Intracellular expression and purification of the c-kit receptor kinase domain in Pichia pastoris.
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Pichia↗
Biomedical subjects
S A Berger
Publications and source records attributed to S A Berger.
Identification of a bacterial inhibitor of protein kinases. Mechanism and role in host cell invasion.
We show that Escherichia coli produce a factor that inhibits the activity of tyrosine and serine/threonine protein kinases. The factor is a protein found in the periplasmic compartment and is also secreted into the culture medium. Using a particle concentration fluorescence immunoassay specific for tyrosine kinase activity and inhibition of the tyrosine kinase p56(lck), we purified this factor to apparent homogeneity. Analysis of trypsin-digested fragments by mass spectrometry identified the inhibitor as the bacterial periplasmic protein UDP-sugar hydrolase, an enzyme with potent and nonspecific 5'-nucleotidase activity. Overexpression of the enzyme in bacteria leads to coordinate increases in both 5'-nucleotidase and p56(lck) inhibitory activity, confirming the identity of the inhibitor. The kinase inhibitory activity appears to be due to the formation of adenosine, which we show is inhibitory for p56(lck), cAMP-dependent protein kinase, and casein kinase. Overexpression of UDP-sugar hydrolase leads to an increase in the recovery of enteropathogenic E. coli following infection of HeLa cell monolayers and corresponding alterations in tyrosine-phosphorylated host proteins. These results suggest that UDP-sugar hydrolase may be an important factor affecting host cell function following intracellular bacterial infection.
Adenosine↗
Calculation of the magnetization distribution for fluid flow in curved vessels.
The signal intensity in magnetic resonance angiography (MRA) images reflects both morphological and flow-related features of vascular anatomy. A thorough understanding of MRA, therefore, demands a careful analysis of flow-related effects. Computational fluid dynamics (CFD) methods are very powerful in determining flow patterns in 3D tortuous vessels for both steady and unsteady flow. Previous simulations of MRA images calculated the magnetization of flowing blood by tracking particles as they moved along flow streamlines that had been determined by a CFD calculation. This manuscript describes MRA simulations that use CFD calculations to determine magnetization variation at a fixed point and, therefore, do not require streamline tracking to calculate the distribution of magnetization in flowing fluids. This method inherently accounts for uniform particle density, avoids problems associated with tracking particles close to the wall, and is well-suited to modeling pulsatile flow.
Blood Flow Velocity↗
Molecular genetic analysis of glucocorticoid and mineralocorticoid signaling in development and physiological processes.
To understand the role of glucocorticoid and mineralocorticoid signalling during development and in whole animal physiology, we have disrupted the mouse glucocorticoid and mineralocorticoid receptor gene by gene targeting. Most of the mice with a disrupted glucocorticoid receptor gene die within the first hours after birth due to severe lung atelectasis. Perinatal induction of gluconeogenic enzymes in the liver is impaired. Regulation of the glucocorticoid synthesis via the hypothalamic-pituitary-adrenal axis is perturbed, leading to increased plasma levels of corticosterone and adrenocorticotrophic hormone. Activation of the hypothalamic-pituitary-adrenal axis results in extensive hypertrophy and hyperplasia of the cortical zones of the adrenal and induction of genes involved in steroid biosynthesis. The adrenal medulla is disorganized and severely reduced in size; no cells capable of adrenaline synthesis can be detected. Mineralocorticoid receptor deficient mice die mainly at day 9/10 after birth. Weightloss precedes death of homozygous mutant mice and is correlated with an increase in the haematocrit. As a consequence of this mutation, plasma levels of renin and aldosterone are high elevated.
Adrenal Medulla↗
Central intraluminal saturation stripe on MR angiograms of curved vessels: simulation, phantom, and clinical analysis.
PURPOSE: To investigate the appearance of reduced signal intensity in the center of blood vessels on magnetic resonance (MR) angiograms that can mimic intraluminal thrombus. MATERIALS AND METHODS: Simulations and phantom studies were performed to analyze MR angiogram appearance distal to a pronounced curve. RESULTS: Saturation effects substantially lower the signal strength in the center of the vessel relative to that at the vessel periphery. These effects appeared even though the flow was well ordered and laminar. In curved geometries, secondary flow patterns produced counter-rotating vortices, which moved the fastest-moving particles to the outside of the curve and folded the slow-moving particles to the center of the vessel. CONCLUSION: Imaging parameter choices that reduce saturation, such as acquisition of a two-dimensional section transverse to the vessel and through the questionable region, effectively eliminate the central hypointensity effect in vivo.
