Genetic regulation of Nrxn1 [corrected] expression: an integrative cross-species analysis of schizophrenia candidate genes.

Neurexin 1 (NRXN1) is a large presynaptic transmembrane protein that has complex and variable patterns of expression in the brain. Sequence variants in NRXN1 are associated with differences in cognition, and with schizophrenia and autism. The murine Nrxn1 gene is also highly polymorphic and is associated with significant variation in expression that is under strong genetic control. Here, we use co-expression analysis, high coverage genomic sequence, and expression quantitative trait locus (eQTL) mapping to study the regulation of this gene in the brain. We profiled a family of 72 isogenic progeny strains of a cross between C57BL/6J and DBA/2J (the BXD family) using exon arrays and massively parallel RNA sequencing. Expression of most Nrxn1 exons have high genetic correlation (r>0.6) because of the segregation of a common trans eQTL on chromosome (Chr) 8 and a common cis eQTL on Chr 17. These two loci are also linked to murine phenotypes relevant to schizophrenia and to a novel human schizophrenia candidate gene with high neuronal expression (Pleckstrin and Sec7 domain containing 3). In both human and mice, NRXN1 is co-expressed with numerous synaptic and cell signaling genes, and known schizophrenia candidates. Cross-species co-expression and protein interaction network analyses identified glycogen synthase kinase 3 beta (GSK3B) as one of the most consistent and conserved covariates of NRXN1. By using the Molecular Genetics of Schizophrenia data set, we were able to test and confirm that markers in NRXN1 and GSK3B have epistatic interactions in human populations that can jointly modulate risk of schizophrenia.

Sudden cardiac death in the young: a clinical genetic approach.

The sudden death of a young person is a devastating event for both the family and community. Over the last decade, significant advances have been made in understanding both the clinical and genetic basis of sudden cardiac death in the young. Many of the causes of sudden death in the young are due to genetic heart disorders, which can lead to both structural (e.g. hypertrophic cardiomyopathy) and arrhythmogenic (e.g. familial long QT syndrome) abnormalities. Most commonly, sudden cardiac death in the young can be the first presentation of an underlying heart problem, leaving the family at a loss as to why an otherwise healthy young person has died. Not only is this a tragic event for those involved, but it also presents a medical challenge to the clinician involved in the management of the surviving family members. Evaluation of families requires a multidisciplinary approach, which should include cardiologists, a clinical geneticist, a genetic counsellor and the forensic pathologist directly involved in the sudden death case. This multifaceted cardiac genetic service is crucial in the evaluation and management of the clinical, genetic, psychological and social complexities observed in families in which there has been a young sudden cardiac death.

Compound and double mutations in patients with hypertrophic cardiomyopathy: implications for genetic testing and counselling.

OBJECTIVE: To report the frequency of single and multiple gene mutations in an Australian cohort of patients with hypertrophic cardiomyopathy (HCM). METHODS: Genetic screening of seven HCM genes (beta-MHC, MyBP-C, cTnT, cTnI, ACTC, MYL2, and MYL3) was undertaken in 80 unrelated probands. Screening was by denaturing high performance liquid chromatography and direct DNA sequencing. Clinical data were collected on all patients and on genotyped family members. RESULTS: 26 mutations were identified in 23 families (29%). Nineteen probands (24%) had single mutations (11 beta-MHC, 4 MyBP-C, 3 cTnI, 1 cTnT). Multiple gene mutations were identified in four probands (5%): one had a double mutation and the others had compound mutations. Six of 14 affected individuals from multiple mutation families (43%) experienced a sudden cardiac death event, compared with 10 of 55 affected members (18%) from single mutation families (p = 0.05). There was an increase in septal wall thickness in patients with compound mutations (mean (SD): 30.7 (3.1) v 24.4 (7.4) mm; p<0.05). CONCLUSIONS: Multiple gene mutations occurring in HCM families may result in a more severe clinical phenotype because of a "double dose" effect. This highlights the importance of screening the entire panel of HCM genes even after a single mutation has been identified.

Effectiveness of attention rehabilitation after an acquired brain injury: a meta-analysis.

