Ahch, the mouse homologue of DAX1: cloning, characterization and synteny with GyK, the glycerol kinase locus

We cloned the murine full-length cDNA encoding Ahch, the mouse homologue of DAX1 (DSS-AHC Region on Human X Chromosome, Gene1) which is the gene responsible for human X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Sequence analysis revealed that the murine and human cDNAs have 65% aa identity and 75% aa similarity overall. The cysteine residues in the putative DNA binding domain, which may interact with Zn2+ ions to form zinc fingers, are 100% conserved between the two species, indicating that the novel zinc-finger structures in DAX1 may be functional. In addition, mouse interspecific backcrosses show that the Ahch gene is closely linked to the glycerol kinase locus, GyK, on the mouse X chromosome, indicating that the order of the loci is conserved in this syntenic region between mouse and human.     

Effects of sleeping in a chemical protective mask on sleep quality and cognitive performance

PURPOSE: We wanted to determine whether sleep is disrupted when soldiers sleep in a new chemical protective mask, the M40. Sleep quantity and quality, extent of protection provided by the mask during sleep, and next day performance were assessed. METHOD: After several days of training, 9 male soldiers slept with and without the M40 mask on four occasions. RESULTS: Soldiers were able to tolerate the mask for most or all of the night. However, sleep, as assessed by wrist-worn activity monitors, was significantly disturbed. Minutes (mean +/- SEM) of waking significantly increased, from 25 +/- 2.1 to 86 +/- 8.5 per night (p < 0.001), and number of awakenings rose from 8 +/- 0.6 to 20 +/- 0.9 (p < 0.0001). Soldiers reported that it took longer and was more difficult to fall asleep when wearing the mask. Errors on a choice reaction time task increased significantly and subjects reported greater fatigue and sleepiness the day after sleeping in the mask. Protection provided by the masks varied substantially among subjects and declined over the course of the study. Some soldiers were protected throughout the night but others were only protected intermittently. CONCLUSION: We conclude that sleeping in the chemical protective mask should only be done when necessary, given the adverse effects on sleep and daytime function, as well as the variability of protection, of the mask.     

Differential responses in anterior pituitary luteinizing hormone (LH) content and LH beta- and alpha-subunit mRNA, and plasma concentrations of LH and testosterone, in bulls treated with the LH-releasing hormone agonist deslorelin

Tooth loss diminishes oral function and quality of life, and national health targets aim to reduce population levels of tooth loss. OBJECTIVES: The purpose of this study was to determine tooth loss incidence and predictors of tooth loss among older adults in South Australia. METHODS: Data were obtained from a cohort study of a stratified random sample of community-dwelling dentate people aged 60+ years. Interviews and oral examinations were conducted among 911 individuals at baseline and among 693 of them (76.1%) 2 years later. Incidence rates and relative risks were calculated for population subgroups and multivariate logistic regression was used to construct risk prediction models. A method was developed to calculate 95% confidence intervals (95% CI) for relative risks (RR) from logistic regression models using a Taylor series approximation. RESULTS: Some 19.5% (95% CI = 15.4-23.6%) of people lost one or more teeth during the 2 years. Men, people with a recent extraction, people who brushed their teeth infrequently, smokers and people born outside Australia had significantly (P < 0.05) greater risk of tooth loss. Baseline clinical predictors of tooth loss included more missing teeth, retained roots, decayed root surfaces, periodontal pockets and periodontal recession. In a multivariate model that controlled for baseline clinical predictors, former smokers (RR = 2.55, 95% CI = 1.48-4.40) and current smokers (RR = 2.06, 95% CI = 0.92-4.62) had similarly elevated risks of tooth loss compared with non-smokers. CONCLUSIONS: The findings from this population suggest that a history of smoking contributes to tooth loss through mechanisms in addition to clinical disease processes alone.     

Basal ganglia organization in amphibians: catecholaminergic innervation of the striatum and the nucleus accumbens

The aim of the present study was to determine the origin of the catecholaminergic inputs to the telencephalic basal ganglia of amphibians. For that purpose, retrograde tracing techniques were combined with tyrosine hydroxylase immunohistochemistry in the anurans Xenopus laevis and Rana perezi and the urodele Pleurodeles waltl. In all three species studied, a topographically organized dopaminergic projection was identified arising from the posterior tubercle/mesencephalic tegmentum and terminating in the striatum and the nucleus accumbens. Although essentially similar, the organization of the mesolimbic and mesostriatal connections in anurans seems to be more elaborate than in urodeles. The present study has also revealed the existence of a noradrenergic projection to the basal forebrain, which has its origin in the locus coeruleus. Additional catecholaminergic afferents to the striatum and the nucleus accumbens arise from the nucleus of the solitary tract, where catecholaminergic neurons appear to give rise to the bulk of the projections to the basal forebrain. In other regions, such as the olfactory bulb, the anterior preoptic area, the suprachiasmatic nucleus, and the thalamus, retrogradely labeled neurons (after basal forebrain tracer-applications) and catecholaminergic cells were intermingled, but none of these centers contained double-labeled cell bodies. It is concluded that the origin of the catecholaminergic innervation of the striatum and the nucleus accumbens in amphibians is largely comparable to that in amniotes. The present study, therefore, strongly supports the existence of a common pattern in the organization of the catecholaminergic inputs to the basal forebrain among tetrapod vertebrates.     

