Different critical perinatal periods and hypothalamic sites of oestradiol action in the defeminisation of luteinising hormone surge and lordosis capacity in the rat.

Female rats show a gonadotrophin-releasing hormone (GnRH)/luteinising hormone (LH) surge in the presence of a preovulatory level of oestrogen, whereas males do not because of brain defeminisation during the developmental period by perinatal oestrogen converted from androgen. The present study aimed to identify the site(s) of oestrogen action and the critical period for defeminising the mechanism regulating the GnRH/LH surge. Animals given perinatal treatments, such as steroidal manipulations, brain local implantation of oestradiol (E(2) ) or administration of an NMDA antagonist, were examined for their ability to show an E(2) -induced LH surge at adulthood. Lordosis behaviour was examined to compare the mechanisms defeminising the GnRH/LH surge and sexual behaviour. A single s.c. oestradiol-benzoate administration on either the day before birth (E21), the day of birth (D0) or day 5 (D5) postpartum completely abolished the E(2) -induced LH surge at adulthood in female rats, although the same treatment did not inhibit lordosis. Perinatal castration on E21 or D0 partially rescued the E2-induced LH surge in genetically male rats, whereas castration from E21 to D5 totally rescued lordosis. Neonatal E(2) implantation in the anterior hypothalamus including the anteroventral periventricular nucleus (AVPV)/preoptic area (POA) abolished the E(2) -induced LH surge in female rats, whereas E(2) implantation in the mid and posterior hypothalamic regions had no inhibitory effect on the LH surge. Lordosis was not affected by neonatal E(2) implantation in any hypothalamic regions. In male rats, neonatal NMDA antagonist treatment rescued lordosis but not the LH surge. Taken together, these results suggest that an anterior hypothalamic region such as the AVPV/POA region is a perinatal site of oestrogen action where the GnRH/LH regulating system is defeminised to abolish the oestrogen-induced surge. The mechanism for defeminisation of the GnRH/LH surge system might be different from that of sexual behaviour, in terms of the site(s) of oestrogen action and critical period, as well as the neurotransmitter system involved.

Gonadotrophin-releasing hormone pulse generator activity in the hypothalamus of the goat.

Pulsatile release of gonadotrophin-releasing hormone (GnRH) is indispensable to maintain normal gonadotrophin secretion. The pulsatile secretion of GnRH is associated with synchronised electrical activity in the mediobasal hypothalamus (i.e. multiple unit activity; MUA), which is considered to reflect the rhythmic oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion. However, the cellular source of this ultradian rhythm in GnRH activity is unknown. Direct input from kisspeptin neurones in the arcuate nucleus (ARC) to GnRH cell bodies in the medial preoptic area or their terminals in the median eminence could be the intrinsic source for driving the GnRH pulse generator. To determine whether kisspeptin signalling could be responsible for producing pulsatile GnRH secretion, we studied goats, measured plasma levels of luteinising hormone (LH) and recorded MUA in the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located. Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in the ARC, where kisspeptin neuronal cell bodies were found. Exogenous administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of the network of GnRH neurones, and could plausibly reflect the pacemaker activity of kisspeptin neurones, whose projections reach the median eminence where GnRH fibres project. These observations suggest that the kisspeptin neurones in the ARC may be the intrinsic source of the GnRH pulse generator.

Possible role of oestrogen in pubertal increase of Kiss1/kisspeptin expression in discrete hypothalamic areas of female rats.

