Caenorhabditis elegans SUR-5, a novel but conserved protein, negatively regulates LET-60 Ras activity during vulval induction.

The let-60 ras gene acts in a signal transduction pathway to control vulval differentiation in Caenorhabditis elegans. By screening suppressors of a dominant negative let-60 ras allele, we isolated three loss-of-function mutations in the sur-5 gene which appear to act as negative regulators of let-60 ras during vulval induction. sur-5 mutations do not cause an obvious mutant phenotype of their own, and they appear to specifically suppress only one of the two groups of let-60 ras dominant negative mutations, suggesting that the gene may be involved in a specific aspect of Ras activation. Consistent with its negative function, overexpressing sur-5 from an extragenic array partially suppresses the Multivulva phenotype of an activated let-60 ras mutation and causes synergistic phenotypes with a lin-45 raf mutation. We have cloned sur-5 and shown that it encodes a novel protein. We have also identified a potential mammalian SUR-5 homolog that is about 35% identical to the worm protein. SUR-5 also has some sequence similarity to acetyl coenzyme A synthetases and is predicted to contain ATP/GTP and AMP binding sites. Our results suggest that sur-5 gene function may be conserved through evolution.

Cloning of a brain-type isoform of human Rab GDI and its expression in human neuroblastoma cell lines and tumor specimens.

Rab proteins, a family of Ras-related small GTP-binding proteins, play a key role in regulating intracellular vesicle trafficking. Rab GDP dissociation inhibitor (GDI3) forms a soluble complex with Rab proteins and thereby prevents the exchange of GDP for GTP. Recently, two isoforms of Rab GDI cDNA were isolated from rats and mice. In this study, we have isolated a brain-type isoform of human Rab GDI cDNA and examined its expression in neuroblastoma. We tentatively designate it as human Rab GDI alpha (hu GDI alpha) and another human Rab GDI, as human Rab GDI beta (hu GDI beta). Hu GDI alpha cDNA encodes a protein of 447 amino acids with a deduced molecular weight of 50,200. Northern blot analysis revealed that hu GDI alpha gene is expressed abundantly in the brain but much less in other tissues, while hu GDI beta gene is ubiquitously expressed. All human neuroblastoma cell lines and tumor specimens examined express hu GDI alpha gene to various extents, while a human T cell leukemia cell line, MOLT3, does not. The levels of both hu GDI alpha and beta mRNA were constant in a human neuroblastoma cell line, NB1, during its neuronal differentiation, while Rab3A and neurofilament-L gene expression and the number of neurosecretory granules were elevated at this condition. These results suggest that hu GDI alpha gene expression is not related to the differentiation state of neuronal cells.

Diffusion tensor imaging of radiculopathy in patients with lumbar disc herniation: preliminary results.

AIMS: The aim of this study was to evaluate the time course of changes in parameters of diffusion tensor imaging (DTI) such as fractional anisotropy (FA) and apparent diffusion coefficient (ADC) in patients with symptomatic lumbar disc herniation. We also investigated the correlation between the severity of neurological symptoms and these parameters. PATIENTS AND METHODS: A total of 13 patients with unilateral radiculopathy due to herniation of a lumbar disc were investigated with DTI on a 1.5T MR scanner and underwent micro discectomy. There were nine men and four women, with a median age of 55.5 years (19 to 79). The changes in the mean FA and ADC values and the correlation between these changes and the severity of the neurological symptoms were investigated before and at six months after surgery. RESULTS: The mean FA values were significantly lower (p = 0.0005) and mean ADC values were significantly higher (p = 0.0115) in compressed nerves than in intact nerves. Although the FA values increased significantly at six months after surgical treatment (p = 0.020), the ADC values decreased but not significantly (p = 0.498). There were strong correlations between the DTI parameters such as the FA value and the severity of the neurological symptoms as assessed using the Japanese Orthopaedic Association (JOA) score and the Roland-Morris Disability Questionnaire (RDQ). CONCLUSION: This preliminary study suggests that it may be possible to use DTI to diagnose, quantitatively evaluate and follow-up patients with lumbar nerve entrapment. TAKE HOME MESSAGE: DTI is a potential tool for functional diagnosis of lumbar nerve damage.

