Low molecular weight factor in bovine caudal epididymal fluid that stimulates calcium uptake in caput spermatozoa

Secretions from the mammalian epididymis contain proteins that bind to developing sperm and are presumed to play a role in sperm maturation. The biochemical functions in sperm of most of these proteins are not known. In this report we describe the presence of a low molecular weight compound in bovine caudal epididymal luminal fluid (CF) that has a potent stimulatory effect on calcium (⁴⁵Ca²⁺) uptake in immature caput epididymal spermatozoa. The studies were initially undertaken to characterize the effect of the protein caltrin, present in bovine seminal plasma (BSP), on calcium uptake into caput spermatozoa. Caltrin is known to block calcium influx into mature bovine sperm. Unexpectedly, the kinetics of calcium uptake into caput sperm showed a biphasic response when treated with BSP, namely, a stimulation of uptake at 1 to 5 min and inhibition of uptake after this time. Since caudal sperm do not show this biphasic response, we reasoned that BSP contained a factor derived from CF that must interact with developing sperm before the binding of caltrin to sperm can prevent further calcium uptake. We first demonstrated that preincubation of caput sperm with CF eliminated the biphasic calcium uptake effect induced in caput sperm by BSP and that caudal fluid alone had a potent stimulatory effect on calcium uptake in caput sperm. Half-maximal stimulation (fivefold over control) occurred at a caudal fluid protein concentration of 0.27 mg/ml. Partial purification of the factor indicates that it is of low molecular weight (MW ～ 1,000), but further chemical characterization has not been carried out and its epididymal site of origin is not known. The results indicate that the regulation of intracellular calcium levels in sperm differs in immature and mature bovine sperm in that an epididymal factor promotes calcium uptake during epididymal maturation, and the seminal fluid protein caltrin prevents it at ejaculation.     

An integrated physical and genetic map of the nematode<Emphasis Type="Italic"> Pristionchus pacificus</Emphasis>

The free-living nematode Pristionchus pacificus is one of several species that have recently been developed as a satellite system for comparative functional studies in evolutionary developmental biology. Comparisons of developmental processes between P. pacificus and the well established model organism Caenorhabditis elegans at the cellular and genetic levels provide detailed insight into the molecular changes that shape evolutionary transitions. To facilitate genetic analysis and cloning of mutations in P. pacificus, we previously generated a BAC-based genetic linkage map for this organism. Here, we describe the construction of a physical map of the P. pacificus genome based on AFLP fingerprint analysis of 7747 BAC clones. Most of the SSCP markers used to generate the genetic linkage map were derived from BAC ends, so that the physical genome map and the genetic map can be integrated. The contigs that make up the physical map are evenly distributed over the genetic linkage map and no clustering is observed, indicating that the physical map provides a valid representation of the P. pacificus genome. The integrated genome map thus provides a framework for positional cloning and the study of genome evolution in nematodes.Communicated by G. Jürgens     

Nerve Growth Factor as a Mitogen for a Pancreatic Carcinoid Cell Line

Abstract: Carcinoid tumors are a group of neuroendocrine neoplasms distributed widely throughout the body but most commonly occurring in the gut. These tumors retain many characteristics of their neural crest origin, including secretion of neuroactive peptides and responsiveness to neurotrophic substances. Nerve growth factor (NGF), a neurotrophic protein involved in maintenance and differentiation of peripheral sympathetic and sensory neurons, regulates growth of several neural tumor cells by inducing a differentiated phenotype and subsequent inhibition of cell growth rate. We examined the actions of NGF in a functioning human pancreatic carcinoid cell line (termed BON). NGF has no effect on the cytoarchitecture or constitutive secretion of bioamines in this carcinoid cell line. NGF, however, stimulates the in vitro cellular proliferation of BON cells. BON cells possess mRNA for the NGF receptors (p75^LNGFR and p140^trkA) and membrane-associated tyrosine kinase activity is increased in response to NGF. Both the mitogenic activity of NGF, as well as the receptor-linked tyrosine kinase activity, can be abrogated in BON cells by the trkA inhibitor K-252a and specific anti-NGF antibody. Our studies demonstrate that NGF is a mitogen for this carcinoid cell line without effect on cellular phenotype or cytoarchitecture. NGF may play a role in the development and progression of human carcinoid tumors.     

Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae

Background

Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum β-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance.

Principal Findings

Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology.

Conclusions and Significance

The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae.     

