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Biomedical subjects

B A Johnson

Publications and source records attributed to B A Johnson.

At least 145 records · Page 8Linked to original sources
PubMed1990

Derivation and validation of a clinical diagnostic model for chlamydial cervical infection in university women.

We developed and prospectively tested a logistic regression model for chlamydial cervical infection. Study subjects included 2271 women receiving gynecologic care in our student health clinic. Clinical data were collected in a standardized fashion. We identified cell culture--isolated Chlamydia trachomatis from 133 (9%) of 1458 subjects in the derivation set and 73 (10%) of 729 subjects in the validation set. Model variables included a new sexual partner within 2 months or more than one sexual partner within 6 months; cervical ectopy; cervical friability; at least 20 polymorphonuclear leukocytes per high-power field in cervical secretions; white blood cells in vaginal secretions; and use of an antibiotic active against C trachomatis within a month. This model can distinguish women with low, medium, and high risks of chlamydial infection (on derivation set: receiver operating characteristic curve area, 0.710; SE, 0.026; on validation set: area, 0.698; SE, 0.035) using simple clinical information obtained in the office.

Adolescent↗
PubMed1990

Fragmentation of isoaspartyl peptides and proteins by carboxypeptidase Y: release of isoaspartyl dipeptides as a result of internal and external cleavage.

Isoaspartate-containing versions of sea urchin sperm-activating peptide, delta sleep-inducing peptide, and lactate dehydrogenase (231-242) were cleaved at internal sites by carboxypeptidase Y. Cleavage occurred between the isoaspartate and the preceding amino acid, and it was accompanied by sequential digestion of amino acids from the two resulting carboxyl termini. Because the isoaspartyl bonds were not cleaved, isoaspartyl dipeptides were among the final products. The rate of release of isoaspartyl dipeptides was different for the three peptides, a 24-h digestion yielding 0.32 mol of isoaspartylglycine/mol of isoaspartyl sperm-activating peptide, 0.50 mol of isoaspartylalanine/mol of isoaspartyl delta sleep-inducing peptide, and 1.15 mol of isoaspartylserine/mol of isoaspartyl lactate dehydrogenase (231-242). The different rates could be explained by the slow cleavage of amino acids preceded by glycine. Isoaspartyl dipeptides were not detected in digests of the corresponding aspartate- or asparagine-containing forms of the peptides. Release of isoaspartyl dipeptides by carboxypeptidase Y was used to demonstrate the presence of isoaspartylglycine sequences in deamidated adrenocorticotropin (0.54 mol/mol), in a mixture of trypic fragments of base-treated calmodulin (0.20 mol/mol), and in a mixture of tryptic fragments of base-treated triosephosphate isomerase (0.08 mol/mol). These results confirm earlier work suggesting that isoaspartylglycine formation is prevalent in proteins exposed to alkaline conditions. They also provide a methodology that should prove useful in the characterization of natural substrates for protein L-isoaspartyl methyltransferase.

Animals↗
PubMed1990

Increased sodium-lithium countertransport in black non-insulin-dependent diabetic hypertensives.

Black hypertensive diabetics have been shown to exhibit elevated intracellular calcium which may stimulate Na/H antiport. Since Na/Li countertransport appears to be a functional mode of Na/H antiport, we measured erythrocyte Na/Li countertransport in nondiabetic normotensive and hypertensive blacks and whites and hypertensive non-insulin-dependent diabetic blacks. Na/Li countertransport was significantly lower in the blacks than in the whites (0.170 +/- 0.017 v 0.230 +/- 0.017 mmol/L/h, P less than .02), but there was no significant difference between the black hypertensives and normotensives. In contrast, black hypertensive diabetics exhibited a significant increase in Na/Li countertransport compared to normotensive and hypertensive nondiabetic blacks (0.252 +/- 0.032 v 0.170 +/- 0.017, P less than .02. Thus, these data indicate that the activity of this antiporter is elevated in black diabetics, possibly further contributing to the development of hypertension in this population.

Adult↗
PubMed1990

Psychopharmacological effects of cannabis.

