Complete nucleotide sequence of mouse mammary tumor virus from jyg chinese wild mice: Absence of bacterial insertion sequences in the cloned viral gag gene

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.     

Inhibition of endotoxin- and antigen-induced airway inflammation by fudosteine, a mucoactive agent

We evaluated the effect of a mucoactive agent (-)-(R)-2-amino-3-(3-hydroxypropylthio) propionic acid (fudosteine), on airway inflammation using endotoxin- and antigen-induced models. Time courses of growth related oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) production, neutrophil migration and goblet cell hyperplasia were examined in endotoxin-induced rat airway inflammation. GRO/CINC-1 in bronchoalveolar lavage fluid (BALF) increased in response to intratracheal instillation of endotoxin and peaked within 4 h. Neutrophils in BALF and goblet cells on trachea peaked 24 and 96 h after endotoxin instillation, respectively. Fudosteine significantly inhibited increases in GRO/CINC-1 at 10-100 mg/kg, and neutrophils and goblet cells at 30 and 100 mg/kg. These results suggest that inflammatory events including neutrophil chemoattractant production and neutrophil migration play important roles for goblet cell hyperplasia in endotoxin-induced airway inflammation, and fudosteine inhibits goblet cell hyperplasia by inhibiting GRO/CINC-1 production and/or neutrophil migration. Furthermore, fudosteine (100 mg/kg) inhibited ovalbumin-induced eosinophil infiltration into BALF, suggesting it attenuates asthmatic inflammation.     

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.     

Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors

Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery.     

Putative tumor-suppressor gene regions responsible for radiation lymphomagenesis in F1 mice with different p53 status

Regions of allelic loss on chromosomes in many tumors of human and some experimental animals are generally considered to harbor tumor-suppressor genes involved in tumorigenesis. Allelotype analyses have greatly improved our understanding of the molecular mechanism of radiation lymphomagenesis. Previously, we and others found frequent loss of heterozygosity (LOH) on chromosomes 4, 11, 12, 16 and 19 in radiation-induced lymphomas from several F1 hybrid mice. To examine possible contributions of individual tumor-suppressor genes to tumorigenesis in p53 heterozygous deficiency, we investigated the genome-wide distribution and status of LOH in radiation-induced lymphomas from F1 mice with different p53 status. In this study, we found frequent LOH (more than 20%) on chromosomes 4 and 12 and on chromosomes 11, 12, 16 and 19 in radiation-induced lymphomas from (STS/A X MSM/Ms)F1 mice and (STS/A X MSM/Ms)F1-p53KO/+ mice, respectively. Low incidences of LOH (10-20%) were also observed on chromosomes 11 in mice with wild-type p53, and chromosomes 1, 2, 9, 17 and X in p53 heterozygous-deficient mice. The frequency of LOH on chromosomes 9 and 11 increased in the (STS/A X MSM/Ms)F1-p53KO/+ mice. Preferential losses of the STS-derived allele on chromosome 9 and wild-type p53 allele on chromosome 11 were also found in the p53 heterozygous-deficient mice. Thus, the putative tumor-suppressor gene regions responsible for lymphomaganesis might considerably differ due to the p53 status.     

A susceptibility locus for radiation lymphomagenesis on mouse chromosome 16

BALB/cHeA (BALB/c) mice are sensitive to radiation lymphomagenesis, while STS/A (STS) mice are resistant. We have selected a recombinant mouse, R1, with a STS-derived D16Mit165-D16Mit34 segment in the vicinity of the centromere of chromosome 16 among progeny from a (CcS-7/Dem x BALB/c)F1 x BALB/c backcross. To test the susceptibility to radiation lymphomagenesis, we generated a genetic cross by mating the male and female R1 progeny obtained by 4-6 backcrosses of R1 to BALB/c. The mice were subjected to 4 x 1.7 Gy of X-irradiation. Of 120 mice analyzed, 94 developed lymphomas (91, of thymic type; 3, of nonthymic type) within 315 days of observation. The analysis indicated a link between the susceptibility to lymphomagenesis and the marker D16Mit34 on chromosome 16. The mice heterozygous for the BALB/c and STS alleles at D16Mit34 were less sensitive to lymphomagenesis than the mice homozygous at this locus. There was no significant difference in latency among the genotypes. Our study showed the existence of a susceptibility locus for radiation lymphomagenesis on chromosome 16 and revealed aspects of the genetics of lymphoma susceptibility.     