The efficacy of attention rehabilitation after an acquired brain injury was examined meta-analytically. Thirty studies with a total of 359 participants met the authors' selection criteria. Studies were categorized according to whether training efficacy was evaluated by comparing pre- and posttraining scores only or included a control condition as well. Performance improved significantly (using the d+ statistic) after training in pre-post only studies but not in pre-post with control studies. Further analyses showed that specific-skills training significantly improved performance of tasks requiring attention but that the cognitive-retraining methods included in the meta-analysis did not significantly affect outcomes. These findings demonstrate that acquired deficits of attention are treatable using specific-skills training. Implications of these results for rehabilitation theory and future research are discussed.

The Caenorhabditis elegans mel-11 myosin phosphatase regulatory subunit affects tissue contraction in the somatic gonad and the embryonic epidermis and genetically interacts with the Rac signaling pathway.

Caenorhabditis elegans embryonic elongation is driven by cell shape changes that cause a contraction of the epidermal cell layer enclosing the embryo. We have previously shown that this process requires a Rho-associated kinase (LET-502) and is opposed by the activity of a myosin phosphatase regulatory subunit (MEL-11). We now extend our characterization and show that mel-11 activity is required both in the epidermis during embryonic elongation and in the spermatheca of the adult somatic gonad. let-502 and mel-11 reporter gene constructs show reciprocal expression patterns in the embryonic epidermis and the spermatheca, and mutations of the two genes have opposite effects in these two tissues. These results are consistent with let-502 and mel-11 mediating tissue contraction and relaxation, respectively. We also find that mel-11 embryonic inviability is genetically enhanced by mutations in a Rac signaling pathway, suggesting that Rac potentiates or acts in parallel with the activity of the myosin phosphatase complex. Since Rho has been implicated in promoting cellular contraction, our results support a mechanism by which epithelial morphogenesis is regulated by the counteracting activities of Rho and Rac.

Fatigue after stroke.

OBJECTIVE: To determine the frequency and outcome of fatigue, its impact on functioning, and its relationship with depression in patients 3 to 13 months poststroke. DESIGN: Survey. SETTING: Community. PARTICIPANTS: Eighty-eight individuals from a pool of 181 consecutive patients previously admitted to an acute stroke service who were willing and able to complete the self-report questionnaires, and 56 elderly controls living independently in the community. MAIN OUTCOME MEASURES: Fatigue Impact Scale (a self-report measure of the presence and severity of fatigue and its impact on cognitive, physical, and psychosocial functions) and the Geriatric Depression Scale. RESULTS: The frequency of self-reported fatigue problems was greater in the stroke group (68%) than in the control group (36%, p < .001) and was not related to time poststroke, stroke severity, or lesion location. Forty percent of the stroke group reported that fatigue was either their worst or one of their worst symptoms. Patients attributed more functional limitations to their fatigue than did control subjects with fatigue. Although the presence of fatigue was independent of depression, the impact of fatigue on functional abilities was strongly influenced by depression. CONCLUSION: Fatigue can contribute to functional impairment up to 13 months after stroke, and its recognition and treatment are important for maximizing recovery.

Peripheral blood CD34+ cell immunomagnetic selection in breast cancer patients: effect on hematopoietic progenitor content and hematologic recovery after high-dose chemotherapy and autotransplantation.

