Search PubMed⌕ Search

Biomedical subjects

J B Jones

Publications and source records attributed to J B Jones.

At least 55 records · Page 3Linked to original sources
PubMed1998

Site-directed mutagenesis combined with chemical modification as a strategy for altering the specificity of the S1 and S1' pockets of subtilisin Bacillus lentus.

By combining site-directed mutagenesis with chemical modification, we have altered the S1 and S1' pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation of unnatural amino acid moieties, in the following manner: WT --> Cysmutant + H3CSO2SR --> Cys-SR, where R may be infinitely variable. A paradigm between extent of activity changes and surface exposure of the modified residue has emerged. Modification of M222C, a buried residue in the S1' pocket of SBL, caused dramatic changes in kcat/KM, of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S1 pocket and is partially surface exposed, effected more modest activity changes. Introduction of a positive charge at S166C does not alter kcat/KM, whereas the introduction of a negative charge results in lowered activity, possibly due to electrostatic interference with oxyanion stabilization. Activity is virtually unaltered upon modification of S156C, which is located toward the bottom of the S1 pocket and surface exposed and whose side chain is solvated. An unexpected structure-activity relationship was revealed for S166C-SR enzymes in that the pattern of activity changes observed with increasing steric size of R was not monotonic. Molecular modeling analysis was used to analyze this unprecedented structure-activity relationship and revealed that the position of the beta-carbon of Cys166 modulates binding of the P1 residue of the AAPF product inhibitor.

Amino Acid Substitution↗
PubMed1998

Differences in binding modes of enantiomers of 1-acetamido boronic acid based protease inhibitors: crystal structures of gamma-chymotrypsin and subtilisin Carlsberg complexes.

In order to probe the structural basis of stereoselectivity in the serine protease family, a series of enantiomeric boronic acids RCH2CH(NHCOCH3)B(OH)2 has been synthesized and kinetically characterized as transition-state analog inhibitors using alpha-chymotrypsin and subtilisin Carlsberg as model systems. When the R-substituent in this series was changed from a p-chlorophenyl to a 1-naphthyl group, alpha-chymotrypsin, but not subtilisin, reversed its usual preference for l-enantiomers and bound more tightly to the D-enantiomer [Martichonok, V., & Jones, J. B. (1996) J. Am. Chem. Soc. 118, 950-958]. The structural factors responsible for the differences in stereoselectivity between the two enzymes have been explored by X-ray crystallographic examination of subtilisin Carlsberg and gamma-chymotrypsin complexes of the L- and D-enantiomers of p-chlorophenyl and 1-naphthyl boronic acid derivatives. In both enzymes, the L-isomers of the inhibitors, which are more closely related to the natural L-amino acid substrates, form tetrahedral adducts, covalently linking the central boron atom and Ogamma of the catalytic serine. The d-isomers, however, differ in the way they interact with subtilisin or gamma-chymotrypsin. With subtilisin, both the D-p-chlorophenyl and D-1-naphthyl inhibitor complexes form covalent Ser Ogamma-to-boron bonds, but with gamma-chymotrypsin, the same inhibitors lead to novel tetrahedral adducts covalently linking both Ser195 Ogamma and His57 Nepsilon2 covalently via the boron atom.

Binding Sites↗
PubMed1998

The efficacy of sublingual hyoscyamine sulfate and intravenous ketorolac tromethamine in the relief of ureteral colic.

A prospective, randomized, open-label, single-dose study was conducted in an emergency department (ED) of a tertiary care teaching hospital to evaluate the efficacy of hyoscyamine sulfate as compared to ketorolac tromethamine for the reduction of pain from ureteral colic in the ED. Patients were included if they were at least 18 years of age and presented to the ED with an initial history and physical examination consistent with ureteral colic. Ureteral calculi were confirmed by ultrasound or intravenous urogram. Consecutive patients were randomized to either a single sublingual dose of 0.125 mg of hyoscyamine sulfate or a single intravenous dose of 30 mg of ketorolac tromethamine given over 1 minute. After 30 minutes, if analgesia was inadequate, patients were given rescue medication. Baseline pain scores were obtained using a horizontal, 100-mm visual analog scale. Additional pain scores were obtained at 10-minute intervals for 30 minutes. Upon completion of the study, both patients and physicians completed a global assessment score questionnaire. Fifty-four evaluable patients were randomized. Demographics and baseline pain scores were similar for each group. Decreasing trends in pain over time were observed for both treatment groups, with significantly greater pain reduction observed with ketorolac tromethamine as compared to hyoscyamine sulfate. Global evaluations of pain relief revealed better results in the ketorolac tromethamine group than in the hyoscyamine sulfate group, although this result was not statistically significant.