Kisspeptin, a peptide encoded by the Kiss1 gene, has been considered as a potential candidate for a factor triggering the onset of puberty, and its expression in the hypothalamus was found to increase during peripubertal period in rodent models. The present study aimed to clarify the oestrogenic regulation of peripubertal changes in Kiss1 mRNA expression in the anteroventral periventricular nucleus (AVPV) and hypothalamic arcuate nucleus (ARC), and to determine which population of kisspeptin neurones shows a change in kisspeptin expression parallel to that in luteinising hormone (LH) pulses at the peripubertal period. Quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry revealed an apparent increase in the ARC Kiss1 mRNA expression and kisspeptin immunoreactivity around the time of vaginal opening in intact female rats. The AVPV Kiss1 mRNA levels also increased at day 26, but decreased at day 31, and then increased at day 36/41. In ovariectomised (OVX) rats, ARC Kiss1 mRNA expression did not show peripubertal changes and was kept at a high level throughout peripubertal periods. Apparent LH pulses were found in these prepubertal OVX rats. Oestradiol replacement suppressed ARC Kiss1 mRNA expression in OVX prepubertal rats, but not in adults. Similarly, LH pulses were suppressed by oestradiol in the prepubertal period (days 21 and 26), but regular pulses were found in adulthood. The present study suggests that a pubertal increase of Kiss1/kisspeptin expression both in the ARC and AVPV is involved in the onset of puberty. These results also suggest that both LH pulses and ARC Kiss1 expression are more negatively regulated by oestrogen in prepubertal female rats compared to adult rats.

Inhibitory effects of macrocarpals on the biological activity of Porphyromonas gingivalis and other periodontopathic bacteria.

BACKGROUND/AIMS: Macrocarpals, which are phloroglucinol derivatives contained in eucalyptus leaves, exhibit antimicrobial activity against a variety of bacteria including oral bacteria. This study examined effects of macrocarpals A, B, and C on periodontopathic bacteria, especially Porphyromonas gingivalis. METHODS: Macrocarpals A, B, and C were purified from a 60% ethanol-extract of Eucalyptus globules leaves. To investigate antibacterial activity, representative periodontopathic bacteria were cultured in media with or without various amounts of macrocarpals; subsequently, the optical density at 660 nm was measured. Macrocarpal inhibition of P. gingivalis Arg- and Lys-specific proteinases was assessed by spectrofluorophotometric assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The effect of macrocarpals on P. gingivalis binding to saliva-coated hydroxyapatite beads was examined with (3)H-labeled P. gingivalis. RESULTS: Growth of P. gingivalis was inhibited more strongly than growth of Prevotella intermedia or Prevotella nigrescens and Treponema denticola by macrocarpals, however, Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum were much more resistant. Macrocarpals inhibited P. gingivalis Arg- and Lys-specific proteinases in a dose-dependent manner. The enzyme-inhibitory effect of macrocarpals was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in which hemoglobin degradation by P. gingivalis proteinase was inhibited by macrocarpals. P. gingivalis binding to saliva-coated hydroxyapatite beads was also strongly attenuated by macrocarpals. CONCLUSIONS: Macrocarpals A, B and C demonstrated antibacterial activity against periodontopathic bacteria. Among tested bacteria, P. gingivalis displayed the greatest sensitivity to macrocarpals; additionally, its trypsin-like proteinase activity and binding to saliva-coated hydroxyapatite beads were inhibited by macrocarpals. These results indicate that eucalyptus leaf extracts may be useful as a potent preventative of periodontal disease.

Anti-diabetic potentials of Momordica charantia and Andrographis paniculata and their effects on estrous cyclicity of alloxan-induced diabetic rats.

Momordica charantia and Andrographis paniculata are the commonly used herbs by the diabetic patients in Pampanga, Philippines. While the anti-diabetic potential of Momordica charantia is well established in streptozocin- or alloxan-induced diabetic animals, the anti-diabetic potential of Andrographis paniculata in alloxan-induced diabetic rat is not known. Neither the effects of these herbs on estrous cyclicity of alloxan-induced diabetic rats are elucidated. Thus, in these experiments, Momordica charantia fruit juice or Andrographis paniculata decoction was orally administered to alloxan-induced diabetic rats. Rats that were treated with Momordica charantia and Andrographis paniculata had higher body weight (BW) compared with diabetic positive control (P < 0.01) from day 22 to day 27 (D27) but exhibited lower BW than the non-diabetic control (P < 0.05). These rats had lower feed (P < 0.05) and liquid intakes (P < 0.01) compared with diabetic positive control from day 17 to D27, but similar with the non-diabetic control. The blood glucose levels in these groups were significantly reduced from day 12 to D27 compared with diabetic positive control (P < 0.01), however, comparable with non-diabetic control. The diabetic positive control had extended mean estrous cycles (8 days) compared to Momordica charantia and Andrographis paniculata-treated diabetic rats (5 days; P < 0.05). Our results suggest that the anti-diabetic potentials of Momordica charantia and Andrographis paniculata could restore impaired estrous cycle in alloxan-induced diabetic rats.

