Spike patterning by Ca2+-dependent regulation of a muscarinic cation current in entorhinal cortex layer II neurons

In entorhinal cortex layer II neurons, muscarinic receptor activation promotes depolarization via activation of a nonspecific cation current (I(NCM)). Under muscarinic influence, these neurons also develop changes in excitability that result in activity-dependent induction of delayed firing and bursting activity. To identify the membrane processes underlying these phenomena, we examined whether I(NCM) may undergo activity-dependent regulation. Our voltage-clamp experiments revealed that appropriate depolarizing protocols increased the basal level of inward current activated during muscarinic stimulation and suggested that this effect was due to I(NCM) upregulation. In the presence of low buffering for intracellular Ca(2+), this upregulation was transient, and its decay could be followed by a phase of I(NCM) downregulation. Both up- and downregulation were elicited by depolarizing stimuli able to activate voltage-gated Ca(2+) channels (VGCC); both were sensitive to increasing concentrations of intracellular Ca(2+)-chelating agents with downregulation being abolished at lower Ca(2+)-buffering capacities; both were reduced or suppressed by VGCC block or in the absence of extracellular Ca(2+). These data indicate that relatively small increases in [Ca(2+)](i) driven by firing activity can induce upregulation of a basal muscarinic depolarizing-current level, whereas more pronounced [Ca(2+)](i) elevations can result in I(NCM) downregulation. We propose that the interaction of activity-dependent positive and negative feedback mechanisms on I(NCM) allows entorhinal cortex layer II neurons to exhibit emergent properties, such as delayed firing and enhanced or suppressed responses to repeated stimuli, that may be of importance in the memory functions of the temporal lobe and in the pathophysiology of epilepsy.     

Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.     

Fusion and Matrix Protein Gene Sequence Analysis of Paramyxoviruses of Type 1(PMV-1) Isolated from Pigeons in Slovenia

Paramyxoviruses of type 1 (PMV-l) isolated from pigeons were genetically analyzed. A part of the fusion and the matrix protein genes were amplified and sequenced, Typical amino acid sequences associated with virulence were determined at the fusion protein cleavage site in all PMV-1 isolates. All Slovene pigeon PMV-1 strains share high amino acid sequence similarity with other pigeon strains. In the phylogenetic tree, they are clustered together with pigeon PMV-1 isolates with moderate pathogenicity. Phylogenetic analysis obtained from the fusion and the matrix protein gene alignments showed the same branching order. Viruses circulating among pigeons were found to form quite unique lineage of virulent NDV strains.     

Phage types and genotypes of shiga toxin-producing Escherichia coli O157:H7 isolates from humans and animals in spain: identification and characterization of two predominating phage types (PT2 and PT8)

Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused.     

Effects of a brisk walk on lipoprotein lipase activity and plasma triglyceride concentrations in the fasted and postprandial states

This study aimed to determine whether changes in plasma heparin-releasable lipoprotein lipase (LPL) activity following a brisk walk were associated with decreases in fasting and/or postprandial triglyceride (TG) concentrations. Two groups of pre-menopausal women participated. In one group (fasting study group, n=10), TG concentrations and post-heparin plasma LPL activity were measured in the fasted state on two occasions: ~18 h after a 2-h treadmill walk at 50% maximal oxygen uptake (exercise trial); and after a day of no exercise (control trial). The other group (postprandial study group, n=9) undertook two oral fat tolerance tests (blood samples taken fasting and for 6 h after a high-fat meal), with plasma LPL activity measured 6 h after meal ingestion. Pre-conditions were the same as for the fasting study group (i.e. control and prior exercise). Prior exercise reduced fasting TG concentrations by 23 (7)% (fasting study group) [mean (SEM)] and by 18 (9)% (postprandial study group) (both P<0.05), and the postprandial TG response by 23 (6)% (postprandial study group) (P<0.01). Plasma LPL activity was not significantly increased by exercise in either the fasting or postprandial study groups. However, exercise-induced changes in both fasting and postprandial LPL activity were significantly correlated with the respective exercise-induced changes in fasting TG concentration and the postprandial TG response (r=−0.70 and −0.77 respectively, P<0.05 for both). These data suggest that increased LPL activity may contribute to the hypotriglyceridaemic effect of moderate exercise, although other mechanisms are also likely to be involved. Electronic Publication     

Regulation of tumor necrosis factor (TNF)-alpha synthesis and TNF receptors expression in T lymphocytes through the CD2 activation pathway.

