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Biomedical subjects

J B Jones

Publications and source records attributed to J B Jones.

At least 19 recordsLinked to original sources
PubMed2026

Characterization of putatively lytic bacteriophages able to infect Xanthomonas citri subsp. citri and identification of novel putative exopolysaccharide depolymerases.

Asiatic citrus canker (ACC), caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (X. citri), leads to substantial economic losses in the global citrus industry, necessitating sustainable alternatives to conventional copper-based bactericides. In this study, we isolated and sequenced 72 putatively lytic bacteriophages (including 65 previously uncharacterized isolates from São Paulo, Brazil) and characterized their host range, stability and biocontrol potential. Genomic analysis revealed highly successful but low-diversity phage genomic signatures; 70 isolates shared ~95% DNA similarity and were closely related to the Japanese phage CP2, mirroring the clonal nature of the endemic X. citri population. These phages primarily belong to the Autographiviridae family, with the exception of the Schitoviridae isolate XacP77. Using HHsearch and AlphaFold structural modelling, we identified conserved tail-fibre genes predicted to encode putative exopolysaccharide depolymerases with structural homology to carbohydrate-binding modules (CBMs), which may facilitate the degradation of the bacterial xanthan gum capsule during infection. While the phages exhibited robust stability across a wide pH range (4-11) and temperatures up to 55 °C, they were highly sensitive to UV exposure, reaching total inactivation after 160 s. Greenhouse assays demonstrated that treatment with phage P27 reduced ACC lesion production by 60%, pointing to the potential of these viruses and their candidate CBM-containing proteins as components of a sustainable biocontrol development within integrated pest management strategies for X. citri.

Xanthomonas↗
PubMed1992

THR246 mutations decrease substrate inhibition in lactate dehydrogenase.

Threonine 246 in Bacillus stearothermophilus L-lactate dehydrogenase has been changed to valine, serine, and alanine by site-directed mutagenesis. Kinetic analyses show a decrease in substrate inhibition for pyruvate reduction with the T246S mutant and virtual elimination of substrate inhibition for the T246A and T246V mutants. The results indicate that the absence of substrate inhibition in the 246A/V-catalyzed reactions is due to the elimination of a key hydrogen bond between the hydroxyl group of threonine and pyruvate in the wild-type complex that is an important contributor in the formation of the abortive enzyme-NAD(+)-pyruvate complex responsible for substrate inhibition.

Escherichia coli↗
PubMed1992

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.

Bacterial Proteins↗
PubMed1992

Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase.

Lactate dehydrogenases are of considerable interest as stereospecific catalysts in the chemical preparation of enantiomerically pure alpha-hydroxyacid synthons. For such applications in synthetic organic chemistry it would be desirable to have enzymes which tolerate elevated temperatures for prolonged reaction times, to increase productivity and to extend their applicability to poor substrates. Here, two examples are reported of significant thermostabilizations, induced by site-directed mutagenesis, of an already thermostable protein, the L-lactate dehydrogenase (EC 1.1.1.27, 35 kDa per monomer subunit) from Bacillus stearothermophilus. Thermal inactivation of this enzyme is accompanied by irreversible unfolding of the native protein structure. The replacement of Arg171 by Tyr stabilizes the enzyme against thermal inactivation and unfolding. This stabilizing effect appears to be based on improved interactions between the subunits in the core of the active dimeric or tetrameric forms of the enzyme. The thermal stability of L-lactate dehydrogenase variants with an active site Arg residue, either in the 171 (wild-type) or in the 102 position, is further increased by sulfate ions. The two stabilizing effects are additive, as found for the Arg171Tyr/Gln102Arg double mutant, for which the stability of the protein in 100 mM sulfate solution reaches that of L-lactate dehydrogenases from extreme thermophiles. All mutant proteins retain significant catalytic activity, both in the presence and absence of stabilizing salts, and are viable catalysts in preparative scale reactions.

Calorimetry↗
PubMed1991

Expression of a foreign gene in cats reconstituted with retroviral vector infected autologous bone marrow.

