Cryopreservation of competent Schizosaccharomyces pombe protoplasts.
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Biomedical subjects
Publications and source records attributed to J Jimenez.
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The present study was undertaken to characterize the immune recognition of pancreatic cholecystokinin receptor by an anti-cholecystokinin antibody. Cholecystokinin receptor from pancreatic plasma membranes was photoaffinity labelled using the specific, cleavable probe 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK(25-33) [CCK(25-33) is the C-terminal nonapeptide of the 33-amino-acid form of cholecystokinin]. Labelled receptor was then solubilized and subsequently prepurified on immobilized wheat-germ agglutinin. The C-terminal-directed anti-cholecystokinin serum (8E) specifically immunoprecipitated a fraction of affinity-labelled cholecystokinin receptor which was identified at Mr 85,000 - 100,000 on SDS/PAGE. The binding affinity of antiserum 8E for covalently labelled cholecystokinin receptor was lower (Kd 0.11 +/- 0.02 nM) than for cholecystokinin (Kd 3.65 +/- 0.55 pM). The compound L364-718, an A-subtype cholecystokinin-receptor antagonist did not interfere with the immune recognition of cholecystokinin. However, the recognition of affinity-labelled cholecystokinin receptor was enhanced as a result of an increasing availability of cholecystokinin molecules. Indeed, the amount of immunoprecipitated receptor was doubled in the presence of 10 microM L364-718. This study offers the possibility of using an anti-cholecystokinin antibody for cholecystokinin-receptor purification and demonstrates that prepurified affinity-labelled cholecystokinin receptor retains A-subtype specificity.
Linkage analysis has been performed on a large Australian family segregating for the autosomal dominant form of retinitis pigmentosa (ADRP). The majority of patients had no subjective symptoms of night blindness until their second decade and good visual acuity until late in life. The disease in this family has been classified as Type II ADRP according to the subdivisions provided by both Massof and Finkelstein and Fishman and colleagues. Linkage (Omax:0.08 at Zmax:4.78) is here demonstrated between the disease locus and D3S47 (a marker locus on the long arm of chromosome 3), which showed in an earlier study very close linkage without recombination to the disease locus in an Irish pedigree with a clinically more severe and early onset (Type I) ADRP.
We have exploited the universality of the molecular mechanisms that control entry into mitosis to clone the Drosophila melanogaster homologues of fission yeast Schizosaccharomyces pombe cell division control (cdc) genes by the complementation of temperature sensitive mutations. The Drosophila genes were expressed in S.pombe as cDNAs from the SP6 promoter. Successful recovery of complementing plasmids required that we first 'adapt' pooled plasmids from a Drosophila embryonic cDNA library for propagation in fission yeast by introducing an ars1-LEU2 DNA fragment into the vector. This library was introduced into S.pombe cdc2 and cdc25 mutants, and plasmids isolated carrying cDNAs that complement these mutations. The gene that encodes the Drosophila cdc2 homologue maps to a single locus in the Drosophila genome at 31E on chromosome 2. It is expressed maternally to provide mRNA in syncytial embryos, and appears to be zygotically expressed in mitotically active regions of the cellularized embryo. We have isolated two different cDNAs that complement cdc25-22. One corresponds to a transcript of string, previously described as the Drosophila homologue of cdc25, and the other to a gene that has not been previously characterized.
Having previously demonstrated the hypoglycemic action of Salvia lavandulifolia Vahl, subspecies oxyodon, the mechanism of action was further investigated. The hypoglycemic action may be the result of several synchronous mechanisms: a) potentiation of insulin release induced by glucose, b) increased peripheral uptake of glucose, c) decreased intestinal absorption of glucose and d) hyperplasia of the pancreatic islet beta cells (seen after chronic treatment).
