Centrifugation stress reduces the responsiveness of spermatozoa to a capacitation stimulus in in vitro-aged semen.

Density gradient centrifugation of semen is commonly used in many assisted reproduction techniques. Although gradients have the potential to isolate and enrich motile and viable spermatozoa, the centrifugation force presents a stress factor to cell organelles and membranes. The objective of the study was to evaluate the impact of density gradient centrifugation stress on sperm capacitation dynamics, cell stability and the ability of spermatozoa to specifically respond to bicarbonate in extended semen undergoing in vitro ageing. Extended boar semen (n = 7) was stored for 12, 24, 72 and 120 h respectively at 17 °C before centrifugation and incubation in variations of an in vitro capacitation medium. The number of viable, acrosome intact sperm and motility parameters as assessed by computer-assisted semen analysis did not change during storage. Kinetic changes in viability (plasma membrane integrity) and intracellular calcium levels (calcium influx) during in vitro capacitation were assessed after preparation of semen samples with both, a Percoll and a sucrose gradient centrifugation, either only Percoll, only sucrose centrifugation or no centrifugation. Changes in the viable sperm population that could be specifically attributed as a response to either bicarbonate or calcium were determined. In in vitro-aged (>12 h stored) spermatozoa, centrifugation reduced the proportion of spermatozoa which specifically responded to the capacitating stimulus bicarbonate. Concomitantly, centrifugation increased the proportion of spermatozoa responding to calcium in absence of bicarbonate, thus indicating an increased sensitivity to incubation per se. Absence of centrifugation steps during semen preparation, revealed a highly conserved ability of in vitro-aged spermatozoa to specifically respond to bicarbonate. In conclusion, density gradient centrifugation alters the physiological property of spermatozoa for controlled capacitation, which may influence the success rates of centrifuged semen in assisted reproductive technologies and confound interpretation of capacitation assays.

Slow binocular rivalry in bipolar disorder.

BACKGROUND: The rate of binocular rivalry has been reported to be slower in subjects with bipolar disorder than in controls when tested with drifting, vertical and horizontal gratings of high spatial frequency. METHOD: Here we assess the rate of binocular rivalry with stationary, vertical and horizontal gratings of low spatial frequency in 30 subjects with bipolar disorder, 30 age- and sex-matched controls, 18 subjects with schizophrenia and 18 subjects with major depression. Along with rivalry rate, the predominance of each of the rivaling images was assessed, as was the distribution of normalized rivalry intervals. RESULTS: The bipolar group demonstrated significantly slower rivalry than the control, schizophrenia and major depression groups. The schizophrenia and major depression groups did not differ significantly from the control group. Predominance values did not differ according to diagnosis and the distribution of normalized rivalry intervals was well described by a gamma function in all groups. CONCLUSIONS: The results provide further evidence that binocular rivalry is slow in bipolar disorder and demonstrate that rivalry predominance and the distribution of normalized rivalry intervals are not abnormal in bipolar disorder. It is also shown by comparison with previous work, that high strength stimuli more effectively distinguish bipolar from control subjects than low strength stimuli. The data on schizophrenia and major depression suggest the need for large-scale specificity trials. Further study is also required to assess genetic and pathophysiological factors as well as the potential effects of state, medication, and clinical and biological subtypes.

Raf-induced transformation requires an interleukin 1 autocrine loop.

The c-Raf-1 serine/threonine protein kinase plays a critical role in the proliferation of most cell types that have been examined. As such, the Raf proto-oncogene is thought to play a central role in the development of human tumors. Although the c-raf-1 gene itself rarely appears to be mutated in human tumors, the kinase activity of Raf is frequently found to be more active in tumor cells, likely through constitutive activation of upstream activators of Raf. The downstream events triggered by Raf that are involved in transformation have been studied less extensively. We show in this study that Raf-induced transformation of NIH 3T3 cells requires the activation of the ubiquitously expressed transcription factor, nuclear factor-kappaB, by Raf. Furthermore, through the use of CrmA, interleukin 1 (IL-1) receptor antagonist, and a dominant-negative form of TRAF6, we demonstrate a requirement for IL-1 production and signaling from the IL-1 receptor as necessary components of Raf-induced transformation. These results indicate that IL-1 may be used as an autocrine growth factor by a number of tumors in which activation of Raf plays an important role in transformation and suggest that blockade of IL-1 signaling may be an approach to limiting the growth of certain tumors.

