Electrical stimulation of denervated rat skeletal muscle retards trabecular bone loss in early stages of disuse musculoskeletal atrophy.

OBJECTIVES: We aimed to determine the intensity of muscle stimulation required to prevent structural failure as well as bone and skeletal muscle loss after denervation-induced disuse. METHODS: Seven-week-old rats (weight, 198-225 g) were randomly assigned to age-matched groups comprising control (CON), sciatic nerve denervation (DN) or direct electrical stimulation (ES) one day later [after denervation] with 4, 8 and 16 mA at 10 Hz for 30 min/day, six days/week, for one or three weeks. Bone architecture and mean osteoid thickness in histologically stained tibial sections and tension in tibialis anterior muscles were assessed at one and three weeks after denervation. RESULTS: Direct ES with 16 mA generated 23-30% maximal contraction force. Denervation significantly decreased trabecular bone volume fraction, thickness and number, connectivity density and increased trabecular separation in the DN group at weeks one and three. Osteoid thickness was significantly greater in the ES16 group at week one than in the DN and other ES groups. Trabecular bone volume significantly correlated with muscle weight. CONCLUSIONS: Relatively low-level muscle contraction induced by low-frequency, high-intensity electrical muscle stimulation delayed trabecular bone loss during the early stages (one week after DN) of musculoskeletal atrophy due to disuse.

Central PACAP mediates the sympathetic effects of leptin in a tissue-specific manner.

We previously demonstrated that the peptidergic neurotransmitter pituitary adenylate cyclase-activating polypeptide (PACAP) affects the autonomic system and contributes to the control of metabolic and cardiovascular functions. Previous studies have demonstrated the importance of centrally-mediated sympathetic effects of leptin for obesity-related hypertension. Here we tested whether PACAP signaling in the brain is implicated in leptin-induced sympathetic excitation and appetite suppression. In anesthetized mice, intracerebroventricular (ICV) pre-treatment with PACAP6-38, an antagonist of the PACAP receptors (PAC1-R and VPAC2), inhibited the increase in white adipose tissue sympathetic nerve activity (WAT-SNA) produced by ICV leptin (2µg). In contrast, leptin-induced stimulation of renal sympathetic nerve activity (RSNA) was not affected by ICV pre-treatment with PACAP6-38. Moreover, in PACAP-deficient (Adcyap1-/-) mice, ICV leptin-induced WAT-SNA increase was impaired, whereas RSNA response was preserved. The reductions in food intake and body weight evoked by ICV leptin were attenuated in Adcyap1-/- mice. Our data suggest that hypothalamic PACAP signaling plays a key role in the control by leptin of feeding behavior and lipocatabolic sympathetic outflow, but spares the renal sympathetic traffic.

The cellular level of histone H3 lysine 4 dimethylation correlates with response to adjuvant gemcitabine in Japanese pancreatic cancer patients treated with surgery.

BACKGROUND: To search for biomarkers identifying pancreatic cancer patients likely to benefit from adjuvant gemcitabine chemotherapy, we investigated the status of several histone modifications in pancreatic tumors and their relationship to clinicopathological features and outcomes. METHODS: Sixty one pancreatic cancer patients, primarily treated by surgical removal of tumors, were involved in the study. Thirty patients completed postoperative adjuvant gemcitabine, and in 31 it was discontinued. Tumor specimens were examined using immunohistochemistry for di- and tri-methylation of histone H3 lysine 4 (H3K4me2 and H3K4me3), dimethylation and acetylation of histone H3 lysine 9 (H3K9me2 and H3K9ac), and acetylation of histone H3 lysine 18 (H3K18ac). Positive tumor staining for each histone modification was used to classify patients into low- and high-staining groups, which were examined for relationships to clinicopathological features and clinical outcomes. RESULTS: High expression of H3K4me3 was related to the well and moderately differentiated tumor histological type (p = 0.012) and low expression of H3K4me2 was related to the presence of perineural invasion (p = 0.007). No cellular histone modifications were associated with overall or disease-free survival of patients as a whole. In the subgroup analyses, a low level of H3K4me2 was significantly associated with worse disease free survival in patients that completed adjuvant gemcitabine (p = 0.0239). Univariate and multivariate hazard models also indicated that a low level of H3K4me2 was a significant independent predictor of disease-free survival (p = 0.007). CONCLUSION: H3K4me2 was found to be a predictor of response to adjuvant gemcitabine in Asian patients with pancreatic cancer.

