Survival of Blastocystis hominis clones after exposure to a cytotoxic monoclonal antibody

Our previous studies have shown that monoclonal antibodies (MAbs) to Blastocystis hominis react mainly with carbohydrate epitopes, while 1 MAb (1D5) reacts specifically with a protein of 30.5 kDa. In the present study, 3 monoclonal antibodies (1D5, 1E7 and 4F7) were used in immunogold localization. 1E7 and 4F7 were found to react primarily with the surface coat, while 1D5 was plasma membrane-specific. In the presence of complement, only 1D5 exhibited a cytotoxic effect on B. hominis whereas 1E7 and 4F7 did not, suggesting that the surface coat of B. hominis could serve as an immunological barrier against host antibodies. Using a recently described agar plating method, only 1D5 exhibited significant (P < 0.01) complement-independent cytotoxicity to B. hominis, inhibiting colony growth at low concentrations. Parasites that had been exposed to 1D5 were morphologically smaller than those that were not exposed to this MAb. Colonies that grew in the presence of 1D5 were isolated and grown in liquid medium containing increasing amounts of the cytotoxic MAb. Two clones that grew well in liquid medium containing 1D5 were also able to develop into colonies in soft agar. This study has shown that the 30.5 kDa protein found on the plasma membrane of B. hominis is a functionally important protein and that not all cells within a certain population would be susceptible to the cytotoxic effects of 1D5. These findings suggest that a heterogenous population exists in continuously maintained cultures of B. hominis.     

In vivo cross-linking of the SecA and SecY subunits of the Escherichia coli preprotein translocase

Precursor protein translocation across the Escherichia coli inner membrane is mediated by the translocase, which is composed of a heterotrimeric integral membrane protein complex with SecY, SecE, and SecG as subunits and peripherally bound SecA. Cross-linking experiments were conducted to study which proteins are associated with SecA in vivo. Formaldehyde treatment of intact cells results in the specific cross-linking of SecA to SecY. Concurrently with the increased membrane association of SecA, an elevated amount of cross-linked product was obtained in cells harboring overproduced SecYEG complex. Cross-linked SecA copurified with hexahistidine-tagged SecY and not with SecE. The data indicate that SecA and SecY coexist as a stable complex in the cytoplasmic membrane in vivo.     

Agreement between face-to-face and telephone-administered mood ratings in patients with rapid cycling bipolar disorder

BACKGROUND AND PURPOSE: Clinical trials have demonstrated that high dose radiation therapy and daily cisplatin (CDDP) could increase local control and survival in carcinoma from various sites. The present phase I-II study has combined high dose radiation therapy and daily CDDP at escalating dosages. METHODS: From August 1994 to December 1995, 23 patients with non-resectable carcinoma of the pancreas were enrolled in a phase I-II multicentric, pilot study to test the toxicity and the effectiveness of high dose radiotherapy and daily cisplatin (CDDP) at escalating dosages. A dose of 6 mg/sqm/day of CDDP was selected for the phase II step since no grade IV toxicity occurred in any patient in the phase I step. RESULTS: Toxicity was considered fairly acceptable. At the time of analysis, the 23 patients who entered the study had clear evidence of evolutive disease either locally or distantly in the liver. It is suggested that high dose radiotherapy (60 Gy continuously) and daily CDDP have little effect on local control of the tumor and survival, and only a moderate effect on pain. CONCLUSIONS: In unresectable, apparently non-metastatic cancers of the pancreas, there is an urgent need for new agents or new combinations of agents to be tested.     

Airway versus transbronchial biopsy and BAL in lung transplant recipients: different but complementary

Lung transplantation is now an established therapeutic intervention for end-stage cardiopulmonary disease in humans. Chronic rejection, in the form of bronchiolitis obliterans syndrome (BOS), remains the commonest cause of morbidity and mortality in those surviving more than 3 months. The pathology of BOS involves airway changes. We have evaluated the potential for endobronchial biopsies (EBB) to complement existing sampling methods used in allograft monitoring and have compared the results of EBB findings with those of bronchoalveolar lavage (BAL) and transbronchial biopsy (TBB) in 18 clinically stable patients. We found that all the EBB had inflammatory cells present but that only five TBB specimens had evidence of inflammation, with airway material being present in 78% of the TBB. Paired BAL and EBB yielded different results, with no correlations between total macrophages, lymphocytes, CD4+ cells or CD8+ cells. We conclude that endobronchial biopsies are potentially useful as an additional sample for the monitoring of inflammation in lung allografts, since they yield different, and potentially complimentary, information to bronchoalveolar lavage and transbronchial biopsy.     

Buspirone metabolite structure profile using a standard liquid chromatographic-mass spectrometric protocol

A rapid and systematic LC-MS protocol is utilized to profile buspirone metabolites. Analysis of rat bile, urine and liver S9 samples using a standard LC-MS method provides structural information for 25 metabolites. The resulting buspirone metabolite structure database contains characteristic retention time, molecular mass and MS-MS product ion information for each compound. Metabolites are categorized according to profile groups, which illustrate that substitution reactions are primarily associated with the azaspirone decane dione and pyrimidine substructures. Structures of new buspirone metabolites are reported and include the despyrimidinyl, despyrimidinylpiperazine, glucuronide, hydroxyglucuronide (four isomers), methoxyglucuronide and hydroxymethoxyglucuronide (two isomers) buspirone metabolites.     

