The G-protein on cholesterol-rich membrane microdomains mediates mucosal sensing of short- chain fatty acid and secretory response in rat colon.

AIM: Short-chain fatty acids (SCFA) stimulate colonic contraction and secretion, which are mediated by an enteric reflex via a mucosal sensing and cholinergic mechanisms. The involvement of G-protein signal transduction was examined in the secretory response to luminal propionate sensing in rat distal colon. METHODS: Mucosa-submucosa and mucosa preparations were used to measure short-circuit current (I(sc)) and acetylcholine (ACh) release respectively. Cholesterol-rich membrane microdomains, lipid rafts/caveolae, were fractionated using a sucrose gradient ultra-centrifugation after detergent-free extraction of the isolated colonic crypt. RESULTS: Luminal addition of methyl-ß-cyclodextrin (10 mm) and mastoparan (30 µm), lipid rafts/caveolae disruptors, significantly inhibited luminal propionate-induced (0.5 mm) increases in I(sc) , but did not affect increases in I(sc) induced by serosal ACh (0.05 mm) or electrical field stimulation (EFS). Luminal addition of YM-254890 (10 µm), a Gα(q/11) -selective inhibitor, markedly inhibited propionate-induced increase in I(sc) , but did not affect I(sc) responses to ACh and EFS. Both methyl-ß-cyclodextrin and YM-254890 significantly inhibited luminal propionate-induced non-neuronal release of ACh from colonocytes. Real-time PCR demonstrated that in mRNA expression of SCFA receptors, GPR 43 was far higher than that of GPR41 in the colon. Western blotting analysis revealed that the cholesterol-rich membrane microdomains that fractionated from colonic crypt cells were associated with caveolin-1, flotillin-1 and Gα(q/11) , but not GPR43. Uncoupling of Gα(q/11) from flotillin-1 in lipid rafts occurred under desensitization of the I(sc) response to propionate. CONCLUSIONS: These data demonstrate that the secretory response to luminal propionate in rat colon is mediated by G-protein on cholesterol-rich membrane microdomains, provably via Gα(q/11) .

A motion of forearm supination with maintenance of elbow flexion produced by electrical stimulation to two elbow flexors in humans.

Motions of the forearm induced by electrical stimulation to two elbow flexors (brachioradialis: BR, biceps brachii: BB) were examined in five healthy human subjects. Stainless steel wire electrodes were implanted percutaneously into each motor point of the muscles. The muscles were stimulated separately with a computer-controlled multi-channel stimulator. The motions were taken with a digital video system. Angular changes of the motions in elbow flexion/extension and forearm pronation/supination were measured. Electromyograms (EMG) of BR, BB, and the triceps brachii (TB) were recorded. Electrical stimulation to BR induced a motion of flexion and that to BB motions of flexion and supination. The stimulation to BR with an adequate intensity provided holding of flexion with the prone forearm in all the subjects. In this situation, additional stimulation to BB resulted in motions of flexion and supination. However, the additional stimulation accompanied with a decrease of the stimulation intensity for BR provided a motion of supination with maintenance of the flexion in all the subjects. Since during the stimulation BR, BB, and TB showed no voluntary contraction in EMG, it is suggested that modulation of contraction between BR and BB by the stimulation can produce force in supination with keeping constant force in flexion to support the weight below the elbow.

Bacterial translocation in neonatal rats: the relation between intestinal flora, translocated bacteria, and influence of milk.