A Moloney murine leukemia virus based retroviral vector was used to transfer the bacterial neomycin resistance gene (neoR) into feline hematopoietic cells. We reconstituted four cats that had been lethally irradiated with autologous bone marrow that had been infected with the N2 or SAX retroviral vector. Bone marrow cells from all four cats expressed the neoR gene 30 days posttransplant and three of four cats still had the neoR gene and a low level of drug resistant colony-forming unit granulocyte-macrophage after more than 200 days. Two of the four cats unexpectedly developed diabetes mellitus 90 days posttransplantation. The expression of a foreign gene in cats, albeit at a low level, demonstrates that retroviral vectors can be used for gene transfer in noninbred large animal species and may be useful for gene therapy of humans. The development of diabetes mellitus in two of the subjects emphasizes the value of animal models for the study of possible deleterious effects of retroviral vector-mediated gene transfer.

Animals↗
PubMed1991

Probing the specificity of the S1 binding site of subtilisin Carlsberg with boronic acids.

The binding properties and limitations of the key S1 site of subtilisin Carlsberg have been probed with boronic acid inhibitors bearing structurally varied substituents ranging from small alkyl to large aromatic groups. The data permit structural features favoring, and disfavoring, good S1 binding to be clarified. In addition, applications of electrostatic energy calculations have identified a hitherto unsuspected region of positive potential in the fundamentally hydrophobic S1 pocket, whose interactions with electronegative substituents of inhibitors can make significant binding contributions.

Binding Sites↗
PubMed1991

Platelet aggregation, storage pool deficiency, and protein phosphorylation in mice with Chediak-Higashi syndrome.

The beige (bgJ/bgJ) mouse is a well-described murine model of Chediak-Higashi syndrome. Platelet function was examined in normal and beige mice to better characterize the defective aggregation response in platelets from mice with Chediak-Higashi syndrome. Platelet aggregation after collagen, thrombin, and phorbol-12-myristate 13-acetate stimulation was significantly (P less than 0.025) decreased in platelets from beige mice, relative to platelets from normal mice. Compared with beige and normal mice, those heterozygous for the bg trait had intermediate responses to collagen and thrombin, but not phorbol-12-myristate 13-acetate. The defect(s) in aggregation of platelets from beige mice was associated with a dense granule storage pool deficiency and decreased stores of serotonin and adenine nucleotides in platelets. Mice heterozygous for the bg trait had normal platelet serotonin and adenine nucleotide concentrations. Platelets from beige mice were approximately 10 times more sensitive to prostacyclin inhibition of collagen-induced aggregation than were platelets from control mice. However, a significant difference in platelet cyclic AMP concentration was not apparent between beige and normal mice after prostacyclin stimulation. Platelet endoperoxide synthesis measured by quantification of thromboxane B2, was normal in beige mice. Protein phosphorylation patterns in mouse platelets were similar to those seen in human platelets. Thrombin and collagen-induced [32P] phosphorylation of 40- and 20-kD proteins in platelets from normal and beige mice was similar. Results indicate that the biochemical defect(s) in platelet function in beige mice is partially attributable to storage pool deficiency and does not result in an absolute defect in phosphorylation of 40- and 20-kD proteins.

Animals↗
PubMed1991

Characterization of platelet function in cyclic hematopoietic dogs.

Platelet aggregation to incremental doses of eight different platelet agonists (collagen, thrombin, platelet-activating factor [PAF], arachidonic acid [AA] plus epinephrine, the calcium ionophore A23187, ADP, phospholipase C [PLC], and 12-O-tetradecanoyl phorbol-13-acetate [TPA]) was compared in normal (N) and cyclic hematopoietic (CH) dogs. Platelet aggregation was defective with collagen, PAF, TPA, and possibly thrombin as agonists but normal when ADP, PLC, arachidonic acid plus epinephrine, and A23187 were used as agonists with CH platelets. In heterozygous CH dogs, platelet aggregation was intermediately defective when tested with collagen and PAF as agonists. Thromboxane B2 (TXB2) concentrations (mean +/- SD; pg/10(6) platelets), as measured by RIA, were similar in CH and normal dogs both prior to (CH: 7.6 +/- 7.0; N: 5.5 +/- 3.9) and after collagen stimulation (collagen: 141.3 +/- 42.5; 123.1 +/- 38.4). Granule storage pools of serotonin and platelet adenine nucleotides were markedly decreased in homozygous CH but not heterozygous CH dogs. Thrombin stimulated phosphorylation of 40- and 20-kd proteins in platelets from CH and normal dogs to an equal extent. However, collagen-stimulated phosphorylation of the 40- but not the 20-kd protein was significantly decreased in platelets from CH dogs. These data suggest that there is a biochemical defect in platelets from CH dogs that results in storage pool disease and decreased phosphorylation of a 40-kd protein.