Twenty-four children with chronic active hepatitis due to hepatitis B virus (HBV) infection, who were positive for HBeAg and had increased levels of transaminases, were included in a controlled study of treatment using recombinant interferon-alpha (rIFN-alpha), 10 MU/m2 body surface, intramuscularly, 3 times a week over a period of 3 months. During therapy, a significant decrease in HBV-DNAp was observed in the 12 patients treated. By the end of therapy, the HBV-DNA had disappeared in 3 children, the same occurring in 1 child (33% overall) during the course of the 4th month. By this time, all the controls remained with HBV replication markers (p less than 0.05). The 4 treated patients who responded became HBeAg-negative, developing anti-HBe during the first 12 months after therapy. In the control group, the HBV-DNA disappeared in 3 children in the 7th month of follow-up. All of the children remained HBsAg-positive. The therapy with rIFN-alpha was well tolerated, secondary effects consisting of a flu-like syndrome and a slight decrease in leukocytes and platelets. At the second biopsy, 15 months after the beginning of therapy, a significant decrease in Knodell's index of histological activity was observed in the responders. In the light of these results and since treated children lost viral replication markers in a shorter period of time than the controls, who seroconverted spontaneously, we consider that rIFN-alpha may be useful in the treatment of chronic hepatitis B in childhood.
The in vitro antibacterial activity of the essential oil obtained from the flowering apex of Thymus serpylloides ssp. gadorensis against various micro-organisms is reported. Moreover, the main chemical groups found in this essential oil are described.
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We report a study of 27 patients with small cell osteosarcoma (SCO), 17 from the M. D. Anderson Cancer Center (MDAH) and ten from the Pediatric Oncology Group (POG). There were 12 male patients and 15 female patients; 19 were white, five were black, and three were Hispanic. They ranged from 6 to 28 years of age with a median of 14 years. Histologically there were three patterns: Ewing's-like, lymphoma-like, and spindle cell. All cases showed osteoid formation and a few had chondroid areas. There was cytoplasmic glycogen in ten cases. Initial treatment for MDAH patients included intraarterial infusion of cisplatin in ten, amputation in four, partial mandibulectomies in two, and biopsy with local radiotherapy and systemic chemotherapy in one. All POG patients had resection or amputation followed by adjuvant chemotherapy. Twelve patients are alive, of whom nine have had significant follow-ups for 25 to 90 months. Fourteen patients are dead of lung, spine, and brain metastases from 1 to 23 months after initial diagnosis. One patient is alive with lung relapse at 4 months. In summary, SCO is a high-grade variant of osteosarcoma, with an incidence of up to 4% of all osteosarcomas, that affects patients of the same age group and has the same anatomic location as conventional osteosarcoma. Currently, SCO appears to have a prognosis that is the same as or slightly worse than that of conventional osteosarcoma. Furthermore, although intraarterial infusion is effective for the primary tumors in the bone, distant metastases are difficult to control.
Biochemical studies on receptors for peptides are most often carried out on affinity-labelled (peptide-receptor) complexes. Necessarily, the assumption is made that a covalent (peptide-receptor) complex behaves as the native receptor. The validity of this assumption is dependent on both the affinity-labelling technique and the resolution of the analytical method used for biochemical characterization. We designed a new affinity-labelling probe in order to minimize structural modifications occurring within the affinity-labelled cholecystokinin (CCK) receptor protein. The probe was 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK-25-33, (125I-ASD-[Thr28,Ahx31]CCK-25-33), the peptide moiety of which was released from its binding site by reduction. It was obtained by coupling a photoactivable chemical to [Thr28,Ahx31]CCK-25-33 via its N-terminus. The resulting peptide was HPLC purified and radioiodinated in the presence of chloramine T. Binding of 125I-ASD-[Thr28,Ahx31]CCK-25-33 was time- and temperature-dependent and reversible. At 25 degrees C, a steady-state level was reached after 60 min and half-maximal dissociation after 38 min. Binding was inhibited by [Thr28,Ahx31]CCK-25-33 and L-364-718 antagonist with IC50 0.4 nM and 0.9 nM, respectively. Photoaffinity labelling of pancreatic plasma membranes by 125I-ASD-[Thr28,Ahx31]CCK-25-33 identified a glycoprotein of Mr 85,000-100,000 which was retained on immobilized wheat germ agglutinin. Enzyme cleavage by endoproteinase Glu-C generated a main fragment of Mr 30,000-34,000. The same glycoprotein was photoaffinity labelled with 125I-DTyr-Gly-[Ahx28,31,pNO2Phe33]CCK-26-33 (Ahx, 2-aminohexanoic acid; pNO2Phe,p-nitrophenylalanine) an intrinsic probe having its photolabile group sited in the binding domain of cholecystokinin. 125I-ASD-[Thr28,Ahx31]CCK-25-33 is a potentially powerful tool for biologically and biochemically studying cholecystokinin receptors.