The use of cannabis can lead to an acute toxic psychosis, 'flashbacks', depersonalization, derealization and marked cognitive and psychomotor impairment. Further research is needed to establish whether a functional psychosis can be provoked, aggravated or prolonged by cannabis intake. Perhaps of greatest significance among the physical sequelae is the potential to suppress the immune system, impair reproduction, produce respiratory disease and increase the risk of lung cancer.

Cannabinoids↗
PubMed1990

Rapid purification of synthetic oligonucleotides: a convenient alternative to high-performance liquid chromatography and polyacrylamide gel electrophoresis.

A new method has been developed to purify and detritylate milligram amounts of synthetic oligonucleotides. Dimethoxytrityl oligonucleotides from 15 to 100 nucleotides in length are applied in triethylammonium acetate or concentrated ammonium hydroxide to a disposable chromatographic cartridge, the NENSORB PREP Nucleic Acid Purification Cartridge. Salts, failure sequences and synthetic by-products are washed away while the desired, full-length, dimethoxytrityl oligonucleotide remains bound to the cartridge. The trityl group is hydrolyzed from the 5'-end of the oligonucleotide with an acid wash and then the purified oligonucleotide is eluted with 35% methanol. Oligonucleotides are recovered salt-free with purities greater than 95%. NENSORB PREP-purified primers provide superior sequence data compared to similar primers used without purification and equivalent data to primers purified by polyacrylamide gel electrophoresis when used in manual radiometric Sanger sequencing.

Chromatography, High Pressure Liquid↗
PubMed1989

Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone.

In vitro aging at pH 7.4, 37 degrees C causes natural sequence recombinant human growth hormone (rhGH), methionyl rhGH, and human pituitary growth hormone to become substrates for bovine brain protein carboxyl methyltransferase, an enzyme that modifies the "side chain" alpha-carboxyl group present at atypical isoaspartyl linkages. The substrate capacity of rhGH increased at a rate of 1.8 methyl-accepting sites/day/100 molecules of hormone. Reversed-phase high performance liquid chromatography (HPLC) of trypsin digests of aged rhGH revealed two altered peptides not present in digests of control rhGH. These two fragments, which had the amino acid compositions of residues 128-134 (Leu-Glu-Asp-Gly-Ser-Pro-Arg) and 146-158 (Phe-Asp-Thr-Asn-Ser-His-Asn-Asp-Asp-Ala-Leu-Leu-Lys), contained the majority of the induced methylation sites, 22 and 58%, respectively. Isoaspartate can result from deamidation of asparagine or isomerization of aspartate. Isomerization of Asp-130, the only candidate site in 128-134, was corroborated by coelution of the altered fragment with the synthetic isoaspartyl peptide upon reversed-phase HPLC. Evidence is presented that the altered 146-158 fragment is a mixture of two peptides resulting from deamidation of Asn-149 to form 70-80% isoaspartate and 20-30% aspartate at this position. The position of isoaspartate in the altered 146-158 fragment was deduced from mass spectrometry, which indicated a single deamidated asparagine; from methylation stoichiometry, which indicated only one methylation site; and from automated Edman degradation, which showed an absence of asparagine and a low yield of aspartate at position 149. These results show that isoaspartate formation from both aspartate and asparagine is a significant, and possibly the major, source of spontaneous covalent alteration of rhGH and that enzymatic carboxyl methylation provides a powerful tool for assessing this type of modification.

Amino Acid Sequence↗
PubMed1989

NMR spectroscopy of 113Cd(II)-substituted gene 32 protein.

