Interactions between canine RAD51 and full length or truncated BRCA2 BRC repeats.

In humans, mutations in the gene for the breast cancer susceptibility protein BRCA2 affect its interactions with the recombinase RAD51 and are associated with an increased risk of cancer. This interaction occurs through a series of eight BRC repeat sequences in BRCA2. A mammalian two-hybrid assay using individual BRC repeats demonstrated that BRC6 did not bind to RAD51, whereas there was strong (BRC1, 2 and 4), intermediate (BRC8), or weak (BRC3, 5 and 7) binding of other BRC repeats to RAD51. In serial deletion mutation experiments, binding strengths were increased when the C-terminal BRC repeat was removed from BRC1-8, BRC1-5 and BRC1-3. These results may provide an insight into the effects of missense or truncation mutations in BRCA2 in canine tumours.

Anti-GM1 antibody IgG subclass: a clinical recovery predictor in Guillain-Barré syndrome.

OBJECTIVE: To determine whether the anti-GM1 antibody IgG subclass (IgG1 to 4) is associated with clinical profiles and patterns of recovery in Guillain-Barré syndrome (GBS). METHODS: The IgG subclassification of anti-GM1 antibody was examined and compared with clinical data on 42 GBS patients positive for the antibody. RESULTS: Frequent anti-GM1 antibody subclasses were IgG1 (76%) and IgG3 (31%). IgG1 antibody was associated with preceding gastroenteritis and Campylobacter jejuni serology, whereas IgG3 antibody was associated with preceding respiratory infection. Although the severity at nadir was similar for IgG1- and IgG3-positive patients, the percentage of patients who could not walk independently was greater for the IgG1-positive group 1 month (42 vs 0%; p = 0.02), 3 months (28 vs 0%), and 6 months (25 vs 0%) after onset. Rapid recovery within 1 month occurred frequently in the patients with the IgG3 antibody but rarely in those with the IgG1 antibody (67 vs 11%; p = 0.003). CONCLUSIONS: The IgG1 subclass of anti-GM1 antibody is a major subtype indicative of slow recovery, whereas isolated elevation of IgG3 subclass antibody titer suggests rapid recovery. Variation in subclass patterns may depend on which pathogen precipitates GBS.

Zolmitriptan is effective and well tolerated in Japanese patients with migraine: a dose-response study.

This phase II study investigated the efficacy, tolerability and dose-response relationship of oral zolmitriptan in the treatment of a single migraine attack in Japanese patients. A bridging analysis then assessed the validity of extrapolating western clinical data to these Japanese patients. In this multicentre, randomized, double-blind, placebo-controlled study, patients received a single dose of placebo or zolmitriptan 1, 2.5 or 5 mg. The primary endpoints were 2-h headache response and the tolerability of zolmitriptan. A statistically significant dose-response relationship was observed for the 2-h headache response (P=0.003). The 2.5 mg group had significantly greater 2-h headache response than the placebo group (P=0.032). The adverse event profile was similar to that reported in western patients, and no adverse events unique to the Japanese population were observed. The bridging analysis report confirmed similar efficacy and tolerability of zolmitriptan in Japanese and western populations. In the Japanese patients, the estimated response rates were 34.3%, 45.2%, 57.7% and 66.2% for placebo, and zolmitriptan 1, 2.5 and 5 mg, respectively, while in the western population the corresponding rates were 39.9%, 49.6%, 61.2% and 71.7%. Zolmitriptan is effective and well tolerated in the acute treatment of migraine in Japanese patients. The optimal dose was 2.5 mg, although the 5 mg dose may provide further benefit for some patients. The bridging analysis supports extrapolation of data from western to Japanese patients.

Anti-GT1a IgG in Guillain-Barré syndrome.

OBJECTIVE: To investigate the presence of serum anti-GT1a IgG in Guillain-Barré syndrome (GBS) and its relation to clinical manifestations. BACKGROUND: Several patients with GBS and bulbar palsy have been reported to have serum anti-GT1a IgG. Most, however, also have anti-GQ1b IgG. A previous study failed to detect GT1a in human cranial nerves, but GQ1b was abundant in human ocular motor nerves. Whether anti-GT1a IgG itself determines the clinical manifestations is not yet clear. METHODS: The association of clinical manifestations with the presence of anti-GT1a IgG and with its cross reactivity was investigated. An immunochemical study was performed to determine whether GT1a is present in human cranial nerves. RESULTS: Anti-GT1a and anti-GQ1b IgG were positive in 10% and 9% respectively of 220 consecutive patients with GBS. Patients with anti-GT1a IgG often had cranial nerve palsy (ophthalmoparesis, 57%; facial palsy, 57%; bulbar palsy, 70%), and 39% needed artificial ventilation. These features were also seen in patients with anti-GQ1b IgG. There was no significant difference between the two groups with respect to the frequency of clinical findings. An enzyme-linked immunosorbent assay showed that anti-GT1a IgG cross reacted with GQ1b in 75% of the patients, GD1a in 30%, GM1 in 20%, and GD1b in 20%. All five patients who carried anti-GT1a IgG that did not cross react with GQ1b had bulbar palsy, neck weakness, absence of sensory disturbance, and positive Campylobacter jejuni serology. Thin-layer chromatography with immunostaining showed that GT1a is present in human oculomotor and lower cranial nerves. CONCLUSIONS: These findings provide further evidence that anti-GT1a IgG itself can determine clinical manifestations. The distinctive clinical features of patients with anti-GT1a IgG without anti-GQ1b activity distinguish a specific subgroup within GBS.

