Juzentaihoto extract suppresses adipocyte hypertrophy and improves hyperglycemia in KKAy mice.

Juzentaihoto is a herbal medicine with reported anti-inflammatory effects, and it is predicted to improve inflammation and insulin sensitivity within obesity. In the present study, juzentaihoto hot water extract (JTT) was administered to obese type 2 diabetic model mice (KKAy) for 56 days. In addition, the effects of JTT on the adipose tissue, glucose metabolism, and blood lipids were evaluated for examining its impact on insulin sensitivity and obesity. As a result of JTT administration, KKAy mice exhibited suppressed adipocyte hypertrophy, decreased the mRNA levels of tumor necrosis factor α, and increased the mRNA levels of adiponectin in epididymal fat tissue. In addition, fasting blood glucose levels, blood triglyceride, and total cholesterol decreased. In summary, these data indicated that JTT administration suppressed the production of inflammatory cytokines and increased adiponectin levels in the adipose tissue. Therefore, with improved insulin sensitivity, blood glucose, and lipid decreased.

Quality control of hospital preparations: Establishment of a simple and rapid method for quantifying ulinastatin in vaginal suppositories.

Ulinastatin vaginal suppositories, used to prevent threatened premature delivery, are frequently used in hospitals. However, there is no established method for quantifying ulinastatin contained in suppositories. Therefore, we investigated a simple and efficient method for quantifying ulinastatin contained in suppositories. Our analytical method involved removal of the base; optimising the enzyme inhibition reaction time and enzyme reaction time; and measuring the absorbance. The modified method was reproducible, operation time was significantly shortened, and cost was reduced to approximately 1/17 of that of the previously reported method. This simple and rapid quantitative method could contribute to the improvement of quality control of ulinastatin vaginal suppositories as an extemporaneous hospital preparation.

Distribution of respiration-related neuronal activity in the thoracic spinal cord of the neonatal rat: An optical imaging study.

The inspiratory motor outputs are larger in the intercostal muscles positioned at more rostral segments. To obtain further insights into the involvement of the spinal interneurons in the generation of this rostrocaudal gradient, the respiratory-related neuronal activities were optically recorded from various thoracic segments in brainstem-spinal cord preparations from 0- to 2-day-old rats. The preparation was stained with a voltage-sensitive dye, and the optical signals from about 2.5s before to about 7.7s after the peak of the C4 inspiratory discharge were obtained. Respiratory-related depolarizing signals were detectable from the ventral surface of all thoracic segments. Since the local blockage of the synaptic transmission in the thoracic spinal cord induced by the low-Ca(2+) superfusate blocked all respiratory signals, it is likely that these signals came from spinal neurons. Under the-low Ca(2+) superfusate, ventral root stimulation, inducing antidromic activation of motoneurons, evoked depolarizing optical signals in a restricted middle area between the lateral edge and midline of the spinal cord. These areas were referred to as 'motoneuron areas'. The respiratory signals were observed not only in the motoneuron areas but also in areas medial to the motoneuron areas, where interneurons should exist; these were referred to as 'interneuron areas'. The upper thoracic segments showed significantly larger inspiratory-related signals than the lower thoracic segments in both the motoneuron and interneuron areas. These results suggest that the inspiratory interneurons in the thoracic spinal cord play a role in the generation of the rostrocaudal gradient in the inspiratory intercostal muscle activity.

Methylxanthines do not affect rhythmogenic preBötC inspiratory network activity but impair bursting of preBötC-driven motoneurons.

Clinical stimulation of preterm infant breathing with methylxanthines like caffeine and theophylline can evoke seizures. It is unknown whether underlying neuronal hyperexcitability involves the rhythmogenic inspiratory active pre-Bötzinger complex (preBötC) in the brainstem or preBötC-driven motor networks. Inspiratory-related preBötC interneuronal plus spinal (cervical/phrenic) or cranial hypoglossal (XII) motoneuronal bursting was studied in newborn rat en bloc brainstem-spinal cords and brainstem slices, respectively. Non-respiratory bursting perturbed inspiratory cervical nerve activity in en bloc models at >0.25mM theophylline or caffeine. Rhythm in the exposed preBötC of transected en bloc preparations was less perturbed by 10mM theophylline than cervical root bursting which was more affected than phrenic nerve activity. In the preBötC of slices, even 10mM methylxanthine did not evoke seizure-like bursting whereas >1mM masked XII rhythm via large amplitude 1-10Hz oscillations. Blocking A-type γ-aminobutyric (GABAA) receptors evoked seizure-like cervical activity whereas in slices neither XII nor preBötC rhythm was disrupted. Methylxanthines (2.5-10mM), but not blockade of adenosine receptors, phosphodiesterase-4 or the sarcoplasmatic/endoplasmatic reticulum ATPase countered inspiratory depression by muscimol-evoked GABAA receptor activation that was associated with a hyperpolarization and input resistance decrease silencing preBötC neurons in slices. The latter blockers did neither affect preBötC or cranial/spinal motor network bursting nor evoke seizure-like activity or mask corresponding methylxanthine-evoked discharges. Our findings show that methylxanthine-evoked hyperexcitability originates from motor networks, leaving preBötC activity largely unaffected, and suggest that GABAA receptors contribute to methylxanthine-evoked seizure-like perturbation of spinal motoneurons whereas non-respiratory XII motoneuron oscillations are of different origin.

