in infectious illness, or delayed recovery from infectious illness among humans as a result of stressful events. This study was undertaken to determine if children who experienced the greatest difficulty adjusting to kindergarten entry, a common event which is differentially stressful, would have the highest frequency of school absence (an index of illness). A randomly selected sample of 49 children entering kindergarten for the first time, and their families (patients of a suburban pediatric practice), were studied over a 6-month period utilizing health diaries, interviews with parents, parent ratings, physicians' records and ratings, and teacher ratings of children's behavior. We were unable to demonstrate a relationship between psychosocial factors and school absence among our sample of children. A possible explanation for this finding may be the relatively nondeviant character of the subjects studied. Another possibility is that kindergarten entry was not experienced as a stressful event for the children, most of whom had attended nursery school.

Absenteeism↗
PubMed1987

Conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes.

The conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes were studied by using near- and far-ultraviolet circular dichroism. The far-ultraviolet spectrum of MIP26 solubilized with octyl beta-D-glucopyranoside indicates an alpha-helical content of approximately 50% and a beta-structure content of approximately 20%. A detergent-free membrane suspension of MIP26 produced a typically distorted far-ultraviolet spectrum which was caused by differential light scattering and absorption flattening. However, decreasing the size of the membrane fragments by sonication produced a far-ultraviolet spectrum free of distortion, and with a rotatory strength profile similar to that obtained for MIP26 solubilized with octyl beta-D-glucopyranoside. This implies similar secondary structure properties for the protein in both the suspension and the sugar detergent. The cleavage of MIP26 with Staphylococcus aureus protease, which results in removal of a 5-kilodalton peptide and which mimics the age-dependent posttranslational changes that take place in the lens, did not significantly affect the conformation of the core protein as judged by the near-ultraviolet circular dichroism spectra.

Animals↗
PubMed1987

Cleavage from the N-terminal region of beta Bp crystallin during aging of the human lens.

Polyclonal antisera have been made to synthetic peptides that correspond to the N-terminal (residues 1-12) and C-terminal (residues 195-204) sequences of bovine beta Bp crystallin. Both anti-beta Bp1-12 and anti-beta Bp195-204 recognize specifically the beta Bp component of bovine lens. In the young human lens, anti-beta Bp195-204 recognizes predominantly the 26,000 MW form of beta Bp, while in older lenses this same antiserum recognizes mainly the 22,000 MW in vivo proteolysis product. In contrast, during aging of the normal human lens anti-beta Bp1-12 recognizes only decreasing amounts of the 26,000 MW form of beta Bp, with no binding to the 22,000 MW form of this polypeptide. These results suggest that during aging of the normal human lens, the N-terminus of beta Bp is the preferred site of in vivo proteolysis.

Adolescent↗
PubMed1987

Primary and secondary structure of bovine retinal S antigen (48-kDa protein).

The complete amino acid sequence of bovine S antigen (48-kDa protein) has been determined by cDNA and partial amino acid sequencing. A 1623-base-pair (bp) cDNA contains an open reading frame coding for a protein of 404 amino acids (45,275 Da). Tryptic peptides and cyanogen bromide peptides of native bovine S antigen were purified and partially sequenced. All of these peptides were accounted for in the long open reading frame. Searching of the National Biomedical Research Foundation data bank revealed no extensive sequence homology between S antigen and other proteins. However, there are local regions of sequence similarity with alpha transducin, including the sites subject to ADP-ribosylation by Bordetella pertussis and cholera toxins and the phosphoryl binding-sites. Secondary structure prediction and circular dichroic spectroscopy show that S antigen is composed predominantly of beta-sheet conformation. Acid-catalyzed methanolysis suggests the presence of low levels of carbohydrate in the molecule.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence for the cDNA of the bovine beta B2 crystallin and assignment of the orthologous human locus to chromosome 22.

We have identified and characterized two over-lapping bovine cDNA clones corresponding to the bovine crystallin beta Bp. The longer of the two clones, which contains the entire coding and 3' untranslated region as well as 54 nucleotides of the 5' untranslated sequence was used to identify and map an orthologous human gene, Hu beta B2, to chromosome 22, q11.2-q12.2. As one other human beta-crystallin, Hu beta A3/A1, has been mapped to chromosome 17, our results indicate that, unlike the tightly linked gamma-crystallins, the human beta-crystallins are not syntenic within the genome.

Amino Acid Sequence↗
PubMed1987

Glutathione reductase in human lens epithelium: FAD-induced in vitro activation.

Glutathione reductase (GR) activity was measured with and without the addition of flavin adenine dinucleotide (FAD) in freshly excised human lens epithelium following cataract surgery, and in control eye bank lens epithelium. Large individual variations in activity were found in both groups. Out of 32 epithelia obtained from cataractous lenses, 14 showed no measurable GR activity. Activity in eight of these 14 epithelia was restored after FAD was added, implying that the apo-enzyme in these 8 epithelia was functional but FAD was not available. Another group of 8 active epithelia showed a significant increase in GR activity upon the addition of FAD, suggesting a mixed population of active enzyme and available fraction of apo-enzyme which was reactivated by the addition of FAD. Unusually high GR activities were observed in epithelia of several patients. This correlated principally with daily intake of thyroxine which is known to have a direct effect on the metabolism of riboflavin. The fact that in a significant number of cataract patients epithelial GR was not active, and the observation that activity could be restored by adding in vitro FAD, demands that more attention should be given to riboflavin nutritional status and FAD synthesis in the eye.

