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Biomedical subjects

J Horwitz

Publications and source records attributed to J Horwitz.

At least 37 records · Page 2Linked to original sources
PubMed2000

Expression of betaB(2)-crystallin mRNA and protein in retina, brain, and testis.

PURPOSE: To evaluate the expression of betaB(2)-crystallin mRNA and protein in rat, bovine, and human nonlens and nonocular tissues. METHODS: betaB(2)-crystallin mRNA levels were detected by RT-PCR. betaB(2)-crystallin protein was purified from rat and bovine tissues by FPLC chromatography. FPLC fractions were analyzed by immunoblotting. The identity of betaB(2)-crystallin protein, isolated from the retina, was confirmed by protein microsequencing. RESULTS: betaB(2)-crystallin transcript was detected in rat brain, rat testis, and human retina by RT-PCR. betaB(2)-crystallin transcript was not found in rat lung, heart, ovary, spleen, thymus, kidney, and liver or in human brain and testis. betaB(2)-crystallin protein was partially purified from and its identity confirmed in rat brain, rat testis, and bovine retina. The bovine retinal protein was further confirmed to be authentic betaB(2)-crystallin by protein microsequencing. CONCLUSIONS: These results establish that betaB(2)-crystallin mRNA and protein are expressed in tissues outside of the lens and outside of the eye including retina, brain, and testis. Extralenticular and extraocular expression of betaB(2)-crystallin, coupled with its participation in phosphorylation pathways, suggests that it has nonrefractive functions in these tissues.

Animals↗
PubMed1999

Cloning, sequencing and differential expression of alphaB-crystallin in the zebrafish, Danio rerio.

Here we report the cloning and expression of alphaB-crystallin from the zebrafish. 5'- and 3'-RACE was used to isolate a 900-bp transcript that contained insertions and deletions that differentiate it from both alphaA-crystallin and HSP-27. The deduced amino acid sequence of zebrafish alphaB-crystallin revealed that it lacked four residues in the C-terminus implicated in protein-protein interactions in other vertebrate species. In addition, the sequence contained two substitutions at sites implicated in phosphorylation in other vertebrate species. Northern analysis and semi-quantitative RT-PCR indicate that zebrafish alphaB-crystallin is expressed at extremely low levels outside of the lens.

Amino Acid Sequence↗
PubMed1999

Identification of a missense mutation in the alphaA-crystallin gene of the lop18 mouse.

PURPOSE: The mouse lop18 (lens opacity 18) mutation causes a white cataract obvious at weaning age. It soon progresses to a large white nuclear cataract with mild cortical changes. The mutation maps to mouse Chromosome 17 in close linkage to the alphaA-crystallin (Crya) gene, which encodes one of the major vertebrate eye lens proteins. Here we report the identification of a missense mutation in the alphaA-crystallin gene of lop18/lop18 mutant mice. METHODS: PCR primers were designed based on the alphaA-crystallin gene sequence from GenBank and PCR products were sequenced. RESULTS: We have analysed the sequence of the alphaA-crystallin gene from the lop18/lop18 mouse and identified a missense mutation. This mutation is tightly associated with the cataract phenotype, as no recombination was detected in 112 meioses. CONCLUSIONS: Our results suggest that a missense mutation in the alphaA-crystallin gene is responsible for the lop18/lop18 phenotype and Cryalop18 should be used as a gene symbol for the lop18 mutation.

Animals↗
PubMed1999

Mutation R120G in alphaB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function.

alphaB-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in alphaB-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M., et al. (1998) Nat. Genet. 20, 92-95]. By using alpha-lactalbumin, alcohol dehydrogenase, and insulin as target proteins, in vitro assays indicated that R120G alphaB-crystallin had reduced or completely lost chaperone-like function. The addition of R120G alphaB-crystallin to unfolding alpha-lactalbumin enhanced the kinetics and extent of its aggregation. R120G alphaB-crystallin became entangled with unfolding alpha-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G alphaB-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G alphaB-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G alphaB-crystallin has decreased beta-sheet secondary structure and an altered aromatic residue environment compared with wild-type alphaB-crystallin. The apparent molecular mass of R120G alphaB-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type alphaB-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G alphaB-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of alphaB-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.