Administration, Sublingual↗
PubMed1998

The tolerability of lactated Ringer's solution and BSS plus for ocular irrigation with and without the Morgan therapeutic lens.

OBJECTIVE: To evaluate two solutions, lactated Ringer's (LR) and a balanced salt solution (BSS Plus, Alcon Laboratories, Ft. Worth, TX), compared with normal saline solution (NSS), for ocular irrigation in healthy adult volunteers with and without the Morgan therapeutic lens (MTL). METHODS: This was a prospective, double-blind, randomized study of healthy volunteers who were at least 18 years of age. Exclusion criteria included the use of analgesics within four hours of the study. A complete ophthalmologic examination was performed at baseline and at the completion of the study. Following randomization and prior to any intervention, baseline discomfort scores were obtained by means of a verbally administered, horizontal, 100-mm, unnumbered analog discomfort scale. Both eyes of each volunteer were irrigated simultaneously for 15 minutes, with additional discomfort scores being recorded every 5 minutes using the same 100-mm, unnumbered analog discomfort scale. A global evaluation to assess the method of irrigation and the solutions used for irrigation was completed by both the physician blinded to the treatment groups and the volunteers. The volunteers were continuously monitored for any adverse effects resulting from the irrigation solutions or MTL. RESULTS: Sixty-three volunteers were recruited into the study, with 61 entered in the final analysis. Age and gender were balanced within each group. There was no significant difference in discomfort scores between the two groups; however, all discomfort scores decreased over time (p = 0.008). A lens-solution interaction was identified, with LR being the most tolerated when administered with the MTL. A statistically higher ocular pH difference was seen between the pre- and postirrigation readings for the control eye in volunteers irrigated with MTL (p = 0.046). Analysis of the global evaluations for each group revealed no difference in the distributions of physician and volunteer scores. No adverse event was reported in either group. CONCLUSION: There does not appear to be any difference in discomfort scores between the ocular irrigation fluids when used without the MTL. Overall, the use of the MTL appears well tolerated by healthy, adult volunteers. However, there does appear to be a significant lens-solution effect on volunteers' discomfort scores, with LR having significantly lower discomfort scores when used for ocular irrigation with the MTL. The authors conclude that the use of the MTL for ocular irrigation is well tolerated and recommend using LR as the irrigation solution for maximal patient comfort.

Acetates↗
PubMed1998

Diversity among xanthomonads pathogenic on pepper and tomato.

Xanthomonas campestris pv. vesicatoria, causal agent of bacterial spot of tomato and pepper, had been considered for nearly 70 years to be a relatively homogeneous organism. However, in the past decade this bacterium was determined to be composed of two genetically and phenotypically distinct groups. The two groups, designated A and B, were distinguished based on amylolytic activity, expression of unique protein bands, reaction on differential hosts (tomato races T1 and T2), reaction patterns with monoclonal antibodies, DNA restriction profiles, and DNA:DNA hybridization. The A and B groups were placed into X. axonopodis pv. vesicatoria and X. vesicatoria, respectively. A third group, designated C, was pathogenically (race T3) and serologically distinct from A and B strains, and formed unique DNA restriction profiles. DNA:DNA hybridization data suggest that C is distinct but related to A strains and may represent a subspecies of A. A final group, designated D, consisted of X. gardneri, an organism identified in Yugoslavia in 1957, and also found in Costa Rica. Group D was determined to be genetically distinct from strains within the other two groups; it represents a third Xanthomonas species pathogenic on tomato and pepper.

