Unbudded G2 as well as Gj arrest in the stationary phase of the basidiomycetous yeast Cryptococcus neoformans

Abstract Stationary-phase cells of Cryptococcus neoformans displayed two morphological characteristics: virtually all the cells were unbudded even in the early stationary phase and even when grown in rich media, and average cell size increased from that of exponential-phase cells. DNA contents for small and large stationary-phase cells were determined by quantitative fluorescence microscopy after DNA staining with propidium iodide or DAPI. Small cells contained G, DNA, whereas large unbudded cells had either a G₂ or G₁ DNA content, indicating that Cr. neoformans can enter into the stationary phase from either the G₁ or G₂ period.     

Adherence of Human Oral Spirochetes by Collagen-Binding Proteins

The ability of spirochetes to adhere to collagens was compared among three species of human oral treponemes. Immunoblot analysis demonstrated that type I-, IV-, and V-collagen-binding polypeptides (CBPs) were detected in the heated and unheated preparations from both Treponema denticola ATCC 33520 and T. socranskii subsp. buccale ATCC 35534. Few CBPs, however, were detected in the heated and unheated preparations from a recently characterized isolate, T. medium strain G7201. Immunoelectron microscopy using rabbit antisera against the CBPs from the unheated preparations demonstrated that four CBPs, a 27 kDa type V-CBP of T. denticola ATCC 33520, a 95 kDa type IV-CBP and a 110 kDa type I-CBP of T. socranskii subsp. buccale ATCC 35534, and a 95 kDa type IV-CBP of T. medium strain G7201, were located on the outer envelopes of the individual cells. The adherence of T. denticola to the collagen-coated surfaces was significantly greater than that of T. medium, suggesting that the CBPs on the oral spirochetal cells play an important role in their adherence to collagen-rich connective tissues of the host.     

Central inhibition of the aortic baroreceptors-heart rate reflex at the onset of spontaneous muscle contraction.

Animals decerebrated at the precollicular-premammillary body level exhibit spontaneous locomotion without any artificial stimulation. Our laboratory reported that the cardiovascular and autonomic responses at the onset of spontaneous locomotor events are evoked by central command, generated from the caudal diencephalon and the brain stem (Matsukawa K, Murata J, and Wada T. Am J Physiol Heart Circ Physiol 275: H1115-H1121, 1998). In this study, we examined whether central command and/or a reflex resulting from muscle afferents modulates arterial baroreflex function using a decerebrate cat model. The baroreflex was evoked by stimulating the aortic depressor nerve (ADN) at the onset of spontaneous muscle contraction (to test the possible influence of central command) and during electrically evoked contraction or passive stretch (to test the possible influence of the muscle reflex). When the ADN was stimulated at rest, heart rate and arterial blood pressure decreased by 40 +/- 2 beats/min and 11 +/- 1 mmHg, respectively. The baroreflex bradycardia was attenuated to 55 +/- 4% at the onset of spontaneous contraction. The attenuating effect on the baroreflex bradycardia was not observed at the onset and middle of electrically evoked contraction or passive stretch. The depressor response to ADN stimulation was identical among resting and any muscle interventions. The inhibition of the baroreflex bradycardia during spontaneous contraction was seen after beta-adrenergic blockade but abolished by muscarinic blockade, suggesting that the bradycardia is mainly evoked through cardiac vagal outflow. We conclude that central command, produced within the caudal diencephalon and the brain stem, selectively inhibits the cardiac component, but not the vasomotor component, of the aortic baroreflex at the onset of spontaneous exercise.     

Changes of renal lesion-related parameters in FGS/Nga and the parental mouse strains, CBA/N and RFM/Nga.

The FGS/Nga mouse strain, established from an outcross between CBA/N and RFM/Nga mice strains, has previously been reported as a spontaneous mouse model for focal glomerular sclerosis (FGS) and is considered to have two pairs of autosomal recessive genes associated with FGS. In this study, we examined the changes of seven renal lesion-related parameters, blood urea nitrogen (BUN), creatinine, albumin and total protein in plasma, urinary protein, systolic blood pressure, and a glomerulosclerosis index on histological observation, in 20-week-old FGS/Nga mice and their age-matched two parental strains, CBA/N and RFM/Nga. The levels of plasma BUN and creatinine, urinary protein and systolic blood pressure were significantly increased in FGS/Nga, compared with those of the parental strains. RFM/Nga mice showed slightly elevated levels of all biochemical makers. In histological analysis, a higher glomerulosclerosis index was observed in FGS/Nga than the two parental strains. RFM/Nga mice appeared to have slight sclerotic lesions of glomeruli, but no renal failure was observed in CBA/N mice. These results suggest that at least one mutant gene that causes the progression of renal lesion in FGS/Nga mice is derived from RFM/Nga.     

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10⁵ cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10⁷ cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Development and characterization of CATS markers for genetic linkage mapping in the house musk shrew, Suncus murinus.