Molecular characterization of a 10-kDa buckwheat molecule reactive to allergic patients' IgE.

BACKGROUND: Using the sera from buckwheat (BW)-allergic patients, several putative causative molecules were reported. However, few molecules were determined on the molecular structure. We demonstrated in 2000 that the major allergen with 24 kDa (BW24KD) is a legumin-like storage protein. OBJECTIVE: The aim of this study was to isolate and characterize further a major allergen with 10 kDa by molecular cloning. METHODS AND RESULTS: Buckwheat allergens were identified by immunoblotting analysis using sera from 14 allergic and two nonallergic individuals. We identified a protein with 10 kDa (BW10KD) that reacted with immunoglobulin E (IgE) more strongly than with IgG and IgA in 57% of the allergic patients but not with IgE in nonallergic individuals. Analyses were performed by N-terminal amino acid sequencing and molecular cloning. Physiological significance was assessed by an immunoblotting experiment showing that the reactivity of an allergic patient's serum IgE to BW10KD was competitively inhibited by natural BW extracts. CONCLUSION: Molecular cloning experiments indicated that BW10KD as a BW allergen was a member of the 2S-albumin multigene family.

Glucoprivation increases estrogen receptor alpha immunoreactivity in the brain catecholaminergic neurons in ovariectomized rats.

Estrogen-dependent enhancement of glucoprivic-induced luteinizing hormone (LH) suppression is hypothesized to be due to increased estrogen receptor alpha (ERalpha)-immunoreactive (ir) cells in specific brain nuclei in a manner similar to fasting. ERalpha expression in various brain areas was determined in ovariectomized rats after systemic 2-deoxy-D-glucose (2DG)-induced glucoprivation. Expression of ERalpha in catecholaminergic neurons in the lower brainstem was also examined. ERalpha-ir cells increased in hypothalamic paraventricular and periventricular nuclei, and A1 and A2 regions of the brainstem 1 h after 2DG injection. The percentage of ERalpha in the tyrosine hydroxylase (TH)- and dopamine-beta-hydroxylase (DBH)-ir neurons was higher in A1 and A2 regions of 2DG-treated rats, but the number of TH- and DBH-ir cells did not change. Thus, 2DG induces ERalpha expression in specific brain nuclei and expression of ERalpha in catecholaminergic neurons of the brainstem indicates a role for estrogen in activating those neurons projecting to the hypothalamic paraventricular nucleus to suppress LH secretion during glucoprivation.

Association of Helicobacter pylori infection with atrophic gastritis and intestinal metaplasia.