Alcohol Dehydrogenase↗
PubMed1999

Lens alpha-crystallin: function and structure.

alpha-Crystallin is a major lens protein, comprising up to 40% of total lens proteins, where its structural function is to assist in maintaining the proper refractive index in the lens. In addition to its structural role, it has been shown to function in a chaperone-like manner. The chaperone-like function of alpha-crystallin will help prevent the formation of large light-scattering aggregates and possibly cataract. In the lens, alpha-crystallin is a polydisperse molecule consisting of a 3:1 ratio of alpha A to alpha B subunits. In this study, we expressed recombinant alpha A- and alpha B-crystallin in E. coli and compared the polydispersity, structure and aggregation state between each other and native bovine lens alpha-crystallin. Using gel permeation chromatography to assay for polydispersity, we found native alpha-crystallin to be significantly more polydisperse than either recombinant alpha A- or alpha B-crystallin, with alpha B-crystallin having the most homogeneous structure of the three. Reconstructed images of alpha B-crystallin obtained with cryo-electron microscopy support the concept that alpha B-crystallin is an extremely dynamic molecule and demonstrated that it has a hollow interior. Interestingly, we present evidence that native alpha-crystallin is significantly more thermally stable than either alpha A- or alpha B-crystallin alone. In fact, our experiments suggest that a 3:1 ratio of alpha A to alpha B subunit composition in an alpha-crystallin molecule is optimal in terms of thermal stability. This fascinating result explains the stoichiometric ratios of alpha A- and alpha B-crystallin subunits in the mammalian lens.

Aging↗
PubMed1999

Effect of aging on the negative chronotropic and anti-beta-adrenergic actions of adenosine in the rat heart.

The effect of aging on the antiadrenergic actions of adenosine was studied in vitro and in vivo by using adult (6-month-old) and old (24-month-old) male Fischer 344 rats. In anesthetized animals, adenosine (0.01-0.1 micromol/kg), given as a rapid bolus into the right atrium, exerted a negative chronotropic effect manifested by a dose-dependent transient prolongation of sinus cycle length (SCL). This effect was similar in both age groups (n = 6, each; i.e., the percentage maximal prolongation of SCL (%deltaSCL) ranged from 12 +/- 2% to 63 +/-14% in the adult and from 20 +/- 7% to 57 +/- 15% in the old rats. In the presence of isoproterenol (0.2 microg/kg/min), the negative chronotropic action of adenosine was potentiated in the adult rats much more than in the old rats [i.e., %deltaSCL ranged from 60 +/- 28% to 183 +/- 48% vs. 40 +/- 12% to 70 +/- 13%, respectively (p < 0.05, adult vs. old)]. In the isolated perfused hearts, isoproterenol (1 microM for 1 min) exerted similar chronotropic and inotropic effects in adult (n = 9) and old hearts [n = 6; i.e., heart rate, left ventricular pressure (LVP), and LVdp/dt increased by 56 +/- 3%, 17 +/- 1%, and 37 +/- 2%, and 57 +/- 2%, 17 +/- 1%, and 35 +/- 3%, respectively, in the absence of, and by 27 +/- 2%, 7 +/- 1%, and 19 +/- 2% and 41 +/- 3%, 12 +/- 1%, and 25 +/-2% in the presence of adenosine (5 microM for 1 min)]. Adenosine administration after isoproterenol caused only an insignificant increase in coronary blood flow. Finally, the adenosine attenuation of either isoproterenol- or forskolin-induced production of 3',5'-cyclic adenosine monophosphate (cAMP) was significantly less in atrial membranes isolated from old versus adult rats (n = 6, each). It was concluded that in the old Fischer 344 rat hearts, the antiadrenergic action of adenosine is attenuated as compared with its action in adult rat hearts.

Adenosine↗
PubMed1999

Antibodies to a microbial peptide sharing sequence homology with betaA3-crystallin damage lens epithelial cells in vitro and in vivo.

