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Biomedical subjects

B M Katz

Publications and source records attributed to B M Katz.

At least 19 recordsLinked to original sources
PubMed1994

Probing of the retinal binding site of bacteriorhodopsin by affinity labeling.

The position of the chromophore within bacteriorhodopsin has been identified by cross-linking a cysteine group, introduced by site-specific mutagenesis, with a chromophore suitably derivatized with an active leaving group. Since bacteriorhodopsin has no cysteines, a site-specific cysteine mutant will contain only one free sulfhydryl group capable of reacting with the retinal analog. Met118, Thr121, and Ser141 were selected to be mutated to cysteine. No pigment absorbing in the visible region was obtained for the Ser141Cys mutant. The Met118Cys and Thr121Cys mutants have similar absorption maxima, proton pumping efficiencies and photocycles to those of the wild-type pigment. 4-Bromoretinal, in which the reactive allylic halide readily undergoes nucleophilic displacement, was used as the reactive chromophore. Pigments were obtained on reaction of all-trans-4-bromoretinal with the apoproteins of Met118Cys, Thr121Cys, and wild-type bacteriorhodopsin (lambda max = 464-470 nm). Analysis of the denatured pigments on SDS-polyacrylamide gels showed incorporation of tritiated chromophore into the Met118Cys mutant but not into the wild-type or Th4121Cys pigments. Met118Cys apoprotein which was preincubated with the cysteine-specific reagent N-ethylmaleimide formed a pigment with 4-bromoretinal but no cross-linking was observed, providing evidence that the cross-linking of the chromophore is to the cysteine at 118. We conclude that Met118 is positioned in the chromophore binding pocket, proximal to the C-4 position of cyclohexyl ring of retinal.

Affinity Labels↗
PubMed1993

Noncovalent occupancy of the retinal-binding pocket of opsin diminishes bleaching adaptation of retinal cones.

Bright light bleaches visual pigment and leads to a persistent desensitization of isolated rod and cone photoreceptors called bleaching adaptation. Bleaching adaptation results from the combined effects of pigment depletion and adaptational modulation of certain cellular reactions in the visual transduction cascade. Here, we present evidence that in solitary cone photoreceptors isolated from the salamander retina, the latter effect is due to the presence of free opsin in the outer segment. Also, we demonstrate that this "opsin adaptation" can be reversed by treating the cells with synthetic retinoids similar to 11-cis retinal but having polyene chains too short to form protonated Schiff base attachments to opsin.

Adaptation, Physiological↗
PubMed1993

Efficacy of progressive muscle relaxation for reducing state anxiety among elderly adults on memory tasks.

Cognitively intact anxious elderly subjects were randomly assigned to either a progressive muscle relaxation-training condition or control condition (ns = 15) and then completed selected subtests from the Wechsler Memory Scale--Revised. Despite significant reductions in state anxiety in the relaxation group, no significant differences were detected between the two groups on memory measures. These results are discussed within the context of previous research, and suggestions for further research are made.

Aged↗
PubMed1991

A putative role for extracellular ATP: facilitation of 67copper uptake and of copper stimulation of the release of luteinizing hormone-releasing hormone from median eminence explants.

We have previously shown that extracellular copper stimulates the release of the luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA), that chelated copper (Cu) but not ionic Cu is the active form of the metal, and that there is a direct correlation between the ligand specificity for 67Cu uptake and Cu action. In this study, we examined the possibility that extracellular ATP can serve as a ligand facilitating Cu action on the LH-RH neuron. Hypothalamic slices or MEA explants of adult male rats were used. It was found that ATP facilitates 67Cu uptake by hypothalamic slices when Cu:ATP molar ratio was 1:2000 but not 1:2. Keeping the [Cu] constant (150 microM) and varying [ATP], ATP facilitation of Cu stimulation of LH-RH release from MEA explants was found to be a saturable function of [ATP]; maximal facilitation occurred with 2.5 mM ATP. When the nucleotide phosphate specificity for facilitation of Cu action was assessed, ADP, ATP, alpha, beta-methylene-ATP (the non-hydrolyzable analogue of ATP) and GTP were equally effective, whereas AMP and adenosine were ineffective. These results indicate that extracellular ATP can facilitate Cu action on the LH-RH neuron and they are consistent with two mechanisms: (1) ATP facilitating Cu uptake and hence, Cu action and (2) ATP facilitating Cu action via an interaction with a purinergic receptor.

