Stretch-dependent sensitization of post-junctional neural effectors in colonic muscles.

BACKGROUND: The colon undergoes distension-induced changes in motor activity as luminal contents or feces increase wall pressure. Input from enteric motor neurons regulates this motility. Here we examined stretch-dependent responses in circular muscle strips of murine colon. METHODS: Length ramps (6-31µm s(-1) ) were applied in the axis of the circular muscle layer in a controlled manner until 5 mN isometric force was reached. KEY RESULTS: Length ramps produced transient membrane potential hyperpolarizations and attenuation of action potential (AP) complexes. Responses were reproducible when ramps were applied every 30 s. Stretch-dependent hyperpolarization was blocked by TTX, suggesting AP-dependent release of inhibitory neurotransmitter(s). Atropine did not potentiate stretch-induced hyperpolarizations, but increased compliance of the circular layer. N(ω)-nitro-L-arginine (L-NNA) inhibited stretch-dependent hyperpolarization and decreased muscle compliance, suggesting release of NO mediates stretch-dependent inhibition. Control membrane potential was restored by the NO donor sodium nitorprusside. Stretch-dependent hyperpolarizations were blocked by L-methionine, an inhibitor of stretch-dependent K(+) (SDK) channels in colonic muscles. Loss of interstitial cells of Cajal, elicited by Kit neutralizing antibody, also inhibited responses to stretch. In presence of L-NNA and apamin, stretch responses became excitatory and were characterized by membrane depolarization and increased AP firing. A neurokinin-1 receptor antagonist inhibited this stretch-dependent increase in excitability. CONCLUSIONS AND INFERENCES: Our data show that stretch-dependent responses in colonic muscles require tonic firing of enteric inhibitory neurons, but reflex activation of neurons does not appear to be necessary. NO causes activation of SDK channels, and stretch of muscles further activates these channels, explaining the inhibitory response to stretch in colonic muscle strips.

Evolving fisher kernels for biological sequence classification.

Fisher kernels have been successfully applied to many problems in bioinformatics. However, their success depends on the quality of the generative model upon which they are built. For Fisher kernel techniques to be used on novel problems, a mechanism for creating accurate generative models is required. A novel framework is presented for automatically creating domain-specific generative models that can be used to produce Fisher kernels for support vector machines (SVMs) and other kernel methods. The framework enables the capture of prior knowledge and addresses the issue of domain-specific kernels, both of which are current areas that are lacking in many kernel-based methods. To obtain the generative model, genetic algorithms are used to evolve the structure of hidden Markov models (HMMs). A Fisher kernel is subsequently created from the HMM, and used in conjunction with an SVM, to improve the discriminative power. This paper investigates the effectiveness of the proposed method, named GA-SVM. We show that its performance is comparable if not better than other state of the art methods in classifying secretory protein sequences of malaria. More interestingly, it showed better results than the sequence-similarity-based approach, without the need for additional homologous sequence information in protein enzyme family classification. The experiments clearly demonstrate that the GA-SVM is a novel way to find features with good performance from biological sequences, that does not require extensive tuning of a complex model.

Motility disorder in experimentally obstructed intestine: relationship between muscularis inflammation and disruption of the ICC network.

We designed a model of small intestinal obstruction in rats to investigate changes in intestinal contractility associated with the immunologically activated components in the tunica muscularis. Although histochemical study did not reveal any typical inflammatory signs such as leucocyte infiltration in the distended intestinal regions of model rats 2-3 weeks after surgical induction of intestinal obstruction, the number of ED2-positive macrophages appeared to be increased in the tunica muscularis. Expression of tumour necrosis factor (TNF)-alpha mRNA was also significantly increased, and the level of CD14 was also increased significantly in the tunica muscularis. Functional studies of distended intestinal muscle segments showed a marked decrease in absolute force stimulated by a cholinergic agent. In addition, the number of spontaneous rhythmic contractions was also reduced in the distended intestinal regions of the obstructed intestine, and this decrease was associated with a reduction in the number of interstitial cells of Cajal (ICC), as revealed by Kit-like immunoreactivity. These results suggest that, under the pro-inflammatory conditions of the tunica muscularis associated with intestinal obstruction, the release of bioactive substances, possibly from activated resident macrophages, may affect smooth muscle contractility. Furthermore, under these conditions, both the number and the function of neighbouring ICC may also be affected.

Quantitation of niflumic acid in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection and its application to a bioequivalence study of talniflumate tablets.

A rapid and simple HPLC method with UV detection (288 nm) was developed and validated for quantitation of niflumic acid in human plasma, the active metabolite of talniflumate. After precipitation with 100% methanol containing the internal standard, indomethacin, the analysis of the niflumic acid level in the plasma samples was carried out using a reverse phase C18 CAPCELL PAK (5 microm, 4.6 mm x 250 mm) column. The chromatographic separation was accomplished with an isocratic mobile phase consisting of a mixture of 0.1M sodium acetate in water and acetonitrile (37:63, v/v), adjusted to pH 6.4. This HPLC method was validated by examining its precision and accuracy for inter- and intra-day runs in a linear concentration range of 0.02-5.00 microg/mL. Stability of niflumic acid in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method was successfully applied to the bioequivalence study of talniflunate in healthy volunteers.