Journal Article↗
PubMed1997

Cysteine proteases such as papain are not inhibited by substrate analogue peptidyl boronic acids.

Peptidyl boronic acids that are close structural analogues of good substrates have been prepared and evaluated as potential transition state analogue inhibitors of the representative cysteine protease, papain. However, no inhibition could be detected at concentrations up to 10 mM. The reasons for the lack of inhibition were sought from molecular modeling. Molecular mechanics and semi-empirical quantum mechanics calculations indicated that the absence of inhibition was due to boronic acid-cysteine protease tetrahedral complexes being 0.79 kcal mol-1 less stable than their preceding noncovalent EI-complexes. In contrast, an analogous boronic acid-serine protease tetrahedral complex was calculated to be 2.74 kcal mol-1 more stable than its precursor Michaelis EI-complex. It thus appears that boronic acids are ineffective inhibitors of cysteine proteases due to the thermodynamic favoring of a weak EI-complex preceding tetrahedral intermediate formation, and that any oxyanion hole stabilization of the subsequent tetrahedral intermediate cannot overcome this energy handicap.

Boronic Acids↗
PubMed1997

Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue.

In vitro and in planta sensitivity of an indirect enzyme-linked immunoassay technique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria, was increased 10-fold by using a new extraction buffer (gl of: KH2PO4, 2; NaHPO4, 11.5; EDTA disodium, 0.14; thimerosal, 0.02; and lysozyme, 0.2). The procedure improved sensitivity without increasing background levels. In vitro, the limit of detection was between 1 x 10(7) and 1 x 10(8) cells ml-1 with the conventional extraction buffer phosphate-buffered saline (PBS) and less than 1 x 10(6) cells ml-1 when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c. vesicatoria strains, absorbance readings were increased close to three-fold with the lysozyme extraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, the limit of detection was 1 x 10(7) cfu ml-1 and 1 x 10(8) cfu ml-1 with the lysozyme solution and PBS, respectively, as the extraction buffers. When using the lysozyme extraction buffer in combination with a commercial amplification system, the limit of detection was decreased to less than 1 x 10(5) cfu ml-1 in leaf tissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of a significant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure, termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS is the reacting epitope.

Antigens, Bacterial↗
PubMed1997

oroshigane, a new segment polarity gene of Drosophila melanogaster, functions in hedgehog signal transduction.

Here we describe a new segment polarity gene of Drosophila melanogaster, oroshigane (oro). Identified as a dominant enhancer of Bar (B), oro is also recessive embryonic lethal, and homozygous oro embryos show variable substitution of naked cuticle with denticles. These patterns are distinctly similar to those of hedgehog (hh) and wingless (wg) embryos, which indicates that oro functions in determining embryonic segment polarity. Evidence that oro function is involved in Hh signal transduction during embryogenesis is provided by its genetic interactions with the segment polarity genes patched (ptc) and fused (fu). Furthermore, ptcIN is a dominant suppressor of the oro embryonic lethal phenotype, suggesting a close and dose-dependent relationship between oro and ptc in Hh signal transduction. oro function is also required in imaginal development. The oroI allele significantly reduces decapentaplegic (dpp), but not hh, expression in the eye imaginal disc. Furthermore, oro enhances the fui wing phenotype in a dominant manner. Based upon the interactions of oro with hh, ptc, and fu, we propose that the oro gene plays important roles in Hh signal transduction.

Animals↗
PubMed1996

Cyclin-dependent kinase inhibitor p27 as a mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60 cells.