BACKGROUND: A high incidence of bacterial translocation in neonates results not only from immaturity of host-defense functions, but also from the dominant colonization of aerobic bacteria in the intestine. Bacterial colonization develops differently among breast-fed, formula-fed, premature, and full-term infants. The purpose of this study was to examine the incidence of bacterial translocation and to identify the translocated bacterial species, relating these findings to the intestinal microflora and to the type of feeding in neonatal rats. METHODS: Animals were divided into three groups: breast-fed normal pups (MR group), formula-fed pups fed via an intragastric cannula implanted esophageally (AR group), and breast-fed pups after the removal of the cannula (Sham group). Artificial rearing was achieved using a machine feeding system. Culture and identification of the bacteria in the intestine, mesenteric lymph nodes, liver, portal blood, and lungs were made using a simplified version of Mitsuoka's method. RESULTS: At 14 days of age, the dominant bacteria in the feces of the MR and Sham Groups were Enterobacteriaceae, Lactobacillus, and Enterococcus, but Enterobacteriaceae and Clostridium were significantly more common in the AR group than in the MR group. The dominant bacteria in the mesenteric lymph nodes were Enterobacteriaceae, Lactobacillus, and Staphylococcus. The extent of systemic bacterial translocation decreased earlier in the Sham group than in the AR group. CONCLUSIONS: The frequency with which species of bacteria were cultured from mesenteric lymph nodes and other peripheral sites did not mirror the composition of the intestinal flora. Among the translocated bacteria, Staphylococcus may be especially hard to recognize and difficult for the host-defense systems to destroy. Breast-feeding inhibited systemic bacterial translocation in the suckling period of the rat.

[Testicular malignant lymphoma: report of two cases].

One patient was a 79-year-old man, who exhibited right scrotal swelling and the other patient was a 73-year-old man, who exhibited left scrotal swelling. Both patients received high orchiectomy under the diagnosis of testicular tumor and the histopathological diagnosis in both patients was non-Hodgkin's lymphoma. Case 1 was diffuse, medium-sized B cell type, and case 2 was diffuse, mixed B cell type. Several examinations revealed no apparent additional involvement. Neither patient received any adjuvant chemotherapy nor postoperative irradiation. In case 1, for a period of 4 years following high orchiectomy, the patient has been doing well. In case 2, 2 years and 6 months postoperatively, para-aortic lymph node swelling occurred, and chemotherapy was initiated with THP-COP but the patient died at 3 years and 3 months after high orchiectomy.

Distinct involvement of NF-kappaB and p38 mitogen-activated protein kinase pathways in serum deprivation-mediated stimulation of inducible nitric oxide synthase and its inhibition by 4-hydroxynonenal.

Cytokine-induced expression of inducible nitric oxide synthase (iNOS) and concomitant production of nitric oxide (NO) involve activation of mitogen-activated protein (MAP) kinases and are in most cases mediated by the transcription factor NF-kappaB. We investigated the role of p38 MAP kinase activation and IkappaB phosphorylation in iNOS expression in a novel iNOS-inducing model in mouse macrophages. Deprivation of serum from the culture medium of RAW 264.7 cells up-regulated iNOS and NO production, which were inhibited by 4-hydroxy-2-nonenal (HNE), a component of oxidatively modified low-density lipoprotein (oxLDL). Serum withdrawal induced phosphorylation of Akt, IkappaB, and p38 MAP kinase. Pretreatment with the potent PI3 kinase inhibitor wortmannin, the NF-kappaB inhibitor PDTC or the specific p38 MAP kinase inhibitor SB203580 each partially attenuated the induction of iNOS and NO production, demonstrating that both p38 activation and IkappaB phosphorylation are required for iNOS expression. SB203580, however, did not prevent the phosphorylation of Akt and IkappaB, suggesting that the p38 MAP kinase signal contributes to iNOS gene expression through an IkappaB-phosphorylation-independent pathway. HNE, which markedly inhibited iNOS expression and NO production, prevented the serum withdrawal-triggered IkappaB phosphorylation but not that of Akt or p38 MAP kinase. A high concentration of HNE stimulated dephosphorylation of IkappaB but promoted activation of p38 MAP kinase. Taken together, these results suggest that NF-kappaB and p38 MAP kinase lie in separate signal pathways for serum deprivation-stimulated iNOS expression and NO production. HNE selectively suppresses the former pathway, targeting a site downstream of Akt.

Light and Electron Microscopic Study of the Retina in a Patient with Congenital Glaucoma.