Identification of a site that mediates transcriptional response of the human beta-actin gene to serum factors.

In quiescent fibroblasts, low-level transcription of the beta-actin gene occurs, and it is rapidly induced by growth factors in serum. Deletion analysis of the 5' upstream region of the human beta-actin gene using a CAT assay showed that the region between +765 and +783 downstream from the cap site, which had enhancer activity in growing NIH3T3 cells, also acted as a serum-response element in quiescent cells. The data suggest that the 19-bp element acts positively as a serum response element. This sequence contains a CC(A/T)6GG sequence which is found in the promoter region of many cytoskeletal and muscle-specific actin genes.

A novel alternatively spliced viral mRNA transcribed in cells infected with human T cell leukemia virus type 1 is mainly responsible for expressing p21X protein.

The pX sequence of human T cell leukemia virus type 1 (HTLV-1) has been thought to be expressed as a doubly spliced mRNA that codes for p40tax, p27rex and p21X. However, we identified a novel alternatively spliced mRNA in the HTLV-1 infected cells by using reverse transcription followed by the polymerase chain reaction. This mRNA contains only the first and third exons of the doubly spliced mRNA and encodes only p21X. Our data that this mRNA is responsible for expressing p21X exists in most of HTLV-1 infected cells strongly suggests that p21X may play a crucial role for HTLV-1 replication.

Lack of involvement of p53 gene mutations in N-methyl-N-nitrosourea-induced bladder tumor progression in N-butyl-N-(4-hydroxybutyl)nitrosamine-treated rats and no suppression by indomethacin.

The relevance of p53 mutations to rat bladder cancer progression induced by a single injection of N-methyl-N-nitrosourea (MNU) and the chemopreventive effects of indomethacin (IM) were investigated in male F344 rats, initially given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) at a dose of 500 ppm in the drinking water for 10 weeks. The animals were subsequently treated with a single intraperitoneal injection of MNU at a dose of 50 mg/kg b.w. at week 20. A subgroup was then given IM dissolved in the drinking water at a concentration of 20 ppm for 20 weeks. The experiment was terminated at week 40 when transitional cell carcinomas (TCC) were observed in all animals given BBN, regardless of the administration of MNU and/or IM (incidences ranged from 80 to 100%). The extent of invasion was significantly greater with the additional MNU treatment but no inhibitory effects of IM were noted. A low frequency of p53 mutations was detected without relation to the extent of tumor invasion. Thus, only two mutations were found, one in a Ta and the other in a T1 carcinoma. The present study thus demonstrated that p53 mutations are not involved in MNU-induced progression in rat urinary bladder cancers, suggesting that they are not critical for malignancy.

p53 gene mutations in rectal cancer associated with schistosomiasis japonica in Chinese patients.

Mutations in p53 tumor suppressor gene were examined in 44 Chinese patients with rectal cancer, including 22 cases with advanced schistosomiasis japonica and 22 cases without schistosomiasis. In schistosomal rectal cancer (SRC), 13 mutations were found in 10 cases, which included 11 base-pair substitutions and two deletions. Of 11 base substitutions, nine were transitions and two were transversions and seven of them were located at CpG dinucleotides. In non-schistosomal rectal cancer (NSRC), 13 mutations were found in nine cases, all of which were base-pair substitutions. Of 13 substitutions, 10 were transitions and three were transversions and three of them were located at CpG dinucleotides. The proportion of base-pair substitutions at CpG dinucleotides was higher in SRC patients than in NSRC patients, although this was not statistically significant (P = 0.054). Point mutation was frequent at codon 248 in SRC. A higher frequency of arginine missense mutations was observed in SRC than in NSRC. These observations suggest that the mutations in SRC are the result of genotoxic agents produced endogenously through the course of schistosomiasis japonica.

Relationship between CD44 expression and differentiation of human prostate adenocarcinomas.