Progression of mammalian cells through G1 is controlled by the concerted action of protein kinases, the activities of which are modulated in both positive (cyclins) and negative [cyclin-dependent kinase inhibitors (CDIs)] manners by families of regulatory proteins. In differentiation of leukemia cells, a G1 arrest is a common, if not invariable, occurence and takes place after the appearance of markers of monocytic differentiation in human leukemia HL60 cells treated with 1,25 dihydroxyvitamin D3 (1,25D3) at low to moderately high concentrations (F. Zhang et al., Cell Proliferation 27: 643-654, 1994). In the present study, we investigated the protein levels of several G1 regulatory proteins that are potential mediators of the 1,25D3-induced G1 block. During the first 24 h of exposure to a high concentration (4 x 10(-7) M) of 1,25D3, no increase was noted in the immunodetectable levels of cyclins D1 or E, or CDIs p16Ink4, p21Cip1/Waf1, or p27Kip1, even though monocytic differentiation markers were evident, and a prolongation of G1 was noted. After 48 h of exposure 4 x 10(-7) M to 1,25D3, a G1 to S-phase block progressively increased in parallel with the abundance of the p27Kip1 CDI. A transient increase in p21Cip1/Waf1 was noted only at 48 hr. The increase in p27Kip1 protein level was dependent on the concentration of 1,25D3 and was accompanied by an increase in cyclin D and E proteins, which normally peak in mid-G1 and at the G1 to S-phase transition, respectively. These results indicate that p27Kip1 protein is a strong candidate for the cell cycle regulator that blocks the entry into the S-phase in 1,25D3-treated HL60 cells.

Animals↗
PubMed1996

Increased stringency of the 1,25-dihydroxyvitamin D3-induced G1 to S phase block in polyploid HL60 cells.

Treatment of mammalian cells with 1,25-dihydroxyvitamin D3 (1,25D3) produces a G1 to S (G1/S) phase cell cycle block. In addition, it has been noted that a smaller proportion of cells accumulates in the G2/M compartment in 1,25D3-treated cultures. Since cyclins have a major influence on the regulation of cell cycle progression, we determined the expression of cyclins A and B as markers of the G2 phase and of cyclin E as the marker of G1/S transition. No increase in the steady-state levels of cyclin A or cyclin B mRNA was detected in the total cell population or in the cyclin B1 protein in the G2/M cell cycle compartment. In contrast, immunodetectable cyclin E protein was increased in cell cultures as a whole and specifically in the G2/M compartment cells. Determination of BrdU incorporation into DNA by flow cytometry showed marked inhibition of DNA replication in cells with DNA content higher than 4C, and autoradiography of 3H-TdR-pulsed cells showed that polynucleated cells did not replicate DNA after 96 h of treatment with 1,25D3 or analogs. Taken together, these experiments show that at least a portion of the G2/M compartment in 1,25D3-arrested cultures of HL60 cells represents G1 cells at a higher ploidy level, which are blocked from entering the high ploidy S phase.

Calcitriol↗
PubMed1996

Structure-based design of a potent transition state analogue for TEM-1 beta-lactamase.

The structure of the plasmid-mediated beta-lactamase TEM-1 has been solved in complex with a designed boronic acid inhibitor (1R)-1-acetamido-2-(3-carboxyphenyl)ethane boronic acid at 1.7 A resolution. The boronate inhibitor was designed based on the crystallographic coordinates of the acyl-enzyme intermediate of TEM-1 bound to the substrate penicillin G. The boronate-TEM-1 complex is highly ordered and defines a novel transition state analogue of the deacylation step in the beta-lactamase reaction pathway. The design principles of this highly effective inhibitor (Ki = 110 nM) and the resulting structural and mechanistic implications are presented.

Acetamides↗
PubMed1995

Use of a low-profile gastrostomy device for administering nutrients in two dogs.

Percutaneous endoscopic gastrostomy (PEG) tubes are an excellent means of providing nutritional support via the enteral route. Most problems with PEG tubes are associated with their maintenance. A low-profile feeding device has been developed for use in human beings to replace standard PEG tubes in situations in which long-term nutritional support is necessary. We adapted a low-profile feeding device for use in 2 dogs. The device offered the advantage of being easier to maintain than standard PEG tubes. It appeared to have been well tolerated. Its low-profile design is likely to result in few problems resulting from an animal chewing on the device or from accidental dislodgment.

Animals↗
PubMed1995

Aziridine analogs of [[trans-(epoxysuccinyl)-L-leucyl]amino]-4-guanidinobutane (E-64) as inhibitors of cysteine proteases.