Purpose: The morphological observation of the human retinal changes caused by severely elevated intraocular pressure (IOP) in congenital glaucoma.Method: Light and electron microscopy.Subjects: One eye of a 15-year-old boy diagnosed as having congenital glaucoma was obtained after he had suffered from severe visual disturbance and ocular pain because of extensive elevated IOP, corneal leucoma, and exophthalmos.Results: Ganglion cells and nerve fibers in the inner layer were lost and replaced by Müller cell processes. The cellular elements in the outer layer were regularly arranged, but these cytoplasmic organelles were not very well developed. Most of the cellular elements of the whole retina contained many dense bodies. The retinal capillaries in the nerve fiber layer were lost or severly damaged, but some of these in the inner plexiform layer were intact. In the retinal arteries and veins marked thickening of the adventitial tissues was observed. These tissues consisted of remarkably increased and irregularly arranged collagen fibers, and a few elastic fibers scattered in some parts.Conclusion: It appeared that the pathological changes in the inner layer of the retina were caused by ischemia. The retinal vascular abnormality was thought to be the result of protective reaction against the severly elevated IOP.

[Clinical study on renal trauma].

A total of 64 patients with renal trauma were treated at the Department of Urology, St. Marianna University School of Medicine, from January 1988 to December 1996. Patients were between 3 and 66 years old (mean 28.2); 56 (87.5%) of them were male and 8 (12.5%) were female. The main cause of injury was a traffic accident in 33 cases (51.6%). According to the Classification of Renal Injury by the Japanese Association for the Surgery of Trauma in Japan, there were 27 cases (42.2%) of Type I (subcapsular injury), 12 cases (18.8%) of Type II (superficial injury), 16 cases (25.0%) of Type III (Deep injury), 6 cases (9.4%) of Type IV (Pedicle injury). Surgical treatment was performed in 6 cases (9.3%). The major associated injuries were bone fracture in 33 cases (51.6%), lung injury in 20 cases (31.3%) and liver injury in 16 cases (25.0%). The literature is also reviewed briefly.

Reactivity of 2-methylene-1,3-dicarbonyl compounds. 1,3-dipolar cycloaddition reaction with ethyl diazoacetate.

The reaction of 2-methylene-1,3-dicarbonyl compounds (1) with ethyl diazoacetate gave 4,5-dihydro-1H-pyrazole derivatives (2), which were stable for several months at room temperature in good yields.

[Differences between gynecological cancer patients taken care for at home and those taken care for at the hospital].

Thirty-five patients with end-stage gynecological cancer, twelve taken care for at home care and 23 taken care for at our hospital, were compared. The life span after recurrence was significantly (p < 0.05) longer in the group taken care for at home. The number of persons living with the patient was also significantly (p < 0.01) larger in the group taken care for at home. Moreover, the hospitalization in the end stage of the disease was significantly (p < 0.01) shorter in the group taken care for at home. The average hospitalization in the end was for 6.5 days. Four patients died at their home. Average period the 12 patients stayed at home was 52.7 days. Following matters are important in order to provide home care. 1. We must know the biological characteristics of the cancer. 2. It is important that it does not lose the timing to which shift to the home care, keeping good doctor-patient relationship. 3. Limit of the therapy against cancer and importance of treatment at home should be carefully and clearly explained to the patient.

Clinico-histopathological findings of Buruli ulcer.

Here, we report the clinico-pathological findings of Buruli ulcer. The patients were 2 females, 9 and 23 years of age and one male, 47 years of age from the Ashanti Country of Ghana. Clinically, cutaneous lesions were classified as nodular, ulcero-nodular and ulcerative. Histopathologically, lesions involved cutaneous and subcutaneous tissue, which showed lympho-epithelioid cell proliferation and panniculitis with characteristic fat necrotic changes. Vascular inflammation, with the nerve tissue involvement, are prominent features on the chronological spectrum of the 3 cases. In all but the early case, Mycobacterium ulcerans could be visualized from the mid dermal area to the subcutis by Fite-Faraco and Harada stain. The ulcerated lesions were also immunoreactive to phenolic glycolipid-1 (PGL-1). These findings suggest Mycobacterium ulcerans infection with lesions of different ages. Further, we also show the need to identify distinct characteristics for differential diagnosis with lesions caused by other mycobacteria.

Effect of testosterone on the number of NADPH diaphorase-stained nerve fibers in the rat corpus cavernosum and dorsal nerve.