It is well documented that CD44 plays an important role in tumor metastasis. We investigated whether there is a correlation between the expression of its isoforms in prostate cancer cells and patient prognosis using 72 cases with biopsy specimens. Immunohistochemistry demonstrated expression of CD44H (68.1%), v6 (36.1%) and v9 (68.1%) to be relatively more frequent than that of other isoforms. A positive correlation between CD44H expression and tumor differentiation was found but this did not extend to clinical staging or prognosis. Likewise, results for CD44v6 or v9 expression suggest that they may be useful markers for prostate adenocarcinoma differentiation but not prognosis.

Molecular cloning of mouse Doc2alpha and distribution of its mRNA in adult mouse brain.

We have previously isolated from a human brain cDNA library, a new protein having two C2-like domains which interact with Ca2+ and phospholipid, and named Doc2alpha. Doc2alpha is abundantly expressed in brain, where it is highly concentrated on the synaptic vesicle fraction, and is implicated in Ca2(+)-dependent exocytosis. We have isolated here a mouse Doc2alpha cDNA and determined the localization of its mRNA in adult mouse brain. The amino acid sequence of the mouse Doc2alpha cDNA is 92% identical with that of the human counterpart. Northern blot analysis and in situ hybridization on adult mouse brain sections have revealed that Doc2alpha is predominantly expressed in mouse brain, where it is expressed in neuronal cells, but not in non-neuronal cells. Doc2alpha is highly expressed in the olfactory bulb, cerebral cortex, hippocampus, amygdaloid complex, and ventromedial hypothalamus nucleus, but not in the cerebellum, caudate-putamen, or ventral thalamus. These results indicate that Doc2alpha is expressed heterogeneously in mouse brain, where it is predominantly expressed in neuronal cells, and suggest that Doc2alpha plays a specific role in the area where it is expressed.

A spontaneous point mutation in the human T-cell leukemia virus type 1 pX gene leads to expression of a novel doubly spliced pX-mRNA that encodes a 25-kD, amino-terminal deleted rex protein.

Primary RNA transcripts of the human T-cell leukemia virus type 1 (HTLV-1) are processed into mature mRNA by a complex series of splicing events. In this paper, we report the finding of a novel doubly spliced pX mRNA in two out of eight HTLV-1-infected cell lines and in one out of 13 peripheral blood mononuclear cells from HTLV-1-infected individuals. The second splicing for this novel pX mRNA is different from that for the known doubly spliced pX mRNA. A novel acceptor site in this splicing was generated by a single point mutation (G to A) at nucleotide 7,337 of the pX gene. This mRNA contained a complete open reading frame that encodes an amino-terminal truncated p27rex protein with 189 amino acids. A new 25-kD protein was detected in the cell lines expressing the novel pX mRNA by an antibody against the carboxy-terminal peptide of p27rex and was termed p25rex. Although the function of p25rex is not clear, we clarified that p25rex is a cytoplasmic phosphoprotein and its function is different from the transcriptional regulator function of p27rex. The possibility that the mutated virus is replicable only in cells coinfected with the wild type HTLV-1 may explain why the incidence of the mutants observed here is low.

Presence of antibodies to p21X and/or p27rex proteins in sera from human T-cell leukemia virus type I-infected individuals.

The human T-cell leukemia virus type I (HTLV-I) pX gene encodes three nonstructural proteins, p40tax, p27rex and p21X. So far, natural antibodies to p27rex and/or p21X have not been found in sera from HTLV-I-infected individuals, although antibodies to p40tax have been found. Recently, the viral transcripts specific for these proteins were detected in fresh peripheral blood mononuclear cells from HTLV-I-infected individuals by the polymerase chain reaction coupled to reverse transcription, showing the in vivo expression of these proteins. We detected antibodies to p21X and p27rex by an enzyme-linked immunosorbent assay (ELISA) system using a recombinantly produced p21X protein as a common antigen, because p21X is identical to the C-terminal portion of p27rex. The sensitivity of the ELISA was determined to be approximately 100 times greater than that of Western blotting. From the analyzed sera of 31 ATL patients, 30 asymptomatic carriers, 18 HAM patients and 100 healthy donors, three specimens from one ATL patient and two carriers were found to be positive for anti-p21X/p27rex antibodies. The specificity of the ELISA reaction was confirmed by the competitive ELISA test with the highly purified recombinant p21X protein. As of result, we first determined the presence of anti-p21X/p27rex antibodies in a small percentage (3.8%) of the sera from HTLV-I-infected individuals. Even sera from the ATL patients, whose fresh PBMCs contained the transcripts for these proteins, were not found to contain these antibodies, suggesting that the immune response to these proteins is low in HTLV-I-infected humans.