Cataract↗
PubMed1987

Characterization of water-insoluble proteins in human lens nuclei.

Water-insoluble proteins from the nuclei of human normal and cataractous lenses were analyzed by solubilizing the lens material in various organic solvent/water mixtures. Gel permeation chromatography with TSK-3000 and TSK-4000 columns and a high performance liquid chromatography system were utilized to fractionate the water-insoluble proteins. The effective solvent systems were a) acetonitrile:tetrahydrofuran: water = 20:60:20, v/v%; b) tetrahydrofuran:water = 50:50, v/v%; and c) methanol:water = 25:75, v/v%. Different protein fractions could be selectively extracted with different solvent mixtures. The fractions obtained were partially identified using antibodies made against the soluble alpha-, beta-, and gamma-crystallins. Protein profiles obtained from cataractous nuclei differed markedly from those obtained from normal nuclei of the same age. In particular, an approximately 350K protein aggregate was found to increase with age in cataractous nuclei. This 350K aggregate is only a minor component in the nuclei of normal lenses.

Aged↗
PubMed1986

Assignment of a human beta-crystallin gene to 17cen-q23.

The gene map assignment of a human beta-crystallin gene to 17cen-q23 has been made using a bovine probe in the study of human-mouse and human-Chinese hamster somatic cell hybrids containing parts of human chromosome 17.

Animals↗
PubMed1986

Patterns of pigmentation in the eye lens of the deep-sea hatchetfish, Argyropelecus affinis Garman.

The present study is a morphological, biochemical and spectrophotometric characterization of the eye lens pigmentation in 45 specimens (11-88 mm in standard length) of the deep-sea hatchetfish, Argyropelecus affinis (Stomiiformes: Sternoptychidae). For comparison, we also examined available lenses of other members of the family Sternoptychidae, including three other species of the genus Argyropelecus, and two species of the genus Sternoptyx. Lens pigmentation was observed in all specimens of Argyropelecus spp. larger than about 36 mm in standard length, but was absent in all Argyropelecus spp. individuals less than 36 mm. However, lens pigmentation was not observed in Sternoptyx specimens of any size. Detailed studies of A. affinis indicated that at 36 mm the nascent lens fiber cells, which are continually laid down over preexisting, unpigmented cells, begin incorporating pigment, and the pigment concentration increases steadily as pigmented cells are added during lens growth. Spectrophotometric and biochemical data suggested that the pigment is a carotenoprotein complex, the carotenoid-like chromophore being strongly associated with a specific soluble lens protein, alpha crystallin. While the lens coloration in these fishes is age-related, analyses of the retinal visual pigment revealed no concomitant age-related change in the peak wavelength of retinal sensitivity in these fishes. Our data on the spectral absorbance of the lens and visual pigment of these fishes suggest that the lens pigmentation acts as a short-wave filter to improve acuity of the visual system.

Animals↗
PubMed1986

Age-dependent changes in the heat-stable crystallin, beta Bp, of the human lens.

The present study examined the effects of aging and cataractogenesis on the biochemical properties of the uniquely heat-stable lens crystallin, beta basic principle polypeptide (beta Bp). Using the techniques of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot immunoassay, we analyzed cortical and nuclear lens sections from normal lenses of individuals aged 0 to 91 years for beta Bp content in the soluble fraction, and retention of heat stability with aging. In addition, we compared the characteristics of beta Bp in cataractous lenses with those of normal lenses of approximately the same age. While beta Bp is synthesized in new cortical cells throughout life, the beta Bp of the nucleus, which had been laid down early in life, decreased significantly in both absolute concentration and in its proportion of the total soluble protein fraction during the normal aging process. In addition, posttranslational changes in the nuclear soluble beta Bp result in a gradual loss of approximately 3000 d in the apparent mass of the beta Bp molecule; i.e., as a result of the aging process, the single heat-stable band of an apparent mass of 26 kd on SDS-PAGE of the young lens nucleus becomes two heat-stable bands, one at 26 kd and one at 23 kd. Normal lenses up to 91 years of age always retain some of the 26 kd subunit, whereas lenses with severe nuclear cataracts had only the 23 kd subunit in the soluble fraction.

Adolescent↗
PubMed1986

Further studies on low molecular weight crystallins: relationship between the bovine beta s, the human 24kD protein and the gamma-crystallins.

Three classes of monomeric crystallins separable by SDS gel electrophoresis or by gel filtration have been demonstrated in human lens. In previous studies we have concluded from physico-chemical and immunological data that all three polypeptides are related and should be classified as gamma-crystallins. The present paper presents further evidence supporting this conclusion, but also demonstrates that the 24,000 dalton (24kD) polypeptide corresponds to the beta s-crystallin. beta s-crystallin was purified by classical techniques from bovine lens and was shown to cross-react with a monoclonal antibody specific for the human 24kD polypeptide. This antibody exhibited no reactivity to other crystallin fractions from either bovine or human lenses. The identification of the 24kD polypeptide as beta s was further supported by analysis of the tertiary structures of the molecules by near UV-circular dichroism and by the finding of a blocked amino terminus on the 24kD polypeptide. Our finding that the human beta s (24kD polypeptide) should actually be classified as a gamma-crystallin is fully consistent with recently reported sequence data on bovine beta s-crystallin.

Animals↗