To serve as an initial step in developing an ideal genetic marker map for the house musk shrew, Suncus murinus, 318 comparative anchor tagged sequence (CATS) primer pairs were assessed for polymorphism ascertainment and linkage mapping. Of the 112 (35.2%) CATS primer pairs that were successfully amplified by PCR in the shrew, 18 (16.1%) showed polymorphism between two mutant strains, BAN-kc, oeb and WZ. Linkage analysis of the polymorphic CATS markers and three visible mutant genes, kc, oeb and wz, genotyped in a 77 F2 mapping panel from a cross of the two mutant strains, assigned wz and five CATS markers into three linkage groups. Sequence analysis revealed that two (ADA and TXN) out of nine CATS amplified sequences had a total of six deletions of varying sizes and 17 single nucleotide polymorphisms (SNPs). BLAST search identified three CATS (ADA, CYP1A2, and TXN) products matching the genes from which they were originally designed, while the remaining six markers could not be identified. Together with the use of the detected SNPs as genetic markers, the five CATS markers linkage mapped in this species will serve as anchors in establishing the first framework map for locating loci affecting all heritable qualitative and quantitative traits in the musk shrew.     

Muscle mechanosensitive receptors close to the myotendinous junction of the Achilles tendon elicit a pressor reflex.

Feedback regulation by activation of mechanosensitive afferents in the exercising muscle causes the cardiovascular and sympathetic nerve responses, which follow tension development and are almost identical between static contraction and passive stretch. The precise location of the mechanoreceptors contributing to the exercise pressor reflex, however, remained unknown. To test the hypothesis that the mechanoreceptors will be located around the myotendinous junction to monitor a change in muscle tension than a change in muscle length, we examined the reflex cardiovascular responses to passive stretch of the triceps surae muscle in anesthetized rats with three interventions; systemic injection of gadolinium, cutting the Achilles tendon, and local injection of lidocaine into the myotendinous junction. Gadolinium (42 micromol/kg iv) blunted the increases in heart rate and mean arterial blood pressure during passive stretch by 36 and 22-26%, respectively, suggesting that the reflex cardiovascular responses were evoked by stimulation of muscle mechanosensitive receptors. The cardiovascular responses to passive stretch were not different between the cut Achilles tendon and the intact tendon in the same rats, suggesting that any mechanoreceptors, terminated in the more distal part of the tendon, did not contribute to the reflex cardiovascular responses. Lidocaine (volume, 0.04-0.1 ml) injected into the myotendinous junction blunted the stretch-induced increases in heart rate and mean arterial blood pressure by 37-49 and 27-34%, respectively. We conclude that the muscle mechanosensitive receptors evoking the reflex cardiovascular responses at least partly locate at or close to the myotendinous junction of the Achilles tendon.     

Delayed development of reflexes and hyperactive locomotion in the spontaneous mutant "waltzing" of the musk shrew, Suncus murinus.

The autosomal recessive mutation waltzing (wz), displaying abnormal circling and head-shaking behavior, has previously been reported in the musk shrew (Suncus murinus). Postnatal development of reflexes and locomotor patterns in an open arena were examined in wz/wz mutant shrews. The wz/wz shrews showed extreme developmental delays in surface-righting reflex and negative geotaxis until 10-16 days after birth, but both reflexes eventually recovered to the levels of +/wz normal. Nevertheless, the wz/wz adults exhibited bi-directional circling behavior 59 times, head-tossing behavior 22 times and horizontal head-shaking behavior 6 times more frequent than in the +/wz controls. Although the wz/wz adult shrews were extremely hyperactive with daily spontaneous locomotor activity exceeding 4-7 times control shrew activity, they appeared to have a normal circadian rhythm. This shrew mutant may therefore be useful as a model for hyperactivity syndromes in humans.     

Activation of Akt Kinase Inhibits Apoptosis and Changes in Bcl-2 and Bax Expression Induced by Nitric Oxide in Primary Hippocampal Neurons

Emerging data indicate that growth factors such as insulin-like growth factor-1 (IGF-1) prevent neuronal death due to nitric oxide (NO) toxicity. On the other hand, growth factors can promote cell survival by acting on phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, serine-threonine kinase Akt, in various types of cells. Here, we examined the mechanism by which IGF-1 inhibits neuronal apoptosis induced by NO in primary hippocampal neurons. IGF-1 was capable of preventing apoptosis and caspase-3-like activation induced by a NO donor, sodium nitroprusside or 3-morpholin-osydnonimine. Incubation of neurons with a P13-kinase inhibitor, wortmannin or LY294002, blocked the effects of IGF-1 on NO-induced neurotoxicity and caspase-3-like activation. In addition, the P13-kinase inhibitors blocked the effect of IGF-1 on down-regulation in Bcl-2 and upregulation in Bax expression induced by NO. Adenovirus-mediated overexpression of the activated form of Akt significantly inhibited NO-induced cell death, caspase-3-like activation, and changes in Bcl-2 and Bax expression. Moreover, expression of the kinase-defective form of Akt almost completely blocked the effects of IGF-1. These findings suggest that activation of Akt is necessary and sufficient for the effect of IGF-1 and is capable of preventing NO-induced apoptosis by modulating the NO-induced changes in Bcl-2 and Bax expression.