BACKGROUND: Tumor cells have been detected in mobilized peripheral blood of breast cancer patients, and they may contribute to tumor recurrence after the transplantation of peripheral blood progenitor cells. One of the most widespread technologies for tumor purging of the graft is immunomagnetic hematopoietic progenitor cell selection. STUDY DESIGN AND METHODS: The study assessed the effectiveness of a magnetic cell-separation system in selecting functional subpopulations of hematopoietic progenitors from 14 blood-derived harvests of 11 patients with high-risk breast cancer after mobilization following cytotoxic chemotherapy supported by granulocyte--colony-stimulating factor, as well as the feasibility of transplanting these selected subpopulations. RESULTS: CD34(+)-enriched cell fractions had a median purity of 93.0 percent (72.7-98.5%). The procedure yielded 52.6 percent of the CD34+ cell input (39.4-116.8%). Median recoveries of colony-forming units (CFUs) (36.87%) and cobblestone area-forming cells (CAFCs) (152.5%) were, respectively, 0.70 and 2.87 times those of CD34+ cells (52.6%). Moreover, CAFC efficiency in the positive cell fraction was 2.57 times that in the starting cell fraction. Peripheral blood neutrophil counts of 0.5 x 10(9) per L and platelet counts of 20 x 10(9) per L were reached after median times of 9 and 11 days, respectively. The number of transfused CAFCs per kg, CD34+ cells per kg, and postthaw CFU-granulocyte-macrophage per kg was correlated, respectively, with the speed of engraftment of neutrophils, platelets, or both. Tumor cells detected in one patient's peripheral blood were not found after CD34+ cell selection. CONCLUSION: Transplantation of immunomagnetically purified peripheral blood CD34+ cells does not increase transplantation-related morbidity. It induces a selective enrichment of more immature hematopoietic progenitors, which makes it suitable for use in cell expansion and gene therapy protocols.

Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape.

We have identified two genes associated with the hypodermal cell shape changes that occur during elongation of the Caenorhabditis elegans embryo. The first gene, called let-502, encodes a protein with high similarity to Rho-binding Ser/Thr kinases and to human myotonic dystrophy kinase (DM-kinase). Strong mutations in let-502 block embryonic elongation, and let-502 reporter constructs are expressed in hypodermal cells at the elongation stage of development. The second gene, mel-11, was identified by mutations that act as extragenic suppressors of let-502. mel-11 encodes a protein similar to the 110- to 133-kD regulatory subunits of vertebrate smooth muscle myosin-associated phosphatase (PP-1M). We suggest that the LET-502 kinase and the MEL-11 phosphatase subunit act in a pathway linking a signal generated by the small GTP-binding protein Rho to a myosin-based hypodermal contractile system that drives embryonic elongation. LET-502 may directly regulate the activity of the MEL-11 containing phosphatase complex and the similarity between LET-502 and DM-kinase suggests a similar function for DM-kinase.

Evidence for multiple routes of speech production in a case of fluent aphasia.

A case study is reported of a 24-year old woman who developed fluent aphasia with superior reading relative to auditory comprehension following herpes simplex encephalitis. Her language disturbance showed exceptional features: oral reading, repetition and naming to confrontation were severely impaired and yet her spontaneous speech recovered to be relatively intact. These features are not consistent with Wernicke's aphasia, pure word deafness or any classic aphasic syndromes. These findings indicate the presence of several routes for phonological output that may be differentially impaired.

Scopolamine differentially affects memory of 8- and 16-month-old rats in the double Y-maze.

The present study investigated the effects of scopolamine on working and reference memory in the same rats at 8 and 16 months of age. Rats were trained in the double Y-maze until a criterion of > or = 88% correct was reached on both memory components. Doses of scopolamine (0.1, 0.4, 0.8 mg/kg for rats at 8 months; 0.05, 0.1, 0.4 mg/kg for rats at 16 months) were administered in a counterbalanced order 30 min before test sessions which also included delays of 0, 5, or 30 s prior to both memory components. Results showed that at both ages the 0.1 mg/kg scopolamine dose selectively impaired working memory, whereas higher doses impaired both working and reference memory. Delays selectively decreased working memory choice accuracy and enhanced the effect of scopolamine. Rats at 16 months performed less well on both reference and working memory and showed greater impairments with scopolamine and delays. The present findings support the hypothesis that a decrease in cholinergic neurotransmission contributes to age-related memory deficits.

Picolinic acid blocks the neurotoxic but not the neuroexcitant properties of quinolinic acid in the rat brain: evidence from turning behaviour and tyrosine hydroxylase immunohistochemistry.