Preparation of tartary buckwheat protein product and its improving effect on cholesterol metabolism in rats and mice fed cholesterol-enriched diet.

Tartary buckwheat protein product (TBP) was prepared from buckwheat flour by alkali extraction and isoelectric precipitation. The protein content of TBP was 45.8%, and its amino acid composition of TBP was similar to that of common buckwheat protein product (BWP). SDS-PAGE analysis showed that the protein profile of TBP was partially different from that of BWP. TBP contained more quercetin (1710 mg/100 g) than BWP (5.4 mg/100 g), while there was a small difference in the contents of rutin between them. In experiment 1, the consumption of BWP and TBP at 20% net protein level for 13 d caused 32% and 25% reductions in serum cholesterol of rats fed cholesterol, respectively, when compared to the consumption of casein (P < 0.05). The reduction of serum cholesterol by BWP and TBP was associated with enhanced excretion of fecal neutral sterols. In experiment 2, the consumption of BWP and TBP for 27 d caused 62% and 43% reductions in the lithogenic index in mice fed cholesterol, respectively (P < 0.05). The reduction in lithogenic index was associated with enhanced excretion of fecal bile acids. Taken together, these results suggest a potential source of TBP as a functional food ingredient as well as BWP.

The effect of fexofenadine on pruritus in a mouse model (HR-ADf) of atopic dermatitis.

Fexofenadine, a histamine H1-receptor antagonist, is approved for the treatment of pruritus associated with atopic dermatitis. The effects of fexofenadine on scratching behaviour, and plasma levels of histamine and eotaxin were assessed in a new model of atopic dermatitis. Mice fed a diet low in Mg2+ and Zn2+ (special diet S) were compared with mice on a normal diet (N) or diet S plus fexofenadine HCl for weeks 0-10 (S + F(0-10)), 0-5 (S + F(0-5)) or 6 - 10 (S + F(6-10)) (seven mice per group). Compared with group N, group S mice showed significantly greater scratching frequency, and plasma histamine and eotaxin concentrations; these three variables were significantly lower in group S + F(0-10) than in group S. Scratching frequency increased when fexofenadine was discontinued. Fexofenadine significantly reduced mast cell and eosinophil numbers. Histamine may be important in the pathological changes seen in this model of atopic dermatitis, suggesting that it might aid future development of antihistamines for the treatment of atopic dermatitis.

A phase I study of pemetrexed (LY231514) supplemented with folate and vitamin B12 in Japanese patients with solid tumours.

The purpose of this study was to determine the maximum tolerated dose (MTD) and recommended dose (RD) of pemetrexed with folate and vitamin B12 supplementation (FA/VB(12)) in Japanese patients with solid tumours and to investigate the safety, efficacy, and pharmacokinetics of pemetrexed. Eligible patients had incurable solid tumours by standard treatments, a performance status 0-2, and adequate organ function. Pemetrexed from 300 to 1,200 mg m(-2) was administered as a 10-min infusion on day 1 of a 21-day cycle with FA/VB(12). Totally, 31 patients were treated. Dose-limiting toxicities were alanine aminotransferase (ALT) elevation at 700 mg m(-2), and infection and skin rash at 1,200 mg m(-2). The MTD/RD were determined to be 1,200/1,000 mg m(-2), respectively. The most common grade 3/4 toxicities were neutropenia (grade (G) 3:29, G4:3%), leucopenia (G3:13, G4:3%), lympopenia (G3:13%) and ALT elevation (G3:13%). Pemetrexed pharmacokinetics in Japanese were not overtly different from those in western patients. Partial response was achieved for 5/23 evaluable patients (four with non-small cell lung cancer (NSCLC) and one with thymoma). The MTD/RD of pemetrexed were determined to be 1,200/1,000 mg m(-2), respectively, that is, a higher RD than without FA/VB(12) (500 mg m(-2)). Pemetrexed with FA/VB(12) showed a tolerable toxicity profile and potent antitumour activity against NSCLC in this study.