Adenosine Triphosphate↗
PubMed1991

The Maillard reaction in vivo.

The Maillard or browning reaction between reducing sugars and protein contributes to the chemical deterioration and loss of nutritional value of proteins during food processing and storage. This article presents and discusses evidence that the Maillard reaction is also involved in the chemical aging of long-lived proteins in human tissues. While the concentration of the Amadori adduct of glucose to lens protein and skin collagen is relatively constant with age, products of sequential glycation and oxidation of protein, termed glycoxidation products, accumulate in these long-lived proteins with advancing age and at an accelerated rate in diabetes. Among these products are the chemically modified amino acids, N epsilon-(carboxymethyl)lysine (CML), N epsilon-(carboxymethyl)hydroxylysine (CMhL), and the fluorescent crosslink, pentosidine. While these glycoxidation products are present at only trace levels in tissue proteins, there is strong evidence for the presence of other browning products which remain to be characterized. Mechanisms for detoxifying reactive intermediates in the Maillard reaction and catabolism of extensively browned proteins are also discussed, along with recent approaches for therapeutic modulation of advanced stages of the Maillard reaction.

Aging↗
PubMed1990

The ligand specificity for uptake of complexed copper-67 by brain hypothalamic tissue is a function of copper concentration and copper:ligand molar ratio.

It has been previously shown that complexation of Cu2+ is essential for effective uptake of Cu2+ by brain tissues and that 67Cu complexed to His is taken up by a high affinity and a low affinity saturable process (Hartter, D. E., and Barnea, A. (1988) J. Biol. Chem. 263, 799-805). Using rat hypothalamic tissue slices, we defined the ligand specificity for these two uptake processes. The effectiveness of stereoisomers or methyl (Me) derivatives of His in facilitating 67Cu uptake by the high affinity process was in this decreasing order: L-His = D-His = Me-3-N-His greater than Me-ester-His greater than Me-alpha-N-His greater than or equal to Me-1-N-His. By the low affinity process it was: L-His = D-His = Me-3-N-His = Me-ester-His = Me-alpha-N-His greater than Me-1-N-His. When facilitation of 67Cu uptake by 14 different amino acids was evaluated using copper:ligand (Cu:L) ratios of 1:2,000 (high affinity process) or 1:2 (low affinity process), His stood out as the most effective. However, when [Cu2+] was 0.1 microM and the Cu:L ratio was increased from 1:2,000 to 1:20,000, Ala, Gly, Lys, Ser, or Thr was each as effective as His; when [Cu2+] was 10 microM and the Cu:L ratio was increased from 1:2 to 1:2,000, Gln, Glu, Gly, Lys, or Ser was each superior to His in facilitating 67Cu uptake. Moreover, by comparison to 67Cu uptake at a Cu:L ratio of 1:2, increasing the ratio attenuated (His) or enhanced (Gln, Glu, Gly, Lys, Ser) 67Cu uptake. These results indicate that 1) coordination of Cu2+ with the 1-N-imidazole and the alpha-amino (but not with the carboxyl) is essential for His facilitation of 67Cu uptake, and 2) the amino acid specificity for uptake of complexed Cu2+ is a function of both [Cu2+] and the molar ratio of copper to amino acid. These results are consistent with coordination of Cu2+ with at least three nitrogens being a primary factor facilitating copper uptake by brain tissue.

Amino Acids↗
PubMed1990

Negative endocrine control system for inflammation in rats.

