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Philanthotoxins are uncompetitive antagonists of Ca2+-permeable AMPA receptors presumed to bind to the pore-forming region, but a detailed molecular mechanism for this interaction is missing. Here a small library of novel philanthotoxins was designed and synthesized using a solid-phase strategy. The biological activities were investigated at cloned and "native" AMPA receptors using electrophysiological techniques. A distinct relationship between length of the polyamine moiety and the location of a secondary amino group was observed. Fitting the data to the Woodhull equation allowed the first experimental demonstration of the relative location and orientation of the philanthotoxin molecule in the receptor. These results were corroborated by in silico studies using a homology model of the AMPA receptor ion channel. Together these studies provide strong evidence for a molecular mechanism by which polyamine toxins antagonize the AMPA receptor ion channel and provide the basis for rational development of uncompetitive antagonists of AMPA receptors.

A major neurotransmitter, L-Glutamate must be stored, transported and received, and these processes are mediated by proteins that bind this simple yet essential amino acid. Detailed evidence continues to emerge on the structure of Glu binding proteins, which includes both receptors and transporters. It appears that receptors and transporters bind to Glu in different conformations, which may present a pharmacological opportunity. This review will compare and contrast information available on Glu Receptors (AMPA, NMDA, KA and mGlu), excitatory amino acid transporters (EAATs), the system Xc- transporter (XCT) and the vesicular Glutamate transporter (GVT). The cross-reactivity of ligands which have been previously used to characterize the glutamate binding proteins with system Xc- raises some fundamental interpretational issues regarding the mechanisms through which these analogues produce CNS damage. Although at one time it was thought that unraveling selectivity among glutamate binding proteins was an intractable problem, recently the NMDA antagonist (memantine) has been approved for general medical practice for treatment of Alzheimer's disease. Two other agents are in advanced clinical trials: an Ampakine for potential improvement of cognitive disorders, and a selective mGlu agonist for treatment of anxiety. The prospects for unraveling cross-reactivity will be weighed in light of a critical comparison of the glutamate binding protein targets.

The growth factor receptor c-Kit has several well-characterized functions during the development of numerous cell types, including red blood cells, mast cells, and melanocytes. Its role in Schwann cells has been described in transformed cells derived from malignant peripheral nerve sheath tumors from patients with neurofibromatosis type 1 (NF1 MPNST; Badache et al. [1998] Oncogene 17:795-800). However, c-Kit functions have not been investigated in normal Schwann cells. We report here that neonatal rat Schwann cells express low c-Kit levels, whereas expression levels for c-Kit are high for Schwann cells derived from MPNST of NF1 patients. In addition, c-Kit expression is not detectable in normal adult human Schwann cells. Although the c-Kit ligand stem cell factor (SCF) induces the phosphorylation of protein kinase B (or Akt) and prevents apoptosis in Schwann cells, SCF has no effect on the proliferation or differentiation of Schwann cells., (Copyright 2005 Wiley-Liss, Inc.)

Age-related differences in cognitive performance are well documented. These differences are most pronounced during tasks with high demands on cognitive control, and it has been proposed that selective alteration of prefrontal activity is associated with cognitive changes in old age. Here, differences in the neural systems underlying selection requirements for older and younger adults were investigated using functional magnetic resonance imaging (fMRI). A verb generation task was used, and selection requirements were varied with regard to whether each noun could be associated with either few (scissors-cut) or many (ball-bounce, kick, throw...) competing alternatives. The two age groups showed statistically equivalent behavioral performance across the task conditions but marked differences in activation. Across both age groups, high selection demands activated several regions including bilateral frontal, left anterior frontal, left inferior temporal regions, and the dorsal anterior cingulate cortex (ACC). Between-group comparisons using region-of-interest analyses revealed less activation for senior adults in left inferior frontal gyrus (IFG), left inferior temporal gyrus, and the anterior cingulate and higher activation in right inferior frontal gyrus compared to young adults. These findings indicate age-related changes in multiple regions contributing to aspects of selection requirements during verb generation.

