Sex differences in oxidant/antioxidant balance under a chronic mild stress regime.

The deterioration of homeostasis between oxidant/antioxidant species may represent an important mechanism linking psychological stress to cardiovascular risk despite the many sex differences in stress responsiveness. The goal of the present study was to investigate the influence of chronic mild stress (CMS), a widely accepted animal model of depression, on oxidative homeostasis-allostasis markers and sICAM-1, a marker of endothelial injury, in the serum of Wistar rats, by taking into account the effect of sex. After six weeks of exposure to mild unpredictable environmental stressors, both male and female rat groups displayed typical changes in hedonic status (anhedonia), which is a core symptom of human depression. Control female rats had higher (nitrite and nitrate) NOx, lower malondealdehyde (MDA) levels with lower activity of antioxidant enzymes and sICAM-1 levels than did control males. CMS induced oxidant/antioxidant responses in both sexes. Females tended to increase their nitric oxide (NO) levels further, while MDA levels did not reach those of males, thus retaining significantly higher NO bioavailability than in males. Concerning the antioxidant enzymes, CMS-females exhibited significantly higher glutathione peroxidase (GPx) activity and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity compared to CMS-males. The CMS response in females was accompanied by lower sICAM-1 levels than in males, suggesting lower endothelial injury. In conclusion, the results of the present study showed that CMS induces different oxidative stress and compensatory responses in both sexes probably due to differences in the mechanisms regulating oxidant/antioxidant pathways.

Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy.

Electron microscopy of biofilms and the localization of extracellular polymers at high resolution require the adaptation of conventional electron microscopic preparation and imaging techniques. A method developed for in situ fixation and embedding of biofilms, imaging of unstained thick sections with electron spectroscopic imaging and the application of lectin or antibody-based marker systems allowed interpretation of extracellular polymer distribution at micrometer scale. By this way, it is possible to discriminate in situ between extracellular polymers produced by different organisms.

Temperature optima of enzyme-catalysed reactions in microemulsion systems.

Ternary phase systems (water/surfactant/organic solvent) were utilised to increase and broaden the temperature optima of enzyme-catalysed reactions. Alcohol dehydrogenases from yeast and Thermoanaerobium brockii (EC 1.1.1.1 and EC 1.1.1.2), lactate dehydrogenase from Lactobacillus delbrueckii (EC 1.1.1.28) and the particulate hydrogenase from Ralstonia eutropha (EC 1.18.99.1) were used as model enzymes in microemulsions, consisting of the surfactant Aerosol OT, and various alkane solvent and aqueous phases. All enzymes exhibited, besides an increase in specific activity, an upshift of the temperature optimum of the catalysed reaction. The temperature optimum could be further shifted by variation of the chain length of the solvent used and/or the addition of compatible solutes to the aqueous phase. Under optimised conditions, catalytic reactions of enzymes from mesophilic microorganisms had temperature optima in the range generally obtained with enzymes from thermophilic organisms.

Feasibility of paramedic treatment and referral of minor illnesses and injuries.

BACKGROUND: Approximately 40% of Hennepin County Medical Center's (HCMC's) ambulance runs are for minor medical conditions as defined by billing criteria ["ALS minor," i.e., no advanced life support (ALS) procedures done in the field]. Current metropolitan guidelines mandate that all such patients must be transported to a hospital unless they refuse this service. It has been proposed that some patients with minor medical conditions could be better served by treatment in the field by paramedics and referred to a clinic or hospital for early follow-up care. It is proposed that this approach would save costs and improve paramedic availability for patients with more serious conditions. OBJECTIVE: To evaluate the feasibility and safety of implementing such a program by identifying high-volume, low-complexity groupings of cases. Such high-volume, low-complexity cases would serve as the topics for curriculum development for paramedic training in field treatment and referral. METHODS: Data were obtained from ambulance run sheets and emergency department (ED) records for all patients transported by the HCMC ambulance service in 1996 who were covered by the Metropolitan Health Plan (MHP) and who were categorized for billing purposes as "ALS minor" transports. The data included demographic information, vital signs, presenting problem, diagnoses in the ED, and procedures, laboratory studies, or x-rays done in the ED. Patients were classified as "potentially treatable" in the field if they were treated and discharged from the ED without undergoing any procedures or diagnostic studies. Patients who required more extensive evaluation in the ED, or who were admitted, were classified as likely too "complex" to be treated at the scene and then referred for early follow-up. The data were analyzed to find the most common presenting problems and the numbers, characteristics, and dispositions of "potentially treatable" and "complex" patients in each group. This information was used to determine what, if any, types of patients could potentially be treated safely and effectively according to this scheme. RESULTS: The study group comprised 1,103 patients, representing 127 different presenting medical problems. There were 523 (47%) "potentially treatable" patients and 580 (53%) "complex" patients. The 127 medical problems were grouped and the 15 most common presenting problem groups were identified. Within these groups there was no single medical problem with high volume. Each of these 15 most common problem groups contained a substantial proportion of "complex" patients, ranging from 24% to 100%. CONCLUSIONS: None of the 15 most frequently encountered problem groups consisted of a high enough proportion of "potentially treatable" cases to serve as a high-volume, low-complexity category for paramedic treatment in the field with early follow-up. Without any identified high-volume, low-complexity categories, a treatment and referral program as proposed in this article would require a substantial investment in development of appropriate criteria and in training paramedics to apply the criteria for numerous clinical entities. This would limit any cost saving, and require great care to avoid compromising patient safety accompanied by substantial professional liability exposure.