Adult↗
Penetration of clindamycin and metronidazole into inflamed appendiceal tissue.
OBJECTIVE: To calculate the penetration of clindamycin and metronidazole into inflamed appendiceal tissue. DESIGN: Prospective study. SETTING: Teaching hospital, Israel. SUBJECTS: 20 Consecutive men and women operated on for acute appendicitis. INTERVENTIONS: Appendicectomy. Each patient was given three intravenous injections of gentamicin 80 mg combined with either clindamycin 600 mg or metronidazole 500 mg immediately before operation over a period of 15 minutes. MAIN OUTCOME MEASURES: Serum and tissue concentrations of the antibiotics. RESULTS: There was no significant difference between the mean serum concentrations of the drugs (clindamycin 17.86 micrograms/ml and metronidazole 9.75 micrograms/ml) but the mean tissue concentrations of clindamycin (10.41 micrograms/g in the base and 9.86 micrograms/g in the tip of the appendix) were significantly higher than those of metronidazole (5.65 micrograms/g in the base and 5.89 micrograms/g in the tip; p = 0.02 and p = 0.05, respectively). Tissue concentrations of clindamycin and serum concentration of both drugs were more than twice their MIC90. The tissue concentrations of metronidazole were close to its MIC90. CONCLUSIONS: Clinical trials are necessary before any conclusion about therapeutic superiority of one or other agent can be drawn.
Adult↗
c-KIT expression enhances the leukemogenic potential of 32D cells.
The growth of human leukemic cells in culture and in vivo is dependent upon the presence of hematopoietic growth factors. Most populations of human leukemic acute myeloblastic leukemia (AML) cells express c-Kit on their surface and respond to Kit ligand (KL) in culture. To determine if this interaction was of potential significance in vivo we used a mouse model system. 32D cells, a murine IL-3-dependent myeloid cell line, were rendered KL responsive by transfection of the murine c-Kit. After injection of 32D or 32D-Kit cells into syngeneic hosts, animals bearing 32D-Kit cells, but not 32D cells, became moribund and were killed. These animals had circulating leukemic blast cells, infiltration of bone marrow, spleen, brain, liver, lung, and kidney. Cells recovered from some of the animals continued to be dependent upon IL-3 or KL for growth while in other cases the cells were factor independent. This model illustrates that the constitutive expression of c-Kit enhances the leukemic potential of 32D cells. The model will be useful for studying the progression of leukemia in vivo and testing whether interruption of the interaction of Kit and KL can affect the growth of leukemic cells.
Amino Acid Sequence↗
Leukocyte common antigen (CD45) is required for immunoglobulin E-mediated degranulation of mast cells.
We demonstrate using primary mast cell cultures derived from wild-type and CD45-deficient mice that mast cell triggering through the high-affinity immunoglobulin E (IgE) receptor requires the cell surface tyrosine phosphatase CD45. Unlike wild-type cells, cross-linking of surface-bound IgE in mast cells deficient in CD45 does not induce degranulation. Degranulation in these mutant cells does occur after treatment with the calcium ionophore A23187 indicating that the degranulation machinery is intact in these cells. We also demonstrate that the tyrosine phosphatase inhibitors orthoVanadate and perVanadate inhibit degranulation in wild-type mast cells, as does cross-linking of CD45 by anti-CD45 antibodies. Finally, we show that CD45-deficient mice are resistant to IgE-dependent systemic anaphylaxis. These results show that, like the T cell receptor and the antigen receptor on B cells, there is an absolute requirement for CD45 in signaling via the high affinity IgE receptor, expanding the number of receptors for which CD45 is an essential component.
Anaphylaxis↗
[Health concerns of Israelis traveling to Third World countries--experience of a travel advisory clinic].
Following army service young Israelis often travel to remote third world regions for prolonged periods. During such tours, they are exposed to a variety of medical problems, including infections, environmental diseases, political violence and inability to obtain proper medical care. From May 1991 to Nov. 1992, 1,969 travelers aged 20-25 years old, and 926 older than 25, were given medical advice, vaccines and medications prior to travel. A clinic specializing in "emporiatrics" has been established to deal with pre- and post-travel medical problems associated with tourism.
Adult↗
Fifty years of language maintenance and language dominance in bilingual Hispanic immigrants.