Interhemispheric switching mediates perceptual rivalry.

BACKGROUND: Binocular rivalry refers to the alternating perceptual states that occur when the images seen by the two eyes are too different to be fused into a single percept. Logothetis and colleagues have challenged suggestions that this phenomenon occurs early in the visual pathway. They have shown that, in alert monkeys, neurons in the primary visual cortex continue to respond to their preferred stimulus despite the monkey reporting its absence. Moreover, they found that neural activity higher in the visual pathway is highly correlated with the monkey's reported percept. These and other findings suggest that the neural substrate of binocular rivalry must involve high levels, perhaps the same levels involved in reversible figure alternations. RESULTS: We present evidence that activation or disruption of a single hemisphere in human subjects affects the perceptual alternations of binocular rivalry. Unilateral caloric vestibular stimulation changed the ratio of time spent in each competing perceptual state. Transcranial magnetic stimulation applied to one hemisphere disrupted normal perceptual alternations when the stimulation was timed to occur at one phase of the perceptual switch, but not at the other. Furthermore, activation of a single hemisphere by caloric stimulation affected the perceptual alternations of a reversible figure, the Necker cube. CONCLUSIONS: Our findings suggest that interhemispheric switching mediates perceptual rivalry. Thus, competition for awareness in both binocular rivalry and reversible figures occurs between, rather than within, each hemisphere. This interhemispheric switch hypothesis has implications for understanding the neural mechanisms of conscious experience and also has clinical relevance as the rate of both types of perceptual rivalry is slow in bipolar disorder (manic depression).

Co-stimulation of promoter for low density lipoprotein receptor gene by sterol regulatory element-binding protein and Sp1 is specifically disrupted by the yin yang 1 protein.

Sterol regulation of gene expression in mammalian cells is mediated by an interaction between the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs) and promoter-specific but generic co-regulatory transcription factors such as Sp1 and NF-Y/CBF. Thus, sterol-regulated promoters that require different co-regulatory factors could be regulated independently through targeting the specific interaction between the SREBPs and the individual co-regulatory proteins. In the present studies we demonstrate that transiently expressed yin yang 1 protein (YY1) inhibits the SREBP-mediated activation of the low density lipoprotein (LDL) receptor in a sensitive and dose-dependent manner. The inhibition is independent of YY1 binding directly to the LDL receptor promoter, and we show that the same region of YY1 that interacts in solution with Sp1 also interacts with SREBP. Furthermore, other SREBP-regulated genes that are not co-regulated by Sp1 are either not affected at all or are not as sensitive to the repression. Thus, the specific interaction that occurs between SREBPs and Sp1 to stimulate the LDL receptor promoter is a specific target for inhibition by the YY1 protein, and we provide evidence that the mechanism can be at least partially explained by the ability of YY1 to inhibit the interaction between SREBP and Sp1 in solution in vitro. The LDL receptor is the key gene of cholesterol uptake, and the rate-controlling genes of cholesterol synthesis are stimulated by the concerted action of SREBPs along with coregulators that are distinct from Sp1. Therefore, repression of gene expression through specifically targeting the interaction between SREBP and Sp1 would provide a molecular mechanism to explain how cholesterol uptake can be regulated independently from cholesterol biosynthesis in mammalian cells.

Rapid streptococcal testing in Vietnamese children with pharyngitis.

Streptococcal pharyngitis has been a significant public health problem in Vietnam for many years. Accurate diagnosis of the infection, however, has been difficult. We carried out a clinical trial of a rapid streptococcal antigen detection test (Quick-Vue (R) Flex Strep A) on a population of 777 children with pharyngitis seen at the Institute for the Protection of Children's Health (Children's Hospital) in Hanoi, Vietnam. Bacterial culture was performed in parallel with the rapid test on simultaneously obtained throat swab specimens. The rapid test was found to be 89% sensitive and 92% specific (96% in children not on prior antibiotics) compared to culture. The test was also found to be convenient and acceptable to patients and clinicians. A significant benefit of the test is that those children found positive are more likely to be treated with penicillin rather than a broad spectrum antimicrobial, which in turn will reduce the likelihood of resistant infections in the future.

Unusual protein-binding specificity and capacity of aza-arenophilic gels.

