Determination of inorganic phosphate in the presence of Triton X-100

Two methods have been developed for the rapid and accurate estimation of orthophosphate in the presence of Triton X-100. The first is unaffected by up to 2.0–2.5% (v/v) of the detergent in the assay samples, while the second method is essentially unaffected by Triton X-100 and is also suitable for use in the presence of acid labile organic phosphate. Both are proportional in the range 0.05–1.0 μmole of orthophosphate in the assay.     

N-estimation from retrospectively ascertained events with applications to AIDS.

It is a common sampling scheme in retrospective studies that the data set includes only individuals who satisfy a certain sampling criterion. In this paper we consider the situation when the sampling criterion is a specified event, and assume that an earlier event can be retrospectively identified given the occurrence of the specified event. A semiparametric method, which is a compromise between nonparametric and parametric methods, is employed for the estimation of the expected number of the specified events (namely, the N-estimation) occurring in arbitrarily given intervals. A number of statistical properties of the estimates are developed. Due to the limitation of semiparametric models, our estimates should be regarded as conservative estimates since in general they underestimate the actual number of the specified events. This type of limitation, however, cannot be avoided with nonparametric or semiparametric models. Applications to acquired immunodeficiency syndrome (AIDS) cases are considered. The blood transfusion AIDS cases reported to the Centers for Disease Control are analyzed in detail.     

Investigation of Cigarette Smoking among Male Schizophrenia Patients

Male schizophrenia patients are known to have a heavier smoking pattern compared with the general population. However, the mechanism for this association is not known, though hypothesis that smoking could alleviate symptomatology of schizophrenia and reduce side effects of antipsychotics has been suggested. The aims of this study were to validate the heavier smoking pattern among male schizophrenia patients and to investigate the possible mechanisms for the association. To enhance the reliability of the study, we recruited two large independent samples with 604 and 535 male Chinese schizophrenia patients, and compared their smoking pattern with that of 535 healthy male controls recruited from general population. Validated multiple indicators and multiple causes structure equation model and regression models were used to investigate the association of smoking with factors of schizophrenia symptomatology and with the usage of antipsychotics and their extra-pyramidal side effects (EPS). Schizophrenia patients had significantly heavier smoking pattern compared with healthy controls in our sample (42.4% vs. 16.8%, p<0.001 for current smoking prevalence; 23.5% vs. 43.3%, p<0.001 for smoking cessation rate; 24.5% vs. 3.0%, p<0.001 for heavy smoker proportion). Their smoking status was also found to be consistently and significantly associated with reduced negative factor scores for schizophrenia symptomatology (β = −0.123, p = 0.051 for sample-A; β = −0.103, p = 0.035 for sample-B; β = −0.082, p = 0.017 for the combined sample). However, no significant association was found between smoking and antipsychotics usage or risk of EPS. These results support that smoking is associated with improved negative symptoms, which could account for the heavier smoking pattern among schizophrenia patients.     

Biochemical Characterization of the Cytochrome P450 CYP107CB2 from <Emphasis Type="Italic">Bacillus lehensis</Emphasis> G1

The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS–PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.     

COMBINING NEW TECHNOLOGIES FOR DETERMINATION OF PHYTOPLANKTON COMMUNITY STRUCTURE IN THE NORTHERN GULF OF MEXICO 1

In situ analysis of phytoplankton community structure was determined at five stations along the Texas Gulf coast using two instruments, the Fluoroprobe and FlowCAM. Results were compared with traditional methods to determine community structure (pigment analysis and microscopy). Diatoms and small nanoplankton (most likely haptophytes) dominated the phytoplankton community at all stations. Estimated chl concentrations for diatoms+dinoflagellates obtained via the Fluoroprobe were not significantly different for three of the five stations sampled when compared with HPLC‐chemical taxonomy analysis, whereas the concentrations of green algal and cryptophyte chl were overestimated. The FlowCAM estimates of overall nanoplankton and microplankton cell abundance were not significantly different when compared with epifluorescence microscopy, and recorded images of phytoplankton cells provided a representative population of the phytoplankton community at each station. The Fluoroprobe and FlowCAM, when used in tandem, are potentially capable of determining the general characteristics of phytoplankton community structure in situ and could be an important addition to biological observing systems in the coastal ocean.     

