Strain differences in adrenal microsomal steroid metabolism in guinea pigs

We recently reported that CYP2D16, a xenobiotic-metabolizing P450 isozyme, was expressed at higher levels in adrenal microsomes from inbred Strain 13 guinea pigs than in those from outbred English Short Hair (ESH) animals. Studies were done to determine if there also were strain differences in adrenal microsomal steroid metabolism. In both inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zone preparations of the adrenal cortex, 21-hydroxylase activities were greater in microsomes from ESH than from Strain 13 guinea pigs. By contrast, 17alpha-hydroxylase activities were similar in the two strains. In both strains, 21-hydroxylase activities were greater in inner than outer zone microsomes, but the opposite was found for 17alpha-hydroxylase activities (outer>inner). Northern and Western analyses revealed higher levels of CYP21 mRNA and protein in adrenals from ESH than Strain 13 guinea pigs, but there were no strain differences in CYP17 mRNA or protein concentrations. Despite the zonal differences in adrenal 17alpha-hydroxylase and 21-hydroxylase activities, CYP17 and CYP21 mRNA and protein levels were similar in the inner and outer zones within each strain of guinea pig. The results demonstrate strain differences in microsomal steroid metabolism that are explained by differences in CYP21 expression. By contrast, the zonal differences in steroid hydroxylase activities may be attributable to post-translational mechanisms.     

A PC-based software test for measuring alcohol and drug effects in human subjects

A new software-based visual search and divided-attention test of cognitive performance was developed and evaluated in an alcohol dose-response study with 24 human subjects aged 21-62 years. The test used language-free, color, graphic displays to represent the visuospatial demands of driving. Cognitive demands were increased over previous hardware-based tests, and the motor skills required for the test involved minimal eye movements and eye-hand coordination. Repeated performance on the test was evaluated with a latin-square design by using a placebo and two alcohol doses, low (0.48 g/kg/LBM) and moderate (0.72 g/kg/LBM). The data on 7 females and 17 males yielded significant falling and rising impairment effects coincident with moderate rising and falling breath alcohol levels (mean peak BrALs = 0.045 g/dl and 0.079 g/dl). None of the subjects reported eye-strain or psychomotor fatigue as compared with previous tests. The high sensitivity/variance relative to use in basic and applied research, and worksite fitness-for-duty testing, was discussed. The most distinct advantage of a software-based test that operates on readily available PCs is that it can be widely distributed to researchers with a common reference to compare a variety of alcohol and drug effects.     

Learning disabilities, low achievement, and mild mental retardation: more alike than different?

Children identified with learning disabilities (LD), low achievement (LA), or mild mental retardation (MMR) were contrasted on 41 measures of ability, academic achievement, social skills, problem behavior, academic engaged time, perceptual-motor skills, and school history. Both multivariate, univariate, and meta-analytic comparisons among the three groups showed relatively large differences on measures of aptitude and achievement, with the LD group scoring higher on measures of cognitive ability than the LA and MMR groups and the LA group showing higher tested academic achievement than the LD and MMR groups. Teacher ratings of academic competence showed similar levels of functioning for the LD and LA groups. No differences among the groups were found on measures of social skills, problem behaviors, or academic engaged time, or on most indices reflecting school history. Results were interpreted in light of studies contrasting LD and LA groups. Comparisons with earlier studies were difficult in light of demographic differences in samples and the lower cognitive and academic functioning of children in the present study. The current study showed that 61% of the LD group could be differentiated from the LA group, with LD-MMR and LA-MMR differentiation levels being 68.5% and 67.5%, respectively.     

Investigation of the calcium-mediated association between the carbohydrate head groups of galactosylceramide and galactosylceramide I3 sulfate by electrospray ionization mass spectrometry

Calcium has been shown previously to cause aggregation of phosphatidylcholine/cholesterol liposomes containing galactosylceramide (GalCer) with similar liposomes containing cerebroside sulfate (galactosylceramide I3 sulfate) (CBS), suggesting that it mediates a carbohydrate-carbohydrate association between these two glycolipids. In order to determine if such an association occurs, the noncovalent complexes formed on addition of calcium chloride to GalCer and CBS in methanol were examined by positive and negative ion spray mass spectrometry. Monomeric Ca2+ complexes of both lipids were observed. In addition, Ca2+ also caused oligomerization of GalCer. Oligomerization of CBS anion was not seen, but dimers would not have been observed, as they would be neutral. However, Ca2+ caused heterotypic complexation of GalCer and CBS. Although these heterotypic complexes were of low abundance in methanol compared with the other monomeric and homotypic oligomeric positive ions formed at low declustering potentials, the heterotypic dimer [GalCer.CBS.Ca2+-H]+ had the greatest stability of all oligomers formed and was the only one to survive at high declustering potentials. Na+ did not cause oligomerization of GalCer in methanol indicating that the complexes of GalCer with Ca2+ are not caused by van der Waals interactions between the lipid moieties. GalCer and CBS are present in high concentrations in myelin. This Ca2+-mediated carbohydrate-carbohydrate interaction, which can bridge apposing bilayers, may be involved in adhesion of the extracellular surfaces of the myelin sheath.     