Early invasive colorectal carcinomas with submucosal invasion: radiographic characteristics with barium double contrast images.

BACKGROUND: We assessed the radiographic characteristics of early colorectal carcinomas with submucosal invasion (CCSI) with the use of double-contrast images. METHODS: From 1989 to 1997, 193 patients with 196 CCSI lesions underwent double-contrast barium enema examinations. Three gastrointestinal radiologists retrospectively reviewed the radiographic characteristics of the lesions and classified them as protruding and depressed types by consensus. Further, subclassifying the protruding into lobular and smooth types was accomplished on the basis of surface structure. Each type was compared with pathologic findings of resected specimens. RESULTS: The incidence of the protruding type was 98.0%, and that of the depressed type was only 2.0%. The proportion of smooth lesions was 49.0% for the protruding type; these had a mean diameter of 17.9 mm, which was significantly smaller than the 23.1 mm mean observed for lobular lesions (p < 0.01). Of the smooth lesions, 44.7% demonstrated massive invasion, whereas 91.8% of lobular lesions exhibited only slight or moderate invasion into the submucosa (p < 0.01). The extent of invasion of the smooth lesions was greater than that for their lobular counterparts in terms of venous and lymph node involvement. CONCLUSION: Almost all CCSIs could be identified radiologically as protruding lesions; these had a smooth rather than a lobulated surface and demonstrated greater malignancy, despite the smaller size. It is clinically important to discriminate these from other polypoid lesions in establishing patient treatment. Double-contrast imaging is useful for evaluation of the surface characteristics of CCSIs in barium enema studies.

Regulation of aquaporin-4 expression in astrocytes.

Aquaporin-4 (AQP4), a mercury-insensitive water channel protein, is abundant in the central nervous system and is localized in astrocytes and ependymal cells. AQP4 is speculated to maintain the homeostasis of intracellular and extracellular water in the brain, but little is known about the mechanism of induction of its expression. To investigate the expressional regulation of AQP4, we analyzed changes in its expression during chemically induced differentiation of embryonal carcinoma cells (P19) to neuronal and astrocytic cells, and during the cell cycle of glioma cells. After exposure to retinoic acid for 4 days AQP4 mRNA expression started at the initiation of astrocytic differentiation of P19 cells at 6 days, and increased markedly by 21 days. AQP4 expression was parallel to that of GFAP, a marker intermediate filament of astrocytes. In glioma cell lines, AQP4 mRNA was not detected in the growing phase, but was induced when the cell cycle was arrested at G0/G1 by transient expression of p21. Although quiescent astrocytes in the G0/G1-phase cultured under the serum-free condition exhibited a high expression of AQP4, serum supplement moved them to the S-phase and markedly decreased the AQP expression. These results suggest that AQP4 expression may be induced not only at the initiation of astrocytic differentiation of neural stem cells, but also at the G0/G1-phase during the cell cycle of astrocytes.

[Usefulness of ambulatory adjuvant chemotherapy with low-dose epirubicin in patients with axially-node positive breast cancer: Chiba Epirubicin Cooperative Study Group].