Ectopic expression of Smad7 inhibits transforming growth factor-beta responses in vascular smooth muscle cells.

Vascular injury stimulates the cytokine-growth factor network in the vascular wall, including transforming growth factor-beta (TGF-beta). Reportedly, the intracellular signaling of TGF-beta is mediated by Smad proteins. We tested the effects of the ectopic expression of inhibitory Smads in cultured rat smooth muscle cells (SMC) to identify the role of TGF-beta/Smad signaling on the phenotypic modulation of SMC. The cells exposed to human recombinant TGF-beta1 (10 ng/ml) were stimulated Smad2 phosphorylation. Infection with the replication-deficient adenovirus vector expressing Smad7, but not bacterial beta-galactosidase or Smad6, was found to inhibit TGF-beta-induced Smad2 phosphorylation in a dose-dependent manner. TGF-beta suppressed the serum-induced proliferation of SMC from 36.3% to 51.0% (p<0.01), as measured by hand-counting, and this inhibition was attenuated by the ectopic expression of Smad7 (from 30.7% to 74.8% of the reduction of TGF-beta-response, p<0.05), but not Smad6. A BrdU incorporation assay also showed that TGF-beta-mediated growth inhibition was attenuated by exogenous Smad7 and that this inhibition can be reversed by an additional expression of exogenous Smad2. TGF-beta increased the expression of alpha-smooth muscle actin and myosin heavy chain by 1.3-fold and 1.6-fold in comparison to the control, respectively, and these increases were attenuated by exogenous Smad7, but not Smad6. Our data indicate that Smads mediate TGF-beta responses on SMC phenotypes. Smad7, but not Smad6, may specifically act as an inhibitor of TGF-beta responses.

Cloning and sequencing full length of canine Brca2 and Rad51 cDNA.

Mammary tumors are the most common neoplasm in female dogs, Canis canis, and in women. Mutations in human Brca2 confer an increased risk of female breast cancer. Previous studies have shown that the Brca2 tumor suppressor protein interacts with the recombinational repair protein Rad51. We cloned the full-length cDNA of the canine homologues of Brca2 and Rad51 to obtain a basis for studying their relationship with susceptibility to mammary tumors. The canine Brca2 and Rad51 cDNAs are 11 and 1.5 kb long, encoding 3.471 and 339 amino acids, respectively. The amino acid sequence of canine Brca2 showed 68% homology with the human protein, and 58% homology with a murine protein. There were highly conserved regions in the C-terminus of all three proteins, where the Rad51 interacting domain and putative nuclear localization signals are located. Comparing with the partial genomic sequence previously reported, we found possible nuclear polymorphisms in exon 11, some of which result in amino acid substitutions. On the other hand, canine Rad51 protein had extremely high homology (99%) to the human and murine proteins. Expression of both Brca2 and Rad51 was detected in the mammary gland, suggesting that these two genes interact in the canine mammary gland.

Diltiazem, a calcium antagonist, inhibits matrix metalloproteinase-1 (tissue collagenase) production and collagenolytic activity in human vascular smooth muscle cells.

Calcium antagonists (CAs) are widely prescribed for patients with cardiovascular diseases. CAs have been reported to inhibit smooth muscle cell (SMC) proliferation in addition to their effects on vascular tone. To determine whether CAs potentially affect vascular remodeling, we measured the expression of matrix-degrading enzymes in growth factor-stimulated SMC. Human cultured SMC were stimulated with 10 ng/ml of platelet-derived growth factor (PDGF)-BB with or without a calcium antagonist, diltiazem. In the cell counting assay, diltiazem (10-5 M) alone had no effect on the proliferation of quiescent SMC, however 10-6-10-5 M of diltiazem dose-dependently inhibited PDGF-stimulated SMC proliferation. The inhibitory effects of diltiazem on SMC proliferation were further confirmed by a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and flow cytometry. In Western blotting, matrix metalloproteinase (MMP)-1 (tissue collagenase) but not MMP-2 (72-kDa gelatinase) expression was upregulated by PDGF and phorbol ester (TPA), which were reduced by diltiazem in a dose-dependent manner. The downregulation of MMP-1 expression was consistent with the reduction of collagenolytic activity measured by a FITC-conjugated type I collagen breakdown assay. PDGF-stimulated c-Jun/AP-1 expression, a major transcriptional factor for MMP-1, was not affected by diltiazem. In contrast, intracellular calcium ions measured with a fluorometric assay of Fluo-3AM-loaded cells revealed that the PDGF-stimulated increase in the intracellular calcium content was dose-dependently reduced by diltiazem. Our data suggest that diltiazem inhibits not only proliferation but also MMP-1 expression and collagenolytic activity in PDGF-stimulated SMC. The administration of CAs potentially influences the process of vascular remodeling, and this possibility should be further verified in vivo.