A number of synthetic aza-arenophilic gels have been synthesized from Sepharose CL-4B, 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine. These show high binding capacities for immunoglobulins and enzymes. Under high-salt buffer binding conditions, IgG can be effectively eluted, essentially free of contamination by BSA, using acidic conditions (pH 2.5) or phosphate buffers (pH 7.4) containing nucleophiles. Enzymes can also be readily adsorbed and desorbed. Thus these gels can be reused as supports for the immobilization of enzymes.

Patient selection may affect gene therapy success. Dominant negative effects observed for ornithine transcarbamylase in mouse and human hepatocytes.

We have achieved significant improvement of ornithine transcarbamylase deficiency (OTCD) in a mouse model through adenoviral-mediated gene transfer of the human ornithine transcarbamylase cDNA. Substantial reduction in orotic aciduria was observed within 24 h of treatment. Metabolic correction was later associated with phenotypic correction and moderate increase in enzymatic activity. In an effort to identify the level of gene expression required to achieve wild-type levels of enzyme activity we uncovered a dominant negative effect of the endogenous mutant protein on the activity of the delivered recombinant wild-type protein. This phenomenon is relevant to homomultimeric protein defects such as OTCD, represent a challenging category of disorders for gene therapy. Thus, although our findings indicate that adenoviral-mediated gene transfer may have potential as a short-term treatment for OTCD in humans and may be effective especially during catabolic crisis, the observations in this study suggest that careful patient selection based on mutation class may be essential for initial OTCD gene therapy trials, and perhaps, for other homomultimeric enzyme deficiencies being considered as gene therapy targets.

Infant feeding practices in Viet Nam.

PIP: Data from the 1988 Vietnam Demographic and Health Survey and the 1994 Demographic Survey are used to determine the trends in breast feeding and amenorrhea among ever married women of reproductive age. Life table procedures are used to calculate monthly probabilities of weaning. Then five month moving averages of equal weight are computed for observed monthly probabilities of weaning. The smoothed probabilities are used to calculate the cumulative proportion weaned at successive monthly ages. Breast feeding is universal in Vietnam. Infants are put to the breast earlier when delivery occurs at home. Almost all children are breast fed through the first six months, and 80% are breast fed for a year. The median duration was 15.3 months in the 1988 survey and 15.9 months in the 1994 survey based on life table methods. Calculations based on current status methods were slightly higher for both years. Rural women tended to breast feed longer than urban women. Children who had mothers working in agriculture were breast fed longer than children whose mothers had other occupations. Socioeconomic factors did not correlate well with breast feeding duration. Findings indicate that over 66% of breast fed infants aged under 3 months were given plain water, and over 90% of infants aged 3-5 months were given plain water. Fresh cow's milk is not given to Vietnamese infants. Juices were given to children aged older than 6 months. Sugar water was given to younger infants. The introduction of supplemental liquids was more common in urban areas. Few infants during the first few months of life were given solid or mushy foods. But by 4 months of age, 50% of infants were given solid or mushy foods, and the practice was more common in rural areas. The urban-rural gap closed by 6 months of age. Over 90% of infants received solids at 9 months. It is expected that modernization will negatively impact on breast feeding.^ieng

Rapid purification of immunoglobulin G using aza-arenophilic chromatography: novel mode of protein-solid phase interactions.

A derivative of aza-arenophilic gel having a dichlorosubstituent and an hydroxy ion as a capping nucleophile has been prepared. The properties of this gel in relation to IgG purification have been investigated in details. In the presence of high salt (1.5 M), albumin and some other serum proteins did not bind to the gel. IgG and some other minor proteins, however, were bound to the gel. The bound proteins can be eluted with an acidic buffer. SDS-PAGE showed that the fraction eluted with 0.1 M sodium acetate pH 4.2 consisted mainly of IgGs.

Flow-injection analysis of serum urea using o-phthalaldehyde and naphthylethylenediamine.

An analytical procedure is developed for the determination of urea using flow injection analysis. The methodology is based on the color that develops (lambda(max), 517 nm) when urea reacts with o-phthalaldehyde in the presence of naphthylethylenediamine under acidic conditions. Calibration curves are found to be linear up to 51 mg urea N/dl when the FIA manifold is operated at 42 degrees , utilizing 90-mul samples and a flow-rate of 0.44 ml/min. By manual injection up to 40 samples can be analysed per hour. Recovery yields varied between 95-99%. The within day CV was 0.5-2.2% whereas the day to day CV was 2.1-3.9%. When applied to the analysis of urea in serum samples, excellent correlations (0.998) are obtained when the FIA results are compared with those obtained for the same serum samples analysed by the free urease/Berthelot's and the hospital method (employing the Abbot Bichromatic Analyser). Interferences are observed with sulphur compounds such as sulphanilamide (0.76 mg/dl) as well as with many amino acids but at relatively high concentrations (21 mg/dl).