Previous results suggest that the tryptophan metabolite, picolinic acid may have the unusual properties of antagonizing the neurotoxic but not the neuroexcitant effects of another tryptophan metabolite, quinolinic acid in the central nervous system. The present experiments tested this possibility utilizing behavioural and tyrosine hydroxylase immunohistochemical techniques. In the first series of experiments, rats received injections of relatively high concentrations of 6-hydroxydopamine (12 micrograms in 1 or 2 microliters), quinolinic acid (120 nmol in 0.5 microliters), picolinic acid (480 nmol in 0.5 microliters) or co-treatments (0.5 microliters) with quinolinic (120 nmol) plus picolinic acid (480 nmol) into the region of the substantia nigra. Results revealed that 6-hydroxydopamine and quinolinic acid alone produced a large loss of tyrosine hydroxylase-positive cells in the pars compacta of the substantia nigra. Behavioural results for all 6-hydroxydopamine (n = 10) and for some quinolinate-treated rats (n = 5) revealed ipsi- and contraversive circling following amphetamine (1 mg/kg, i.p.) and apomorphine (0.5 mg/kg, s.c.), respectively, consistent with unilateral loss of dopamine cells in the substantia nigra. The remaining quinolinate-treated rats (n = 9) circled ipsiversively following either stimulant suggesting damage to the pars reticulata. Groups treated with picolinic acid alone (n = 6) or co-injected (n = 6) showed no loss of tyrosine hydroxylase-positive cells in the substantia nigra and no circling response to the stimulants. In the second series of experiments, low concentrations of quinolinic acid (2.5, 5.0, 7.5 nmol), picolinic acid (10, 20, 30 nmol), or the two together (7.5 plus 30 nmol, respectively) were microinjected (0.5 microliter) into the dorsal striatum and circling behaviour evaluated. These results revealed dose-dependent contralateral circling with either quinolinate or picolinate; co-injection of the two tryptophan metabolites also produced contralateral circling. It was concluded that picolinic acid blocks the neurotoxic but not the neuroexcitant effects of quinolinic acid.

Mnemonic deficits in the double Y-maze are related to the effects of nucleus basalis injections of ibotenic and quisqualic acid on choline acetyltransferase in the rat amygdala.

Many researchers have reported that the magnitude of decrease in cortical choline acetyltransferase (ChAT) following excitotoxic lesions of the nucleus basalis magnocellularis (nbm) is unrelated to the degree of cognitive impairment. Recently, an explanation has been offered for this lack of correlation: different excitotoxins, when injected into the nbm, differentially affected cholinergic projections to the cortex and amygdala, and those excitotoxins previously reported to produce the greatest mnemonic deficits produced the largest decreases in amygdaloid ChAT. The present study evaluated the role of amygdalofugal cholinergic projections in memory by comparing the effects of intra-nbm ibotenic and quisqualic acid on cortical and amygdaloid ChAT and on mnemonic performance in the double Y-maze. Rats were trained in the double Y-maze until working and reference memory choice accuracy stabilized to a criterion of > or = 78% correct. Rats then were given either bilateral quisqualic acid (60 nmol in 0.5 microliter), bilateral ibotenic acid (50 nmol in 0.5 microliter), or sham (0.9% saline in 0.5 microliter) lesions of the nbm, and again were tested on the maze. Quisqualate produced a selective impairment of working memory, a large (51%) decrease in cortical ChAT and a small (17%) decrease in amygdaloid ChAT; ibotenate, on the other hand, produced a greater impairment of working memory, an impairment of reference memory, a similar (51%) decrease in cortical ChAT, but a greater (30%) decrease in amygdaloid ChAT. These results suggest that the cholinergic projections from the nbm to the cortex and amygdala play an important role in memory. They suggest that excitotoxins producing greater depletions of amygdaloid ChAT produce greater mnemonic deficits.

Scopolamine injected into the rat amygdala impairs working memory in the double Y-maze.