The binding of vasoactive intestinal peptide (VIP) and stimulation of adenylate cyclase by VIP were studied in intestinal epithelial cells during hypothyroidism. Experimental hypothyroidism was induced in rats by the administration of KC10(4). The binding capacity, but not the affinity, of VIP receptors decreased in the hypothyroid rats. Besides, the stimulation of cyclic AMP production by VIP was also diminished in cells from hypothyroid rats. These observations indicate a decrease of the responsiveness of intestinal epithelial cells to VIP in the hypothyroid status, suggesting a role of the peptide in the pathophysiologic mechanism of intestinal manifestations during hypothyroidism.
Gas chromatography with electron-capture detection was used in the determination of diaminopimelic acid (as the N-heptafluorobutyryl isobutyl derivative) and 3-hydroxymyristic acid (as the O-pentafluorobenzoyl methyl derivative) in Gram-negative bacterial cells in infected human urine. Use of the column-switching (two-dimensional gas chromatography) technique greatly enhanced the selectivity of the detection and simplified the processing of samples. The system described should prove useful for trace detection of specific bacterial constituents in complex environments.
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1. The effect of chronic intake of ethanol on the binding of vasoactive intestinal peptide (VIP) to rat spleen lymphoid cells was investigated. 2. The intake chronic of ethanol elicited an increase in specific VIP binding. 3. This increase was due to an increase in binding capacity of both the high and the low affinity binding sites. 4. There was a decrease in the affinities of both classes of VIP binding sites.
The essential oil of Bupleurum gibraltaricum showed considerable anti-inflammatory activity against carrageenan-produced pedal edema in rats. This appeared to be due to the delta-3-carene component. The essential oil and delta-3-carene both produced qualitatively similar changes in rat uterine contractions caused by oxytocin (a progressive dose-related modification of the concentration-response curves) and acetylcholine (non-progressive modification of the concentration-response curves).
Staphylococcus aureus isolated from clinically diagnosed cases of toxic shock syndrome (TSS) showed susceptibility to phage types belonging to both I and III groups (90.5%). Phage typing patterns showed a wide diversity among 87 toxic shock syndrome toxin-1 (TSST-1) positive strains isolated from different non TSS clinical sources. Toxin producing strains isolated from both TSS and non TSS showed a remarkable ability to bind to crystal violet (pattern C/D, 97.2%) incorporated into brain heart infusion agar media at subinhibitory concentrations and these isolates were traced to biotype var. hominis. The cellular fatty acid compositions of TSS and non-TSS strains belonging to the three biotypes S. aureus var. hominis, S. aureus var. bovis and S. aureus var. canis did not differ. TSST-1 producing strains demonstrated a high salt aggregation test value (above 1.5) indicating a low cell surface hydrophobicity. Both TSS and non TSS strains demonstrated a high lipolytic activity. TSST-1 positive strains in general, showed significantly higher lipase activity than strains isolated from septicemia (p less than 0.0001) and superficial (p less than 0.0001) infections. The proteolytic activity is higher among TSS (median value 0.075 U/ml) than to non TSS (median value 0.045 U/ml) strains. There was no correlation with the quantity of toxin production in vitro and to the properties described.
A method was developed for determining two characteristic mycobacterial lipid constituents, tuberculostearic acid (as its pentafluorobenzyl ester) and 2-eicosanol (as its pentafluorobenzoyl ester), by using gas chromatography with electron capture detection. A microprocessor-controlled column-switching system (two-dimensional gas chromatography) facilitated sample preparation and increased specificity. The usefulness of the technique was illustrated by its ability to reveal picogram amounts of tuberculostearate in a suspension of Mycobacterium leprae isolated from a naturally infected armadillo. Two-dimensional gas chromatography with electron capture detection may in some instances provide a convenient alternative to gas chromatography-mass spectrometry for use in demonstrating the presence of mycobacteria in a complex environment.
The essential oil of Thymus longiflorus Boiss was tested for its in vitro antibacterial activity. The results showed antibacterial effects against Gram-positive and Gram-negative bacteria, especially against Pseudomonas fluorescens and Mycobacterium phlei.