Inflammatory processes may be suppressed by endogenous mechanisms such as release of adrenocorticosteroid hormones through stimulation of the hypothalamus-pituitary-adrenal axis. In the present study, the relationship between the temporal development of carrageenan-induced edema in the hindlimb of the rat and release in plasma of the principal endogenous adrenocorticosteroid of the rat corticosterone was investigated. Suplantar injection of carrageenan produced a biphasic increase in basal plasma corticosterone levels that was not attributed to diurnal variation. The plasma level of corticosterone increased rapidly after injection of carrageenan and peaked 12-fold at 20 min. This first phase increase was attributed to the stress of the injection since it was mimicked by subplantar injection of saline. The second phase of corticosterone release was gradual and peaked 12-fold 7 hr after injection of carrageenan. The second phase was not elicited by subplantar injection of saline. When the hypothalamus-pituitary-adrenal axis is impaired via hypophysectomy, carrageenan-induced edema is more intense and lasts longer than in control rats. The results demonstrate that adrenocorticosteroid hormones are released as a result of the stress of injection and by the inflammatory response. Release of adrenocorticosteroids acts as a feedback mechanism to suppress the inflammatory response.

Animals↗
PubMed1990

Uptake of 67copper complexed to 3H-histidine by brain hypothalamic slices: evidence that dissociation of the complex is not the only factor determining 67copper uptake.

It was previously shown that complexation of 67Cu with His facilitates 67Cu uptake by hypothalamic slices and that His, in a concentration that is 1000-fold greater than Cu(His)2, inhibits 67Cu uptake (D. E. Hartter and A. Barnea, J. Biol. Chem. 263, 799-805 (1988)). We addressed the question: Does dissociation of the Cu(His)2 complex occur during the process of Cu2+ uptake and if so, is dissociation the only factor determining uptake? Rat hypothalamic slices were incubated with 67Cu(3H-His)2 and the kinetic profiles of 67Cu and 3H-His uptake were evaluated. 67Cu uptake was linear for up to 60 min, Vo vs S [0.1-160 microM Cu(His)2] was sigmoidal, Lineweaver-Burk plot was non-linear, Scatchard plot was bell-shaped, and Hill plot had multiple slopes. In contrast, 3H-His uptake was linear for up to 30 min, Vo vs S was biphasic, Lineweaver-Burk plot was linear, Scatchard plot was biphasic, and Hill plot had a single slope. Keeping [67Cu] constant and increasing [3H-His] resulted in a dose-dependent inhibition of 67Cu uptake which was not accompanied by an inhibition of 3H-His uptake. Substituting His in the complex with Phe or Lys resulted in a marked shift to the right in Vo vs S for 67Cu uptake and at S less than 40 microM, only His facilitated 67Cu uptake relative to ionic 67Cu2+. However, Vo vs S for 3H-His, 3H-Phe, and 3H-Lys uptake were superimposeable, indicating comparable dissociation of the complexes. In summary, we demonstrate that, although complexation of Cu2+ is essential for 67Cu uptake by hypothalamic tissue, 67Cu and 3H-His are taken up by distinct processes, which implies dissociation of the complex at the level of the membrane. Moreover, even though dissociation occurs, it is not the only factor that determines Cu2+ uptake by the hypothalamic tissue. It is suggested that the physicochemical properties of the Cu complex is an important factor determining Cu uptake by brain tissue.

Animals↗
PubMed1990

Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain.

We have previously demonstrated that hypothalmic slices obtained from adult male rats accumulate 67Cu by two ligand-dependent, saturable processes: a high and low affinity process. To further establish the generality of these uptake processes, we defined the ligand requirements and the saturation kinetics of 67Cu uptake by tissue slices obtained from the newborn hypothalamus (HT); adult male hypothalamus, hippocampus, cortex, median eminence, and caudate nucleus; hypothalamus and hippocampus of castrated (14 days) males and of pregnant (19 days) and ovariectomized (14 days) females. It was found that ionic 67Cu2+ was poorly taken up by newborn HT and adult caudate, complexation with His enhanced 67Cu uptake 3-4-fold, and complexation with albumin inhibited 67Cu uptake. These ligand requirements are identical to those we have previously shown for the adult HT. When 67Cu uptake was evaluated under conditions optimal for the high or the low affinity process, for each process the dose response curves generated from these various tissues were very similar. In addition, we assessed the uptake of both components of the CuHis2 complex by incubating tissues with 67Cu3 H-His2 and found that the tissue ratio of 67Cu:3H was a sigmoidal function of the concentration of the Cu complex such that at greater than 5 microM, the ratio was about 3-fold greater than the medium ratio; indicating preferential uptake of 67Cu relative to 3H-His. The changes in isotope ratios were observed in newborn HT and adult HT, as well as caudate. These similarities in the ligand requirements and saturation kinetics of 67Cu uptake establish the generality of these two processes of in vitro uptake of copper in the rat brain.