Mating in Heliothis virescens: Transfer of juvenile hormone during copulation by male to female and stimulation of biosynthesis of endogenous juvenile hormone

Studies were undertaken to determine whether adult males of Heliothis virescens transfer juvenile hormone (JH) to females during copulation, and an in vitro radiochemical assay was used to determine whether mating causes an allatotropic effect, i.e., stimulation of JH biosynthesis by corpora allata (CA). In vitro, CA from 3-day-old mated females synthesized and released approximately 2.5 times total JH as that of CA from comparably aged virgin females. Of the homologues, JH II exhibited significant increase in mated females; JH I also increased but not significantly. JH III remained similar to that of virgin females. This is the first demonstration of an allatotropic effect of mating in moths. In contrast to the female, CA of virgin males did not produce any JH, but accessory sex glands (ASG) in 3-day-old males synthesized small amounts of JH. Immediately after adult emergence, male ASG contained approximately 1.5 ng JH I and II, which increased by 12 h after emergence and remained at this high level up to 54 h after emergence. JH III was barely detected in ASG. JH in ASG of mated male immediately after uncoupling was depleted almost completely, and 24 h later recovered to levels comparable to that of 54-h-old virgin male. Virgin female bursa copulatrix did not contain any JH, but mated female bursa, immediately after uncoupling, had JH at levels comparable to that observed in virgin male ASG. By 6 h after uncoupling, JH levels decreased dramatically in mated female bursa. These data suggest the transfer of JH to females by the male. Arch. Insect Biochem. Physiol. 38:100–107, 1998. © 1998 Wiley-Liss, Inc.     

Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly.

Two invariant tryptophan residues on the N-terminal extracellular region of the rat alpha1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA(A) (gamma-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA(A) receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant alpha1 subunits, coexpressed with beta2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [(3)H]muscimol, a GABA site ligand, but no binding to [(35)S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [(3)H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with beta2 and gamma2 polypeptides, the mutant alpha1 polypeptides did not form [(3)H]flunitrazepam binding sites though wild-type alpha1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant alpha1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between alpha and beta subunits, but not of the GABA binding site.     

Separation and nucleation control of α and β polymorphs of l-glutamic acid by swift cooling crystallization process

Separation of crystal nucleation of the two known polymorphs of l-glutamic acid, the metastable α and the stable β, from pure aqueous solution is attained by following a swift cooling crystallization process. Results elucidate a clear distinction of the preferred nucleation regions of α, β and combinations of α and β in the temperature range between 1 and 40°C. Also, the type of nucleation is supersaturation dependent: higher supersaturation favours α and lower supersaturation favours β. Morphology and structure of the polymorphs confirm their form of crystallization.     

Diversity analysis of pseudomonas in rice rhizosphere for multifaceted plant growth promotion

This investigation was carried out based on the hypothesis that there may be some pseudomonad strains, which could exist in rhizosphere of plant species contributing multifaceted beneficial activities. For this purpose, 21 pseudomonad isolates from the rhizosphere of rice, cultivated in western parts of Tamil Nadu were screened. All the 21 isolates were authenticated as pseudomonads by a genus-specific PCR screening. The molecular diversity of these isolates was investigated by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and the dendrogram obtained from the analysis revealed that all the 21 isolates clustered into seven groups. Further, these isolates were screened for plant growth promoting activities such as diazotrophy (PCR amplification of nifH gene and acetylene reduction assay), Indole acetic acid (IAA) and siderophore production (spectrometrically), 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase for ethylene regulation (PCR screening), mineral solubilization (biochemically) and antagonistic potential against soil pathogenic fungi (dual culture assay). Based on the results, two elite Pseudomonas isolates (S9 and O3) were chosen as multi-functional plant growth-promoting rhizobacteria, paving way for potential use as bioinoculants in rice.     

Beaming into the Rat World: Enabling Real-Time Interaction between Rat and Human Each at Their Own Scale

Immersive virtual reality (IVR) typically generates the illusion in participants that they are in the displayed virtual scene where they can experience and interact in events as if they were really happening. Teleoperator (TO) systems place people at a remote physical destination embodied as a robotic device, and where typically participants have the sensation of being at the destination, with the ability to interact with entities there. In this paper, we show how to combine IVR and TO to allow a new class of application. The participant in the IVR is represented in the destination by a physical robot (TO) and simultaneously the remote place and entities within it are represented to the participant in the IVR. Hence, the IVR participant has a normal virtual reality experience, but where his or her actions and behaviour control the remote robot and can therefore have physical consequences. Here, we show how such a system can be deployed to allow a human and a rat to operate together, but the human interacting with the rat on a human scale, and the rat interacting with the human on the rat scale. The human is represented in a rat arena by a small robot that is slaved to the human’s movements, whereas the tracked rat is represented to the human in the virtual reality by a humanoid avatar. We describe the system and also a study that was designed to test whether humans can successfully play a game with the rat. The results show that the system functioned well and that the humans were able to interact with the rat to fulfil the tasks of the game. This system opens up the possibility of new applications in the life sciences involving participant observation of and interaction with animals but at human scale.