We evaluated the usefulness of adjuvant chemotherapy with low-dose epirubicin (EPI) as a key drug in patients with axially-node positive breast cancer. All the 24 patients who were entered in the study between January 1991 and December 1992 were shown histologically to have more than 4 axially-node involvement and underwent curable resection for the breast lesions. Twenty mg/m2 of EPI was administered intravenously every 4 weeks as ambulatory treatment for 1 year and 5-fluorouracil (5-FU) and tamoxifen (TAM) were concomitantly administered at a dose of 150 mg/day and 20-40 mg/day, respectively, daily for 2 years. The median follow-up period was 70 months with a 55.1% 5-year relapse-free survival and 67.4% 5-year survival rate. One patient developed Grade 3 nausea.vomiting, anorexia and general fatigue; however, the other toxicities were mild, such as Grade 1 leukopenia, liver dysfunction, nausea.vomiting, anorexia and general fatigue. This adjuvant therapy with low-dose EPI was safely administered to outpatients, and is considered to be useful for the treatment of axially-node positive breast cancer.

The soluble interleukin 6 receptor: mechanisms of production and implications in disease.

Interleukin 6 (IL-6) performs a prominent role during disease and has been described as both a pro- and anti-inflammatory cytokine. A key feature in the regulation of IL-6 responses has been the identification of a soluble interleukin 6 receptor (sIL-6R), which forms a ligand-receptor complex with IL-6 that is capable of stimulating a variety of cellular responses including proliferation, differentiation and activation of inflammatory processes. Elevated sIL-6R levels have been documented in numerous clinical conditions indicating that its production is coordinated as part of a disease response. Thus, sIL-6R has the potential to regulate both local and systemic IL-6-mediated events. This review will outline the central role of sIL-6R in the coordination of IL-6 responses. Details relating to the mechanisms of sIL-6R production will be provided, while the potential significance of sIL-6R during the development of clinical conditions will be emphasized. We want to convey, therefore, that when thinking about the inflammatory capability of IL-6, it is essential to consider not only the action of IL-6 itself, but also the effect sIL-6R may have on cellular processes.

Ventricular echo beats and retrograde atrioventricular nodal exits in the dog heart: multiplicity in their electrophysiologic and anatomic characteristics.

INTRODUCTION: A single ventricular echo beat frequently is induced in the dog heart by ventricular pacing, but it has not been investigated using a concomitant ablative technique. We studied the effects of ablating the anterior atrial input to the AV node on ventricular echo beats induced in the dog heart to evaluate their electrophysiologic characteristics, the anatomic reentrant circuit, and the retrograde AV nodal exits. METHODS AND RESULTS: In 20 dogs, an epicardial radiofrequency current was applied to the right anterior septum in an attempt to ablate the anterior input to the AV node. Ventricular programmed stimulation was performed to evaluate the ventricular echo beat and the retrograde AV nodal exit before and after ablation. The AV junction was examined with light microscopy. Seventeen dogs in which the PR interval was prolonged significantly from 108+/-17 msec to 153+/-19 msec (P < 0.001) were selected for ventricular echo evaluation; 3 dogs in which persistent second- or third-degree AV block was induced by ablation were excluded. Ventricular echo beats, which were induced in 13 of 17 dogs, were classified into the anterior type (n = 6) or posterior type (n = 7) according to the earliest atrial activation site during the echo beat. The retrograde AV nodal exit site showed anterior-exit only (n = 10), posterior-exit only (n = 2), and dual-exit (n = 5) patterns. After ablation, the anterior-type ventricular echo beat was noninducible in all 6 dogs, whereas the posterior-type ventricular echo beat was noninducible in only 3 of 7 dogs. In 17 dogs, VA conduction was not demonstrated after ablation in 3 dogs, all of which showed the anterior-exit only pattern. CONCLUSION: The effect of ablation on the ventricular echo beats and retrograde AV nodal exit site suggests multiplicity in their electrophysiologic and anatomic characteristics in the dog heart.

Hypercholesterolemia inhibits angiogenesis in response to hindlimb ischemia: nitric oxide-dependent mechanism.

