A rapid urease test for presumptive identification of Cryptococcus neoformans

A rapid method to evidence urease activity is described. Urea hydrolysis and consequent production ammonia are detected by a chemical reaction producing a blue phenol compound (indophenol blue). Three hundred and three yeast were tested. Out of 107 urease-positive organisms detected by Christensen's Urea Agar Test (CUAT) 102 were positive by our method. No false negatives were observed by this method when testing 87 Cryptococcus strains. Ths practical screening test for presumptive identification of Cryptococcus neoformans is simple, unaffected by pH changes and requires 15 minutes to be performed.     

Relationship between the levels of wheat-rye metaphase I chromosomal pairing and recombination revealed by GISH

The metaphase I and anaphase I stages of meiosis of wheat×rye hybrids carrying the ph1b mutation were analyzed by genomic in situ hybridization. This technique allows distinction between three different types of wheat-rye associations in metaphase I configurations as well as detection of wheat-rye recombinant chromosomes in anaphase I cells. The frequency of associations between wheat and rye chromosomes greatly exceeded the level of wheat-rye recombination found in the three hybrids examined. Extremely distal associations, which account for about 50% of the total wheat-rye metaphase I chromosomal pairing, can explain such a discrepancy between metaphase I and anaphase I data. It is further discussed whether these associations reflect very distally located chiasmata or nonchiasmatic pairing. The sizes of the segments exchanged in wheat-rye recombinant chromosomes provide cytological evidence that wheat-rye recombination is restricted to the distal chromosomal regions. Received: 24 August 1995; in revised form: 27 February 1996 / Accepted: 28 March 1996     

Brain ATP Depletion Induced by Acute Ammonia Intoxication in Rats Is Mediated by Activation of the NMDA Receptor and Na+, K+-ATPase

Abstract: Injection of large doses of ammonia into rats leads to depletion of brain ATP. However, the molecular mechanism leading to ATP depletion is not clear. The aim of the present work was to assess whether ammonium-induced depletion of ATP is mediated by activation of the NMDA receptor. It is shown that injection of MK-801, an antagonist of the NMDA receptor, prevented ammonia-induced ATP depletion but did not prevent changes in glutamine, glutamate, glycogen, glucose, and ketone bodies. Ammonia injection increased Na⁺,K⁺-ATPase activity by 76%. This increase was also prevented by previous injection of MK-801. The molecular mechanism leading to activation of the ATPase was further studied. Na⁺,K⁺-ATPase activity in samples from ammonia-injected rats was normalized by "in vitro" incubation with phorbol 12-myristate 13-acetate, an activator of protein kinase C. The results obtained suggest that ammonia-induced ATP depletion is mediated by activation of the NMDA receptor, which results in decreased protein kinase C-mediated phosphorylation of Na⁺,K⁺-ATPase and, therefore, increased activity of the ATPase and increased consumption of ATP.     

Induction of Nitric Oxide Synthase in Rat C6 Glioma Cells

Abstract: We have examined the induction of nitric oxide syhthase (NOS) activity in the rat astrocyte-derived C6 glioma cell line. In contrast to the previous results with primary astrocyte cultures, incubation of C6 cells with bacterial endotoxin lipopolysaccharide (LPS; 1 μg/ml for 24 h) did not stimulate NO₂ production. However, addition of either tumor necrosis factor-a (TNF-α) or interferon-γ (IFN-γ), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose-dependent manner (EC₅₀ values of 39 ng/ml of TNF-α and 9.4 U/ml of IFN-γ), and the effect of TNF-α could be further potentiated (twofold) by the presence of interleukin-1β. The simultaneous presence of TNF-α and IFN-γ yielded a greater response than either cytokine alone; however, the respective EC₅₀ values were not affected. A cytoplasmic extract from induced C6 cells catalyzed the Ca²⁺-independent conversion of l -arginine to l - citrulline, with an apparent K _m of 51.2 n M , and this activity could be blocked by l -arginine analogues in the potency order amino > methyl > nitroarginine. Immunoblot analysis revealed an apparent molecular mass of 125 kDa for the NOS protein induced in C6 cells. These results indicate that the combination of LPS plus cytokines can induce NOS activity in C6 glioma cells with properties similar to those of the enzyme expressed in primary astrocyte cultures.     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse     