OBJECTIVES: To elucidate the effect of testosterone on penile nerve supply. METHODS: Three groups of 10 rats each were assessed; two groups were castrated and the third underwent a sham operation (control). After castration, one group received subcutaneous injection of testosterone while the others received sesame oil. At 8 weeks, the rats underwent a functional analysis. The evaluation included a subcutaneous injection with apomorphine to study centrally mediated erection, and cavernous nerve electrostimulation and papaverine injection to study peripherally mediated erection. At death, a penile midshaft specimen was taken for nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining. RESULTS: In the apomorphine study, castrated rats had no erections, but the erectile function of those receiving testosterone was restored to the level of the controls. The mean numbers of NADPH-diaphorase-stained nerve fibers in the copora cavernosa and both dorsal nerves of castrated rats, at 165.8 +/- 20.0 and 271.3 +/- 21.1, respectively, were significantly lower than those of the controls, at 271.7 +/- 14.6 and 471.2 +/- 27.6, respectively. Those of the testosterone replacement group, at 290.7 +/- 10.1 and 500.7 +/- 23.9, respectively, recovered to the control level. The intracavernosal pressure decreased significantly in the absence of testosterone, both after electrostimulation and intracavernosal papaverine injection, and recovered to the control level after testosterone replacement. CONCLUSIONS: Our results indicate that testosterone acts on the nervous system to mediate erection. When it is absent, there may be downregulation of both the production and activity of nitric oxide (NO), thereby decreasing the response to peripheral stimulation via the NO pathway. Testosterone replacement may upregulate NO activity to the control level.

[Light and electron microscopic study of the retina in a patient with congenital glaucoma].

PURPOSE: The morphological observation of the human retinal changes caused by severely elevated intraocular pressure (IOP) in congenital glaucoma. METHOD: Light and electron microscopy. SUBJECTS: One eye of a 15-year-old boy diagnosed as having congenital glaucoma was obtained after he had suffered from severe visual disturbance and ocular pain because of extensive elevated IOP, corneal leukoma, and exophthalmos. RESULTS: Ganglion cells and nerve fibers in the inner layer were lost and replaced by Müller cell processes. The cellular elements in the outer layer were regularly arranged, but these cytoplasmic organelles were not very well developed. Most of the cellular elements of the whole retina contained many dense bodies. The retinal capillaries in the nerve fiber layer were lost or severely damaged, but some of these in the inner plexiform layer were intact. In the retinal arteries and veins marked thickening of the adventitial tissues was observed. These tissues consisted of remarkably increased and irregularly arranged collagen fibers, and a few elastic fibers scattered in some parts. CONCLUSION: It appeared that the pathological changes in the inner layer of the retina were caused by ischemia. The retinal vascular abnormality was thought to be the result of protective reaction against the severely elevated IOP.

[Clinical study of transurethral surgery for benign prostatic hyperplasia using a thick loop (VaporTome)].

From December 1996 to June 1998, we performed transurethral surgery of the prostate using a thick loop, VaporTome developed by Circon ACMI on 23 patients with symptomatic benign prostatic hyperplasia (BPH). The mean international prostate symptom score decreased significantly, from 22.6 at baseline to 9.0, 6.5, 4.9 and 5.3 at 1, 3, 6 and 12 months after operation, respectively. The mean quality of life index also decreased significantly, from 5.5 to 1.8, 1.8, 1.4 and 1.2, respectively. The mean peak flow rate increased significantly, from 6.2 preoperatively to 15.8, 17.1, 16.6 and 17.4 ml/sec at 1, 3, 6 and 12 months after operation, respectively. The mean prostate volume decreased significantly, from 66.9 to 24.8, 17.3, 19.0 and 18.2 ml. The mean residual urine decreased significantly, from 167.9 to 11.4, 9.4, 12.8 and 20.4 ml. As for overall efficacy, the rate of excellent and good cases at 1, 3, 6 and 12 months was 80.9, 89.4, 94.7 and 85.8%, respectively. No serious complications were observed. Our clinical results suggest that transurethral surgery for BPH using VaporTome has several potential advantages including high efficacy, minimal morbidity compared with standard transurethral resection of the prostate and lower cost compared with other minimally invasive procedures.