BACKGROUND: Endothelium-derived nitric oxide (EDNO) plays an important role in the regulation of angiogenesis, whereas hypercholesterolemia (HC) impairs EDNO release. We examined the hypothesis that HC may inhibit ischemia-induced angiogenesis by inhibition of EDNO in a rat model of unilateral hindlimb ischemia and that oral L-arginine supplementation, a substrate for NO synthase, may prevent HC-related impairment of angiogenesis. METHODS AND RESULTS: Male Sprague-Dawley rats were fed (A) standard diet (control), (B) 2% high-cholesterol diet (HC group), or (C) high-cholesterol diet with oral L-arginine (2.25% in drinking water) (HC+L-arg group). At 2 weeks of the dietary intervention, unilateral limb ischemia was surgically induced in all animals. Dietary HC groups (B and C) revealed elevated total and LDL cholesterol levels compared with control animals. Laser Doppler blood flow analyses showed significant decreases in the ischemic/normal limb blood flow ratio in the HC group compared with controls (P:<0.05) when followed up until 4 weeks after surgery. Selective angiography and immunohistochemical analyses in the ischemic limb at postoperative day 14 revealed significantly lower angiographic scores (P:<0.01) and capillary densities (P:<0.01) in the HC group than controls, which were associated with decreased tissue contents of NO(x) and cGMP. Oral L-arginine supplementation (HC+L-arg) significantly improved all parameters of the laser Doppler blood perfusion ratio, angiographic scores, and capillary densities (P:<0.01 versus HC group), which were accompanied by significant elevations in serum L-arginine levels and tissue NO(x) and cGMP contents. CONCLUSIONS: Collateral vessel formation and angiogenesis in response to hindlimb ischemia were significantly attenuated in rats with dietary HC. The mechanism may be related to the reduced NO bioactivity in the ischemic tissues. Augmentation of the tissue NO activity by oral L-arginine supplementation restored the impaired angiogenesis in HC.

A survey of some Indian medicinal plants for anti-human immunodeficiency virus (HIV) activity.

BACKGROUND & OBJECTIVES: India has an extensive area of forest enriched with plant diversity. Several of these plants have been used as folklore medicines. However, the medicinal plants have rarely been investigated for anti-human immunodeficiency virus activity. Hence, some Indian medicinal plants were screened in vitro against human immunodeficiency virus (HIV). METHODS: The inhibitory effect of plant extracts on HIV replication was monitored in terms of inhibition of virus induced cytopathogenicity in MT-4 cells. The MT-4 cells were infected with HIV. The HIV infected or mock infected MT-4 cells were incubated at 37 degrees C in a CO2 incubator in the presence of the plant extracts. After five days, cell viability was measured by tetrazolium based colorimetric assay. RESULTS & INTERPRETATION: Of the 69 plant species screened, 16 were effective against HIV-1 and 4 were against both HIV-1 and HIV-2. The most effective extracts against HIV-1 and HIV-2 are respectively Cinnamomum cassia (bark) and Cardiospermum helicacabum (shoot + fruit). The findings provide a rationale for further studies on isolation of active principles and pharmacological evaluation.

Fiber connections of the corpus mamillare in a percomorph teleost, tilapia Oreochromis niloticus.