A novel serum chitinase that is expressed in bovine liver.

Chitinases are ubiquitous chitin-fragmenting hydrolases. They are synthesized by a vast array of organisms, including those not composed of chitin. Here, we describe a novel serum chitinase (chitin-binding protein b04, CBPb04), which is expressed in bovine liver. Although CBPb04 is secreted as an endocrine chitinase, it shows higher homology with human gastrointestinal tract exocrine chitinase (AMCase) than with macrophage endocrine chitinase (human chitotriosidase). This suggests that cows have a specific defense against chitin-containing microorganisms. CBPb04 mRNA is expressed in hepatocytes. This is the first report of a hepatogenic mammalian chitinase.

Findings in a patient with herpes simplex viral meningitis associated with acute retinal necrosis syndrome.

We report the case of a 41-year-old man with clinical findings of viral meningitis associated with acute retinal necrosis syndrome in his right eye. MR images showed right optic nerve enlargement and high-intensity signal abnormalities in the region of the left lateral geniculate body and the left occipital lobe.

[Anti-GD1b IgG antibody-related Guillain-Barré syndrome initially mimicking brainstem infarction].

We described a 58-year-old woman with Guillain-Barré syndrome, who initially showed rapid progression of brainstem infarction-like signs. She developed superficial sensory disturbance on the left side, dysarthria, and left-predominant limb weakness within a few hours. She showed bilateral extensor plantar responses and head CT scan detected no abnormality. It was difficult to be distinguished from brainstem infarction until symmetrical limb weakness and generalized areflexia appeared. Serum anti-GD1b IgG antibody with cross-reactivity with GM1b was detected. Cerebrospinal fluid examination revealed albuminocytologic dissociation on day 5. After 5 sessions of immunoadsorption therapy, her symptoms gradually lessened. Anti-GD1b antibody has been detected in patients with sensory ataxic neuropathy. Our patient, however, was characterized with early involvement of brainstem with ataxia of cerebellar type. Our case suggests that anti-GD1b antibody-associated neuropathy has a broad spectrum of clinical features, which are related to cross-reactivity of this antibody.

Muscle weakness in Parkinson's disease: a follow-up study.

We compared isokinetic muscle strength between initial and subsequent measurements in 10 patients who could repeat the same testing later among 23 previously described patients with Parkinson's disease. Patients were divided into two groups according to changes in clinical condition between the times of the first and the subsequent measurements. For patients who had improved, both extension and flexion on the more affected side showed significantly greater torque at 15 rpm in the later than the earlier measurement. For patients who had worsened, both extension and flexion on the more affected side showed significantly less torque at 5 and 15 rpm in the later than the earlier measurement. Although isokinetic muscle strength is likely to depend on movement velocity in the early stages of Parkinson's disease, it may be influenced by bradykinesia, as the disease progresses. Speed-force correlation seen in these patients may give clues to the understanding of the pathophysiology of bradykinesia.

Changes in sodium or glucose filtration rate modulate expression of glucose transporters in renal proximal tubular cells of rat.

Renal glucose reabsorption is mediated by luminal sodium-glucose cotransporters (SGLTs) and basolateral facilitative glucose transporters (GLUTs). The modulators of these transporters are not known, and their substrates glucose and Na+ are potential candidates. In this study we examined the role of glucose and Na+ filtration rate on gene expression of glucose transporters in renal proximal tubule. SGLT1, SGLT2, GLUT1 and GLUT2 mRNAs were assessed by Northern blotting; and GLUT1 and GLUT2 proteins were assessed by Western blotting. Renal cortex and medulla samples from control rats (C), diabetic rats (D) with glycosuria, and insulin-resistant 15-month old rats (I) without glycosuria; and from normal (NS), low (LS), and high (HS) Na+-diet fed rats were studied. Compared to C and I rats, D rats increased (P < 0.05) gene expression of SGLT2 by approximately 36%, SGLT1 by approximately 20%, and GLUT2 by approximately 100%, and reduced (P < 0.05) gene expression of GLUT1 by more than 50%. Compared to NS rats, HS rats increased (P < 0.05) SGLT2, GLUT2, and GLUT1 expression by approximately 100%, with no change in SGLT1 mRNA expression, and LS rats increased (P < 0.05) GLUT1 gene expression by approximately 150%, with no changes in other transporters. In summary, the results showed that changes in glucose or Na+ filtrated rate modulate the glucose transporters gene expression in epithelial cells of the renal proximal tubule.