Galpha12 and Galpha13 mediate differentiation of P19 mouse embryonal carcinoma cells in response to retinoic acid

P19 mouse embryonal carcinoma cells can be stimulated to differentiate into endodermal-like, mesodermal-like, and neuronal-like phenotypes in response to specific morphogens. At low concentrations, retinoic acid stimulates P19 embryonal cells to differentiate to cells displaying an endodermal phenotype, whereas at higher concentrations it stimulates differentiation to neuroectoderm. The Galpha12 and Galpha13 subunits of heterotrimeric G-proteins are expressed in the embryonal P19 cells and stimulated in response to retinoic acid as the cells differentiate to endodermal or neuroectodermal phenotypes. Suppression of the expression of either Galpha12 or Galpha13 by antisense RNA is shown to promote cell detachment from substratum and apoptosis. Expression of the constitutively active, mutant form of Galpha12 (Q229L), in contrast, stimulates loss of the embryonal phenotype. Expression of the constitutively active form of Galpha13 (Q226L) stimulates differentiation of the cells from embryonal to endodermal, in the absence of retinoic acid. Thus, both Galpha12 and Galpha13 are essential to stimulation of cell differentiation by retinoic acid. Deficiency of either Galpha12 or Galpha13 increases programmed cell death.     

Phage typing of Staphylococcus aureus associated with subclinical bovine mastitis

Six hundred and seventeen isolates of Staphylococcus aureus from subclinical clinical mastitis cases in 63 dairy herds in Northern Ireland were typed using a set of 25 phages. Ninety-four per cent of the isolates were typable, with nine phages, predominantly from groups I and III, being responsible for almost all of the lysis. Although 68 phage patterns were found, six of them typed 47.2% of the isolates. One strain accounted for 14.7% of the isolates, but the largest number of strains (44) was restricted to individual farms. The epidemiological significance of these findings for on-farm mastitis control is discussed.     

Secretion of cardiac plasminogen activator during hypoxia-induced right ventricular hypertrophy

In the circulation, fibrinolytic activity is determined to a large degree by the relative levels of tissue plasminogen activator (tPA) and its major inhibitor (PAI-1). Vascular beds in different organs secrete tPA and PAI-1 into the circulation, and the total secretory rate of each protein is balanced by its half-life in the bloodstream. We are testing the hypothesis that in the heart, ventricular hypertrophy will alter the rates of formation of tPA and/or PAI-1 and the rates of their release into the cardiac vasculature. In this study, we have examined the effects of continuous hypoxia on PA activity in extracts of rat heart ventricles, on the activity secreted into the cardiac vasculature of perfused hearts, and on the levels of mRNAs for tPA and PAI-1. Rats were subjected to hypobaric hypoxia at 0.5 atm for 1-21 days. The treatment caused polycythemia within 1-3 days, and right ventricular hypertrophy by 3 days. PA activity in extracts of both right and left ventricles was significantly elevated after 3 days of hypoxia, continued to increase for 4 additional days, and remained elevated for 3 weeks. The actions of inhibitors of urokinase and tPA indicated that the PA activity in heart extracts was exclusively tPA. Fibrin zymography confirmed that result. The mRNAs for tPA and for PAI-1 were elevated after 1 day of hypoxia and then returned to near control levels on days 2 and 3. After 7 days, hearts from hypoxic rats secreted more tPA activity into perfusates than did hearts from controls. The difference in secretory rates was proportional to the differences in the levels of tPA in the corresponding heart extracts.     

Purification and characterization of a neutral, bile salt-independent retinyl ester hydrolase from rat liver microsomes. Relationship To rat carboxylesterase ES-2

A neutral, bile salt-independent retinyl ester hydrolase (NREH) has been purified from a rat liver microsomal fraction. The purification procedure involved detergent extraction, DEAE-Sepharose ion exchange, Phenyl-Sepharose hydrophobic interaction, Sephadex G-100 and Sephacryl S-200 gel filtration chromatographies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated enzyme has an apparent molecular mass of approximately 66 kDa under denaturing conditions on SDS-PAGE. Analysis of the amino acid sequences of four peptides isolated after proteolytic digestion revealed that the enzyme is highly homologous with other rat liver carboxylesterases. In particular, the sequences of the four peptides of the NREH (60 amino acids total) were identical to those of a rat carboxylesterase expressed in the liver (Alexson, S. E. H., Finlay, T. H., Hellman, U., Svensson, L. T., Diczfalusy, U., and Eggertsen, G. (1994) J. Biol. Chem. 269, 17118-17124). Antibodies against this enzyme also react with the purified NREH. Purified NREH shows a substrate preference for retinyl palmitate over triolein and did not catalyze the hydrolysis of cholesteryl oleate. With retinyl palmitate as substrate, the enzyme had a pH optimum of 7 and showed apparent saturation kinetics, with half-maximal activity achieved at substrate concentrations (Km) of approximately 70 microM.     

Long-term phonatory instability in individuals with multiple sclerosis

This paper uses a new approach to describe and quantify the long-term phonatory instability of speakers with MS. Sustained vowel phonations of 20 individuals with a definite diagnosis of multiple sclerosis (MS) and 20 age- and gender-matched individuals with normal speech were recorded. The phonations were f0 and intensity analyzed and subjected to spectral analysis using the Fast Fourier Transform. Three methods for analyzing the instabilities are presented, compared, and related to perceptual judgments: (a) coefficients of variation, (b) magnitude-based analysis of spectral energy, and (c) frequency-based analysis of spectral components. All measures reliably distinguished between individuals with MS and persons with normal speech. A single factor based on a linear discriminant analysis of the frequency-based measures was especially useful in distinguishing these groups. Critical frequency bands of instability, corresponding to wow (1-2 Hz), tremor (around 8 Hz), and flutter (17-18 Hz), distinguished the MS group from those of the control group.