The hypothalamus and perhaps its function appear to be similar among vertebrates. Thus, studying the teleostean hypothalamus could be a good model for understanding common neural circuits and mechanisms retained through the vertebrates. However, connections of the inferior lobe, which is considered the hypothalamus in teleosts, is poorly known. The corpus mamillare (CM) is a nucleus of the inferior lobe named after the mammalian mamillary body based on similarities in external morphology. Afferent connections of the CM have been reported only in cypriniform teleosts. These include projections from the nucleus pretectalis superficialis pars magnocellularis, a nucleus lacking in percomorph teleosts, and projections from the secondary gustatory nucleus. Efferent connections of the CM have not been reported in teleosts. In the present study, the CM and its subdivisions and the connections of these subnuclei were identified in isolated and maintained brains of tilapia Oreochromis niloticus by local DiI and biocytin injection. Afferent connections confirmed by reciprocal injections were from the nucleus diffusus lobi inferioris (NDLI) and the nucleus diffusus tori lateralis (NDTL). Efferent connections of each CM subnuclei were also reciprocally confirmed. These connections were to the area dorsalis pars medialis of the telencephalon, the nucleus ventromedialis (NVM) of the thalamus, the tectum opticum (TO), and the nucleus posterioris periventricularis. Because the NDLI is known to receive gustatory information in tilapia, the CM could relay gustatory inputs to multisensory areas, the TO and NVM, for which there are no current reports regarding gustatory inputs.

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome.

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.

Inhibitory mechanism by polysialic acid for lamina-specific branch formation of thalamocortical axons.

During development, thalamocortical axons form arbors primarily in layer 4 of the neocortex. This lamina-specific branch formation was studied in cultures of rat thalamic explants grown next to chemically fixed cortical slices. After a week in vitro, thalamic axons formed branches specifically in the target layer of fixed cortical slices, regardless of the orientation of the ingrowth. This in vitro system permits a direct assessment of contributions of membrane-associated molecules to thalamic axon branch formation. To this end, the present study uses three enzymatic perturbations: chondroitinase, phosphatidylinositol phospholipase C, or the polysialic acid (PSA)-specific endoneuraminidase (endo N). With endo N pretreatment of cortex, the number of branch points was increased significantly, whereas branch tip length was decreased. In addition, the localization of branch points to the target layer was weakened considerably. These features of branch formation were not altered by the other two enzymatic treatments, except that branch tips were shortened by chondroitinase treatment to the same extent as in endo N treatment. These results suggest that membrane-bound components are involved in lamina-specific branch formation of thalamocortical axons, and in particular that PSA moieties contribute to laminar specificity by inhibiting branch emergence in inappropriate layers.

Characterization of factors regulating lamina-specific growth of thalamocortical axons.

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer-specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina-specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol-linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers.

[Change in skin reactivity to common allergens in allergic patients over a 30-year period. Association with aeroallergen load].

Five-year changes in the percentage of positive intradermal test to allergens in patients with bronchial asthma (BA) and allergic rhinitis (AR) over a period of 30 years from 1969 to 1998 were analyzed, with consideration of their association with the yearly changes in the levels of airborne pollens and molds over 30 years. Allergens used throughout the period were house dust, pollens from various plants (Japanese cedar, pine, oak, Japanese alder, Zelkova, orchard grass, ragweed, mugwort and Japanese hop) and molds (Alternaria, Cladosporium, Aspergillus, Penicillium and Candida). Overall percentages to house dust were higher in BA patients than in AR patients, but those to pollens were distinctly higher in the latter. Those to molds were higher in BA patients, except that those to Alternaria were about the same in both BA and AR patients. Therefore, the clinical expression of allergy may differ according to the type of allergen to which subjects are sensitized. Over the course of 30 years, the increase in the percentage of positive intradermal test to all of the allergens used was noted in both BA and AR patients, although this did not always reflect the aeroallergen load. This includes the two extremes to pollens from Japanese cedar and pine, either of which is the most common source of allergen in Japan. Significant increases in the percentages to Japanese cedar pollen (from 12.5% to 54.4% in BA patients, and from 35.1% to 81.5% in AR patients) appeared to be roughly correlated to marked increases in pollen count. In contrast, that to pine pollen was kept low at about 2%, because of their weak allergenicity, but abruptly increased to 5-6% in the last decade, despite the almost invariable pollen count over the last 30 years. Therefore, the increased sensitivity to aeroallergens may also be associated with other factors besides their load in the air.