New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome.

M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca(2+) concentrations [(Ca(2+) )i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca(2+) )i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca(2+) )i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes.

Excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors involves the AP-1 transcription factor.

Cells preferentially expressing GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are particularly sensitive to excitotoxicity mediated through non-N-methyl-D-aspartate receptors. However, the excitotoxic signalling pathways associated with GluR4-containing AMPA receptors are not known. In this work, we investigated the downstream signals coupled to excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors, using a HEK 293 cell line constitutively expressing the GluR4flip subunit of AMPA receptors (HEK-GluR4). Glutamate stimulation of GluR4-containing AMPA receptors decreased cell viability, in a calcium-dependent manner, when the receptor desensitisation was prevented with cyclothiazide. The excitotoxic stimulation mediated through GluR4-containing AMPA receptors increased activator protein-1 (AP-1) DNA-binding activity. Inhibition of the AP-1 activity by overexpression of a c-Jun dominant-negative form protected HEK-GluR4 cells against excitotoxic damage. Taken together, the results indicate that overactivation of Ca2+-permeable GluR4-containing AMPA receptors is coupled to a death pathway mediated, at least in part, by the AP-1 transcription factor.

Elemental diet modulates the growth of Clostridium difficile in the gut flora.

BACKGROUND AND AIMS: Tube feeding is regarded as a risk factor for Clostridium difficile-associated diarrhoea. Recently, we reported that C. difficile toxin was frequently found in patients receiving an elemental diet. The present study was conducted to clarify whether elemental diets are associated with the growth of C. difficile in the gut flora. METHODS: C. difficile was cultured for 72 h in various concentrations of elemental diet containing 3% thioglycollate, and the growth rate or activity of C. difficile was evaluated by Gram stain or by measuring optical density at 560 nm. Faecal samples from 10 healthy adults were cultured in elemental diet + 3% thioglycollate. RNA was extracted from faeces with glass powder, which can eliminate PCR inhibitors, and mRNA of C. difficile toxin B was measured by reverse transcription PCR. RESULTS: Maximum OD560 value during culture in thioglycollate-containing elemental diet was 2.4 times higher than that in thioglycollate alone (P = 0.0163). Viability of C. difficile was decreased in thioglycollate but not in thioglycollate-containing elemental diet. Toxin B mRNA was detected in five faecal samples (50%) before culture and in all samples after culture. CONCLUSIONS: Our results suggest that an elemental diet can modulate the growth of C. difficile in the gut flora.

Corneodesmosin DNA polymorphisms in MHC haplotypes and Japanese patients with psoriasis.

In order to examine the relationship between corneodesmosin (CDSN) and psoriasis we have determined the presence of CDSN polymorphisms by DNA sequencing in (a) nine B-LCL cell lines of major histocompatibility complex ancestral haplotypes known to be associated with psoriasis vulgaris including 13.1AH, 46.1AH, 46.2 and 57.1AH, and in (b) a group of 267 unrelated individuals comprising Japanese psoriasis patients (n = 101) and Japanese subjects without the disease (n = 166). Three novel CDSN gene sequences were identified. In addition, we have classified the 18 alleles into seven main groups based on phylogeny of non-synonymous substitutions. However, we have found no statistically significant differences between the patients and the unaffected individuals in any of these groups. These findings indicate that CDSN is not a major psoriasis susceptibility gene.

Tsutsugamushi disease in Kanagawa Prefecture, Japan: clinical report of two cases and survey of other incidences in 1998.

Tsutsugamushi disease is characterized by the early appearance of a black crust at the bitten area and the subsequent development of macular or macropapular rush on the whole body with high fever. While treatment with tetracycline derivatives and chloramphenicols is effective, delayed diagnosis or inappropriate treatment will lead to fatality. In this report, we describe two typical cases of tsutsugamushi disease and discuss other incidences in Kanagawa Prefecture, Japan, in 1998. One of the present two patients was diagnosed to be a case of the new type by Kawasaki strain of Rickettsia tsutsugamushi, while responsible strain was not identified for the other case. Since the disease is spreading widely even to suburban areas, we emphasize the need to consider the possible diagnosis of tsutsugamushi disease in patients with generalized eruption and high fever.