Recent neurochemical results suggest the hypothesis that the nucleus basalis magnocellularis (nbm) cholinergic projection to the amygdala may play a role in memory. The present study investigated the effects of intra-amygdaloid injections of the cholinergic antagonist scopolamine on working and reference memory in the double Y-maze. Rats were pretrained until working and reference memory choice accuracy stabilized to a criterion of > or = 86% correct. Bilateral cannulae were then surgically implanted in the basolateral amygdaloid complex. Rats (n = 9) received scopolamine in doses of 8.0, 24.0, and 72.0 micrograms/0.5 microliter and saline (0.5 microliter) in a counterbalanced order with retraining to criterion between injections. Intra-amygdaloid scopolamine produced a dose-dependent and differential impairment of working and reference memory. A dose of 24.0 micrograms impaired working memory without significantly affecting reference memory; doses of 8.0 micrograms and 72.0 micrograms affected neither and both types of memory, respectively. Results implicate amygdaloid acetylcholine in memory.

Muscimol injections into the nucleus basalis magnocellularis of rats: selective impairment of working memory in the double Y-maze.

Anatomical and neurochemical results suggest that the cortico- and amygdalopetal cholinergic neurons of the nucleus basalis magnocellularis (NBM) may receive GABAergic inputs. The present experiments were undertaken to evaluate the possible influence of intra-NBM injections of the GABAA agonist, muscimol, on memory. In two experiments, rats were chronically implanted with guide cannulae placed bilaterally into the NBM. Rats were trained to a criterion of at least 83% correct on each component in a double Y-maze task that allowed a dissociation of working and reference memory. The task began with placement into one of the two end arms of the first Y-maze and the reference memory task was to go to the stem for food. Access to the second Y was then given and the working memory task was to go to the goal arm opposite the arm in the first maze from which that trial began. In experiment 1, pre-trained rats (n = 7) received muscimol (0.5 microliter) in doses of 0, 0.01, 0.1 and 1.0 microgram in a counterbalanced order with re-training to criterion between injections. In experiment 2, pre-trained rats (n = 8) received saline, muscimol (0.1 microgram), the GABAA antagonist, bicuculline (0.01 microgram), and muscimol + bicuculline. Results of experiment 1 revealed that intra-NBM muscimol produced a dose-dependent and differential impairment of working and reference memory. A dose of 0.1 microgram impaired working memory without significantly affecting reference memory; doses of 0.01 microgram and 1.0 microgram affected neither and both types of memory, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired post-transcriptional expression of interleukin-2 receptor in pokeweed mitogen-activated T cells.

The expression and role of interleukin-2/interleukin-2 receptor (IL-2/IL-2R) system in the pokeweed mitogen (PWM)-induced T cell mitogenesis was studied. In the absence of monocytes (Mo), both soluble and Sepharose-bound PWM fail to induce T cell mitogenesis even when exogenous IL-2 or IL-1 or IL-1 + IL-2 or IL-4 are also present. In the presence of Mo, PWM stimulation of T lymphocytes (highly depleted of B lymphocytes) induces as much IL-2 mRNA as phytohemagglutinin (PHA), but results in higher and persistent IL-2 levels in culture supernatants despite the concomitant T cell mitogenesis, suggesting that PWM-activated T cells do not utilize the IL-2 they produce. Confirming this notion, Mo-dependent PWM-preactivated T cells, as compared to PHA-preactivated ones: (a) failed to consume exogenous IL-2 and their mitogenic response did not increase upon exposure to exogenous IL-2; (b) exhibited very low numbers of high-affinity IL-2R; and (c) showed lower expression of IL-2R p55 and undetectable expression of IL-2R p75 on their surface. Moreover, the PWM-induced T cell mitogenesis was not inhibited by anti-IL-2 or CD25 antibodies and only partially (50%-60%) inhibited by cyclosporin A, while these treatments abrogated the PHA-induced one. PWM-activated T cells, as compared to the PHA-activated ones, exhibited as high (p55) or even higher (p75) mRNA expression of both IL-2R p55 and p75 subunits. The possibility that PWM interferes with IL-2R subunits once expressed on the T cell surface was excluded. Thus, intracellular PWM-related events are likely to impair IL-2R expression post-transcriptionally. Possible explanations for this effect and its relation with the capacity of PWM to induce T cell-dependent B cell differentiation are discussed.

Differential responsiveness of human B lymphocytes to phorbol ester and calcium ionophore based on their state of activation.