Animals↗
PubMed1990

Conversion disorder. Camouflage for brain lesions in two cases.

We present two cases of serious central nervous system disease initially misdiagnosed as conversion disorder. In each instance, multiple medical evaluations did not uncover the underlying neurologic disorder. Skepticism on the part of psychiatric evaluators led to eventual diagnosis. Common difficulties in the clinical application of the diagnostic criteria for conversion disorder are discussed in relation to the specific pitfalls of the two cases. We make recommendations to aid in the evaluation of suspected cases of conversion disorder.

Brain Neoplasms↗
PubMed1990

Synchrony of bone marrow proliferation and maturation as the origin of cyclic haemopoiesis.

Cyclic haemopoiesis in Grey Collie dogs is characterized by stable oscillations in all haemopoietic lineages. It is proposed that in these animals, in contrast to normal animals, the maturation process of haemopoietic (in particular granuloid) cells from the primitive progenitors to the functional cells is characterized by an abnormally strong synchrony. It is conjectured that the marrow maturation time has a very small variance compared with non-cyclic normal dogs. With a mathematical model of haemopoiesis it is shown that small fluctuations are amplified via regular feedback processes such that stable granuloid oscillations are established. Erythroid oscillations are induced indirectly by granuloid feedback to the stem cell pool. The model calculations further show that the synchrony hypothesis of bone marrow maturation can quantitatively explain the following experimental results: (1) the maintenance of stable cycles of granuloid and erythroid bone marrow and blood cells with a period of approximately 14 d; (2) the disappearance of granuloid and erythroid cycles during the administration of the colony stimulating factor rhG-CSF; (3) the reappearance of oscillations when the administration of CSF is discontinued; (4) the cessation of cycles during endotoxin application; and (5) the persistence of cycles during erythroid manipulations (bleeding anaemia, hypoxia, hypertransfusion). We therefore conclude that cyclic haemopoiesis is not caused by a defect in the regulatory control system but by an unusual maturation process.

Animals↗
PubMed1979

Ultrastructural studies on the evolution of amyloidosis in the cyclic hematopoietic (CH) dog.

Electron microscopy studies were made on tissues of cyclic hematopoietic (CH) dogs of various ages presenting a high incidence of spontaneous amyloidosis. The distribution and morphologic characteristics of amyloidosis in this animal model closely correspond to the secondary and familial forms of the disease in humans. Plasma cells and, particularly, macrophages presented marked changes during the evolution of amyloid deposition. Residual bodies in the macrophages contained abundant cell debris, a result of both endocytic and autophagocytic activities. Intracellular amyloid fibrils were not observed by conventional electron microscopy. A few reticular cells contained intracytoplasmic fibrils which were morphologically different from amyloid. There was no correlation between the amount of intracellular fibrils and the size of the extracellular amyloid deposits. On the contrary, a temporal association between the magnitude of the amyloid deposits and cytoplasmic changes in the macrophages at sequential stages of the evolution of the disease was evident. It is suggested that the hematopoietic defect in the CH dog could play an important role in the production of amyloidosis, making this animal an excellent experimental model for studies of that disease.

Amyloidosis↗
PubMed1979

Circular dichroism and magnetic circular dichroism of nitrogenase proteins.

