Antimicrobial susceptibility tests for anaerobic bacteria with use of the disk diffusion method.
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Anti-Bacterial Agents↗
Biomedical subjects
M E Cox
Publications and source records attributed to M E Cox.
Techniques for controlling variability in gram staining of obligate anaerobes.
Identification of anaerobes recovered from clinical samples is complicated by the fact that certain gram-positive anaerobes routinely stain gram negative; Peptostreptococcus asaccharolyticus, Eubacterium plautii, Clostridium ramosum, Clostridium symbiosum, and Clostridium clostridiiforme are among the nonconformists with regard to conventional Gram-staining procedures. Accurate Gram staining of American Type Culture Collection strains of these anaerobic bacteria is possible by implementing fixing and staining techniques within a gloveless anaerobic chamber. Under anaerobic conditions, gram-positive staining occurred in all test organisms with "quick" fixing techniques with both absolute methanol and formalin. The results support the hypothesis that, when anaerobic bacteria are exposed to oxygen, a breakdown of the physical integrity of the cell wall occurs, introducing Gram stain variability in gram-positive anaerobes.
Bacteria, Anaerobic↗
C-terminal CTII motif of 2',3'-cyclic nucleotide 3'-phosphodiesterase undergoes carboxylmethylation.
Eukaryotic proteins with a carboxyl-terminal CaaX motif are modified by isoprenylation and subsequently processed by proteolysis of the three terminal amino acids and carboxylmethylation of the exposed cysteine residue. The myelination-associated 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) has a C-terminal CTII sequence and is isoprenylated; however, no examples of subsequent processing exist when threonine, a polar residue, is located adjacent to the cysteine. Here we show that CNP is capable of being carboxylmethylated in both insect cells and glioma cells. This processing is dependent upon isoprenylation of the cysteine and can be inhibited with the isoprenylated cysteine derivative, N-acetyl-S-farnesyl-L-cysteine. Although the role of the methyl group at the C-terminus of other isoprenylated proteins is not fully understood, modulation of signal transduction pathways is strongly indicated. This modification of CNP may similarly regulate cell biological processes in myelinogenesis.
2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
Partial purification of a mitogen-activated protein kinase kinase activator from bovine brain. Identification as B-Raf or a B-Raf-associated activity.
A classical biochemical approach was taken to identify mitogen-activated protein kinase kinase (MEK) activators in bovine brain. Fractionation revealed the presence of one major MEK-stimulating activity that was distinct from c-Raf-1 and MEK kinase. Similar results were obtained using bovine adrenal chromaffin cells, and in both cases, immunoblotting and immunoprecipitation experiments demonstrated co-purification of MEK activator with B-Raf. Partially purified MEK activator stimulated phosphorylation of MEK1 on residues tentatively identified as serine 218 and serine 222. Little or no MEK activator was associated with c-Raf-1 in bovine brain or chromaffin cells, although this protein was expressed, suggesting that B-Raf might be the major MEK activator in cells of neural origin.
Adrenal Glands↗
5-lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-lipoxygenase.
5-Lipoxygenase (5-LO) and its activating protein (FLAP) are both required for cellular leukotriene (LT) synthesis, with 5-LO catalyzing both the synthesis of (5S)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) from arachidonic acid and the subsequent synthesis of LTA4 from 5-HPETE. We have previously expressed both human 5-LO and human FLAP to high levels in Spodoptera frugiperda (Sf9) insect cells, using recombinant baculoviruses. To study the mechanism by which FLAP activates 5-LO, we compared cellular 5-LO activity in Sf9 cells expressing this enzyme to that in Sf9 cells coexpressing FLAP and 5-LO. In this system, FLAP stimulates the utilization of arachidonic acid by 5-LO as a substrate, and increases the efficiency with which 5-LO converts 5-HPETE to LTA4. LT synthesis in cells coexpressing FLAP and 5-LO is inhibited by 3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H-indol-2-yl]-2,2- dimethyl-propanoic acid (MK-886), an LT biosynthesis inhibitor which specifically binds to FLAP. These studies in Sf9 cells, together with our recent demonstration that FLAP specifically binds arachidonic acid, suggests that FLAP activates 5-LO by acting as an arachidonic acid transfer protein.
5-Lipoxygenase-Activating Proteins↗
5-lipoxygenase-activating protein is an arachidonate binding protein.
5-Lipoxygenase-activating protein (FLAP) is an 18-kDa integral membrane protein which is essential for cellular leukotriene (LT) synthesis, and is the target of LT biosynthesis inhibitors. However, the mechanism by which FLAP activates 5-LO has not been determined. We have expressed high levels of human FLAP in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus, and used this system to demonstrate that FLAP specifically binds [125I]L-739,059, a novel photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK-886, an LT biosynthesis inhibitor which specifically interacts with FLAP. These studies suggest that FLAP may activate 5-LO by specifically binding arachidonic acid and transferring this substrate to the enzyme.
5-Lipoxygenase-Activating Proteins↗
Tyrosine phosphorylation of alpha-tubulin is an early response to NGF and pp60v-src in PC12 cells.