Circulating auto-antibodies (Abs) against lens antigens (Ags) are highly prevalent in patients with cataract, but their origin and pathogenic significance are unknown. We hypothesized that Abs raised after exposure to infectious microbes could cross-react with lens Ags. To test this hypothesis, we generated a monoclonal Ab to human betaA3-crystallin. Epitope analysis indicated that the ETQAE sequence in the N-terminus region of betaA3-crystallin was critical for mounting a humoral response. Similar sequences were found in three microbial Ags. Mice injected with a microbial oligopeptide containing ETQAE emulsified with complete Freund's adjuvant (CFA) raised Abs which cross-reacted with betaA3-crystallin and developed lens epithelial cell (LEC) damage in vitro. We also genetically engineered an betaA3-crystallin-expressing E. coli. Mice immunized with the recombinant E. coli developed LEC damage. These results support the hypothesis that exposure to microbes having Ags homologous to self Ags can trigger a humoral immune response that leads to LEC damage in mice.

Amino Acid Sequence↗
PubMed1998

Up-regulation of osteonectin/SPARC in age-related cataractous human lens epithelia.

PURPOSE: To characterize gene expression patterns between epithelia isolated from cataractous and normal human lenses. METHODS: Reverse transcriptase differential display was used to identify differential expression between cataractous and normal epithelia. RT-PCR was used to compare pooled and individual RNA samples. RESULTS: One transcript, up-regulated in cataractous as compared to normal epithelia, was identified as osteonectin which is also known as SPARC (secreted acidic protein rich in cysteines). RT-PCR confirmed over-expression of this RNA. High levels of osteonectin mRNA were also detected in six individual epithelia dissected from cataractous lenses. CONCLUSIONS: The present study provides evidence for up-regulation of osteonectin in human age-related cataract and suggests that osteonectin, a protein involved in cell-cycle control, extracellular matrix and Ca++ binding, plays an important role in human lens homeostasis and may be involved in processes leading to lens opacity.

Adult↗
PubMed1998

Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

The decrement in dopamine levels exceeds the loss of dopaminergic neurons in Parkinson's disease (PD) patients and experimental models of PD. This discrepancy is poorly understood and may represent an important event in the pathogenesis of PD. Herein, we report that the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is a selective target for nitration following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (MPP+). Nitration of TH also occurs in mouse striatum after MPTP administration. Nitration of tyrosine residues in TH results in loss of enzymatic activity. In the mouse striatum, tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 hr post MPTP injection. Striatal TH was not nitrated in mice overexpressing copper/zinc superoxide dismutase after MPTP administration, supporting a critical role for superoxide in TH tyrosine nitration. These results indicate that tyrosine nitration-induced TH inactivation and consequently dopamine synthesis failure, represents an early and thus far unidentified biochemical event in MPTP neurotoxic process. The resemblance of the MPTP model with PD suggests that a similar phenomenon may occur in PD, influencing the severity of parkisonian symptoms.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1998

The small heat-shock protein, alphaB-crystallin, has a variable quaternary structure.

alphaB-crystallin is a major structural protein in the lens that is found in a variety of other tissues and is associated with numerous neurological disorders. It is a member of the small heat-shock protein family and possesses chaperone-like properties. Cryo-electron microscopy has been applied to analyze the quaternary structure of human recombinant alphaB-crystallin, which spontaneously forms roughly spherical multimers 8 to 18 nm in diameter. Class-sum images based on nearly 5000 alphaB-crystallin particles reveal the presence of a large central cavity, weak regions of density within the protein shell, and an asymmetric quaternary structure. The class-sum images are variable in size and shape, and are suggestive of snapshots of a conformationally flexible assembly. As gel-filtration chromatography reveals a range of molecular masses (650 (+/-140) kDa) for the assembly, the class-sum images were further classified on the basis of total molecular mass. A reconstruction at approximately 4 nm resolution was calculated from the images assigned to 32 subunit (approximately 645 kDa) assemblies. Comparison of class-sum images with reprojections of the reconstruction indicates that the resolution is limited by the variable nature of the assembly. A three-dimensional variance map indicates significant structural divergence within the protein shell and on the outer surface of the particle. Some of the strong variance may correspond to the flexible, exposed C-terminal residues of the alphaB-crystallin monomers. The variable quaternary structure of alphaB-crystallin is consistent with the polydisperse size of the assembly and the previously observed subunit exchange between multimers. Thus, we propose that the monomer packing is variable, and that the quaternary structure of the assembly is not completely defined. A variable alphaB-crystallin quaternary structure may facilitate binding of target proteins in up to stoichiometric ratios.