Utilizing our lateral metalation coupled with Jacobsen's catalytic asymmetric amino nitrile synthesis, we have demonstrated the ability to synthesize isoxazole-containing amino acid glutamate analogues in high yield and high enantiomeric excesses. Chiral centers alpha or beta at the C-5 position do not detract from diastereoselectivity of the Jacobsen-Strecker reaction. [reaction: see text]

Cognitive control requires the resolution of interference among competing and potentially conflicting representations. Such conflict can emerge at different points between stimulus input and response generation, with the net effect being that of compromising performance. The goal of this article was to dissociate the neural mechanisms underlying different sources of conflict to elucidate the architecture of the neural systems that implement cognitive control. By using functional magnetic resonance imaging and a verbal working memory task (item recognition), we examined brain activity related to two kinds of conflict with comparable behavioral consequences. In a trial of our item-recognition task, participants saw four letters, followed by a retention interval, and a probe letter that did or did not match one of the letters held in working memory (positive probe and negative probe, respectively). On some trials, conflict arose solely because of the current negative probe having a high familiarity, due to its membership in the immediately preceding trial's target set. On other trials, additional conflict arose because of the current negative probe having also been a positive probe on the immediately preceding trial, producing response-level conflict. Consistent with previous work, conflict due to high familiarity was associated with left prefrontal activation, but not with anterior cingulate activation. The response-conflict condition, when compared with high-familiarity conflict trials, was associated with anterior cingulate cortex activation, but with no additional left prefrontal activation. This double dissociation points to differing contributions of specific cortical areas to cognitive control, which are based on the source of conflict.

A series of 4-isoxazolyl-1,4-dihydropyridines (IDs) were prepared and characterized, and their interaction with the calcium channel was studied by patch clamp analysis. The structure-activity relationship (SAR) that emerges is distinct from the 4-aryldihydropyridines (DHPs), and affinity increases dramatically at higher holding potentials. Thus, among the 3'-arylisoxazolyl analogues p-Br > p-Cl >> p-F, and p-Cl > m-Cl > o-Cl >> o-MeO. Four of the analogues were examined by single-crystal X-ray diffractometry, and all were found to adopt an O-exo conformation in the solid state. The calculated barrier to rotation, however, suggests that rotation about the juncture between the heterocyclic rings is plausible under physiological conditions. A variable-temperature NMR study confirmed the computation. With Striessnig's computational sequence homologation procedure, a working hypothesis was derived from the data that explains the unique SAR for IDs.

The transition from childhood through adolescence to adulthood is a difficult stage, particularly for patients with type 1 diabetes. The yearning for autonomy and independence, as well as the hormonal changes around the time of puberty, can manifest in poor glycaemic control. The focus on diet and weight increases the prevalence of eating disorders, compounding the difficulties in supervising diabetes patients. This can be exacerbated by the realisation that hyperglycaemia induces weight loss and the use of this knowledge to further manipulate diabetes control to gain a desired body image. The management of adolescents with type 1 diabetes is therefore challenging and requires close collaboration between psychological medicine and diabetes teams. This review describes the difficulties frequently encountered, with a description of four cases illustrating these points. Case 1 demonstrates the problem of needle phobia in a newly diagnosed patient with type 1 diabetes leading to persistent hyperglycaemia, the recognition of weight loss associated with this and the development of bulimia. The patient's overall management was further complicated by risk-taking behaviour. By the age of 24 years, she has developed diabetic retinopathy and autonomic neuropathy and continues to partake in risk-taking behaviour. Case 2 illustrates how the lack of parental support shortly after the development of type 1 diabetes led to poor glycaemic control and how teenagers often omit insulin to accommodate lifestyle and risk-taking behaviour. Case 3 further exemplifies the difficulty in managing patients with needle phobia and the fear of hypoglycaemia. Case 4 adds further weight to the need for parental support and the impact of deleterious life events on glycaemic control by manipulation of insulin dosage., (Copyright 2001 John Wiley & Sons, Ltd.)