Neuronal differentiation is accompanied by extensive reorganization of the cytoskeleton to initiate the extension of neuritic processes. We have used the rat PC12 pheochromocytoma cell line to examine the role of protein tyrosine kinase activity in the induction of these events. Immunoblotting with phosphotyrosine antibodies revealed that tyrosine phosphorylation of alpha-tubulin in PC12 cells occurred within 10 min of nerve growth factor (NGF) treatment. Tyrosine phosphorylation of alpha-tubulin also occurred on induction of pp60v-src expression in a PC12 cell line (PC12-B9) harboring an inducible v-src gene under transcriptional control of the mouse metallothionine I gene promoter. Two tyrosine phosphorylated proteins in NGF- and pp60v-src induced PC12 cells were identified as alpha-tubulin isoforms by comigration with alpha-tubulin on two-dimensional gel electrophoresis, and by immunoprecipitation with phosphotyrosine antibodies followed by immunoblotting with a monoclonal antibody specific for alpha-tubulin. These results demonstrate that alpha-tubulin is an in vivo tyrosine kinase substrate, which is phosphorylated as an early event in the neuronal differentiation pathway of PC12 cells in response to NGF or pp60v-src. Tyrosine phosphorylation of alpha-tubulin could conceivably alter microtubule dynamics during induction of neurite extension.
Animals↗
Protein tyrosine kinases in nervous system development.
Protein tyrosine kinases are important mediators of intracellular signaling during nervous system development. Activation of receptor protein tyrosine kinases by neurotrophic factors are initial events in the development of discrete cell populations. The patterns of expression and characterization of substrates for nonreceptor protein tyrosine kinases indicates that they also play a crucial role in neuronal development. The observed functional redundancy among protein tyrosine kinases and their associated intracellular signaling pathways underscores the need for further characterization of these novel interactions to elucidate the mechanisms regulating nervous system development.
Animals↗
Neurite extension and protein tyrosine phosphorylation elicited by inducible expression of the v-src oncogene in a PC12 cell line.
Tyrosine-specific protein kinase activity in neuronal differentiation was studied in a PC12 pheochromocytoma cell line (PC12-B9) produced by stable transfection with an inducible v-src gene encoding an activated tyrosine kinase (pp60v-src) under the transcriptional control of the mouse metallothionine I gene promoter. Induction of pp60v-src expression with Cd2+ and Zn2+ resulted in the reversible differentiation of PC12-B9 cells into neuron-like cells. pp60v-src elicited morphological differentiation with apparent first order kinetics at the same rate as NGF-directed neurite outgrowth in PC12-B9 cells. v-src gene expression enhanced the rate of NGF-directed neurite extension in an additive manner. Induction of pp60v-src alone constitutively increased the levels of phosphotyrosine-modified proteins (130-120, 90, 83, 65, 60/59, 36 kDa) detected by immunoblotting with phosphotyrosine antibodies. NGF treatment of PC12-B9 cells transiently increased the levels of distinct phosphotyrosine-modified proteins (108, 46, 42 kDa), as well as common substrates, including a 59-kDa protein that comigrated with alpha-tubulin. Phosphotyrosine-modified proteins were not synergistically increased in PC12-B9 cells induced for both v-src and NGF. The nonsynergistic effects of v-src gene expression on neurite outgrowth and phosphorylation suggest that pp60v-src induces PC12 cell differentiation by an intracellular signaling pathway that is largely distinct from that induced by NGF.
Adrenal Gland Neoplasms↗
c-src and other proto-oncogenes implicated in neuronal differentiation.
The proto-oncogene c-src has been implicated in the development and mature function of the nervous system. pp60c-src, the protein product of the c-src gene, is a tyrosine protein kinase that is highly enriched in fetal neural tissue. pp60c-src appears in two phases of neuronal development. Neuroectodermal cells of gastrulating embryos first express pp60c-src around the time of commitment to neuronal or glial pathways. Later, committed neuroepithelial cells express pp60c-src near the onset of terminal neuronal differentiation. Immunocytochemical analyses of pp60c-src in developing chick retina, telencephalon, and cerebellum show immunoreactivity concentrated in regions rich in growth cones and neurites. Moreover, pp60c-src is concentrated approximately 10-fold in a biochemical fraction from fetal rat brain that is enriched in nerve growth cone membranes. These results point toward a function for pp60c-src in neurite outgrowth. A functional role for other proto-oncogenes in the development of the nervous system was indicated from a study of the expression of a battery of proto-oncogenes during the retinoic acid-induced differentiation of the mouse embryonal carcinoma cell line P19 to a neuronal phenotype. Nuclear runoff transcription of the proto-oncogenes c-src, c-fms, c-sis, N-ras, c-myc, and c-fos was observed in proliferating and retinoic acid-treated cells.
Animals↗
A tyrosine kinase related to pp60c-src is associated with membranes of Electrophorus electricus electric organ.