Neutron and X-ray reflectivity studies of human serum albumin adsorption onto functionalized surfaces of self-assembled monolayers

Endurance exercise training increases fat oxidation during large muscle mass exercise. Although the source of this fat has been thought to be plasma free fatty acids (FFA) released from adipose tissue, the training-induced decrease in lipolytic hormonal responses to exercise is not consistent with this concept. The purpose of this communication is to review findings, from our laboratory indicating that, in young healthy subjects, endurance exercise training reduces plasma FFA turnover and oxidation during moderate intensity prolonged 2-leg cycling while simultaneously enhancing depletion of triglycerides from the active musculature. Evidence is presented that metabolism of intramuscular triglycerides can explain the increase in total fat oxidation observed in the trained state during large muscle mass exercise. However, these results may not be applicable to exercise involving small muscle groups, a distinction that is likely to be important in explaining the apparent conflict between our findings and those from other laboratories where experimental conditions were different. In summary, for large muscle mass exercise up to 2 h in duration, plasma FFA are a less important fuel source in the trained state, and intramuscular triglycerides supply the major portion of the increase in oxidized fatty acids.     

The prognostic value of NCAM, p53 and cyclin D1 in resected non-small cell lung cancer

Expression of the neural cell adhesion molecule, NCAM, in frozen sections has been associated with decreased postoperative survival in non-small cell lung carcinoma. Of the various isoforms of NCAM described, the highly sialylated isoform plays a role in the migration of embryonal cells from the neural crest and is expressed by highly malignant tumours such as small cell lung carcinomas. We investigated the clinical significance of expression of this NCAM isoform as a prognostic factor in a series of 96 non-small cell lung carcinomas resected with curative intent. We also evaluated the effect of microwave pre-treatment of formalin-fixed, paraffin-embedded sections on the NCAM immunostaining and related the outcome to the postoperative clinical course of disease. In addition, in an attempt to extend our search for possible molecular markers of unfavourable prognosis in lung cancer, we evaluated increased immunostaining for p53 and cyclin D1 in the same series. We did not find a significant relation between expression of NCAM or its highly sialylated isoform and the length of postoperative survival. The numbers of positive cases (9 and 14, respectively) were relatively low. Increased p53 and cyclin D1 immunostaining (50 and 55 of the 96 tumours) failed to show a significant relation with postoperative survival. In our material, tumour stage was the only significant prognostic factor.     

T-cell-mediated cytotoxicity against keratinocytes in sulfamethoxazol-induced skin reaction

BACKGROUND: The incidence of skin rashes or erythema multiforme to sulfamethoxazole in exposed patients is about 3%. Among patients with acquired immunodeficiency syndrome the risk is approximately 10 times higher. The pathogenesis of these reactions and the reason for the increased frequency in HIV infections are not understood. OBJECTIVE: To investigate drug specific T-cell-mediated cytotoxicity in sulfamethoxazole- induced skin reactions. METHODS: Specific T-cell lines and T-cell clones generated from a donor who developed a skin rash to sulfamethoxazole were assessed with a standard 4 h 51Cr cytotoxicity assay in the presence or absence of soluble sulfamethoxazole. B lymphoblasts and keratinocytes with and without interferon gamma pretreatment were used as target cells. Selective blockers of FasL/Fas and perforin-mediated killing and immunostaining for perforin were used to evaluate the involvement of the different cytolytic pathways. RESULTS: CD4+ and CD8+ sulfamethoxazole specific T-cell clones showed a drug-specific and MHC-restricted cytotoxicity against autologous B lymphoblasts in the presence of soluble sulfamethoxazole. Keratinocytes, if pretreated with interferon gamma, were specifically killed predominantly by CD4+ T-cell clones. Specific T-cell clones of both CD4+ and CD8+ phenotype showed a strong immunoreactivity for perforin and the cytotoxicity was blocked by concanamycin A which suggests a perforin-mediated killing. CONCLUSION: Perforin-mediated killing of autologous keratinocytes in the presence of soluble sulfamethoxazole by drug-specific CD4+ lymphocytes may be a pathway for generalized drug-induced delayed skin reactions. The requirement of interferon gamma pretreatment of keratinocytes for efficient specific killing might explain the increased frequency of drug allergies in generalized viral infections like HIV, when interferon gamma levels are elevated.     

Altered hippocampal kainate-receptor mRNA levels in temporal lobe epilepsy patients

This study determined whether hippocampal kainate (KA) receptor mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsies. Hippocampal sclerosis (HS; n = 17), nonsclerosis (non-HS; n = 11), and autopsy hippocampi (n = 9) were studied for KA1-2 and GluR5-7 mRNA levels using semiquantitative in situ hybridization techniques, along with neuron densities. Compared with autopsy hippocampi, HS and non-HS cases showed decreased GluR5 and GluR6 hybridization densities per CA2 and/or CA3 pyramid. Furthermore, HS patients demonstrated increased KA2 and GluR5 hybridization densities per granule cell compared with autopsy hippocampi. These findings indicate that chronic temporal lobe seizures were associated with differential changes in hippocampal KA1-2 and GluR5-7 hybridization densities that vary by subfield and pathology group. In temporal lobe epilepsy patients, these results support the hypothesis that pyramidal cell GluR5 and GluR6 mRNA levels are decreased as a consequence of seizures, and in HS patients granule cell KA2 and GluR5 mRNA levels are increased in association with aberrant fascia dentata mossy fiber sprouting and/or hippocampal neuronal loss.     

Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex

We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.