Alternative splicing, chromosome assignment and subcellular localization of the testicular haploid expressed gene (THEG).

We have previously isolated and characterized the mouse Testicular Haploid Expressed Gene (Theg) that is specifically expressed in haploid germ cells. We now describe the molecular cloning and characterization of the human homologue (THEG) of mouse Theg. Expression studies by using both dot blot and Northern blot techniques revealed that human THEG is expressed specifically in the testis. Additionally, we found two alternatively spliced transcripts (THEG major and THEG minor) for THEG by using reverse transcription-polymerase chain reaction on human testicular RNA. Sequence analysis of these PCR products demonstrated that the smaller transcript (THEG minor) lacks 72 bp which was also observed for the mouse Theg. We have isolated the cDNAs of human THEG major and THEG minor, containing the complete open reading frames, which encode putative nuclear proteins of 379 amino acids and 355 amino acids, respectively. Database searches identified two genomic clones on chromosome 19 harboring the human THEG gene, which is approximately 14 kb pairs in size, contains eight exons, and comparison of the two cDNA sequences with the genomic sequence indicated that the smaller transcript lacks exon 3. Furthermore, we assigned the human THEG gene (THEG) to human chromosome 19ptel--> p13 by fluorescence in situ hybridization. Moreover, we detected mouse THEG protein prominently in the nucleus of round spermatids by using an antibody against THEG on both testicular sections and cellular suspensions. Additionally, the subcellular localization of mouse THEG was confirmed by a green fluorescent protein (GFP) fusion protein of mouse THEG which was found mainly in the nucleus of transfected NIH3T3 cells. These data suggest that both human and mouse THEG are specifically expressed in the nucleus of haploid male germ cells and are involved in the regulation of nuclear functions.

Evaluation of methodologies including immunofluorescent assay (IFA) and the polymerase chain reaction (PCR) for detection of human pathogenic microsporidia in water.

Microsporidia is a term used to describe a group of emerging protozoan pathogens whose environmental occurrence has only recently been documented due to lack of detection methodologies. This study evaluates and describes current methods for detection of microsporidia in water. Standard methods, for the collection and processing of large volumes of water to detect protozoa, showed only a 4.8% recovery, of microsporidia spores, from 100 l volumes of tap. Immunofluorescent assay (IFA) analysis was assessed using two different antibodies specific for human pathogenic microsporidia. Results indicated that the use of IFA for routine screening of water for microsporidia was not an acceptable approach. The antibodies tested for the IFA resulted in false positives and false negatives and did not react with Enterocytozoon bieneusi, which is an important human pathogenic microsporidia. Finally, the small sizes of the human pathogenic microsporidia prevent confirmation and species determination by light microscopic methods. Two methods for isolating microsporidia DNA from water for use in polymerase chain reaction (PCR) amplification of microsporidia target sequences were assessed. Both of these DNA isolation methods when combined with the PCR showed the ability to detect less than ten spores in purified water concentrates. Thus, this study represents the first documentation and evaluation of current methods for the detection of human pathogenic microsporidia in water.

Effects of unilateral lesions of retrotrapezoid nucleus on breathing in awake rats.

In anesthetized rats, unilateral retrotrapezoid nucleus (RTN) lesions markedly decreased baseline phrenic activity and the response to CO2 (E. E. Nattie and A. Li. Respir. Physiol. 97:63-77, 1994). Here we evaluate the effects of such lesions on resting breathing and on the response to hypercapnia and hypoxia in unanesthetized awake rats. We made unilateral injections [24 +/- 7 (SE) nl] of ibotenic acid (IA; 50 mM), an excitatory amino acid neurotoxin, in the RTN region (n = 7) located by stereotaxic coordinates and by field potentials induced by facial nerve stimulation. Controls (n = 6) received RTN injections (80 +/- 30 nl) of mock cerebrospinal fluid. A second control consisted of four animals with IA injections (24 +/- 12 nl) outside the RTN region. Injected fluorescent beads allowed anatomic identification of lesion location. Using whole body plethysmography, we measured ventilation in the awake state during room air, 7% CO2 in air, and 10% O2 breathing before and for 3 wk after the RTN injections. There was no statistically significant effect of the IA injections on resting room air breathing in the lesion group compared with the control groups. We observed no apnea. The response to 7% CO2 in the lesion group compared with the control groups was significantly decreased, by 39% on average, for the final portion of the 3-wk study period. There was no lesion effect on the ventilatory response to 10% O2. In this unanesthetized model, other areas suppressed by anesthesia, e.g., the reticular activating system, hypothalamus, and perhaps the contralateral RTN, may provide tonic input to the respiratory centers that counters the loss of RTN activity.