The involvement of the CD2 (T11) molecule, an alternative activation pathway for T lymphocytes, in the regulation of tumor necrosis factor (TNF)-alpha/TNF receptor system in human T lymphocytes has been investigated. It has been found that both TNF-alpha synthesis and secretion were induced after incubation of purified T lymphocytes with the appropriate mitogenic combination of antibodies specific for two different epitopes on the CD2 molecule. Moreover, TNF-alpha secretion was also observed by activation of T lymphocytes either through CD3 or CD69 molecular pathways, or with other stimulating agents such as Ca2+ ionophore in combination with phorbol esters. The expression of TNF receptors has been studied in both nonactivated and CD2-activated T lymphocytes. Unstimulated T cells weakly expressed a functional 75-kDa receptor form, whereas they lacked detectable levels of the 55-kDa receptor form. Triggering of T cell activation through the CD2 molecule also markedly increased the expression of the p75-kDa TNF receptor form, but did not exert any inductive effect on the expression of the p55-kDa TNF receptor. In addition, we have found that TNF-alpha enhanced the proliferative response triggered by the mixture of anti-CD2 monoclonal antibodies. Taken together, these results support a role for the CD2 activation pathway in the functional regulation of TNF-alpha/TNF receptor system in T lymphocytes, and reinforce the view of CD2 as an alternative pathway for regulation of the cytokine network that modulates the function of T lymphocytes.     

Cell type determines the relative proportions of (-) and (+) strand RNA during poliovirus replication.

To examine the influence of the host cell type on poliovirus RNA synthesis we compared the levels of (-) and (+) strand poliovirus RNA during infection of epithelial (HeLa and HEp-2), leukocytic (U-937, HL-60 and K-562) and nerve (IMR-32) cells. The levels of (-) strand RNA were higher in IMR-32, U-937, K-562 or HL-60 cells than those in HeLa or HEp-2 cells. By contrast, (+) strand RNA content was greater in HeLa or HEp-2 cells. Although significant levels of (+) strand RNA were detected in U-937, K-562 and HL-60 cells, no viral protein synthesis was detected by polyacrylamide gel electrophoretic analysis of metabolically labelled proteins. The molar ratio of poliovirus (-) and (+) RNAs was 2-3 fold higher in IMR-32, U-937 and K-562 cells than in HeLa or HL-60 cells and 5-6 fold higher than in HEp-2 cells. Differentiation of HL-60 cells with a variety of inducers produced differential effects on poliovirus (-) and (+) RNA content and modified the molar ratio of (-)/(+) strand RNAs. These findings indicate that host cell components play a critical role in the regulation of the amount of poliovirus (-) and (+) strand RNAs synthesized during infection.     

DR.lyp/lyp bone marrow maintains lymphopenia and promotes diabetes in lyp/lyp but not in +/+ recipient DR.lyp BB rats

Lymphopenia is due to a frameshift mutation in Gimap5 on rat chromosome 4 and is linked to type 1 diabetes in the diabetes prone (DP) BB rat. The hypothesis that bone marrow derived cells confer the lymphopenia phenotype was tested by reciprocal bone marrow transplantation in 40-day-old lethally irradiated diabetes resistant (DR) congenic DR.lyp/lyp (lymphopenia and diabetes) and DR.+/+ (no lymphopenia and no diabetes) rats. In two independent series of transplants, all DR.lyp/lyp rats (n=5 and 4) receiving DR.lyp/lyp bone marrow retained lymphopenia and developed insulitis (5/5 and 4/4) as well as diabetes in some (2/5 and 3/4). Both DR.+/+ and DR.lyp/lyp rats receiving DR.+/+ bone marrow cells as well as DR.+/+ rats receiving DR.lyp/lyp bone marrow cells showed no lymphopenia or diabetes. In accordance with earlier studies in non-congenic BB rats, the DR.+/+ rats receiving DR.lyp/lyp bone marrow cells recapitulated an intermediary phenotype rather than the +/+ or lyp/lyp phenotypes. Our data demonstrate that BBDP rat lymphopenia and diabetes are transferred by bone marrow transplantation to syngeneic DR.lyp/lyp but not DR.+/+ recipients. The intermediary recapitulation of DR.lyp/lyp T cells in recipient DR.+/-/+/- rats suggests that radiation resistant +/-/+/- T cells, the Gimap5 mutation in bone marrow cells, or both may not support the development of lymphopenia.