The Paramecium circadian clock: synchrony of changes in motility, membrane potential, cyclic AMP and cyclic GMP

The behavior of a ciliate protozoan, Paramecium, is known to represent the electrical state of the cell membrane, and regulation of the membrane potential and ciliary motion are known to involve cAMP and cGMP. The present study shows the synchrony of circadian changes in motility, resting membrane potential and cyclic nucleotides in P. multimicronucleatum. Using an automated system for tracking isolated single microorganisms, the isolated Paramecium cells are confirmed to swim fast and straight during the day (and subjective day) and slowly, with frequent turning, at night (and subjective night). The resting membrane potential is more negative during the day than at night. cAMP and cGMP concentrations oscillate in a manner, such that both cAMP and cGMP are higher during the day (or subjective day) than at night (or subjective night). The ratio of cGMP to cAMP during the light and dark cycle (LD) fluctuates, paralleling the fluctuation of the resting membrane potential measured during the LD. These results suggest that the Paramecium will provide an excellent model to explore daily and circadian orchestration of second messengers mediating signals from ambient light/dark cycles and circadian pacemaker to ion channels and cilia, directly involved in daily and circadian cellular outputs of resting membrane potential and motility. Accepted: 23 January 1997     

Analysis of helicase gene mutations in Japanese Werner’s syndrome patients

The profile of helicase gene mutations was studied in 89 Japanese Werner’s syndrome (WRN) patients by examining the previously described mutations 1– 4 as well as a new mutation found during this study, designated mutation 5. Of 178 chromosomes (89 patients), 89 chromosomes (50%) had mutation 4, 11 (6.2%) chromosomes had mutation 1, and two chromosomes (1.1%) contained mutation 5. Mutations 2 and 3 were not observed in this patient population. The remaining 76 (42.7%) chromosomes had none of these mutations. A significant fraction of all patients (22 total patients, 24.7%) appear to be compound heterozygotes, including those carrying mutations of both types 1 and 4. The genotype analysis of the markers surrounding the WRN helicase gene strongly suggests that most of the chromosomes carrying either mutation 1 or 4 were derived from two single founders. Received: 25 July 1996 / Revised: 20 September 1996     

Highly sensitive analytical method for aluminum movement in soybean root through lumogallion staining

We present highly sensitive aluminum detection method in root using fluorescent lumogallion. Roots treated with 100 μM AlCl₃ including 0.2 mM CaCl₂ (pH 4.5) were stained for 60 min with 10 μM lumogallion fluorescence solution and fluorescence from aluminum complex in root was observed under confocal laser microscope. There was a good correlation between the intensity of fluorescence and aluminum content. When the amount of aluminum lost during each step in staining process was measured, it was found that about 10% of aluminum was lost only at staining stage. Through lumogallion staining method, aluminum accumulation especially at an early stage of aluminum treatment in root was shown. At the beginning (2 hr), aluminum began to be accumulated in root cap. After 4 hr treatment, the aluminum distribution was spread to about 3 mm from root apex in the root cap and outer cortex. When aluminum was found in the outer cortex in 3–5 mm from the root apex, the viability was tended to be decreased in the same area (6 hr). At the same time, aluminum amount in meristem was increased. However the comparison of lumogallion staining method with that of morin, which has been widely used to detect aluminum in root, the sensitivity of lumogallion method was found to be much higher.     

Oxidative Damage to Membranes by a Combination of Aluminum and Iron in Suspension-Cultured Tobacco Cells

Aluminum (Al) and ferrous iron [Fe(II)] are separately non-toxicto cultured tobacco cells in nutrient solution. However, Aland Fe(II) together cause the peroxidation of membrane iipids,the accumulation of Al and Fe, and the loss of viability [Onoet al. (1995) Plant Cell Physiol. 36: 115]. We investigatedthe cause-and-effect relationships of these various responses.In cells exposed to Fe(TT) or Fe(III)-EDTA, both the peroxidationof ipids and the loss of viability were similarly enhanced byAlCl₃ in a dose-dependent manner. During exposure to AlCl₃,the accumulation of Al and the loss of viability became apparentrapidly and simultaneously at 8 h, whereas both the peroxidationof lipids and the accumulation of Fe occurred at later times.However, lipophilic antioxidants protected cells efficientlynot only from the peroxidation of Iipids but also from the lossof viability and the accumulation of Al and Fe. These resultssuggest that the peroxidation of Iipids in the plasma membranethat is caused by both Al and Fe leads to the accumulation ofAl and Fe and the loss of viability. (Received April 18, 1997; Accepted October 1, 1997)     