Spanish language tests of 801 Cuban and Mexican immigrants showed no evidence of language loss during 50 years of U.S. residence; a few years after immigration, their English vocabulary approximated that of English monolinguals. The critical-age hypothesis was not supported for the acquisition of English vocabulary when English schooling and language usage were controlled by multiple regression. Most Ss continued to speak about as much Spanish as English; but read, wrote, and heard (on television and radio) far more English than Spanish. Under these conditions, Ss maintained Spanish dominance on tests of vocabulary recognition, lexical decision, and oral comprehension. Dominance was task specific and shifted to English on a category generation task about 12 years after immigration. No evidence of bilingual language interference was found; this is attributed to the strong Spanish foundation of the participants.
Adult↗
[Serological screening for Lyme disease in Israel].
During 1989-1992, 123 serum samples from patients with dermatological or neurological disorders were tested for Lyme disease antibody. In only 1 case was a diagnosis of autochthonous Lyme disease confirmed. 8.9% of the samples were positive for antibody to the 41kD flagellar antigen of Borrelia burgdorferi (ELISA test) and 7.3% for the 39kD protein (immunodot test). The diagnosis of Lyme disease should be confirmed by carefully timed tests to detect antibody against multiple species-specific bacterial antigens.
Antibodies, Bacterial↗
Effect of inoculum size on antimicrobial susceptibility of Helicobacter pylori.
An agar dilution assay was used to assess the effect of inoculum size and culture period on the susceptibility of 15 clinical isolates of Helicobacter pylori to ampicillin, erythromycin, tetracycline, chloramphenicol, metronidazole and tinidazole. The mean MIC of the isolates increased 2.2- to 21.2-fold as the inoculum size progressed from 10(3) to 10(7) cfu/spot. Identical results were noted when isolates were maintained for two or four days prior to testing. Inoculum size should be carefully controlled when assessing the in vitro susceptibility of Helicobacter pylori.
Helicobacter pylori↗
Effect of surgical mask position on bacterial contamination of the operative field.
The influence of surgical mask usage on bacterial contamination of the operative field was studied during 30 cardiac catheterization procedures. Mask position was varied during each procedure according to a predesigned random table. The number of bacterial colonies recoverable when no mask was worn was significantly higher than that detected when a full mask was worn (P < 0.002). Shedding of Staphylococcus epidermidis was greater when no mask was worn (mean 5.2 colonies 10 min-1) than shedding with full mask (mean 2.7 colonies 10 min-1; P < 0.004). Although mask placement below the nose was associated with higher mean colony counts than that above the nose, these differences were not statistically significant.
Bacteria↗
Lyme disease acquired in Israel: report of a case and studies of serological cross reactivity in relapsing fever.
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Adult↗
Transfer of bacteria associated with cryotherapy.
The aim of this study was to determine whether living bacteria can be transmitted by cryosurgery with carbon dioxide. Cultures were taken from the outer layer of the ice before starting treatment, and again at the end of the working day during ten consecutive working days. Each of ten dry ice blocks was sterile prior to use. Staphylococcus epidermidis was isolated from six of the latter following use. This finding indicates that the dry ice was contaminated during the procedure and that bacteria were transmitted from the patient's skin to the dry ice and to culture media. The medical implications of this finding require serious consideration regarding the risk of infection to both patient and clinical personnel from this procedure.
Cryosurgery↗
Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.
Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.
Adenosine Triphosphate↗
Penetration of ceftriaxone and cefoperazone into bile and gallbladder tissue in patients with acute cholecystitis.
The penetration of ceftriaxone and cefoperazone into bile and gallbladder tissue was prospectively studied in 21 adult patients undergoing early surgery for acute cholecystitis. Comparable tissue, bile, and serum concentrations of the drugs were demonstrable; however, significantly fewer preoperative doses of ceftriaxone were required for adequate perioperative treatment. In view of its higher serum half-life and superior antibacterial activity toward common biliary pathogens, ceftriaxone appears to be a useful drug for the perioperative management of acute cholecystitis.
Acute Disease↗
Fatal necrotizing esophagitis due to Penicillium chrysogenum in a patient with acquired immunodeficiency syndrome.
Although blue-green molds of the genus Penicillium are ubiquitous in the human environment, invasive penicilliosis is uncommon and primarily encountered among immunosuppressed patients. A patient with HIV infection who died of severe necrotizing esophagitis caused by Penicillium chrysogenum is reported and the relevant English language literature on human penicilliosis is reviewed. Although infectious esophagitis is commonly associated with AIDS, Penicillium esophagitis has not been described in such patients.
AIDS-Related Opportunistic Infections↗