Acute focal demyelinating disease simulating brain tumors: histopathologic guidelines for an accurate diagnosis.

The object of the present study was to determine the histopathological guidelines for accurate diagnosis of cases of acute focal demyelinating disease that simulates brain tumors. The surgical pathology of three such cases is assessed. Histopathological keys to the diagnosis of such cases are as follows. First, a pattern of sheets of atypical gemistocytic astrocytes in the white matter that show well-formed processes and that are adequately distanced from each other argues against a diagnosis of neoplasm. Second, uniform distribution of foamy macrophages aligned along axons, with occasional focal collections surrounding blood vessels and in the absence of any associated coagulative necrosis argues against the presence of a tumor. Third, perivascular chronic inflammatory infiltration, especially a mixture of lymphocytes and macrophages, favors the diagnosis of demyelination plaque. In such cases the lymphocytes will be predominantly T cells. Fourth, pleomorphic astrocytic proliferation with a lack of vascular endothelial proliferation should raise the suspicion that the lesion may not be a brain tumor. These diagnostic keys should be followed when diagnosing cases that are suspected to be demyelination processes rather than brain tumors. The presence of demyelination plaque should then be confirmed by imaging modalities such as staining with myelin-and axon-specific stains.

The biochemical analysis of neurotransmitters in central neurocytomas.

Central neurocytomas are rare benign intraventricular tumours composed of small round synaptophysin-positive cells, suggesting a neuronal origin of these tumour cells. Although past electron microscopic studies demonstrated synaptic vesicles in the synapse of central neurocytomas, the kinds of neurotransmitters in central neurocytomas have never been identified. In this study we analyzed neurotransmitters in an attempt to clarify the tumorigenesis of central neurocytomas. We studied frozen central neurocytoma specimens from four patients. The tissue levels of glutamate and GABA (gamma-aminobutyric acid) in the tumours were determined by gas chromatography-mass spectrometry (GC-MS) using a selected ion monitoring method. The tissue levels of acetylcholine, choline, catecholamines and metabolites of catecholamines in the tumours were measured by high-performance liquid chromatography combined with electrochemical detection. Choline was found in extremely high concentration in all central neurocytomas when compared with levels in controls. In one central neurocytoma, GABA was found in extremely high concentration compared with controls. In all central neurocytomas, glutamate was found in lower or identical concentrations compared with controls. In all central neurocytomas and controls, dopamine and catecholamine concentrations were extremely low. These results indicated that the histogenesis of central neurocytomas begins with the subependymal stem cells, which have the potential to differentiate into cholinergic or GABA neurons.

Role of Rho-associated kinase in neointima formation after vascular injury.

BACKGROUND: The Rho/Rho-associated kinase (Rho-kinase) system is implicated in various cellular functions, including migration, proliferation, and apoptosis. Because a possible role of the system is suggested in neointima formation after vascular injury, we sought to examine whether a new specific Rho-kinase inhibitor, Y27632, prevents neointima formation of the balloon-injured rat carotid artery, and if so, to investigate the effects of Y27632 on migration, proliferation, and apoptosis of smooth muscle cells (SMCs) in the injured artery. METHODS AND RESULTS: Y27632 was administered intraperitoneally from 1 day before to 14 days after vascular injury. Treatment with Y27632 inhibited phenylephrine-induced Rho-kinase activation in the carotid artery on the basis of immunoblotting against the phosphorylated myosin-binding subunit of myosin phosphatase. Y27632 markedly prevented neointima formation at days 7 and 14. In controls, BrdU(+) proliferating and TUNEL(+) apoptotic SMCs were transiently and coincidentally increased in the neointima, with a peak at day 7. Y27632 significantly increased the neointimal TUNEL(+) SMCs at days 7 and 14, but not BrdU(+) SMCs. Y27642 significantly decreased the number of intimal SMCs at day 4, while not affecting the number of BrdU(+) or TUNEL(+) SMCs. Reendothelialization after balloon injury was not significantly affected by Y27632 at days 7 and 14. CONCLUSIONS: Y27632 inhibited neointima formation by enhancing SMC apoptosis and probably by suppressing early SMC migration. Therefore, a role of Rho-kinase is suggested in neointima formation after vascular injury.