AIMS: To evaluate the effect of Helicobacter pylori infection and aging on atrophy and intestinal metaplasia of the gastric mucosa. METHODS: One hundred and sixty-three patients were divided into three age groups and underwent an upper gastrointestinal endoscopy where no esophagitis, peptic ulcers, or malignancies were detected. Two biopsy specimens were obtained from the anterior and posterior walls of the antrum and of the fundus. These were used to evaluate the grade of gastritis, bacterial culture and histologic evidence of H. pylori infection. RESULTS: Helicobacter pylori infection was found to be directly associated with an increased risk of gastritis grade (odds ratio (OR) = 90 (95% CI; 30-270)). An age of 60 years and older along with H. pylori infection was also strongly associated with an increased risk of atrophy (OR = 6.6, (95% CI; 2.9-15.2)); OR = 9.8, (95% CI; 2.7-35.4)), as was intestinal metaplasia of the gastric mucosa (OR = 5.5, (95% CI; 1.7-17.6)); OR = 7.9, (95% CI; 2.8-46.1)). The prevalence of atrophic gastritis increased with advancing age in H. pylori-infected patients, but no such phenomenon was observed in H. pylori-uninfected patients. The prevalence of intestinal metaplasia significantly increased with advancing age, irrespective of the presence of H. pylori infection. In addition, H. pylori uninfected female patients had a decreased risk of intestinal metaplasia. CONCLUSIONS: These results suggest that atrophic gastritis is not a normal aging process, but instead is likely to be the result of H. pylori infection, while intestinal metaplasia is caused by both the aging process and H. pylori infection. A decreased risk of intestinal metaplasia found in uninfected female subjects may partly explain the lower prevalence of gastric cancer in females than in males.

Contribution of angiogenesis to the progression of colon cancer: possible inhibitory effect of angiogenesis inhibitor TNP-470 on tumor growth and hepatic metastasis.

The contribution of angiogenesis to tumor growth and hepatic metastasis of colorectal cancer was investigated by means of immunohistochemical study and in vitro and in vivo experiments. Colorectal cancer specimens from 30 patients with hepatic metastasis and 39 patients without hepatic metastasis were studied by staining with antibodies against factor VIII-related antigen. Microvessel count in patients with liver metastasis was significantly higher than in those without liver metastasis (p<0.005). The effect of TNP-470 was evaluated with in vitro and in vivo experiments using human colon cancer cell line, LM and the highly hepatic metastasis cell line, LM-H5. The effect of TNP-470 on the proliferation of the cancer cells and human umbilical vein endothelial cells (HUVECs) was examined. TNP-470 inhibited more sensitively the proliferation of HUVECs than cancer cells in vitro. IC50 was approximately 3 pg/ml in HUVECs and approximately 2 microg/ml in cancer cells. The effect of TNP-470 on the growth of xenografts and liver metastases by LM-H5 in nude mice was examined. TNP-470 (30 mg/kg) was administered by subcutaneous injection every third day for 4 weeks. TNP-470 inhibited both the growth of xenograft and the hepatic metastasis. The number of metastatic foci in the liver was 78.2+/-30.1 in the control group and 20.6+/-16.5 in the treated group. These results suggest that TNP-470 is a potent agent to inhibit tumor growth and hepatic metastasis of colon cancer.

Amino-acid replacements in an internal region of tropomyosin alter the properties of the entire molecule.

Two isoforms of lobster muscle tropomyosin, a fast muscle type, fTm, and a slow muscle type, sTm1, are identical except for 15 residues within the region of amino acids 39-80, which corresponds to exon 2 of the tropomyosin genes of many phyla. Although the difference in the sequence does not include the terminal regions, the two isoforms are extremely different in viscosity, which is a good measure of the head-to-tail interaction strength and should be dependent on the conformation of the terminal 7-9 residues. To determine the influence of amino-acid replacements in the internal region on the overall conformation and the functional properties of the molecule, we compared the physical properties of the two isoforms and their interactions with other proteins, such as actin and myosin subfragment 1 (S1). Limited proteolysis by trypsin and chymotrypsin showed that sTm1 is more susceptible than fTm at the sites outside the region with the replaced residues. Compared with fTm, sTm1 showed higher viscosity, had a higher actin affinity, and inhibited acto-S1 ATPase to a greater extent. Finally, the binding isotherm of S1-ADP to actin-sTm1 is less sigmoidal than that to actin-fTm. These results indicate that the amino-acid replacements in the internal region alter the conformation and the physical properties of the entire molecule as well as its interactions with actin and myosin.

Chinese herb nephropathy in Japan presents adult-onset Fanconi syndrome: could different components of aristolochic acids cause a different type of Chinese herb nephropathy?

