Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g^-1) ] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g^-1, while the threshold for the ST was greater than 0.1.10³ CFU^-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g^-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.     

A pattern recognition machine

Summary A new probabilistic learning machine for pattern recognition is described. The machine operates on the basis of random criteria obtained from special pseudorandom generators. The input, particularly versatile due to the use of an image dissector, allows a random scan for which normal television storage pick-up tubes are not suitable. The computation data section, completely automatic, uses two ferrite core memories with a capacity of 64,000 bits. Consequently it is possible to obtain very quick recognizing cycles for a maximum of 16 classes of 256 learning examples each. Preliminary experiments are reported, some of which, like the recognition of meteorological events by the analysis of absolute baric topography maps, could already be suitable for practical application of the machine.This work was supported by Consiglio Nazionale delle Ricerche (C.N.R.) through the Gruppo Nazionale Cibernetica (G.N.C.).     

Sensitive detection of human growth hormone mRNA in routinely formalin-fixed,paraffin-embedded transgenic mouse tissues by non-isotopic in situ hybridization

A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.     

Actions and interactions of growth hormone and insulin-like growth factor-II: body and organ growth of transgenic mice

To characterize long-term actions and interactions of growth hormone (GH) and insulin-like growth factor-II (IGF-II) on postnatal body and organ growth, hemizygous phosphoenolpyruvate carboxykinase (PEPCK)-human IGF-II transgenic mice were crossed with hemizygous PEPCK-bovine GH transgenic mice. The latter are characterized by two-fold increased serum levels of IGF-I and exhibit markedly increased body, skeletal and organ growth. Four different genetic groups were obtained: mice harbouring the IGF-II transgene (I), the bGH transgene (B), or both transgenes (IB), and non- transgenic controls (C). These groups of mice have previously been studied for circulating IGF-I levels (Wolf et al., 1995a), whereas the present study deals with body and organ growth. Growth curves (week 3 to 12) were estimated by regression with linear and quadratic components of age on body weight and exhibited significantly (p < 0.001) greater linear coefficients in B and IB than in I and C mice. The linear coefficients of male I and C mice were significantly (p < 0.001) greater than those of their female counterparts, whereas this sex-related difference was absent in the bGH transgenic groups. The weights of internal organs as well as the weights of abdominal fat, skin and carcass were recorded from 3.5- to 8- month-old mice. In addition, organ weight-to-body weight-ratios (relative organ weights) were calculated. Except for the weight of abdominal fat, absolute organ weights were as a rule significantly greater in B and IB than in I and C mice. IGF-II overproduction as a tendency increased the weights of kidneys, adrenal glands, pancreas and uterus both in the absence and presence of the bGH transgene. Analysis of relative organ weights demonstrated significant (p < 0.05) effects of elevated IGF- II on the relative growth of kidneys (males and females) and adrenal glands (females), confirming our previous report on organ growth of PEPCK-IGF-II transgenic mice. In females, IGF-II and GH overproduction were additive in stimulating the growth of spleen and uterus, providing evidence for tissue-specific postnatal growth promoting effects by IGF-II in the presence of elevated IGF-I     

Three types of ion channels are present on the plasma membrane of Friend erythroleukemia cells

In Friend murine erythroleukemia cells the presence of ion channels was investigated with the patch-clamp technique. During the first 48 hours after cell seeding, three types of ion channels, with the following order of membrane density, were found: i) a Ca2+-dependent K+ channel, fully activated at a cytosolic Ca2+ concentration of 10(-6) M and moderately activated at 10(-7)M; ii) a monovalent cation channel non voltage-activated, with an open-close kinetics dependent on the pressure gradient across the patch; iii) a chloride channel with a slow open-close kinetics. The latter two channels were labile and did not survive during intracellular perfusion. The membrane potential of the leukemia cells was not constant, but underwent large (tens of millivolts) fluctuations due to the opening of a few channels. The average resting membrane potential recorded in this study agrees with that measured in these cells by means of the accumulation ratio of the lipophilic cation Tetraphenylphosphonium.     

Novel alpha-conotoxins from Conus spurius and the alpha-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.     

A HERG current sustains a cardiac-type action potential in neuroblastoma S cells

From the adrenergic SH-SY5Y human neuroblastoma clone, we isolated a subclone (21S) endowed with a glial-oriented phenotype. At difference from the parental clone, 21S cells responded to depolarizing stimuli with overshooting action potentials, whose repolarization phase was composed of an initial rapid episode, followed by a long-lasting plateau and a slow return to the resting potential (V(REST)). The action potential depolarization phase was sustained by a TTX-sensitive Na(+) current, while the first repolarizing episode was produced by the scanty delayed rectifier potassium current (I(KDR)) expressed in 21S cells. The bulk of repolarization, including the after-hyperpolarization, was sustained by the human eag related (HERG) potassium current (I(HERG)) that also governs V(REST) in 21S cells. This double role of I(HERG), together with the poor expression of I(KDRs), represents a novel finding in electrophysiology, as well as gives a clue to identify a new excitable element of the complex cellular population of neuroblastoma.