Aziridine derivatives of E-64 have been synthesized, and their characterization against the cysteine proteases cathepsin B, cathepsin L, and papain is reported. The inhibition was found to be strongly pH-dependent, with maximum activity observed at pH 4, indicating that the protonated aziridinium ion form of the inhibitor is the more reactive form. At low pH, the peptide aziridine HO-(L)Az-Leu-NH-iAm inactivated papain with a second-order rate constant, kinac/Ki, of 7.0 x 10(4) M-1 s-1, a value very close to that observed with E-64 or with the corresponding epoxysuccinyl analog HO-(L)Eps-Leu-NH-iAm. This demonstrates that with the correct peptide sequence, aziridine analogs of E-64 can be good irreversible inhibitors of cysteine proteases. Substitution of the epoxysuccinyl moiety by an aziridine does not affect the specificity of inhibition against the three proteases used in this study. The D-diastereomer is the preferred (by 10-fold) diastereomer for the inhibition of cysteine proteases. The reactivity of both diastereomers of iBuNH-Az-LeuPro-OH against cathepsin B was also found to be much lower than that of iBuNH-(L)Eps-LeuPro-OH, which is a potent selective inhibitor of cathepsin B. These differences are attributed mainly to the presence of the protonated aziridine ring, which can modify the binding mode of aziridine analogs at the active site of cysteine proteases.

Animals↗
PubMed1995

Effect of interfacial pressure on the binding and phospholipase A2 activity of recombinant human lecithin-cholesterol acyltransferase.

We examined the effect of surface pressure on the interfacial binding and phospholipase A2 activity of lecithin-cholesterol acyltransferase. The enzyme bound to phosphatidylcholine monolayers with an apparent dissociation constant of 1.5 nM was excluded from the interface at pressures > 29 mN/m and exhibited maximal phospholipase activity at pressures between 29-28 mN/m. These data suggest that lipoprotein surface pressure may regulate lecithin-cholesterol acyltransferase activity in vivo.

Humans↗
PubMed1995

Improved specificity toward substrates with positively charged side chains by site-directed mutagenesis of the L-lactate dehydrogenase of Bacillus stearothermophilus.

The substrate specificities of L-alpha-hydroxy acid dehydrogenases, including L-lactate dehydrogenases (L-LDH's), can often be quite broad. However, an LDH with high catalytic activity toward alpha-keto acids with positively charged side chains, such as those containing ammonium groups, has not been described, even though there is evidence from metabolic studies that a natural dehydrogenase with such activity might exist in Nature. L-omega-Amino-alpha-hydroxy acids are important intermediates in the synthesis of pharmacologically active compounds, and enzymatic reduction of omega-amino-alpha-keto acids represents an attractive route to these compounds. Graphics analysis indicated that introduction of acidic amino acids at position 102 of the L-LDH of Bacillus stearothermophilus (BSLDH) would favor binding of such side chain ammonium groups. Accordingly, Q102E and Q102D mutant BSLDH's were constructed and the steady state kinetic parameters determined for these mutants for a broad range of alpha-keto acids, including omega-amino-keto acids. The results obtained show that, compared to WT-BSLDH, these mutants show up to 25-fold improvements in kcat/Km values for omega-amino-alpha-keto acid substrates.

Base Sequence↗
PubMed1995

Phlyctainophora lamnae (Nematoda; Philometridae) from dogfish Squalus acanthias off southern New Zealand.

Coiled globose female nematodes and larvae, identified as Phlyctainophora lamnae Steiner, 1921, were found in tumour-like lesions on the tail fin of spiny dogfish Squalus acanthias L. (Squalidae) caught off southern New Zealand. Nematodes provoke a chronic inflammatory response by the host. No male nematodes were recovered. This is the first record of the genus from New Zealand and the first record of adult Phlyctainophora from the Southern Hemisphere. Phlyctainophora squali Mudrey & Dailey, 1969 is considered a synonym of P. lamnae.

Animals↗