Crystallins↗
PubMed1998

A conserved disulfide motif in human tear lipocalins influences ligand binding.

Structural and functional characteristics of the disulfide motif have been determined for tear lipocalins, members of a novel group of proteins that carry lipids. Amino acid sequences for two of the six isolated isoforms were assigned by a comparison of molecular mass measurements with masses calculated from the cDNA-predicted protein sequence and available N-terminal protein sequence data. A third isoform was tentatively sequence assigned using the same criteria. The most abundant isoform has a measured mass of 17 446.3 Da, consistent with residues 19-176 of the putative precursor (calculated mass 17 445.8 Da). Chemical derivatization of native and reduced/denatured protein confirmed the presence of a single intramolecular disulfide bond in the native protein. Reactivity of native, reduced, and denatured protein with 4-pyridine disulfide and dithiobis(2-nitrobenzoic acid) indicated that access to the free cysteine is markedly restricted by the intact disulfide bridge. Mass measurements of tryptic fragments identified C119 as the free cysteine and showed that the single intramolecular disulfide bond joined residues C79 and C171. Circular dichroism indicated that tear lipocalins have a predominant beta-pleated sheet structure (44%) that is essentially retained after reduction of the disulfide bond. Circular dichroism in the far-UV showed reduced molecular asymmetry and enhanced urea-induced unfolding with disulfide reduction indicative of relaxation of protein structure. Circular dichroism in the near-UV shows that the disulfide bond contributes to the asymmetry of aromatic sites. The effect of disulfide reduction on ligand binding was monitored using the intrinsic optical activity of bound retinol. The intact disulfide bond diminishes the affinity of tear lipocalins for retinol and restricts the displacement of native lipids by retinol. Disulfide reduction is accompanied by a dramatic alteration in ligand-induced conformational changes that involves aromatic residues. The disulfide bridge in tear lipocalins is important in conferring protein rigidity and influencing ligand affinity. The disulfide bond appears highly conserved so that these findings may have implications for the entire lipocalin superfamily.

Amino Acid Sequence↗
PubMed1998

Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.

Amino Acid Sequence↗
PubMed1998

Phospholipase D hydrolyzes short-chain analogs of phosphatidylcholine in the absence of detergent.

Phospholipase D is an important enzyme in signal transduction in neuronal tissue. A variety of assays have been used to measure phospholipase D activity in vitro. The most typical measure of phospholipase D activity is the production of phosphatidylethanol in the presence of ethanol. Phosphatidylethanol is a product of transphosphatidylation activity that is considered a unique property of phospholipase D. To support transphosphatidylation activity, high concentrations of ethanol may be required. Furthermore, most assays in the literature utilize a detergent. These extreme conditions, detergent and ethanol, may alter phospholipase D and hinder the study of its regulation. In this manuscript we describe an assay that eliminates these potentially confounding conditions. It utilizes high specific activity [3H]butanol as a nucleophilic receptor. This eliminates the need for high concentrations of alcohol. The substrate is an analog of phosphatidylcholine that contains short-chain fatty acids, 1,2-dioctanoyl-sn-glycero-3-phosphocholine. Phospholipase D readily hydrolyzes this substrate in the absence of detergent. This novel assay should be useful in the further characterization of phospholipase D.

Animals↗
PubMed1998

Increased levels of methylated intermediates of phosphatidylcholine lead to enhanced phospholipase D activity.