Biogeneration of 2-phenylethanol and 2-phenylethylacetate important aroma components

Summary 2-Phenylethanol and 2-phenylethylacetate important aroma components are obtained in substantial amounts whenHansenula anomala CBS 110 andKloeckera saturnus CBS 5761 are grown with L-phenylalanine as sole nitrogen source. Up to 2 g/L of mixtures of alcohol and ester are obtained. While in the first microorganism the alcohol predominates, in the second the acetate is formed almost exclusively. Experiments with 1-¹³C-d-Glucose show complete incorporation of the label into the methyl group of the acetate.     

Down's syndrome,Edwards' syndrome,Patau's syndrome —synthesis of glycosaminoglycans

Synthesis of glycosaminoglycans (GAGS) by fibroblasts derived from seven patients with Down's syndrome, five patients with Edwards' syndrome, and two patients with Patau's syndrome were studied in cell culture. The aneuploid strains were compared with diploid fibroblasts from age-matched controls. In terms of hyaluronic acid and sulfated GAG synthesis, the amount of synthesized hyaluronic acid was not significantly different between postnatal aneuploid strains and controls.     

Modulation of drug-induced cytotoxicity by a bispecific monoclonal antibody that recognizes the epidermal growth factor receptor and doxorubicin

A hybrid hybridoma secreting a bispecific hybrid mAb (bsmAb), which recognizes both the epidermal growth factor receptor (EGF-R) and the drug doxorubicin, was produced by somatic hybridization of two hybridomas. The bsmAb obtained was able to retarget doxorubicin cytotoxicity in vitro specifically on EGF-R-positive cells exerting at the same time an antidotal effect on cells that did not overexpress the EGF-R. Distribution studies in mice indicate that the bsmAb selectively delivers the drug to tumour cells and modifies doxorubicin biodistribution with a statistically significant decrease of drug concentration in the intestine, which is the main target of early anthracycline toxicity. In keeping with this finding is the remarkable antidotal activity exerted by bsmAb in mice treated with doxorubiein, which is proved by retardation in loss of body weight and mortality. The effectiveness on tumour growth of the mAb followed by the administration of doxorubicin appears to be equal to that of the drug alone; however, the bsmAb exerts a remarkable antidotal activity.     

Osmosensitivity of the 2H+−K+-exchange and the H+-ATPase complex F0F1 in anaerobically grownEscherichia coli

Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H⁺–K⁺-exchange in two steps, the first of which was DCCD¹ sensitive and osmo-dependent and had the 2H⁺/K⁺ stoichiometry. H⁺-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H⁺-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H⁺/K⁺-exchange was absent intrkA anduncA mutants. InuncB mutant 2H⁺/K⁺-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H⁺/K⁺-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H⁺/K⁺-exchange and the F₀F₁ and osmoregulation of the F₀F₁ via F₀ and a periplasmic space are postulated.Abbreviations F₀F₁ H⁺-ATPase complex - F₀ H⁺-channel, proteolipid - F₁ H⁺-ATPase - Trk constitutive system for K⁺ uptake - PV periplasmic protein valve - DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - _H or _K transmembrane electrochemical gradient for H⁺ or K⁺ respectively - membrane potential - upshock or downshock increase or decrease of medium osmolarity, respectively - CGSC E. coli Genetic Stock Center, Yale University, USA     

Sensorimotor transformation from light reception to phototactic behavior inDrosophila larvae (Diptera: Drosophilidae)

In this paper we examine theDrosophila melanogaster larval response to light. We survey the morphology of the larval visual and motor systems in relation to larval locomotory behavior and phototaxis. In addition, this paper proposes a model of sensorimotor transformation and examines the reversal in taxis occurring at theD. melanogaster larval wnadering stage.