Avid AL, a synthetic ligand affinity gel mimicking immobilized bacterial antibody receptor for purification of immunoglobulin G.

Avid AL is an affinity gel designed for the purification of immunoglobulin G (IgG). The gel was prepared by first reacting Sepharose with 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine and then with 2-mercaptoethanol. The IgG purified by Avid AL is about 95% pure. The binding parameters of Avid AL for the whole IgG, Fab and Fc fragment and the stability of gel were investigated. The IgG bound to Avid AL can be eluted with an acidic buffer or with a novel neutral buffer containing electron donors. The development of such a mild neutral elution buffer is described. Application of Avid AL in a rapid gram-scale IgG purification was demonstrated. The possible mechanism of IgG binding is discussed.

Use of microwell plates carrying hydrazide groups to enhance antibody immobilization in enzyme immunoassays.

Using microwell plates functionalized with surface hydrazide groups, we site-specifically immobilized antibodies to the surface of the wells. The procedure for site-specific, oriented immobilization of IgG to hydrazide surfaces is simple. It requires a brief, mild oxidation of the carbohydrate side chains of IgG with NaIO4 at a pH of approximately 5 and incubation of the oxidized IgG with the hydrazide surface. These oxidized immunoglobulins bind to the hydrazide groups of these plates preferentially by the Fc region of the molecule, resulting in improved specific activity. Enzyme immunoassays for horseradish peroxidase and human IgG were tested on both standard (hydrophobic) and hydrazide group-carrying surfaces. The improved antigen binding capacity of the hydrazide-modified plates results in greatly increased sensitivity (approximately 4 x) and improved linearity in both assays when compared to native, nonoxidized antibody used with standard microwell plates. Furthermore, the hydrazide functionalized plates exhibited lower non specific binding.

Rapid and simple isolation of multigram goat IgG from serum using Avid AL and radial flow column.

Multigram quantities (2.5-10 g) of highly purified IgG were obtained within 4 h from serum by using Avid AL packed in a radial-flow column. Avid AL is an affinity gel containing a synthetic, low-mol-wt ligand capable of selectively binding IgG from serum of all animal species tested. By packing the gel in a radial-flow column up to 500 mL, a high flow rate of 50 mL/min can be achieved without adversely affecting the performance of the gel and the purity of the isolated antibody.

Flow injection analysis of lactose using covalently immobilized beta-galactosidase, mutarotase, and glucose oxidase/peroxidase on a 2-fluoro-1-methylpyridinium salt-activated Fractogel support.

Milk samples were analyzed for their lactose content using flow injection analysis and incorporating immobilized beta-galactosidase or beta-galactosidase/mutarotase and glucose oxidase/peroxidase bioreactors. These enzymes were immobilized, under mild conditions, on to a 2-fluoro-1-methylpyridinium salt-activated Fractogel support. The use of a phosphate buffer (0.15 M) was found to facilitate the rapid mutarotation of alpha-D-glucose and hence could obviate the need for the more expensive mutarotase. The chromogenic agents of choice for monitoring the reaction were 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid. Linearity was observed over the concentration range 16-160 micrograms/ml using lactose standards (r = 0.996). Between 30 and 40 milk samples/h can be analyzed. Comparisons are made with existing HPLC and alkaline methylamine methods for a range of milk matrices. The FIA method consistently gives the lowest standard deviations and coefficient of variation for the various milk matrices analyzed.

Simple, quick and efficient site-directed antibody immobilization in a cartridge.

A method is described for the simple, quick and efficient attachment of antibody within a cartridge for use as an immunoaffinity chromatography column. Antibodies are immobilized via their Fc regions through the use of periodate-oxidized carbohydrate functionalities of the immunoglobulin G. The method allows for the in situ coupling of the immunoglobulin G without prior removal of the oxidizing periodate solution. The entire procedure can be completed in 50 minutes. This method is especially useful for quick determinations of a particular monoclonal antibody's functionality or avidity towards a specific antigen. It may also be used in place of a conventional immunoaffinity column for the rapid isolation of small amounts of an antigen. This method will reduce the lengthy process of preparing an immunoaffinity column from several days to less than an hour.