BACKGROUND: We encountered two cases of Chinese herb-induced Fanconi syndrome in Japan. One component of the chinese medicine was "Kan-mokutsu" (Aristolochia manshuriensis) in which aristolochic acids (AAs) were detected. METHODS: Renal biopsy showed flattening of proximal tubular epithelial cells and paucicellular interstitial fibrosis without glomerular lesions, all of which were in accordance with Chinese herb nephropathy (CHN). To date, many cases of CHN have been reported mainly as progressive renal failure in western countries. RESULTS: However, our cases were different from those in that they presented Fanconi syndrome. The detected AAs in our cases consisted of aristolochic acid (AA)-I, II and D. In contrast, in Belgium, the incriminated agent was Aristolochia fangchi which consisted of AA-I, B, C, and aristolactum. CONCLUSION: These findings could indicate that different components of AAs could cause different clinical lesions, or that the amount of ingested AAs might reflect clinical pictures, that is to say, our patients took lower volume of Chinese herbs and might be in an early stage of CHN. Furthermore, it is likely that susceptibility to this substance may be different among races. CHN would include two clinical aspects: subacute renal failure and adult-onset Fanconi syndrome. It is important to bear in mind that CHN could present Fanconi syndrome.

[Weekly hepatic arterial infusion of high dose 5-fluorouracil for liver metastasis from colorectal cancer].

A weekly infusion of high dose 5-fluorouracil by way of the hepatic artery has been performed in 23 cases with synchronous metastasis from colorectal cancer since 1993. The prognosis in these cases was compared with 94 cases treated without infusion chemotherapy in 94 cases before 1992. The overall one-year and three-year survival rate was 64.8% and 30.2%, respectively, in cases with infusion chemotherapy. The one-year and three-year survival rate was 42.8% and 18.6%, respectively, in cases without infusion chemotherapy. Overall survival rate was significantly different between cases with and without infusion chemotherapy (p < 0.05). In conclusion, weekly infusion chemotherapy resulted in a better survival rate than without infusion chemotherapy.

Intussusception of a mucocele of the appendix secondary to an obstruction by endometriosis: report of a case.

We treated a patient with a complete invagination of the cecum which contained a mucocele of the appendix secondary to an obstruction by endometriosis. Preoperatively, a barium enema showed a crab's claw-like area without filling in the oral side of the transverse colon. An emergency laparotomy was performed and revealed a mucocele of the appendix to have induced appendicecal invagination; however, no colonic invagination was observed. An appendicecal resection was thus done. Pathologically, the resected specimen was a mucosal hyperplasia with mucin-secreting lesions of the appendix. The theories regarding the pathogenesis of appendicecal mucocele are reviewed and discussed.

Effects of visual and auditory stimulation on somatosensory evoked magnetic fields.

DESIGN AND METHODS: We investigated the effects of continuous visual (cartoon and random dot motion) and auditory (music) stimulation on somatosensory evoked magnetic fields (SEFs) following electrical stimulation of the median nerve on 12 normal subjects using paired t test and two way ANOVA for the statistics. RESULTS: In the hemisphere contralateral to the stimulated nerve, the middle-latency components (35-60 ms in latency) were significantly enhanced by visual, but not by auditory stimulation. The dipoles of all components within 60-70 ms following stimulation were estimated to be very close each other, around the hand area of the primary sensory cortex (SI). In the ipsilateral hemisphere, the middle-latency components (70-100 ms in latency), the dipoles of which were estimated to be in the second sensory cortex (SII), were markedly decreased in amplitude by both the visual and auditory stimulation. CONCLUSIONS: These changes in waveform by visual and auditory stimulation are thought to be due to the effects of the activation of polymodal neurons, which receive not only somatosensory but also visual and/or auditory inputs, in areas 5 and/or 7 as well as in the medial superior temporal region (MST) and superior temporal sulcus (STS), although a change of attention might also be a factor causing such findings.

Steroid sulfatase expression is an independent predictor of recurrence in human breast cancer.