[A case of recurrent spontaneous vesical rupture subsequent to irradiation for uterine cancer].

The patient, a 68-year-old woman, had undergone radical hysterectomy and post-operative radiotherapy for uterine cancer in 1974. She was admitted to our hospital complaining of abdominal pain in February, 1996. Contrast-enhanced computed tomographic scan and cystography showed leakage of contrast medium around the bladder. We diagnosed her with spontaneous vesical rupture, and performed conservative therapy. Two months later, she was re-admitted with recurrent vesical rupture. We again performed conservative therapy. We consider that conservative therapy can be indicated for spontaneous vesical rupture with good general condition and no severe urinary tract infection.

Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis.

OBJECTIVE: To elucidate the effect of testosterone on penile innervation. Materials and methods Three groups of six rats each were assessed; two groups (1 and 2) were castrated and the third (group 3) underwent a sham operation (control). Eight weeks after castration, group 2 received a subcutaneous injection with testosterone. At 8 weeks, the rats in group 1 and 3 underwent a final functional analysis while those in group 2 did so at 12 weeks. The evaluation included a subcutaneous injection with apomorphine to study centrally mediated erection, and cavernosal nerve electrostimulation and papaverine injection to study peripherally mediated erection. At death a penile mid-shaft specimen was taken for NADPH-diaphorase staining. RESULTS: In the apomorphine study, castration resulted in significantly fewer yawns and erections than in the control, and those in group 2 significantly better central erectile function than in the controls. The mean (SEM) number of nitric oxide synthase (NOS)-containing nerve fibres in the corpora cavernosa and both dorsal nerves of castrated rats, at 46.2 (9.1) and 203 (32.1), respectively, were significantly lower than in rats in group 2, at 84.1 (11.2) and 300.6 (17.1), and than in the controls, at 88.6 (10.9) and 306.3 (22.9), respectively. The intracavernosal pressure decreased significantly in the absence of testosterone, both after electrostimulation and intracavernosal papaverine injection. However, there was no difference between the control and group 2 rats in either the number of NOS-containing nerve fibres or in the peripheral erectile functional study. CONCLUSIONS: Testosterone acts on the nervous system to mediate erection; when it is absent there may be down-regulation of both the production and activity of NO, thereby decreasing the response to peripheral stimulation via the NO pathway. The restoration of erectile function seen in rats in group 2 supports this phenomenon. Delayed testosterone replacement has no detrimental effect on the restoration of the erectile mechanism after castration.

Presence and quantification of neuropeptide Y in pulmonary edema fluids in rats.

The present study was undertaken to determine the contribution of neuropeptide Y to edema occurrence in neurogenic and hydrostatic pulmonary edema. To induce neurogenic pulmonary edema, fibrinogen and thrombin were injected into the cisterna magna; and to evoke hydrostatic pulmonary edema, saline was infused intravenously. Concentrations of neuropeptide Y in serum and edema fluid were measured using enzyme-linked immunosorbent assay (ELISA), which showed a mean value of 158 nM (95% confidence limit 124-202 nM) in the neurogenic edema fluid, significantly higher than that in the hydrostatic one. Using immunohistochemistry, fluorescent signals reactive to neuropeptide Y were found in the alveolar macrophages and edema fluid in case of fibrin-induced pulmonary edema, but were almost absent in hydrostatic edema and absent in normal lungs. Mean ratio of protein concentrations in edema fluid to that in serum was 74.9 +/- 0.9% in fibrin-induced pulmonary edema, being higher than that in hydrostatic one. From these results, we conclude that neuropeptide Y has a relationship to the high protein concentration ratio, i.e., to increased pulmonary vascular permeability, and consequently may contribute to the development of neurogenic pulmonary edema in rats.

Decapitated and decaudated spermatozoa in man, and pathogenesis based on the ultrastructure.