Icreased drug permeability in Chinese hamster ovary cells in the presence of cyanide

In this report we investigated whether the modulation of drug permeability in Chinese hamster ovary (CHO) cells was an energy-dependent process. We observed that (1) in the absence of glucose, metabolic inhibitors such as cyanide, azide, and dinitrophenol stimulated the uptake of [³H]colchicine and other drug; (2) cyanide-induced stimulation of drug uptake could be prevented by the presence of metabolizable sugars such as glucose and ribose; (3) cyanide-treated cells were fully viable; (4) on the addition of cyanide and glucose the kinetics of drug permeability changes were very rapid. These data are consistent with the hypothesis that an energy-dependent membrane barrier against the uptake of a variety of drugs was operative in CHO cells.The nature of this energy-dependent membrane barrier was examined in colchicine-resistant mutants (CH^RC4 and CH^RC5 cells) previously characterized as membrane mutants with greatly reduced drug permeability (Ling and Thompson, (1974) J. Cell Physiol. 83, 103–116). The mutants were more refractile to the cyanide-induced stimulation of drug permeability but more sensitive to the glucose prevention cyanide-induction. In the presence of cyadine, the uptake rate of [³H] colchicine by CH^RC4 cells increased by about 100-fold and approached a rate similar to that of wild-type cells. These results suggest that the colchicine-resistant mutants may be altered in their energy-dependent modulation of drug permeability.     

Partial purification and properties of guinea-pig liver polynucleotide phosphorylase

1. Polynucleotide phosphorylase has been isolated and partially purified from crude preparations of guinea-pig liver nuclei. 2. The enzyme is particulate and associated with RNA and lipids characteristic of membranes. 3. It has phosphorolysis and exchange activities, but the latter may be due to a contaminating enzyme. 4. The phosphorolysis activity is dependent on bivalent cations, preferably Mg(2+), has a pH optimum between 8.6 and 9.2 and is inhibited by potassium chloride and sodium chloride. 5. The enzyme catalyses phosphorolysis of poly A, poly C, poly U, rRNA and tRNA. Poly G is only phosphorolysed to a very small extent and DNA is not a substrate. 6. The enzyme appears to lack nucleoside diphosphate polymerization activity.     

The Glycerol‐3‐Phosphate Acyltransferase TbGAT is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei

Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the glycerol‐3‐phosphate acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored glycerol‐3‐phosphate acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl‐CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N‐terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked glycerol‐3‐phosphate acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and GPI‐anchored protein procyclin. However, the null mutant exhibited a slight decrease in phosphatidylethanolamine biosynthesis that was compensated with a modest increase in production of ether phosphatidylcholine. Our data suggest that an alternative initial acyltransferase takes over TbGAT's function in its absence.     

Mechanistic aspects of chiral discrimination by surface‐immobilized α1‐acid glycoprotein

The immobilization of the globular protein α‐1‐acid glycoprotein (AGP) onto silica gel led to the commercial availability of an AGP column, which has a high enantioselectivity. The enantioselectivity of AGP columns has been demonstrated in numerous applications. Due to potential AGP structural changes occurring upon its immobilization, the interaction between particular pairs of enantiomers and the stationary phase is very difficult to assess. Therefore, in this paper we report a mechanistic study that probes the nature of these types of interactions. As model ligands, we employed two LTD₄ antagonists (L‐708, 738, MK0476, and their enantiomers) which have a rigid backbone consisting of a conjugated aromatic region and a side chain which is terminated with a carboxylic functional group. The difference between the two compounds is a two‐fluorine versus one‐chlorine substituent in the aromatic region of the molecule. To study the interaction between the two homologues and the AGP stationary phase, several parameters were varied, including pH, ionic strength, organic modifier, and temperature. van't Hoff plots were constructed and found to be nonlinear. They could, however, be divided into two linear regions, one from 0°C to ∼30°C, and another from 39°C to 50°C. The region at lower temperature implied that the separation was entropy‐dominated while the separation at higher temperature was enthalpically driven. The transition from the entropic to the enthalpically driven separation region suggested that bound AGP undergoes a conformational change. Fluorescence spectroscopy performed on the AGP stationary phase found evidence for a limited conformational transition at a similar temperature, consistent with this hypothesis. Chirality 11:224–232, 1999. © 1999 Wiley‐Liss, Inc.