[Whole-body MR imaging for evaluation of bone marrow cellularity in aplastic anemia].

The aim of this study was to investigate the usefulness of whole-body MRI(WB-MRI) in the evaluation of cellularity in bone marrow and the distribution of fatty marrow in aplastic anemia. WB-MRI was performed on five patients with aplastic anemia who ranged in age from 62 to 70 years of age, and on four controls with malignant lymphoma who ranged in age from 59 to 67 years. Coronal images were obtained using a body coil with an FOV of 48 cm x 48 cm, and with both fast short T1 inversion recovery(STIR) and spin-echo T1-weighted(T1-WI) in three regions: (1) head to thorax, (2) abdomen to pelvis, and (3) lower extremities. The findings on WB-MRI were compared with those of histological studies of bone marrow at the sternum and the posterior iliac crest. The results were as follows: (1) there was a correlation between the cellularity of histological studies of bone marrow and signal intensity on WB-MRI; (2) WB-MRI could detect the activity of bone marrow; and (3) in a comparison of signal intensity in aplastic anemia and control subjects, there were differences of signal intensity in the central marrow.

The effective size of fluctuating populations.

We consider a Wright-Fisher model whose population size is an autocorrelated stochastic process. Our interest is in the effects of autocorrelated fluctuations of the population size on the effective size. We define the inbreeding effective size and the variance effective size and show that these effective sizes are the same for this model. In the literature, it is said that the effective size is equal to the harmonic mean of population size when the size fluctuates. We will show, however, that the effective size is not the same as the harmonic mean of population size unless the fluctuations of population size are uncorrelated. The effective size is larger (resp. smaller) than the harmonic mean when the fluctuations of population size are positively (resp. negatively) autocorrelated. Further, we obtain some asymptotic expressions for effective size when the population size is very large and/or the autocorrelation of the fluctuation is very strong.

Staphylococcus aureus in inflammatory bowel disease.

BACKGROUND: The potential role of superantigens in inflammatory bowel disease (IBD), particularly Crohn disease, has been broached in studies of the functions of T cell receptors. Staphylococcal cells have been found in intestinal lymph follicles of IBDs. To clarify a role of staphylococcal superantigens in IBD, we attempted to determine whether Staphylococcus aureus could be detected in intestinal mucosa, including surgical specimens and lymph follicles of initial cases. METHODS: One-hundred-and-six colonic and ileal specimens were obtained from 38 Crohn disease, 25 ulcerative colitis and 36 non-IBD patients through therapeutic surgery or endoscopic biopsy. In Crohn disease, 23 surgical specimens and 11 biopsy specimens from initial cases were included. DNA was extracted with phenol-chloroform after homogenization and proteinase K treatment in 73 mucosal specimens. Using an inverted microscope, lymph follicle tissue was microdissected from the remaining 33, mostly biopsy, specimens. DNA was then extracted by freeze-thawing. A coagulase gene characteristic of S. aureus was sought. A nested polymerase chain reaction was performed utilizing primers that amplify a region of the coagulase gene. Polymerase chain reaction products were analyzed with polyacrylamide gel electrophoresis. RESULTS: Only one surgically resected colonic specimen, from a 42-year-old male ulcerative colitis patient, registered positive staphylocoagulase amplification. CONCLUSIONS: Staphylococcal superantigens are not involved in either the early lesions or the established lesions of Crohn disease. However, S. aureus infection occasionally may occur during the course of IBD.

No evidence of persistent mumps virus infection in inflammatory bowel disease.

BACKGROUND AND AIM: There is controversy regarding whether paramyxovirus infection is causally associated with inflammatory bowel disease (IBD). The latest cohort study claimed that atypical measles and mumps infections in childhood may be risk factors for later IBD. This study was conducted to clarify the validity of a causal link between persistent mumps virus infection and IBD. SUBJECTS AND METHODS: (1) Amplification of the mumps virus genome was performed in both intestinal specimens (ulcerative colitis 15, Crohn's disease 15, control 10) and peripheral blood lymphocytes (PBL) (ulcerative colitis seven, Crohn's disease six, control three) by reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern hybridisation using primers specific to the viral genome encoding phosphoprotein or haemagglutinin-neuraminidase. (2) Titre of serum antimumps IgG was measured in 16 patients with ulcerative colitis, in 16 patients with Crohn's disease, and in 16 normal controls using an enzyme linked immunosorbent assay. RESULTS: (1) The mumps virus genome was not detected by RT-PCR in intestinal specimens or PBL in any case. (2) Antimumps IgG titre was positive in 7/16 ulcerative colitis, 10/16 Crohn's disease, and 11/16 control specimens. The mean (SEM) titre of antimumps IgG was 12.281 (7.831) in ulcerative colitis, 7.675 (1.608) in Crohn's disease, and 8.637 (1.969) in controls, with no significant difference between the three groups. CONCLUSION: We could not find any evidence to support a causal link between persistent mumps virus infection and IBD.