For tonsil B cells of a particular high density (below 65% Percoll), both phorbol myristate acetate (PMA) (5 ng/ml) and calcium ionophore A23187 (500 nM) were required to induce RNA synthesis, significant DNA synthesis also occurring in the presence of 12,000 MW B-cell growth factor (BCGF). In contrast, PMA alone, even at 1 ng/ml, was a sufficient stimulus to induce strong DNA synthesis in low-density B cells (45-50% Percoll) and strong proliferative responsiveness to BCGF in intermediate-density B cells (55-65% Percoll). In these latter B-cell populations, A23187 (500 nM), acted synergistically with non-mitogenic PMA doses to induce strong DNA synthesis, the PMA dose required being 5-50 times lower in low-density B cells (0.1-1 ng/ml) than in intermediate-density B cells (5 ng/ml). Preactivation for 30 hr with anti-Ig antibodies plus BCGF, known to drive B cells into late G1, rendered high-density B cells responsive to PMA (1-10 ng/ml) with high, dose-related DNA synthesis. These data indicate that the B-cell mitogenicity of a given nanomolar dose of PMA depends on the more advanced state of activation of B cells. It was also found that the above optimal dose of A23187 (500 nM) paradoxically inhibited the PMA-induced DNA synthesis of low-density B cells and in vitro preactivated high-density B cells. Data obtained with low-density B cells suggest that a calcium influx during the PMA-induced proliferative phase of B cells may provide a negative signal for the DNA synthesis.

Cellular activation without proliferation to B cell growth factor and interleukin 2 in chronic lymphocytic leukaemia B cells stimulated with phorbol ester plus calcium ionophore.

Individual leukaemic B cells of chronic lymphocytic leukaemia (CLL) do not proliferate to B cell growth factor (BCGF) or interleukin 2 (IL-2) when co-stimulated with immunoglobulin (Ig) ligands. To exclude possible defective signalling via surface Ig (sIg), phorbol myristate acetate (PMA) plus calcium ionophore (A23187) were used to activate purified CLL B cells and compared with staphylococcal protein A coupled to sepharose beads (Seph-PA). RNA synthesis and phenotypic changes after PMA plus A23187 stimulation indicate that CLL B cells from (10) different individuals are similarly able to undergo the G0 to the G1 phase transition and express surface activation antigens. In contrast, they are variable in the capacity to show DNA synthesis, which occurred in only six out of 10 cases. Even in the presence of BCGF (10%, v/v) or IL-2 (50 U/ml) four out of nine CLL B cells activated with PMA plus A23187 or PMA alone were still unable to proliferate although they were induced to express CD23, 4F2, CD25 and OKT9 antigens by PMA plus A23187. However, PMA plus A23187 induced IgM secretion which increased further in response to IL-2 even in the absence of DNA synthesis. Moreover, in other CLL B cell populations, the unresponsiveness to growth factors upon co-stimulation with Ig ligands (Seph-PA) may simply reflect a defective signalling via sIg cross-linking which can be circumvented by PMA plus A23187 stimulation. Recombinant Interferon-gamma (50 U/ml) failed to affect DNA synthesis and IgM secretion.

Further studies on the ELISA-spot technique. Its application to particulate antigens and a potential improvement in sensitivity.

Applications of the ELISA-spot technique to particulate antigens (sheep erythrocytes and E. coli) are reported; sheep erythrocytes were used to ensure a rigorous comparison of the spot assay and the haemolytic plaque assay. The spot technique was also applied to soluble antigens (dextran and trinitrophenyl-lipopolysaccharide) to assess the putative occurrence of non-haemolytic antibodies. In most instances, the spot assay disclosed higher numbers of antibody-secreting cells than the plaque assay. An examination of the kinetics of spot formation demonstrated that spot development was most rapid during the first and second hours of enzyme activity and slowed thereafter, although the numbers of spots at 16 h were higher than those at 2 h. To shorten the assay time a redox reaction (which yields an insoluble formazan) was coupled to the enzymatic reaction. In duplicate assays, this improved technique gave larger numbers of spots in a shorter time, than the conventional assay.