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. This intrinsic zinc is retained within the DNA-binding core fragment, g32P-(A+B) (residues 22-253), obtained by limited proteolysis of the intact protein. Ultraviolet circular dichroism provides evidence that Zn(II) binding causes significant changes in the conformation of the peptide chain coupled with alterations in the microenvironments of tryptophan and tyrosine side chains. NMR spectroscopy of the 113Cd(II) derivative of g32P-(A+B) at both 44.4 and 110.9 MHz shows a single 113Cd resonance, delta 637, a chemical shift consistent with coordination to three of the four sulfhydryl groups in the protein. In vitro mutagenesis of Cys166 to Ser166 creates a mutant g32P that still contains 1 Zn(II)/molecule. This mutant protein when substituted with 113Cd(II) shows a 113Cd signal with a delta and a line width the same as those observed for the wild-type protein. Thus, the S-ligands to the metal ion appear to be contributed by Cys77, Cys87, and Cys90. Relaxation data suggest that chemical shift anisotropy is the dominant, but not exclusive, mechanism of relaxation of the 113Cd nucleus in g32P, since a dipolar modulation from ligand protons is observed at 44.4 MHz but not at 110.9 MHz. Complexation of core 113Cd g32P with d(pA)6 or Co(II) g32P with poly(dT) shows only minor perturbation of the NMR signal or d-d electronic transitions, respectively, suggesting that the metal ion in g32P does not add a ligand from the bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cadmium↗
PubMed1989

Deamidation of calmodulin at neutral and alkaline pH: quantitative relationships between ammonia loss and the susceptibility of calmodulin to modification by protein carboxyl methyltransferase.

Measurements of ammonia release provide the first direct evidence that calmodulin becomes extensively deamidated during incubations at 37 degrees C, pH 7.4 or pH 11. A stoichiometry of 0.5 mol of NH3 released/mol of calmodulin is observed after 2 h at pH 11 or after 8-9 days at pH 7.4. These treatments also increase the ability of calmodulin to serve as a substrate for the isoaspartate-specific protein carboxyl methyltransferase from bovine brain. The stoichiometries of methylation are highly correlated with the stoichiometries of ammonia release. Deamidation and increased methyl-accepting capacity also occur in parallel for seven other proteins (aldolase, bovine serum albumin, cytochrome c, lysozyme, ovalbumin, ribonuclease A, and triosephosphate isomerase) upon incubation at pH 11. However, in comparison to calmodulin, these other proteins show very little deamidation and increased methylation capacity following incubation at pH 7.4. Deamidation of calmodulin at pH 7.4 is unaffected by the addition of 10(-7) M Ca2+; however, at 4 X 10(-6) M Ca2+, the rate of deamidation is inhibited by approximately 70%. The Ca2+-protection effect is consistent with the suggestion (B. A. Johnson, N. E. Freitag, and D. W. Aswad, (1985) J. Biol. Chem. 260, 10913-10916) that deamidation occurs preferentially at Asn-60 and/or Asn-97, each of which resides in a distinct Ca2+-binding domain.

Ammonia↗
PubMed1989

Micropigmentation for the treatment of vitiligo.

A technique of permanent dermal micropigmentation using a nonallergenic iron oxide pigment to cover recalcitrant areas of vitiligo is described. The areas included in this study were the distal digits, the lips, hands, wrists, axillae, elbows, hairline, perioral area, and lower legs. The immediate postmicropigmentation results invariably showed dramatic aesthetic improvements. There was a moderate degree of fading in the majority of cases, most of which occurred within the first six weeks. The pigment that remained usually persisted with minimal to no further fading. Short- and long-term complications, which were few, mild, and resolved fully, are discussed. No allergic reactions to the pigment or koebnerization of the vitiligo have been noted.

Adult↗
PubMed1988

Allostery in Callinectes sapidus hemocyanin: cooperative oxygen binding and interactions with L-lactate, calcium, and protons.