Previous work from this laboratory and others has shown that neurotransmitters can activate phospholipase D. Unlike the phospholipase C that specifically hydrolyzes inositol-containing phospholipids, phospholipase D in neuronal tissue specifically hydrolyzes phosphatidylcholine. One route for the synthesis of phosphatidylcholine, is via methylation of phosphatidylethanolamine. Using an in vitro assay, we have previously shown that methylated intermediates are also good substrates for phospholipase D (1). In this manuscript we demonstrate that these intermediates are also substrates in the intact PC12 cells. Cells incubated with methyl and dimethylethanolamine incorporate more [3H]palmitic acid into the corresponding phospholipid, phosphatidyl-N-methylethanolamine and phosphatidyl-N,N-dimethylethanolamine. In these cells bradykinin causes a greater increase in [3H]phosphatidylethanol production. Elevated levels of [3H]phosphatidylcholine do not enhance bradykinin-stimulated [3H]phosphatidylethanol production, therefore, this effect is specific for the methylated intermediates. Finally, this effect is not due to some generalized enhancement of receptor coupling because incubation of the cells with methylethanolamine does not lead to an increase in bradykinin stimulated inositol phosphate production.

Animals↗
PubMed1998

Effect of aging on A1-adenosine receptor-mediated inhibition of norepinephrine release in the rat heart.

Adenosine inhibits norepinephrine (NE) release from cardiac adrenergic nerves and reduces the postsynaptic beta-adrenergic mediated actions of NE, leading to decreased myocardial force of contraction. The actions of adenosine are mediated by pre- and postsynaptic adenosine A1 receptors (A1-AdoR). We reported that adenosine inhibition of postsynaptic beta-adrenergic receptor-mediated cyclic adenosine monophosphate (cAMP) production declines with age in male F344 rat hearts. In this study, cardiac synaptosomes, isolated intact adrenergic nerve terminals, were used to examine the effect of age on adenosine inhibition of NE release. Cardiac synaptosomes were prepared from the hearts of 6- and 24-month-old male F344 rats, loaded with [3H]NE, and placed in a superfusion system. [3H]NE release was induced by high [K+] exposure in the presence of varying concentrations of adenosine or the specific A1-AdoR agonist, N6-p-sulfophenyladenosine (SPA). [3H]NE release was significantly reduced in old rats compared with young rats. Inhibition of [3H]NE release by adenosine and SPA was significantly greater in young rats compared with old rats. The A1-AdoR antagonist, 8-(p-sulfophenyl)-theophylline, blocked the actions of adenosine on [3H]NE release, and the specific adenosine A2-receptor agonist, cyclopropylcarboxamidoadenosine, had no effect on [3H]NE release. Our data suggest that presynaptic A1-AdoR-mediated inhibition of NE release in the rat heart declines with age.

Adenosine↗
PubMed1998

Age-related decline in beta adrenergic and adenosine A1 receptor function in the heart are attenuated by dietary restriction.

Previously published reports from this laboratory have shown that the antiadrenergic effect of adenosine A1 agonists declines with age in the rat heart [ J Mol Cell Cardiol 29:593-602] and that this decline may be caused by a decrease in coupling between adenosine A1 receptors (AdoA1R) and guanine nucleotide-binding proteins [ Circ Res 81:1065-1071]. Dietary restriction (DR; 60% calories of ad libitum) has been shown to attenuate age-related changes in cellular signal transduction pathways. Therefore, the present study investigated whether DR altered the age-related changes in AdoA1R-mediated function in senescent rat hearts. Ventricular membranes were isolated from the hearts of ad libitum (AL) fed and DR male F344 rats that were 6, 12 and 24 months of age. In AL rats, there was an age-related decline in isoproterenol (ISO)-stimulated adenylyl cyclase when compared with the 6-month-old rats. The decline in ISO-stimulated cyclase was attenuated in DR animals. In AL rats, inhibition of ISO-stimulated adenylyl cyclase by the AdoA1R agonist, N6-p-sulfophenyladenosine (SPA) decreased with age. In DR rats, the age-related decline in inhibition was attenuated. Previous results from this laboratory indicated that in AL fed rats, there was an age-related decrease in the percentage of high-affinity binding sites for SPA, from 55% at 6 months to 23% at 24 months. Diet restriction attenuated this age-related shift in high-affinity binding sites so that the percentage of high-affinity sites at 24 months was 42%. Our results suggest that DR maintains AdoA1R function by preventing a loss of high-affinity AdoA1R sites.

Adenylyl Cyclases↗