Lymph folliculitis in ulcerative colitis.

Preferential involvement of the appendix has recently been confirmed in ulcerative colitis. Since the appendix is an aggregate of lymph follicles, this new observation implies a critical role of the lymph follicles, of both the large bowel and the appendix, in an etiopathogenesis of ulcerative colitis. This report presents two cases of ulcerative colitis in which lymph folliculitis and lymphoid hyperplasia were observed. Lymph folliculitis was observed endoscopically in a border between an established lesion and an uninvolved area. Case 1, proctitis type, relapsing remitting, mild in severity, showed lymph folliculitis in a proximal border of an established rectal lesion. Case 2, with left-sided colitis, mild in severity, had a skip appendiceal orifice inflammation. Lymph folliculitis was observed in the cecum surrounding established appendiceal orifice inflammation. In both cases, lymphoid hyperplasia was observed in an uninvolved area with clear vascular patterns. These two cases clearly demonstrate the involvement of gut lymph follicles in ulcerative colitis. Lymph folliculitis and/or lymphoid hyperplasia was proposed to be early lesions in ulcerative colitis. In addition, the need for microbiology targeting lymph follicles of the large bowel and appendix is stressed in order to disclose the casual microbial agents in ulcerative colitis.

Respiratory activity in glossopharyngeal, vagus and accessory nerves and pharyngeal constrictors in newborn rat in vitro.

1. Previously, in a brainstem-spinal cord-rib preparation from neonatal rats we demonstrated that a decrement in extracellular pH (from about 7.4 to 7.1) caused expiratory activity in an internal intercostal muscle (IIM) during the first half of the expiratory phase (Ea). As the initial step in finding nerves or muscles firing during the second half of the expiratory phase (Eb), the patterns of activity in the glossopharyngeal, vagus and accessory nerves were examined in the present study. 2. Since the emerging motor rootlets of these three nerves (> 20; collected into about 10 bundles before the jugular foramen) are distributed in a continuous fashion from rostral to caudal levels of the brainstem, visual identification was impossible. Therefore, antidromic compound action potentials evoked by stimulation of the glossopharyngeal nerve (IX), the pharyngeal branch of the vagus nerve (PhX), the superior laryngeal nerve (SLN), the cervical vagus nerve (CX) and the accessory nerve (XI) were recorded from the peripheral stumps of the various rootlets. Nerve rootlets could be categorised into rostral, intermediate and caudal groups (rostIX-XI, intIX-XI, caudIX-XI). The rostIX-XI rootlets showed their largest potential on IX stimulation, while the intIX-XI and caudIX-XI rootlets showed their largest potentials on CX stimulation. The intIX-XI rootlets showed larger potentials on PhX and SLN stimulation than the caudIX-XI rootlets. 3. Activity was recorded simultaneously from the central stumps of the rootlets in the above three groups. Most rootlets showed inspiratory bursts. Under low pH conditions, all representatives of group rostIX-XI, most of intIX-XI and about half of caudIX-XI showed additional bursts during the Ea phase. Groups intIX-XI and caudIX-XI but not rostIX-XI also showed discrete bursts during the Eb phase in some preparations. In general, expiratory activity was prominent in intIX-XI. The spinal branch of XI showed no consistent respiratory activity. 4. Since the intIX-XI rootlets showed Eb bursts and large antidromic potentials on stimulation of PhX and SLN (which innervate the inferior pharyngeal constrictor muscle (IPC)), electromyograms were recorded from the rostral and caudal parts of IPC (rIPC and cIPC). Under low pH conditions, cIPC showed bursts during the Ea and Eb phases, while rIPC showed bursts predominantly during the Eb phase. 5. These results indicate that recording from rIPC would be a useful way of examining the neuronal mechanisms responsible for Eb phase activity.

Bufadienolides and a new lignan from the bulbs of Urginea maritima.

Thirty three compounds were obtained from the bulbs of Urginea maritima (Liliaceae). The compounds were identified by means of fast atom bombardment mass spectrometry (FAB-MS), nuclear magnetic resonance (1H-NMR), (13C-NMR), nuclear overhauser effects (NOE) and two dimensional (2D) NMR. Ten of them were new natural compounds. Nine were bufadienolides and only one was lignan in these compounds.