Development and evaluation of the polymerase chain reaction to detect Mycoplasma genitalium

The polymerase chain reaction (PCR) was developed to detect Mycoplasma genitalium. Oligonucleotide primers were used to amplify a 374 bp region of the attachment protein of the mycoplasma. DNA from three strains of M. genitalium tested gave a characteristic PCR product which was not seen with DNA from any other source. As little as 10(-15) g of M. genitalium DNA could be detected and it was found in the vagina of progesterone-treated BALB/c mice inoculated with M. genitalium organisms later than they could be cultured from this site, but not in mice that never became colonised vaginally.     

Isolation and characterization of maltose non utilizing (mnu) mutants mapping outside the MAL1 locus in Saccharomyces cerevisiae

The MAL1 locus of Saccharomyces cerevisiae comprises three genes necessary for maltose utilization. They include regulatory, maltose transport and maltase genes designated MAL1R, MAL1T and MAL1S respectively. Using a MAL1 strain transformed with an episomal, multicopy plasmid carrying the MAL2 locus, five recessive and one dominant mutant unable to grow on maltose, but still retaining a functional MAL1 locus were isolated. All the mutants could use glycerol, ethanol, raffinose and sucrose as a sole carbon source; expression of the maltase and maltose permease genes was severely and coordinately reduced. Only the dominant mutant failed to accumulate the MAL1R mRNA.     

The effect of temperature on conduction velocity in human muscle fibers

The effects of variation of intramuscular temperature (T) on conduction velocity (CV) of the action potential along single human muscle fibers of the biceps brachii was studied in situ in 15 normal volunteers (mean age 39 years, range 21–62 years). Cooling was obtained by direct application of ice over a rectangular skin region including the stimulating and recording area. The intramuscular T was monitored by a needle thermocouple (copperconstantane). In all the 24 muscle fibers studied, a linear relationship was observed between CV and T. The slopes of the regression lines, ranging between 0.190 and 0.079 m/s, were positively correlated with the starting CV at 36°C ranging between 2.2 and 5.2 m/s. If conduction changes are expressed as a percentage of the basal CV at 36°C, the CV/T coefficient is the same for all the fibers and independent of the individual CV: 3.4% of CV/°C.     

Effects of cholinergic muscarinic agents on protein kinase C activity in rat pineal gland.

The role of protein kinase C (PKC) on muscarinic regulation of serotonin release in the pineal gland was investigated by measuring the pineal-PKC activity and serotonin secretion in response to muscarinic agents. Pineal slices, short-term incubated (0-15 min) without additions produced a low serotonin release and 20 to 24 percent PKC activity was found associated with membrane fractions. Prolonged exposure of pineal slices (30-180 min) produced further translocation of PKC activity to the membranes and a significant increase of serotonin release. Short-term treatment with pilocarpine and carbachol, stimulated PKC activity of both cytosolic and particulate fractions and the release of pineal serotonin. The pilocarpine effect was blocked by atropine indicating that it was mediated by muscarinic receptors. The present data support that PKC activation correlates with the increase of serotonin release by muscarinic agonist in pineal gland.     

An immune response defect due to low levels of class II cell surface expression. Analysis of antigen presentation and positive selection.

The effects of quantitative differences in class II cell surface expression have been difficult to address in intact animals. This study uses several lines of H-2s/s mice carrying an A beta k transgene that differ significantly in terms of class II cell surface expression. Due to inefficient chain pairing, mice carrying 60 to 65 copies of this transgene express only low levels of A alpha s/A beta k on the cell surface, and cell surface expression of the endogenous A alpha s/A beta s complex (and total Ia) is severely reduced (to 7-15% control levels). The significant decrease in class II cell surface expression in the thymic cortex of these mice did not affect the frequency of peripheral T cells expressing at least 10 distinct TCR V beta chains. However, T cell proliferative responses to the A alpha s/A beta s-restricted peptide MBP 89-101 were abrogated in high copy number A beta k mice. Experiments using bone marrow chimeras demonstrated that both inefficient Ag presentation and failure to positively select appropriate T cells contributed to this lack of response. Inefficient Ag presentation was clearly the dominant defect, and the density of class II cell surface expression required for positive selection appeared to be quite low.     