Crystallization and Preliminary X-Ray Analysis of Neuropsin, a Serine Protease Expressed in the Limbic System of Mouse Brain

Neuropsin (M_r25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that crystal form I belongs to triclinic space groupP1 with unit cell dimensionsa= 97.16 Å,b= 97.12 Å,c= 46.75 Å and α = 99.17°, β = 99.77°, γ = 117.35°. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space groupP1 but has unit cell dimensions ofa= 38.40 Å,b= 55.16 Å,c= 65.37 Å and α = 95.38°, β = 89.98°, γ = 110.46° with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis. Intensity data to 3.1 Å resolution for form I and to 2.2 Å resolution for form II have been collected.     

Spawning behavior and reproductive isolation of two species ofPseudolabrus

The reproductive behavior of two wrasses,Pseudolabrus eoethinus (Richardson, 1846) andP. sieboldi Mabuchi & Nakabo, 1997, was studied on a rocky coast in Shikoku, Japan, where the two species occur sympatrically. In the study area, individual males of each species established mating territories within which pair spawnings occurred. Pair spawnings were observed only between conspecifics, indicating that the two coexisting species were reproductively isolated. Both species usually spawned during the afternoon in early winter and had similar reproductive behavior. Although habitat, temporal and behavioral isolation mechanisms were not apparent, reproductive isolation seemed to occur via premating isolation through visual recognition of conspecific mates because the two species differed in body size and coloration. On one occasion a maleP. sieboldi was observed to perform streaking behavior to join a spawning pair ofP. eoethinus. Thus, postmating isolation between the two species is also discussed.     

Possible role of VEGF in the progression of kidney disease in streptozotocin (STZ)-induced diabetic rats: effects of an ACE inhibitor and an angiotensin II receptor antagonist.

Two endothelium-derived factors, endothelin (ET), a vasoconstrictor, and vascular endothelial growth factor (VEGF), an angiogenic factor are thought to be involved in the pathogenesis of diabetic vascular complications. The aim of this study was to determine the effects of an angiotensin II type I (AT-1) receptor antagonist and an ACE inhibitor on the pathogenesis of VEGF and ET-1-mediated kidney disease in STZ-induced diabetic rats. Two days after STZ administration, diabetic rats were treated for 8 weeks with enalapril maleate, an ACE inhibitor, candesartan cilexetil, an AT-1 receptor antagonist, or saline. Urinary albumin and N-acetyl beta-D glucosaminidase (NAG) excretion as well as the VEGF protein content in the kidney were all found to be elevated in diabetic rats. Administration of enalapril maleate or candesartan cilexetil decreased the level of microalbuminuria and NAG excretion in diabetic rats. Administration of enalapril maleate also suppressed the elevated renal VEGF protein content in these animals while candesartan cilexetil treatment had no effect. Serum ET-1 and VEGF levels were unchanged by these treatments. These data support a role for AT-1 receptor antagonists and ACE inhibitors in the prevention of diabetic nephropathy, and suggest that the former may work by reducing renal VEGF levels.     

Hematological characteristics of rats spontaneously developing eosinophilia.

Hematological and genetic characteristics of newly found eosinophilic rats were studied. Hematologically, high blood eosinophil counts started at 6 weeks of age. Almost all 10-week-old rats had eosinophilia with individual counts above 500/microliter and 5 to 100 times the normal level. Proliferating eosinophils had normal morphology. An increase in lymphocyte counts was observed at 5 weeks of age, one week earlier than the onset of eosinophilosis. In bone marrow, proliferation of eosinophils was also observed at 8 weeks of age and thereafter progressed, suggesting a role in the pathogenesis of eosinophilia in this rat. The results of genetic cross experiments revealed the disease to be hereditary. The spontaneously eosinophilic rat therefore warrants attention as a model for studying the underlying mechanisms of human and animal eosinophilia.