A tyrosine-specific protein kinase immunologically related to pp60c-src, the cellular homolog of the Rous sarcoma virus-transforming protein, was expressed at elevated levels in the electric organ of the electric eel Electrophorus electricus. The electric organ kinase phosphorylated antibodies reactive with pp60c-src at tyrosine residues in immune complex protein kinase assays and was associated with electric organ membranes enriched in acetylcholine receptors. The protein recognized by anti-pp60c-src antibodies was phosphorylated in endogenous membrane phosphorylation reactions and was shown to have a relative molecular mass of 57 kDa by two-dimensional gel electrophoresis. In immune complex protein kinase assays the 57-kDa protein was phosphorylated at threonine by a distinct threonine kinase from the electric organ. The tyrosine kinase was purified 844-fold from electric organ membranes by chromatography on omega-aminohexyl agarose, phosphocellulose, and casein-Sepharose. Threonine kinase activity in immunoprecipitates was not observed in the tyrosine kinase fractions after the first step. Incubation of the casein Sepharose fraction with [gamma-32P]ATP-Mn2+ in solution resulted in phosphorylation of only the 57-kDa protein. Phosphorylation occurred solely at tyrosine, suggesting that the kinase is capable of autophosphorylation. The structural and functional properties of the 57-kDa electric organ kinase indicate that the 57-kDa electric organ protein is a member of the src subfamily of tyrosine kinases and is closely related to pp60c-src.
Animals↗
Neutrophil uptake of vaccinia virus in vitro.
We studied human neutrophils for uptake of vaccinia virus. Uptake was determined radiometrically and by electron microscopy. Vaccinia virus was labeled with 14C or 3H, incubated with neutrophils, and quantified in neutrophil pellets in a new radiometric phagocytosis assay. Better results were obtained from assays of [3H]thymidine-labeled virus; uptake increased through 1 hr and then plateaued. Phagocytosis of 3H-labeled Staphylococcus aureus was normal. Uptake of virus was serum dependent. Hexose monophosphate shunt activity was measured by two methods. No 14CO2 from [14C]1-glucose accompanied uptake of vaccinia virus, in contrast to the respiratory burst accompanying bacterial phagocytosis. Electron microscopy showed intact to slightly digested intraphagolysosomal vaccinia virus. Pock reduction assay showed a decrease in viral content due to neutrophils until 6 hr of incubation, when a modest but significant increase was observed. Thus, neutrophil uptake of vaccinia virus is distinguished from bacterial phagocytosis.
Animals↗
Physicians and dietitians can be a team!
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Dietetics↗
Methanol fixation. An alternative to heat fixation of smears before staining.
Methanol fixation of Gram-stained smears was compared to heat fixation. Smears were prepared in duplicate from direct clinical specimens, blood culture bottles, and bacterial colonies. Results from this study show that methanol fixation is superior in every instance to heat fixation. The morphology of bacteria and tissue cells was not distorted, little or no background debris was observed, and a better Gram reaction was noted.
Bacterial Infections↗
Distribution of fallout cesium-137 in Hawaii.
The concentration of 137Cs was determined by gamma spectrometry in soils and surface-deposited volcanic sublimates from various climatic locations on the islands of Hawaii, Oahu and Maui. A distinct pattern of higher concentrations on the wetter windward sides of the islands and a correlation between 137Cs concentration and amount of rainfall were observed, which confirm the source of the 137Cs as atmospheric fallout. In the soils, the concentrations measured (to 1.93 pCi/g) are within the range reported for the continental United States and other mid-latitude locations in the northern hemisphere. In the basaltic lavas, the concentration of 137Cs was found to be variable but was overall very low, and is apparently controlled by the porosity and permeability of the lavas in addition to rainfall.
Cesium Radioisotopes↗
Zinc and the special senses.
There is evidence that zinc is important for maintenance of the special senses: vision, taste, and smell. Rod function is impaired in zinc deficiency due partly to its role in vitamin A metabolism. However, optic nerve function may also be affected by zinc status. Microophthalmia, anophthalmia, and optic nerve abnormalities have all been found in the offspring of female rats fed zinc-deficient diets. Zinc deficiency clearly decreases taste acuity in both animals and humans. However, other nutritional and non-nutritional conditions also produce hypogeusia. There is limited evidence that zinc deficiency impairs olfactory acuity in humans. New approaches to the assessment of taste and smell abnormalities may provide reliable and reproducible associations between zinc deficiency and taste and smell defects.
Animals↗
Rapid Tween 80 hydrolysis test for mycobacteria.
A rapid Tween 80 hydrolysis test for mycobacteria utilizing gas-liquid chromatography is described. The test requires 1 h of incubation followed by simple extraction and chromatography of nonderivatized oleic acid.
Bacteriological Techniques↗
Radiometric detection of Haemophilus influenzae in cerebrospinal fluid specimens.
The investigation demonstrates the usefulness of a radiometric urease test in the rapid detection of Haemophilus influenzae in cerebrospinal fluid specimens. The test is simple and economical to perform and results are obtained within a one hour incubation period.
Carbon Radioisotopes↗