Interaction of menadione and duroquinone with Q-cycle during DT-diaphorase function]

The interaction of quinones (menadione and duroquinone) with DT-diaphorase and mitochondrial electron transport chain translocators at low (120 mosM) and high (400 mosM) values of the medium tonicity in the quinone concentration range of 6-90 microM was studied. It was shown that with a rise in menadione (K3) concentration the number of electron transport carriers interacting with it increase. At K3 concentration of 6 microM the latter is reduced by DT-diaphorase and fully oxidized via the Q-cycle. At K3 concentration of 15 microM the latter is also reduced by DT-diaphorase via the Q-cycle, but in this case the oxidation is incomplete (about 30% K3H2 is oxidized by the terminal part of the respiratory chain). At 90 microM K3 50% of quinone is reduced by DT-diaphorase and 50% by the respiratory chain NADH dehydrogenase complex enzymes; about 30% of K3H2 is oxidized via the Q-cycle, about 20%--by the terminal part of the respiratory chain and about 50%--by O2 without cytochrome oxidase. Unlike menadione, duroquinone (6-90 microM) is reduced only by DT-diaphorase and is oxidized in all cases by cytochrome oxidase. It was shown that the increase in the mitochondrial matrix volume in low tonicity media decreases the rate of the DT-diaphorase shunt operation.     

Simultaneous Comparisons of Multiple Treatments to Two (or More) Control

DUNNETT (1955) developed a procedure simultaneously comparing k treatments to one control with an exact overall type I error of α when all sampling distributions are normal. Sometimes it is desirable to compare k treatments to m≧2 controls, in particular to two controls. For instance, several new therapies (e.g., pain relievers) could be compared to two standard therapies (e.g., Aspirin and Tylenol). Alternatively, a standard therapy could be very expensive, difficult to apply and/or have bad side effects, making it useful to compare each new therapy to both standard therapy and no therapy (Placebo). Dunnett's method is expanded here to give comparisons of mean values for k treatments to mean values for m≧2 controls at an exact overall type I error of α when all sampling distributions are normal. Tabled values needed to make exact simultaneous comparisons at α = .05 are given for m = 2. An application is made to an example from the literature.     

Sequence of the cDNA encoding ovine tumor necrosis factor-alpha: problems with cloning by inverse PCR.

We have cloned and sequenced the ovine tumor necrosis factor-alpha (TNF-alpha)-encoding cDNA, using gene amplification by polymerase chain reaction (PCR) technology, to aid studies of assorted diseases in this species. We used primers selected from published TnfA sequences of other species on a cDNA template prepared from lipopolysaccharide-stimulated ovine alveolar macrophages, to generate a product representing the central region of the molecule. We then used a novel method based on 'inverse PCR' to generate a product containing the 5' and 3' ends of the molecule. Here, we present the complete sequence of the ovine TNF-alpha cDNA and compare it with other published TNF sequences. The cloned cDNA has a leader sequence of 156 bp followed by a protein-coding sequence of 702 bp and a 3'-untranslated region of 800 bp. The protein product of the gene is a protein of Mr = 25,586, 79% homologous to human TNF-alpha. An mRNA produced by alveolar macrophages, which hybridises to the cloned gene, is induced greatly, with a peak induction time of approx. 135 min, in response to stimulation by lipopolysaccharide and to plating on plastic. We also discuss the resolution of some artefacts of the inverse PCR technique.     

The effect of high hydrostatic pressure on the modulation of regulatory enzymes from spinach chloroplasts.

High hydrostatic pressure enhanced the specific activity of regulatory enzymes of the Benson-Calvin cycle (fructose-1,6-bisphosphatase, glyceraldehyde-3-P dehydrogenase, phosphoribulokinase) which are modulated by the ferredoxin-thioredoxin system. High activity of chloroplast fructose-1,6-bisphosphatase required dithiothreitol, fructose 1,6-bisphosphate, and Ca2+. At 100 bar the A0.5 for fructose 1,6-bisphosphate (0.3 mM) was lower than that at 1 bar (1.5 mM), whereas similar variations of pressure did not alter the A0.5 for Ca2+ (55 microM). The response of chloroplast glyceraldehyde-3-P dehydrogenase exposed to 500 bar was a 4-fold increase in the NADP-linked activity; conversely, the NAD-dependent activity remained unchanged. The concerted action of high pressure and Pi (or ATP), both activators of chloroplast glyceraldehyde-3-P dehydrogenase, led to inactivation. On the other hand, the activity of phosphoribulokinase increased 10-fold when the enzyme was incubated at 1500 bar; the activation process was strictly dependent on the presence of dithiothreitol. At variance with these enzymes, bovine liver fructose-1,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase, and chloroplast ribulose 1,5-bisphosphate carboxylase, whose activities are not modulated by reduced thioredoxin, were inactivated by high pressure. The comparison of oligomeric enzymes revealed that the stimulation of specific activity by high pressure correlated with thioredoxin-mediated activation, and it did not depend on a particular subunit composition. Present results show that high pressure resembled thioredoxin, cosolvents, and chaotropic anions in its action on regulatory enzymes of the Benson-Calvin cycle. The comparison of physiological and non-physiological modulators suggested that thioredoxin-mediated modifications of noncovalent interactions is an important event in light-dependent regulation of chloroplast enzymes.