Human observers can discriminate two attributes from the same object more efficiently than attributes from two different objects even if the retinal locations of the attributes are the same in the single and dual object cases. The single object advantage challenges the spatial spotlight view of attention and suggests that attentional selection can be object based. We report that the single object advantage is reliably reduced when an object working memory task is performed concurrently, whereas concurrent verbal and spatial working memory tasks have no effect. This selective interference effect provides support for the existence of object-based attentional processes that also contribute to the short-term retention of objects in working memory. These results are consistent with the hypothesis that both attentional and memory subsystems are organized along domain-specific lines, and suggest the importance of attention in rehearsal operations. The contributions of inferior temporal and parietal mechanisms that have been implicated in attending to and remembering objects are considered.

Crying is attachment behavior triggered in infancy by separation and throughout life by loss. Adult crying, as reported and demonstrated in psychotherapy, reflects the quality of early attachment experiences as well as current adult bonds. Specific instances, as well as overall patterns, of crying and inhibited crying may be understood in light of attachment styles, biological factors, and socialization. Types of adult crying (healthy crying, crying for no reason, prolonged or frequent crying associated with depression or physiological disorders) and types of inhibited crying (healthy tearlessness, pervasive emotional control, detached tearlessness, and displaced crying) reflect attachment style and may, in addition, be assessed as healthy or symptomatic of psychological or physiological disorders or a combination.

Respiratory viruses are an extremely common cause of childhood morbidity. However, the current seroprevalence of viruses in infant populations is difficult to establish because invasive venipuncture may be technically and ethically unacceptable. This prospective study aimed to establish the seroprevalence of respiratory viruses in an infant population by use of a novel multiantigen fluorescence immunoassay against common respiratory viruses, using heel-prick blood samples collected on filter paper. Mothers and babies were recruited in the immediate peripartum period in the Royal Maternity Hospital, Belfast. Cord blood samples at birth and heel-prick filter paper blood samples at 7 mo were collected for measurement of virus-specific IgG to respiratory syncytial virus, influenza A virus, adenovirus, and parainfluenza virus type 1, type 2, and type 3 by indirect immunofluorescence using a multiviral assay developed for this purpose. Of 386 mothers approached, 325 (84%) permitted follow-up at 7 mo, and of these, 256 (79%) agreed to the heel prick. From 234 paired samples, 125 infections were documented. Adenovirus infections were commonest, 53 (22.6%), followed by respiratory syncytial virus, 32 (13.7%); influenza A virus, 22 (9.4%); parainfluenza virus type 3, 14 (6%); parainfluenza virus type 1, 2 (0.85%); and parainfluenza virus type 2, 2 (0.85%). These results demonstrate the seroprevalence of a range of respiratory viruses in an infant population, using a novel multiviral immunoassay. The filter paper collection of blood samples and multiantigen assay format has implications for easy, widespread viral serodiagnosis in both seroepidemiology studies and in the diagnosis of pediatric viral illnesses. Filter paper permits recovery of respiratory virus-specific IgG and can be used as a simple and acceptable epidemiologic and diagnostic tool.

Frequent violations of the assumption that data are normally distributed occur in exercise science and other life and behavioral sciences. When this assumption is violated, parametric statistical analyses may be inappropriate for data analysis. We provide a rationale for using a generalized form of nonparametric analyses based on the Puri and Sen (1985) L treated as a chi 2 approximation. If data do not meet the assumption of normality, this nonparametric approach has substantial power and is easy to use. An advantage of this generalized technique is that ranked data may be used in standard parametric statistical programs widely available on desktop and mainframe computers, for example, regression, analysis of variance (ANOVA), multivariate analysis of variance (MANOVA) within BioMed, SAS, SPSS. Once the data are ranked and analyzed with these programs, the only adjustment required is to use a standard formula to calculate the nonparametric test statistic, L, instead of the parametric test statistic (e.g., F). Thus, rank-order nonparametric models become parallel with their parametric counterparts allowing the researcher to select between them based on characteristics of the data distribution. Examples of this approach are provided using data from exercise science for regression, ANOVA (including repeated measures) and MANOVA techniques from SPSSPC. Using these procedures, researchers can easily examine data distributions and make an appropriate decision about parametric or nonparametric analyses while continuing to use their regular statistical packages.