Allosteric interactions of Callinectes sapidus hemocyanin have been investigated by generation of precision oxygen-binding curves using a modified Imai apparatus and their subsequent analysis using numerical methods. The Monod-Wyman-Changeux (MWC) model, the MWC model extended to include hybrid R3T3 states, and the phenomenological Adair equation were fit to the experimental data. The hybrid MWC model provided a quantitatively better fit to the data than did the MWC model. The fits of the hybrid model and the Adair equation were statistically similar, supporting the adequacy of the hybrid model as a description of cooperative oxygen binding by C. sapidus hemocyanin. The hybrid model is also supported by an apparent negative cooperativity at the fourth oxygen-binding step, a feature that is not possible with the conventional MWC model. The oxygen-binding curves obtained at various concentrations of three allosteric effectors, Ca, protons, and L-lactate, were analyzed in terms of the hybrid MWC model. All three effectors altered both the affinities of the model states and the equilibrium constants between the states. L-Lactate, which increases affinity and decreases cooperativity, increased the affinity of both the R and T states as well as preferentially stabilizing the hybrid and R states. Protons, which decrease affinity and cooperativity, act by reducing the affinity of both the R and T states and stabilizing the R state. Ca ions, which increase both affinity and cooperativity, act by increasing the affinity of the R state and preferentially stabilizing the T state.

Allosteric Regulation↗
PubMed1988

Requirements in cosmetics for black skin.

As large, well-funded cosmetics houses are taking more interest in the needs of black consumers, so should the dermatologist. The dermatologist should be able to discuss intelligently with patients those products that are intended for black skin and hair. Patients also appreciate a referral to a hair stylist or cosmetologist that the doctor is familiar with. As outlined in this article, the most common cosmetics problems encountered by black consumers include the lack of selection of appropriate shades of cosmetics; greasy and irritating "black" make-up; irritant or allergic reactions to fragrance and other cosmetic ingredients; acne from "oil-free" products; and a shortage of effective products to treat "ashiness." It is hoped that this review will help the reader understand what black patients may expect from their skin and hair cosmetics.

Black People↗
PubMed1987

Determination of L-lactate binding stoichiometry and differences in allosteric interactions of structurally distinct homohexamers from Panulirus interruptus hemocyanin.

The role of structurally distinct subunits from the hemocyanin of Panulirus interruptus was investigated by the analysis of the oxygen-binding properties of reassembled homohexamers. Homohexamers reassembled from subunits a and b exhibited cooperative oxygen binding, whereas subunit c did not. The oxygen affinity of homohexamers from subunits b and c was specifically increased by the addition of L-lactate, whereas that of subunit a was not. Both native hexamers and the homohexamers from subunit b have approximately one oxygen-linked lactate binding site per hexamer.

Animals↗
PubMed1987

Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase.

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-60 and/or Asn-97. Incubation of the A form with methyltransferase and S-adenosyl-L-methionine converts about half of the A form to an electrophoretic band indistinguishable from the B form. The activity of this partly converted calmodulin rises to 30-50% that of native calmodulin. These observations imply that the methyltransferase may have a biological role in restoring activity to proteins which contain abnormal isoaspartyl peptide bonds resulting from asparagine deamidation.

Animals↗
PubMed1987

Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides.

Prolonged incubation of L-isoaspartate-containing forms of lactate dehydrogenase (231-242), sperm activating peptide, and adrenocorticotropin (22-27) at 37 degrees C, pH 7.4, with S-adenosyl-L-methionine and protein carboxyl methyltransferase from bovine brain leads to extensive conversion of the atypical isopeptide bond to a normal peptide bond. For the lactate dehydrogenase-related peptide, conversion was 80% complete after 24 h. For the other two peptides, conversion reached a level of approximately 65% after 48 h. The mechanism of conversion involves (i) rapid enzymatic methylation of the alpha-carboxyl of the L-iso-Asp residue; (ii) nonenzymatic demethylation resulting in formation of an L-aspartyl cyclic imide; and (iii) a slow, nonenzymatic hydrolysis of the cyclic imide to form a mixture of 15-25% normal L-Asp peptide and 75-85% L-iso-Asp peptide. The regenerated L-iso-Asp peptide is remethylated and the cycle is repeated. The extent of conversion is limited by a competing side reaction wherein the L-imide slowly racemizes, leading to the formation of mainly D-iso-Asp peptide, which is not a substrate for the methyltransferase. The ability of protein carboxyl methyltransferase to initiate conversion of L-iso-Asp residues to normal L-Asp suggests a possible role for this enzyme in facilitating the repair or degradation of deamidated proteins in vivo.

Amino Acid Sequence↗