An HRP study of afferent connections of the supracommissural ventral telencephalon and the medial preoptic area in hime´salmon (landlocked red salmon,oncorhynchus nerka)

The supracommissural ventral telencephalon and the medial preoptic area have been shown to play important roles in the sexual behavior of hime´salmon (landlocked red salamon,Oncorhynchus nerka). In the present study, the sites of neurons projecting to these regions were examined by means of the retrograde horseradish peroxidase (HRP) tracing method. The morphology of neurons in these sites of origin was also studied by means of the Golgi method. The nucleus preopticus periventricularis and the rostral part of nucleus preopticus (NPP-rNPO) received bilateral projections from the middle parts of the area ventralis telencephali pars ventralis (Vv) and the area ventralis telencephali pars dorsalis (Vd), NPP and lateral part of the preoptic area (LPOA), ipsilateral projections from the caulal part of Vv, nucleus anteriors periventricularis (NAPv), nucleus ventromedialis thalami (NVM) at the level of the posterior commissure, nucleus lateralis tuberis pars medialis (NLTm), nucleus anterior tuberis (NAT), nucleus saccus vasculosus (NSV), nucleus recessus posterioris (NRP) and midbrain tegmentum (TG), and a projection from the nucleus posterior tuberis (NPT), which is situated on the midline of the brain. The area ventralis telencephali pars supracommissuralis and neighboring caudal ventral telencephalon (Vs-cV) received ipsilateral projections from almost all parts of the Vv, the middle and caudal parts of Vd, almost all parts of the NNP, the NPO at the level between the habenula and the posterior commisure, and the rostral part of the nucleus dorsomedialis thalami (NDM). The Vs-cV also received a projection from NPT. These findings seem to give anatomical bases for understanding the neural mechanisms involved in sexual behavior as well as neuroendocrine functions.     

Ocular Ferning Test: A Qualitative Test for Mucus Deficiency

A new qualitative test for the study of conjunctival mucus has been developed. Conjunctival scrapings were obtained from 196 patients. Microscopic mucus ferning (arborization) was observed in 148 (91%) patients with various forms of acute conjunctivitis. Six patients (18%) with cicatrizing conjunctivitis (diffuse conjunctival cicatrization, ocular pemphigoid, Stevens-Johnson syndrome) had mucus ferning in their conjunctival scrapings specimens. Mucus ferning was significantly absent (P < 0.005) in patients with diffuse conjunctival cicatrization when compared to mucus ferning in other forms of conjunctivitis. Ocular mucus ferning test is a simple inexpensive office test for the evaluation of patients with mucus deficiency.     

Studies on post-transplant dyslipidemia in kidney transplant patients

This study was performed to retrospectively compare changes in the levels of total cholesterol, non-HDL cholesterol, triglycerides, and immunosuppressive drugs, cyclosporine A and steroids in patients with living-relation renal transplants with those from non-heart-beating donors. We experienced 11 cases of kidney transplants from non-heart-beating donors during the period from April 1995 to May 2003. We evaluated 13 cases of kidney transplants from living-relation donors during the same period. The immunosuppressants used included mainly cyclosporine A as well as mycophenolate mofetil or azathioprine, steroid and ALG, or basiliximab. Over-night fasting lipids (total cholesterol, triglycerides and HDL cholesterol) were studied before renal transplantation and repeated after renal transplantation at 1, 3, 6 and 12 months. The levels of total cholesterol and triglycerides remained in the normal range before transplantation. However, the levels of total cholesterol increased siginificantly 1 and 3 months after transplantation from non-heart-beating donors and remained at higher levels up to 12 months after transplantation. A similar pattern in the levels of triglycerides was observed. The levels of HDL cholesterol remained unchanged and stayed in the normal range before and 1, 3, 6, and 12 months after transplantation from non-heart-beating donors. On the other hand, significant increases in non-HDL cholesterol were observed 3 and 6 months after transplantation from non-heart-beating donors. After transplantation from living-relation donors, levels of total cholesterol, triglycerides, and non-HDL cholesterol remained unchanged and remained in the normal range up to 12 months after transplantation. Although there were no significant differences in the total dosage of cyclosporine A between the patients with living-relation donors and those with non-heart-beating donors, a significant increase in the total dosage of methylprednisolone was observed in patients with non-heart-beating donors compared with those in the patients with living-relation donors. Renal function recovery in patients with living-relation donors was better than in those with non-heart-beating donors. These results may suggest that significant increases in total cholesterol, especially non-HDL cholesterol and triglycerides, were probably partly due to an increased use of immunosuppressants, steroids. It is necessary to aggressively control post-transplant hyperlipidemia and important to reduce or withdraw steroids in the selected, low-risk recipients as early as possible from the viewpoint of preventing post-transplant hyperlipidemia.