Previous studies have indicated that porcine reproductive and respiratory syndrome virus (PRRSV) can be identified in and transmitted through boar semen. However, the site(s) of replication indicating the origin of PRRSV in semen has not been identified. To determine how PRRSV enters boar semen, five vasectomized and two nonvasectomized PRRSV-seronegative boars were intranasally inoculated with PRRSV isolate VR-2332. Semen was collected three times weekly from each boar and separated into cellular and cell-free (seminal plasma) fractions. Both fractions were evaluated by reverse transcriptase nested polymerase chain reaction (RT-nPCR) for the presence of PRRSV RNA. Viremia and serostatus were evaluated once weekly, and boars were euthanatized 21 days postinoculation (DPI). Tissues were collected and evaluated by RT-nPCR, virus isolation (VI), and immunohistochemistry to identify PRRSV RNA, infectious virus, or viral antigen, respectively. PRRSV RNA was identified in semen from all vasectomized and nonvasectomized boars and was most consistently found in the cell fraction, within cells identified with a macrophage marker. Viral replication as determined by VI was predominately found within lymphoid tissue. However, PRRSV RNA was widely disseminated throughout many tissues, including the reproductive tract at 21 DPI. These results indicate that PRRSV can enter semen independent of testicular or epididymal tissues, and the source of PRRSV in semen is virus-infected monocytes/macrophages or non-cell-associated virus in serum. PRRSV-infected macrophages in semen may result from infection of local tissue macrophages or may originate from PRRSV-infected circulating monocytes or macrophages.

Objectives: To determine whether vaccine virus is found in serum and semen of vaccinated boars, whether vaccination prevents subsequent shedding of wild-type virus after challenge exposure, and whether semen and blood variables are altered after vaccination or challenge exposure with wild-type virus, or both., Design: Throughout the 50-day postvaccination period, serum and semen from exposed boars were evaluated for the presence of porcine reproductive and respiratory syndrome virus (PRRSV). All boars were then challenge-exposed with PRRSV isolate VR-2332 and evaluated for an additional 27 days. Semen quality variables, serostatus, and blood variables were monitored., Animals: 7 PRRSV-seronegative adult boars., Procedure: Semen was collected 3 times weekly and evaluated by use of a nested reverse-transcriptase polymerase chain reaction for detection of PRRSV RNA. Serum was obtained weekly and evaluated by nested reverse-transcriptase polymerase chain reaction, virus isolation, and PRRSV ELISA. Semen quality variables were evaluated 3 times weekly, and CBC was performed weekly., Results: Vaccine virus was shed in the semen of all vaccinated boars, but shedding was of shorter duration in 4 of 5 vaccinated boars than that generally observed after exposure to wild-type virus. After challenge exposure, shedding of wild-type virus in semen was shortened or eliminated in 4 of 5 vaccinated boars. Percentage of forward movement and normal spermatozoal morphology and motility were significantly reduced in vaccinated boars after challenge exposure., Conclusions: Vaccine virus was shed in semen of vaccinated boars, but vaccination generally reduced or eliminated shedding of wild-type PRRSV after challenge exposure. Semen quality appeared to be less than optimal, particularly after vaccination and subsequent challenge exposure with wild-type virus., Clinical Relevance: Extra-label use of the PRRSV vaccine in boars remains controversial because some boars may still shed wild-type virus in semen after challenge exposure at postvaccination day 50. Semen quality also appeared to be altered after vaccination and subsequent challenge exposure.