Isolation and characterization of resident macrophages from the smooth muscle layers of murine small intestine.

Macrophages within the murine tunica muscularis were isolated and cultured for physiological studies. Following dispersion, macrophages were identified by phagocytotic activity of fluorescein isothiocyanate (FITC)-dextran. Immediately following isolation, macrophages were rounded and possessed fluorescent granula but developed a ramified shape after 3-4 days in culture. Resident and cultured macrophages were immunopositive for F4/80 and I-Ad/I-Ed. Greater than 90% of F4/80 positive cultured cells were FITC-dextran positive. Macrophages had resting membrane potentials (RMP) of -33.3 +/- 1.5 mV after 1 day in culture, which increased to -53.9 +/- 4.4 mV after 3-4 days. The change in RMP was associated with the development of an inward rectifying K+ current, and a decrease in a voltage-dependent, inactivating outward current. After 3-4 days in culture the inflammatory mediated substances adenosine triphosphate (ATP), platelet-activating factor and bacterial lipopolysaccharide induced increases in cytoplasmic Ca2+ ([Ca2+]i). Forskolin suppressed the ATP-induced increase in [Ca2+]i. Macrophages exhibited oxidative bursts, measured by oxidation of dihydrorhodamine-123 to rhodamine-123. Oxidative bursts coincided with a reduction in intracellular pH. Macrophages expressed a proton conductance that may participate in pH maintenance during reactive oxygen production. These results suggest that resident macrophages in the intestine may play a role in the immunological protection of the gut.

Increased smooth muscle contractility of intestine in the genetic null of the endothelin ETB receptor: a rat model for long segment Hirschsprung's disease.

BACKGROUND AND AIMS: The endothelin ETB receptor null rat (ETB(-/-)R) has an intestinal segment without ganglia, and this rat is characterised by intestinal obstruction similar to that observed in human Hirschsprung's disease. In the present study, we have examined the myogenic mechanism responsible for obstruction in the ETB(-/-)R. RESULTS: The ETB(-/-)R had an enlarged belly and the average lifespan was 18.1 days. The bowel from the rectum to the lower part of the small ileum was constricted whereas the upper region was dilated with faecal stasis and thus presented as megaileum. The constricted muscle segments without ganglia had a greater increase in absolute force when stimulated by carbachol, high K+, and endothelin-1 compared with that of normal siblings. In contrast, in the dilated part with ganglia, the absolute contractile force due to these stimulants in the ETB(-/-)R was not different from that in the ETB(+/+)R. Such a functional hypertrophy of the musculature was observed in parts of the colon, caecum, and distal ileum without ganglia but not in the part of the proximal ileum and jejunum with ganglia. Morphological study demonstrated that the thickness of the circular and longitudinal muscle layers was greater in the constricted part of the intestine in the ETB(-/-)R, and these changes were associated with an increase in the number of smooth muscle cells. CONCLUSIONS: Our findings suggest that both increased contractility of smooth muscle and increased thickness of the intestinal muscular wall may contribute to the intestinal obstruction in the ETB(-/-)R.

Upregulation of iNOS by COX-2 in muscularis resident macrophage of rat intestine stimulated with LPS.

We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE(2), and PGI(2). The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by N(G)-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 microM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.

Relationship between normal heart size and body indices in Korean.

We provided a curve-fit equation to predict the normal heart weight (g) in Koreans by examining 422 autopsies (215 males and 207 females, from newborn to age 77 yr) who were relatively in good general condition. Heart weight was well correlated with body surface area (m2), body weight (kg), and body height (cm) but poorly with age in both sex. Heart weight progressively increased from birth to the earlier 3rd and 4th decades in male and female, respectively, and then gradually decreased; mean heart weight of all age group was greater in male than in female and significantly different from birth to 4th decade. In both sex, heart weight exponentially increased in accordance with the increase of body height, body weight, and body surface (in male, heart weight=0.00312 x body height(2.239), r2=0.750, p<0.0001; in female, heart weight=0.00443 x body height(2170), r2=0.781, p<0.0001; in male, heart weight=9.22 x body weight(0.853), r2=0.770, p<0.0001; in female, heart weight=9.00 x body weight0.855, r2=0.820, p<0.0001; in male, heart weight=155.18 x body surface area1.290, r=0.808, p<0.0001; in female, heart weight=124.13 x body surface area1.242, r=0.834, p<0.0001). These results indicate that heart weight is better correlated with body surface area than with body weight; however, body weight should be a better determinant of a predicted heart weight, since body surface area is entirely dependent on body height and body weight.