Animals↗
PubMed1990

Effects of a persuasive communication on beliefs, attitudes, and career choice.

Fishbein's Theory of Reasoned Action was used to formulate a persuasive communication in an attempt to influence unclassified American college students' beliefs, attitudes, intentions, and behaviors regarding signing up for a career as a registered nurse. A two-stage cluster sample was used to assign 90 male and female students to either an experimental or control group. After persuasive communication exposure, the experimental group showed a significantly more positive change in beliefs, attitudes, and intentions than did the control group exposed to a neutral message. Sign-up rate was also statistically significant for the experimental group. With the Fishbein model to predict sign-up behavior, no other scores were found to add to the prediction once behavioral intention was entered into the model. Change in behavioral intention explained 49% of the variation in behavior. Normative belief scores did not approach statistical significance.

Adolescent↗
PubMed1989

Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase.

The minimal structure in the heat-stable inhibitor protein of cAMP-dependent protein kinase required for a low nanomolar potency of inhibition is the peptide Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-+ ++Ile22-NH2 (PKI-(6-22)-amide). While primary structural determinants for interaction with the protein kinase are distributed throughout the 17 residues of this peptide, we have previously shown that phenylalanine 10 in the NH2-terminal portion is a particularly important determinant for high affinity binding (Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810). To investigate this requirement further, peptide analogs of PKI-(6-22)-amide in which various natural and nonstandard amino acids are substituted for phenylalanine 10 have been synthesized and tested for inhibitory potency against the catalytic subunit of the protein kinase. Consistent with the importance of the hydrophobicity of phenylalanine, an alanine 10 substitution analog exhibited a 270-fold decrease in inhibitory potency, whereas the leucine 10 analog lost only 33-fold in activity as compared to the parent peptide PKI-(6-22)-amide. Peptides containing the spatial conformation analogs D-phenylalanine, homophenylalanine, or phenylglycine were 60-120-fold less potent than the parent peptide. Peptides containing various para-substituted phenylalanines at position 10 were only 5-11-fold less potent. One exception to this was (4'-azidophenylalanine 10)PKI-(6-22)-amide, which was nearly equipotent with the parent inhibitor. The most potent analogs were those peptides containing highly aromatic residues at position 10. The 2'-thienylalanine 10, tryptophan (formyl) 10, tryptophan 10, and the 1'-naphthylalanine 10 analogs were 3-fold less potent, equipotent, slightly more potent, and 4-fold more potent than the parent peptide inhibitor, respectively. We conclude that phenylalanine 10 in PKI-(6-22)-amide, and presumably in the native protein inhibitor, interacts through specific hydrophobic and/or aromatic binding to a hydrophobic pocket or cleft near the active site of the protein kinase.

Amino Acid Sequence↗
PubMed1989

Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

Affinity Labels↗
PubMed1980

Characteristics of death education curricula in American medical schools.

To determine the content, structure, objectives, and organization of death education curricula in American medical schools, a cross-sectional survey was developed. The data, representing 100% of the population, demonstrated that such curricula expanded markedly during the past decade. The developmental stimuli for death education, sources of funding, and objectives of death education curricula were relatively consistent across respondents. Conversely, course content, faculty and patient roles, learning resources, and involvement in curriculum research did not evidence a systematic development. The results of the study suggest the need for a further investigation of the clinical implications of death education.

Cross-Sectional Studies↗