Primary and secondary structural homologies between the HIS4 gene product of Saccharomyces cerevisiae and the hisIE and hisD gene products of Escherichia coli and Salmonella typhimurium

Summary A detailed comparative analysis of the Escherichia coli and Salmonella typhimurium hisIE and hisD gene products and the functionally equivalent, single, HIS4 gene product of Saccharomyces cerevisiae permitted several insights concerning the relationship between these genes. Our analysis supports the idea that HIS4 results from the fusion of hisIE and hisD. The comparison permitted a more precise definition of the functional domains of hisI/HIS4A and hisE/HIS4B as well as the two functional domains of hisD/HIS4C. The homologies between the bacterial and yeast sequences suggest a region of the hisD/HIS4C protein that may constitute one of the active centres. A large fragment at the amino terminal region of the yeast protein is missing from the bacterial hisIE gene product and is probably not needed for catalytic activity. Another region of non-homology in the yeast protein is probably a peptide bridge connecting the HIS4AB domain to HIS4C. Although the overall homology at the level of amino acid sequence is modest (about 38%) there is a striking similarity when the hydropathic patterns and predicted secondary structural configurations of these proteins are compared.     

First human isolate of Leptospira interrogans as serovar bratislava in Italy

Abstract A strain of Leptospira interrogans was isolated from a patient suffering from leptospirosis and was typed by the Gross Agglutination Absorption test using monoclonal antibodies prepared against different serovars of the Australis serogroup. This newly isolated strain belonged to serovar bratislava . It is the first reported isolation from man, in Italy, of Leptospira bratislava , thus supporting the emerging role of this serovar in human leptospirosis.     

Effect of temperature on the physiology and cytology of the methylotrophic yeastCandida boidinii growing in methanol-limited chemostat

All deviations from optimum cultivation temperature affect strongly the physiology and morphology of cells ofCandida boidinii strain 2 during growth in methanol-limited chemostat. The optimum cultivation temperature was 28–30 °C at which maximum cell concentration and maximum cell yield (Y _S 0.4 g/g) were achieved. At suboptimal growth temperatures the cells were rich in cell protein, RNA, alcohol oxidase (AO) and in peroxisomes. Formation of cubic peroxisomes and a 20 % decrease of budding cells in the population was observed. At supraoptimal growth temperatures (>30 °C) a sharp decrease in AO activity was accompanied by degradation of peroxisomes in the cells. The culture forms pseudomycelium: at 34 °C the cells stop growing and they are washed out of the bioreactor.     

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

Delayed Excitotoxic Neurodegeneration Induced by Excitatory Amino Acid Agonists in Isolated Retina

Abstract: Evidence from in vitro studies suggests that excitotoxic neuronal degeneration can occur by either an acute or delayed mechanism. Studies of the acute mechanism in isolated chick embryo retina using histological methods indicate that this process is rapidly triggered by activation of glutamate receptors of either the N-methyl-d -aspartate (NMDA) or non-NMDA subtypes. The delayed mechanism, studied primarily in cortical and hippocampal cell cultures prepared from embryonic rodent brain, requires activation of NMDA receptors. In these cell culture systems, stimulation of non-NMDA receptors does not rapidly trigger delayed neuronal degeneration, or does so only indirectly, via activation of NMDA receptors secondary to glutamate release. To provide a more valid basis for comparison of these two mechanisms, we have modified the isolated chick embryo retina model to permit studies of delayed as well as acute excitotoxic neurodegeneration. Retinas maintained for 24 h exhibited no morphological or biochemical signs of damage. Retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium at various times after exposure to agonists and normalized to total LDH in each retina. Glutamate exposure (1 mM, 30 min) did not result in LDH release by the end of the exposure period, but LDH was released over the following 24 h. Briefer periods also led to substantial LDH release. Incubation in the presence of NMDA, or the non-NMDA agonists kainate (KA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), led rapidly to delayed LDH release. NMDA and AMPA were more potent than glutamate, but high concentrations of glutamate led to more LDH release than high concentrations of these agonists. KA was a powerful excitotoxin, providing more LDH release than glutamate, NMDA, or AMPA at every concentration tested. The delayed LDH release induced by glutamate involved activation of both NMDA and non-NMDA receptors, as a combination of receptor-selective antagonists was necessary to provide complete blockade. These results indicate that glutamate, NMDA, AMPA, and KA all cause delayed as well as acute excitotoxic damage in the retina. It is interesting that brief exposure to the non-NMDA receptor agonists, in relatively low concentrations, led to delayed LDH release. This is different than in other in vitro models of delayed excitotoxic neurodegeneration.     