The ultrastructure of decapitated tails and decaudated heads of ejaculated spermatozoa from an infertile man were investigated. The decaudated heads had the nucleus and acrosome, but neither the implantation fossa nor the basal plate were observed at the caudal pole of the nucleus. The nuclear membrane at this portion showed numerous nuclear pores. The decapitated tails contained the proximal centriole at the proximal end. In addition, most decapitated tails had segmented columns, outer coarse fibres, axoneme, mitochondrial sheath and fibrous sheath in the normal position. The ultrastructure reflects the high motility of the decapitated tails of the spermatozoa. Drastic abnormalities were found in the developing spermatids in the biopsied testes. In spermatids later than the Golgi phase, the pair of centrioles were floating in the cytoplasm of the spermatid, but the distal centriole elongated the flagellum. Neither the implantation fossa nor the basal plate were observed at the caudal pole of the nucleus. It is concluded that the tail might be separated from the head before or after completion of tail formation in spermatids. The explanation for why the proximal centriole failed to connect to the nucleus is unknown.

[The virulence for mice of newly described mycobacterial species].

The virulence for mice of some newly described slowly growing mycobacterial species, M. intermedium DSM 44049 (Group I), M. lentiflavum ATCC 51985 and M. interjectum ATCC 51457 (Group II), M. genavense ATCC 51233, M. celatum ATCC 51131, M. branderi ATCC 51788 and M. conspicuum DSM 6333 (Group III) was compared with the virulence of M. intracellulare N-260 (virulent strain). 10(6) CFU cultured in Middlebrook 7H9 medium were given i.v. to the BALB/c mice. Infected animals were sacrificed after one day and 56 days, and observed for the incidence and/or degree of gross lesions of visceral organs. The bacterial load (CFU) in tissue homogenates of the lungs and spleen was counted using 7H11 agar plates. Macroscopically, marked enlargement of the spleen and numerous small whitish nodules in the lungs of mice infected with M. intracellulare were noted, whereas with other test strains no gross disease in the lungs and slight enlargement of the spleen of mice infected with M. genavense and M. branderi were noted. The average log CFU in the lungs and spleen 56 days after the infection was in the order as follows: M. intracellulare (5.90 and 7.57, respectively), M. genavense (4.35, 7.15), M. celatum (5.01, 6.00), M. branderi (4.09, 6.33), M. conspicuum (3.11, 5.10), M. intermedium (2.26, 4.19), M. lentiflavum (1.70, 3.91), and M. interjectum (1.43, 2.93). Microscopically, granulomatous lesions mainly consisted of macrophages with acid-fast bacilli were found in the alveolar walls and splenic red pulp of mice infected with M. intracellulare. No histopathological changes were produced in the lungs of any of mice infected with other test strains, and some milder features were revealed in the spleen of mice infected with M. genavense, M. branderi and M. celatum. Thus, we concluded that all of the test strains of the newly described nontuberculous slowly growing mycobacteria were less virulent for mice than were M. intracellulare.

Purification and some characteristics of enterocin ON-157, a bacteriocin produced by Enterococcus faecium NIAI 157.

Bacteriocin-like activity (BLA) was screened in 690 strains of lactic acid bacteria isolated from plant materials such as silages and fermented vegetables. Among them, a strain identified as Enterococcus faecium NIAI 157 showed a clear BLA against the indicator strain, Ent. faecium IFO 13712. The proteinaceous nature and antimicrobial activity against closely related species strongly indicated that this BLA was a bacteriocin and was designated enterocin ON-157. The bacteriocin activity of this strain was extracellularly produced in the logarithmic growth phase in MRS broth and purified by ultrafiltration, ammonium sulphate precipitation and cation-exchange chromatography. Purified enterocin ON-157 had a molecular weight of approximately 2500 Da in SDS-PAGE analysis and was easily inhibited by treatment with alpha-amylase and proteolytic enzymes. Enterocin ON-157 had a bactericidal mode of action and inhibited the growth of the enterococci, Lactobacillus sake and Listeria monocytogenes. Enterococcus faecium NIAI 157 harboured two plasmids, 49.0 kb and 43.7 kb, and a variant missing a larger plasmid by curing with novobiocin lost the bactriocin activity.