Prevention by antioxidants of heterocyclic amine-induced carcinogenesis in a rat medium-term liver bioassay: results of extended and combination treatment experiments.

The effects of 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ) and other antioxidants on heterocyclic amine (HCA)-induced rat hepatocarcinogenesis were examined in a medium-term liver bioassay. In one study the experimental period was extended for up to 28 weeks to confirm the inhibition of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1)-induction of glutathione-S-transferase placental form (GST-P) positive foci detected earlier in an 8-week experiment. Six-week-old male F344 rats were given a single i.p. injection of diethylnitrosamine (DEN) (200 mg/kg b.w.), and starting 2 weeks later, groups of 20 animals received a diet containing 0.03% Glu-P-1 together with 0.5% HTHQ, Glu-P-1 alone, HTHQ alone or a basal diet alone for 26 weeks. Three weeks after the DEN injection, animals were subjected to partial hepatectomy. The combined incidence of hepatocellular adenomas and carcinomas in the group fed Glu-P-1 alone was 89%, in contrast to 40% with simultaneous HTHQ treatment, and near the control level of 30% without Glu-P-1 and HTHQ. In the second experiment, to assess the effects of HTHQ on HCAs in combination, to mimic the human situation, after DEN initiation groups of 15 rats received diets containing a 0.0155% HCA mixture (0.003% Glu-P-1, 0.0015% 3-amino-1,4-dimethyl-5-H-pyrido[4,3-b]indole (Trp-P-1), 0.004% 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAaC), 0.003% 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 0.004% 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), which are all heptocarcinogens) together with 0.5 or 0.125% HTHQ, HCA alone, 0.5 or 0.125% HTHQ alone, or basal diet alone for 6 weeks. The numbers of GST-P positive foci decreased in a dose-dependent manner to 12.2+/-3.1 and 7.2+/-2.4 by the simultaneous treatment with 0.125 and 0.5% HTHQ, respectively, from a value of 17.6+/-3.6 for the HCA mix alone. In a third experiment, after DEN initiation, groups of 15 rats were placed on diets containing 0.02% MeIQx together with 0.25% HTHQ, 0.05% phenylethyl isothiocyanate (PEITC), 1% green tea catechins (GTC) or a mixture of HTHQ, PEITC and GTC, MeIQx alone, antioxidants alone or in combination, or basal diet alone for 6 weeks. These compounds were previously shown to inhibit HCA-associated GST-P positive foci. The numbers of GST-P positive foci in rats treated with MeIQx together with HTHQ (7.7+/-2.6) or antioxidant mixture (0.4+/-2.8) were significantly lower than with MeIQx alone (12.1+/-3.1), but a clear synergistic effect was not demonstrated. These results confirmed the ability of HTHQ to inhibit hepatocarcinogenesis induced by HCAs.

Mechanism of relaxant response to papaverine on the smooth muscle of non-pregnant rat uterus.