Body composition analysis by DEXA by using dynamically changing samarium filtration

Gotfredsen, Anders, Lene Bæksgaard, and Jannik Hilsted.Body composition analysis by DEXA by using dynamically changing samarium filtration. J. Appl. Physiol.82(4): 1200-1209, 1997.Dual-energy X-ray absorptiometry (DEXA)has a high accuracy for body composition analysis but is influenced bybeam hardening and other error sources in the extremes of measurement.To compensate for beam hardening, the Norland XR-36 introduces adynamically changing samarium filtration system, which depends on thecurrent-absorber thickness. With this system we found a good agreement(r = 0.99) between reference andmeasured amounts of tissue or fat percentages in a plastic phantom andin smaller (~0.5-4 kg) and larger (~5-20 kg) piles oftissue (ox muscle and lard). Scans of six healthy volunteers coveredwith combinations of beef and lard (~5-15 kg) showed a goodagreement (r = 0.99) between referenceand DEXA values of added soft tissue mass and fat percentage. Weconclude that the DEXA method (and, in particular, the Norland XR-36using dynamic filtration) has a high accuracy for body compositionanalysis. It has a potential for gaining status as a reference methodin the future and may presently be used as a supplement to thetraditional methods for body composition analysis.

     

Interaction between advancing ice fronts and erythrocytes

It can be shown theoretically and experimentally that in purely aqueous suspension, cells (as well as microsolutes) areexcluded by advancing freezing fronts. This puts the cells under considerable osmotic stress and may be considered to be the major source of cell destruction upon freezing. It is also shown theoretically and experimentally that in aqueous suspensions, admixed with appropriate concentrations of a cryoprotectant (e.g., glycerol), cells areengulfed by advancing freezing fronts: Under such conditions, cells do not undergo any osmotic stress and remain undamaged when frozen. The influence of various common cryoprotectants is discussed, as is the reason why penetrating as well as nonpenetrating agents can be equally effective cryoprotective agents. The reason why leukocytes require lower cryoprotectant concentrations than erythrocytes is also elucidated.     

CEP Biomarkers as Potential Tools for Monitoring Therapeutics

Background

Carboxyethylpyrrole (CEP) adducts are oxidative modifications derived from docosahexaenoate-containing lipids that are elevated in ocular tissues and plasma in age-related macular degeneration (AMD) and in rodents exposed to intense light. The goal of this study was to determine whether light-induced CEP adducts and autoantibodies are modulated by pretreatment with AL-8309A under conditions that prevent photo-oxidative damage of rat retina. AL-8309A is a serotonin 5-HT_1A receptor agonist.

Methods

Albino rats were dark adapted prior to blue light exposure. Control rats were maintained in normal cyclic light. Rats were injected subcutaneously 3x with 10 mg/kg AL-8309A (2 days, 1 day and 0 hours) before light exposure for 6 h (3.1 mW/cm², λ=450 nm). Animals were sacrificed immediately following light exposure and eyes, retinas and plasma were collected. CEP adducts and autoantibodies were quantified by Western analysis or ELISA.

Results

ANOVA supported significant differences in mean amounts of CEP adducts and autoantibodies among the light + vehicle, light + drug and dark control groups from both retina and plasma. Light-induced CEP adducts in retina were reduced ~20% following pretreatment with AL-8309A (n = 62 rats, p = 0.006) and retinal CEP immunoreactivity was less intense by immunohistochemistry. Plasma levels of light-induced CEP adducts were reduced at least 30% (n = 15 rats, p = 0.004) by drug pretreatment. Following drug treatment, average CEP autoantibody titer in light exposed rats (n = 22) was unchanged from dark control levels, and ~20% (p = 0.046) lower than in vehicle-treated rats.

Conclusions

Light-induced CEP adducts in rat retina and plasma were significantly decreased by pretreatment with AL-8309A. These results are consistent with and extend previous studies showing AL-8309A reduces light-induced retinal lesions in rats and support CEP biomarkers as possible tools for monitoring the efficacy of select therapeutics.