Detection of Coccidioides immitis in Kern County, California, by multiplex PCR

Coccidioides immitis is a fungal human pathogen endemic to semiarid soils in southern California and Baja California (Mexico). Results of culture-dependent detection of C. immitis in the past indicated a spotty distribution and unreliable prediction of C. immitis growth sites and accumulation sites. In this project we investigated bulk soil samples for the presence of the pathogen in nonagricultural loamy soils at nine different locations around Bakersfield, Kern County, California, for almost 2 y (2008-2009). To detect the pathogen we used a multiplex PCR method with optimized soil handling and storage, DNA extraction procedure and PCR protocol. With this method we were able to detect C. immitis in 8.42% of our samples in 2008 (n = 285), mostly from early spring to early summer. In 2009 however the percentage of samples positive for C. immitis from the same sites declined to 2.68% (n = 261). We also were able to distinguish C. immitis growth sites from accumulation sites. One site close to a recreation area (Lake Webb, Buena Vista Lake Basin), not previously known to support the growth of C. immitis, was identified as a strong growth site of the fungus. The cultivation-independent method in this study together with soil parameters can be used to predict and confirm C. immitis growth sites and might be a valuable tool for public health institutions.     

Phase-dependence of elbow muscle coactivation in front crawl swimming

Propulsion in swimming is achieved by complex sculling movements with elbow quasi-fixed on the antero-posterior axis to transmit forces from the hand and the forearm to the body. The purpose of this study was to investigate how elbow muscle coactivation was influenced by the front crawl stroke phases. Ten international level male swimmers performed a 200-m front crawl race-pace bout. Sagittal views were digitized frame by frame to determine the stroke phases (aquatic elbow flexion and extension, aerial elbow flexion and extension). Surface electromyograms (EMG) of the right biceps brachii and triceps brachii were recorded and processed using the integrated EMG to calculate a coactivation index (CI) for each phase. A significant effect of the phases on the CI was revealed with highest levels of coactivation during the aquatic elbow flexion and the aerial elbow extension. Swimmers stabilize the elbow joint to overcome drag during the aquatic phase, and act as a brake at the end of the recovery to replace the arm for the next stroke. The CI can provide insight into the magnitude of mechanical constraints supported by a given joint, in particular during a complex movement.     

Detection of diverse hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translated proteins of HCV

Broadly directed hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been identified from liver-infiltrating lymphocytes but have been more difficult to assess in peripheral blood of infected persons. To enhance the detection of CTL from peripheral blood mononuclear cells (PBMC), we cocultured PBMC with autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines that had been infected with recombinant vaccinia virus constructs so that they expressed the entire translated polyprotein of HCV-H, a type 1a strain. These stimulated cells from HCV-infected as well as exposed seronegative persons were then cloned at limiting dilution and tested for HCV-specific CTL activity using a standard (51)Cr release assay. HCV-specific CTL were detected in PBMC from seven of nine persons with chronic hepatitis, including five of seven in whom CTL had previously been detected from liver biopsy specimens but not PBMC. In a single person with chronic HCV infection, CTL directed against as many as five different epitopes were detected in peripheral blood and were similar in specificity to those detected in liver tissue. This technique was used to evaluate eight subjects identified to be at high risk for HCV exposure due to continued injection drug abuse; no evidence of CTL in PBMC was found. We conclude that CTL can be detected in PBMC from the majority of persons with chronic HCV infection but are present at lower levels or absent in exposed but persistently seronegative persons. The high degree of concordance of HCV epitopes identified from liver and PBMC suggests that this strategy is a reasonable alternative to liver biopsy for characterizing the CTL response to HCV in chronically infected persons.     

Micropatterned substrates for the growth of functional neuronal networks of defined geometry

The in vitro assembly of neuronal networks with control over cell position and connectivity is a fascinating approach not only for topics in basic neuroscience research but also in diverse applications such as biosensors and tissue engineering. We grew rat embryonic cortical neurons on patterned substrates created by microcontact printing. Polystyrene was used as a cell repellent background, onto which a grid pattern of physiological proteins was applied. We printed laminin and a mixture of extracellular matrix proteins and additionally both systems mixed with polylysine. Attachment of cells to the pattern with high fidelity as well as the formation of chemical synapses between neighboring cells on the pattern could be observed in all four cases, but cell attachment was strongly increased on samples containing polylysine. Neurons grown on patterned substrates had a membrane capacity smaller than that of neurons on homogeneously coated controls, which we attributed to the geometrical restrictions, but did not differ either in resting membrane potential or in the quality of synapses they formed. We therefore believe that the cells attach and differentiate normally on the pattern and form functional, mature synapses following the predefined geometry.     

Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes.

The DNA content of the nucleus of the yeast Saccharomyces cerevisiae has been determined by both renaturation kinetics and DNA per cell measurements. Renaturation kinetics experiments were performed by following the decrease of optical hyperchromicity at 260 nm and by hydroxyapatite chromatography. DNA per cell measurements were made by the diaminobenzoic acid method and by the ethidium bromide method of Klotz &; Zimm (1972b). The conclusion from the above experiments is that the S. cerevisiae nucleus contains 9 × 10⁹ ± 2 × 10⁹ daltons of DNA. Previously we (Lauer &; Klotz, 1975) had measured the molecular weight of the largest piece of DNA in the yeast nucleus to be 2 × 10⁹ ± 0.2 × 10⁹. Here we extend this work by using a more highly protein-denaturing buffer system and conclude that the largest piece of DNA in the S. cerevisiae nucleus contains 1.5 × 10⁹ to 2.2 × 10⁹ daltons of DNA in both haploid and diploid cell lysates. From genetics, the largest yeast chromosome should contain 13% of the genome, or 0.9 × 10⁹ to 1.5 × 10⁹ daltons of DNA (using our DNA per cell range). Thus, the large DNA we measure contains from one to two times the amount of the DNA predicted from genetics to be in the largest chromosome. In light of these new data, viscoelastic measurements on yeast DNA are now consistent with the idea that each chromosome contains one piece of DNA.     

The effect of peroxynitrite on the catalytic activity of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a key enzyme of the *NO/cGMP pathway. Many cardiovascular disorders are associated with reduced *NO-mediated effects, while vascular superoxide (O(2)*(-)) production is increased. Both radicals rapidly react to peroxynitrite. We investigated whether peroxynitrite affects the activity and protein expression of sGC in intact vascular preparations. Catalytic sGC activity and expression of the sGC-beta(1) subunit was measured by conversion of radiolabeled GTP and western blot, respectively, using cytosolic extracts from rat aorta that had been incubated for 4 h with *NO/O(2)*(-) systems (devoid of free *NO) generating either 0.13 microM or 7.5 microM peroxynitrite/min. Incubation of rat aorta with 0.13 microM peroxynitrite/min had no effect. In striking contrast, incubation with 7.5 microM peroxynitrite/min resulted in a shift of the concentration-response curve obtained with a *NO donor (p =.0004) and a reduction of maximal specific activity from 3579 +/- 495 to 2422 +/- 265 pmol cGMP/mg/min (p =.036). The expression of the sGC-beta(1) subunit was unchanged. Exposure of aorta to the O(2)*(-) component had no effect, while exposure to the *NO-component reduced sGC expression to 58.8 +/- 7% (p <.001) and maximal sGC activity from 4041 +/- 992 to 1429 +/- 491 pmol cGMP/mg/min (p =.031). These data suggest that continuous generation of extracellular peroxynitrite might interfere with the *NO/cGMP signaling in vascular cells.     

The cerebral ganglia of Milnesium tardigradum Doyère (Apochela, Tardigrada): Three dimensional reconstruction and notes on their ultrastructure

Differential interference contrast micrographs from stretched animals, serially sectioned semi-thin and ultrathin sections revealed that the cerebral ganglia (supraoesophageal mass) of the eulardigrade Milnesium tardigradum lie above the buccal tube and adjacent tissue like a saddle. It has an anterior indentation which is penetrated by two muscles that arise from the cuticle of the forehead. The cerebral ganglia consist of lateral outer lobes bearing an eye on each side, and two inner lobes which extend caudally. Between the inner lobes a cone-like projection tapers into a nerve bundle. Each outer lobe is joined with the first ventral ganglion. From the outer lobe near the eye the ganglion for a posterolateral sensory field extends to the epidermis. Anterior to the supraoesophageal mass are three dorsal ganglia for the upper three peribuccal papillae. Two additional ganglia attached to the cerebral mass supply the lateral cephalic papillae. The cerebral ganglia are covered by a thin neural lamella. The pericarya which surround the neuropil have large nuclei. Near the axons in the centre of the supraoesophageal mass the cytoplasm is crowded with vesicles of different size and appearance. Some of them resemble synaptic vesicles while others resemble dense core bodies. Structurally different types of synapses and axons can be distinguished within the neuropil.