In the present paper, we examined a mechanism of the papaverine induced relaxation in the smooth muscle of non-pregnant rat uterus. The hyperosmotic 65 mM KCl (H-65K+)-or oxytocin-induced contraction in the uterus was inhibited by an addition of papaverine in a concentration-dependent manner. Papaverine did not increase both cAMP and cGMP contents in the uterus in the presence of H-65K+ or oxytocin. In fura 2 loaded muscles, papaverine did not affect an increase of [Ca2+]i level by high K+ or oxytocin. In permeabilized muscles, papaverine had no effect on the Ca2+-induced contraction. H-65K+ and oxytocin increased the rate of oxygen consumption 1.8 and 1.5 times higher than that in the resting condition, respectively. The increase of oxygen consumption in the H-65K+ or oxytocin was significantly inhibited by papaverine (1-100 microM). These results suggested that papaverine inhibits smooth muscle contraction mainly by inhibition of mitochondrial respiration in rat uterus as well as guinea pig ileum, which shows a highly spontaneous activity and a highly metabolic dependency of a contraction.

Spontaneous aortic arteriosclerosis in layer chickens.

Aortas of 21 grower chickens (both sexes) aged 30-130 days and of 51 layer hens aged 500 days were examined histopathologically. Aortic sclerotic lesions were classified histopathologically into six stages. As early as 30 days, the abdominal aortas of growers had cellular-fibrous plaques that became more severe with advancing age. The majority of the abdominal aortas of all the 500-day-old layers had a conspicuous increase of collagen fibres, with atheromatous lesions in 23 (45.1%). These sclerotic changes were most severe in the distal aortas.

Tyzzer's disease complicated with distemper in a puppy.

A Pomeranian puppy which died from diarrhea and nasal discharge showed catarrhal pneumonia, acute enteritis and focal liver necrosis. Slender bacilli were detected within ileal enterocytes and hepatocytes. A double infection with a distemper virus and Tyzzer's organism at a cellular level was seen within the ileal enterocytes.

[Treatment for recurrent paradoxical brain embolism through the patent foramen ovale].

We report here two patients with recurrent paradoxical brain embolism through the patent foramen ovale(PFO). The TIAs, which occurred frequently under antiplatelet therapy, resolved soon after commencing the anticoagulation therapy. Case 1, a 57-year-old woman, was diagnosed as having lacunar brain infarction and was treated with ticlopidine hydrochloride. Eleven months later, she suffered from frequent TIAs. Anticoagulation therapy was started after the presence of PFO was documented on transesophageal echocardiography(TEE). Thereafter, her TIA disappeared. Case 2 was a 67-year-old man with a history of lacunar brain infarction. Although he was treated with aspirin for 9 months, he showed transient monoplegia in his right leg. His TEE study also revealed the presence of PFO. After the anticoagulation therapy reached the proper level(PT-NR = 2), he never experienced the recurrence of TIA. If the antiplatelet therapy failed to prevent the recurrence of TIA, it is recommended to find out the presence of PFO which is a potential source of paradoxical brain embolism. In such a case, anticoagulation therapy should be instituted.

Quantitative evaluation and visualization of lumbar foraminal nerve root entrapment by using diffusion tensor imaging: preliminary results.

BACKGROUND AND PURPOSE: DTI can provide valuable structural information that may become an innovative tool in evaluating lumbar foraminal nerve root entrapment. The purpose of this study was to visualize the lumbar nerve roots and to measure their FA in healthy volunteers and patients with lumbar foraminal stenosis by using DTI and tractography with 3T MR imaging. MATERIALS AND METHODS: Eight patients with lumbar foraminal stenosis and 8 healthy volunteers underwent 3T MR imaging. In all subjects, DTI was performed with echo-planar imaging at a b-value of 800 s/mm(2) and the lumbar nerve roots were visualized with tractography. Mean FA values in the lumbar nerve roots were quantified on DTI images. RESULTS: In all subjects, the lumbar nerve roots were clearly visualized with tractography. In all patients, tractography also showed abnormalities such as tract disruption, nerve narrowing, and indentation in their course through the foramen. Mean FA values were significantly lower in entrapped roots than in intact roots. CONCLUSIONS: We demonstrated that DTI and tractography of human lumbar nerves can visualize and quantitatively evaluate lumbar nerve entrapment with foraminal stenosis. We believe that DWI is a potential tool for the diagnosis of lumbar nerve entrapment.