Four seronegative adult boars were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332. Serum and semen were collected 2-3 times weekly for over 100 days postinoculation (DPI). Serum samples were assayed for PRRSV by virus isolation (VI) and a polymerase chain reaction (PCR) and screened for antibodies to PRRSV using the indirect fluorescent antibody (IFA) and virus neutralization (VN) tests. Semen was assayed for PRRSV RNA by PCR. Virus and viral RNA was detected in the serum of all boars within 1 DPI by Vi and/or PCR. However, VI results indicated that viremia was transient and occurred from 1 to 9 DPI. Viral RNA was detected in serum from 1 to 31 DPI. In the acute stage of the infection, PRRSV RNA was detected in serum by PCR prior to the presence of viral RNA in semen. The PRRSV RNA was detected in semen as early as 3 DPI and persisted for 25 DPI in 2 of the boars and 56 and 92 DPI in the remaining 2 boars. Detection of PRRSV RNA in semen occurred 2-8 and 28-35 days prior to the detection of antibodies by IFA and VN, respectively. PRRSV was isolated from the bulbourethral gland of the boar that shed viral RNA in semen for 92 DPI. These results suggest that PRRSV RNA can be detected by PCR in boar serum and semen, and may persist for variable periods of time. Viremia and the serologic status of the boar are not adequate indicators of when PRRSV or PRRSV RNA is being shed in the semen. Preliminary findings also indicated that neither shipping stress nor reinoculation with homologous PRRSV resulted in viremia or viral RNA shedding in semen.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a devastating disease in swine. The presence and transmission of PRRSV by boar semen has been demonstrated by using a swine bioassay. In this assay, 4- to 8-week-old pigs were inoculated intraperitoneally with semen from PRRSV-infected boars. Seroconversion of these piglets indicated the presence of PRRSV in semen. Seroconversion in gilts has also been demonstrated following artificial insemination with semen from PRRSV-infected boars. These methods of detecting PRRSV in boar semen are time-consuming, laborious, and expensive. The objective of this study was to develop a reliable and sensitive PCR assay to directly detect PRRSV in boar semen. Primers from open reading frames 1b and 7 of the PRRSV genome were used in nested PCRs. Virus was detected at concentrations as low as 10 infectious virions per ml in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a 32P-labeled oligonucleotide probe. The primers did not react with related arteriviruses or other swine viruses. The PCR assay showed good correlation with the swine bioassay, and both methods were superior to virus isolation. To consistently identify PRRSV in boar semen, the cell fraction was separated by centrifugation at 600 x g for 20 min, a lysis buffer without a reducing agent (2-mercaptoethanol) was used, and nondiluted and 1:20-diluted cell fractions were evaluated by PCR. PRRSV was not reliably detected in the seminal plasma fraction of boar semen.

Although defined formula diets may be useful for initial episodes of Crohn's disease, the effects of these diets on subsequent attacks of Crohn's disease or in conjunction with corticosteroids are unknown. To evaluate these issues, we studied 27 patients in a randomized prospective trial. Ten patients received only prednisone (group I), nine received only a defined formula diet (Vital HN [high nitrogen]) (group II), and eight received a combination of prednisone and Vital HN (group III). At the time of entry into the study, the groups were similar with respect to age, sex, Crohn's Disease Activity Index, previous and current treatments, anatomic site of disease, and nutritional status. After 1 month of treatment, we noted seven successes (70%) and three failures in group I (prednisone only), three successes (33%) and six failures in group II (Vital HN only), and six successes (75%) and two failures in group III (combination therapy). Four patients randomized to receive only Vital HN were unable or unwilling to tolerate the defined formula diet. Of the five patients who were able to take the defined formula diet for 1 month, however, three (60%) were successfully treated. The patients who received prednisone (groups I and III) responded better than did the patients who received only the defined formula diet. These results may be attributable to the use of a nonelemental diet or the treatment of patients who were not experiencing an initial attack of Crohn's disease or who had previously received corticosteroids. The expensive and often poorly tolerated defined formula diets should not be considered as a substitute for standard therapy with corticosteroids in Crohn's disease.

Polyacrylamide gel electrophoresis was conducted on genomic RNA extracted from rotaviruses detected in diarrheic pigs from conventional swine herds. Ninety samples contained sufficient virus for RNA band visualization and genome classification. Genome profiles were characteristic of typical group A rotaviruses in 67.8% of the 90 samples, of group B rotaviruses in 10.0%, and of group C rotaviruses in 11.1%. In 11.1% of the samples, the presence of more than 11 bands suggested concurrent infection with more than 1 strain of rotavirus. In infections among nursing pigs, 76.4% were group A rotaviruses, 7.4% were group B, 7.4% were group C, and 8.8% were coinfections. In infections among weaned pigs, 40.9% were group A, 18.2% were group B, 22.7% were group C, and 18.2% were coinfections. Coelectrophoresis with prototype OSU and Gottfried strains revealed a great diversity in electropherotype among field strains of rotavirus.