Ex vivo generation of functional dendritic cells from mobilized CD34+ hematopoietic stem cells

Gene engineering to enhance tumour immunogenicity and elicit curative responses against established tumours and tumour recurrences has become an attractive prospect. Gene engineering enables new genes to be selectively inserted into the genome of a tumour cell, or the construction of new fusion plasmids coding tumour antigens and immunomodulatory molecules. The rationale behind current research is to enhance the immune recognition of tumour antigens through their association with the molecules on which immune recognition depends. The immunotherapy data obtained in many experimental tumour systems provide a realistic assessment of the potential and limits of this technological approach. Experimental vaccination of rodents has been shown to induce a significant immune memory, even against poorly immunogenic tumours, that can prevent tumour growth and cure initial metastases, but is poorly effective against established tumours. Its use in tumour prevention is a fresh dawning perspective.     

Methodology of serial ECG classification using an adaptation of the NOVACODE for Q wave myocardial infarction in the Bypass Angioplasty Revascularization Investigation (BARI)

Serial electrocardiographic (ECG) changes are a critical component of the diagnostic algorithm for classification of myocardial ischemic events in large-scale clinical trials. This study describes a computerized serial ECG classification program developed at the St. Louis University Core ECG Laboratory for use in the Bypass Angioplasty Revascularization Investigation (BARI) trial, in which patients with multivessel coronary artery disease were randomized to receive either coronary artery bypass grafting or percutaneous transluminal coronary angioplasty. The St. Louis University program detects and codes serial changes in Q, ST, and T wave items according to Minnesota code (MC) criteria using a modified NOVACODE hierarchical classification system. Measurements using a seven-power calibrated coding loupe are used to generate the MC from a customized software program. Significant minor or major changes are detected by the serial comparison program and referred to a physician coder for verification. Serial comparison coding rules are used to adjust for weaknesses in the standard MC classification system resulting from instability at decision boundaries. Of 4,244 BARI randomized and registry study participants with follow-up ECGs received at the Core ECG Laboratory as of March 1995, a grade 2 MC Q wave progression was noted in 568 participants (13.4%) using MC criteria alone, as compared with 367 (8.6%) after the St. Louis University coding rules were applied. The incidence of grade 1 MC Q wave progressions was 16.4% (697/4,244) versus 6.1% (259/4,244) when the St. Louis University program was applied. Intraobserver variability for grade 2 Q wave progression codes determined from a sample of 812 serial.     

Recombinant expression and characterization of the major beta-lactamase of Mycobacterium tuberculosis

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta-lactam antibiotics. beta-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses beta-lactam resistance. beta-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of beta-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta-lactamase activity, respectively, were identified. The beta-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the beta-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A beta-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major beta-lactamase of M. tuberculosis is a class A beta-lactamase with predominant penicillinase activity. A second, minor beta-lactamase with relatively greater cephalosporinase activity is also present.     

The Glu318Gly mutation of the presenilin-1 gene does not necessarily cause Alzheimer's disease

In early-onset familial Alzheimer's disease (AD) pathogenic mutations have been found in the amyloid precursor protein (APP) gene and in the presenilin (PS)-1 and PS-2 genes. We screened for mutations in these genes in 20 patients with familial AD from the Finnish population. In addition, we sampled 41 sporadic AD patients and 59 controls to test for mutations identified in our familial AD cases. We detected an A-to-G transition in the PS-1 gene, resulting in a glutamic acid (Glu)-to-glycine (Gly) substitution at codon 318 in 2 familial and 2 sporadic AD patients. The Glu318Gly mutation has previously been reported to cause AD. We also found the Glu318Gly mutation in 4 healthy aged controls (range, 74-87 years). We thus conclude that the mutation is most likely a rare polymorphism not related to AD.