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of nitrogenase components (MoFe protein and Fe protein) from Azotobacter vinelandii (Av) and Klebsiella pneumoniae (Kp) have been obtained in the near infrared-visible-near ultraviolet spectral region. Previously, visible CD was reported to be absent or barely detectable in nitrogenase proteins; MCD spectra have not been reported. The chiroptical spectra can be measured in solution at room temperature, an advantage relative to spectroscopic methods requiring cryogenic sample temperatures. Absorption spectra were also obtained. The CD and MCD are markedly more structured, and thus interpretively more useful, than the corresponding absorption spectra. The dithionite-reduced MoFe proteins (Av1, Kp1) have nearly identical CD and MCD, demonstrating identical numbers and types of metal centers in similar protein environments. The CD and MCD cannot be explained solely in terms of contributions from known 4-Fe or 2-Fe clusters; the near-infrared MCD is inconsistent with the presence of known 4-Fe clusters. CD and MCD spectra of Lauth's violet-oxidized Kp1 are also reported. The reduced Fe proteins (Av2, Kp2) have similar CD and MCD, again indicating significant conservation of chromophore environment. The spectra clearly demonstrate the presence of a reduced bacterial ferredoxin-like (C(3-)) 4-Fe cluster. No obvious evidence of additional chromophores is observed. CD, MCD, and absorption spectra of Av1-oxidized Av2 are reported. The absorption spectrum shows the expected shoulder near 390 nm. The CD and MCD are characteristic of a C(2-) 4-Fe cluster; in particular, the diagnostic near-infrared MCD peak is observed at approximately 8300 cm(-1). The CD of Av2 oxidized in the presence and absence of MgATP are radically different, providing the first direct evidence for MgATP interaction with Fe protein in this oxidation state.

Azotobacter↗
PubMed1979

Some immunological and haematological aspects of human cyclic neutropenia.

In addition to standard peripheral blood cell counts, sequential studies have been made of changes in the T-lymphocyte population and in the serum titres of the presumptive humoral regulators of haematopoiesis, Colony Stimulating Activity (CSA) and Erythroid Stimulating Activity (ESA), in a young woman with cyclic neutropenia (CN). In addition, serum immunoglobulins, C3 and total complement levels and serum protein concentrations were determined on several occasions during the study. Similar tests were done concomitantly on a haematologically normal, age and sex-matched control. Cell counts on peripheral blood from the subject with CN demonstrated a clearly defined periodicity in neutrophil and monocyte concentrations and equivocal fluctuations in reticulocyte numbers. There was no evidence of periodicity in the lymphocyte concentrations and the T-lymphocyte population appeared functionally normal. Spontaneous incorporation of tritiated thymidine into peripheral blood cells showed a highly significant correlation with the monocyte count, suggesting that these cells were responsible for the radioisotope uptake. CSA titres were elevated on all occasions tested and showed no evidence of periodicity. ESA showed some evidence of cycling with elevated levels being observed during the periods of neutropenia. Serum complement levels were within the normal range but all classes of immunoglobulins were elevated and albumin levels were depressed.

Adult↗
PubMed1979

The in vitro growth of erythroid colonies from dog bone marrow.

Enriched methyl cellulose media together with either human urinary erythropoietin or serum collected from phlebotomized dogs exposed to hypoxia was used in the study of the erythroid colony forming (CFU-E) capacity of dog marrow. The dog serum erythropoietin was found to be more efficient in stimulating CFU-E than comparable concentrations of human urinary erythropoietin. Numbers of CFU-E were directly related to the culture concentration of the stimulating serum and to the number of cells per plate. Sheep plasma erythropoietin was also found to be effective in stimulating CFU-E growth. The system described is chemically better defined and produced more consistent results than has been reported for the plasma clot method.

Animals↗
PubMed1979

Evidence that the fetal mouse liver cell assay detects erythroid regulatory factors (ERF) not measured with a standard in vivo assay.

A simplified modification of the fetal mouse liver cell (FMLC) assay has been used to investigate the relationship between in vivo and in vitro detectable Erythroid Regulatory Factors (ERF) in the serum of dogs exposed to hypoxia. The results suggest that the FMLC assay is a useful technique for following changes in serum ERF. However, the data obtained cannot be directly equated with the in vivo erythrocythemic mouse assay unless a correction factor is applied. This is necessary because the in vitro assay, in general, detects 50% more ERF than does the standard mouse assay.

Animals↗