Steroid sulfatase (STS) hydrolyzes several sulfated steroids such as estrone sulfate, dehydroepiandrosterone sulfate, and cholesterol sulfate. In the present study, we have measured STS mRNA levels in 97 breast cancers by reverse transcription-PCR using a fluorescent primer in the presence of an internal standard RNA and evaluated its association with disease-free and overall survival. The median value was 728.0 amol/ng RNA (range, 0-11,778 amol/ng RNA). Levels were significantly higher in tumors demonstrating lymph node metastasis than in those without nodal involvement (P = 0.033) and in patients who experienced a recurrence during the follow-up period (mean, 40.8 months; median, 39 months) compared with those with no evidence of further disease (mean, 49.2 months; median, 48 months; P = 0.029). No significant associations were found between STS mRNA expression and age, menopausal status, tumor size, histological grade, estrogen receptor status, or postoperative adjuvant therapy. High levels of STS mRNA proved to be a significant predictor of reduced relapse-free survival as a continuous variable (log STS mRNA; P = 0.028). As a dichotomous variable with an optimized cutoff point of 1,240 amol/ng RNA, expression was also associated with a significantly shorter relapse-free survival rate (P = 0.002), but no significant correlation was found between the STS mRNA level and overall survival. Expression was found to be an independent factor for predicting relapse-free survival on multivariate analysis. The results thus support a putative role of STS in breast cancer growth and metastasis.

Total synthesis of a new cytotoxic acetogenin, jimenezin, and the revised structure.

The first total synthesis of jimenezin was achieved by using carbohydrates as chiral building blocks, thus revising the proposed structure 1 to 2. The key steps in this synthesis include an efficient construction of the THP-THF fragments 3 and 16 through a stereoselective condensation between the pyranyl aldehyde 5 and the acetylene derivative 6, and a palladium-catalyzed coupling reaction of 3 or 16 with a terminal butenolide 4.

Effects of L-carnitine supplementation on muscular symptoms in hemodialyzed patients.

Various muscle symptoms are well recognized among patients on maintenance hemodialysis. Carnitine deficiency may be an important factor of dialysis-associated muscle symptoms, whereas high-dose L-carnitine supplementation may result in unphysiologically high plasma levels of carnitine and carnitine esters. We studied the effect of low-dose L-carnitine treatment (500 mg/d) on muscle symptoms, plasma carnitine fractions, and lipid profiles in 30 periodically dialyzed patients with muscular weakness, fatigue, or cramps/aches. After 12 weeks of L-carnitine treatment, about two-thirds of patients had at least some improvement in muscular symptoms, whereas carnitine fractions were normal or slightly above normal ranges, but lipid profiles showed no demonstrable changes. This study also showed the correlation between plasma-free carnitine deficiency and months on dialysis. These results suggest that prolonged low-dose L-carnitine treatment can improve dialysis-associated muscle symptoms by restoring carnitine tissue levels and washing out acyl moieties.

Functional quantitative analysis of the genome in cultured human mesangial cells. Technical note.

For normal physiological function, each cell tightly regulates gene expression in a specific fashion so that critical proteins are synthesized in a well-coordinated manner. Therefore, it is very important to uncover which genes are expressed in specific cells. Recent technological advances combined with rapid large-scale DNA sequencing and computerized data processing have allowed us to investigate the expression levels of a variety of transcripts in the mesangial cells, a target of injury in many forms of glomerulonephritis. Utilizing a large scale sequencing of a 3'-directed cDNA library, which allows us to avoid variable cloning efficiencies reflecting the size of cDNA, we investigated expression profiles of various molecules in cultured human mesangial cells. Among the 1,193 sequenced clones, 688 (57.7%) appeared more than once (redundant sequence group), representing 203 different species. Thirty-nine of these appeared more than three times. The most abundant mRNA was that of fibronectin, which consisted of 3.9% of the total mRNA population. Except for mitochondrial or ribosomal genes, calcyclin came next (2.5%), followed by two cytoskeletal genes, gamma-actin gene and calpactin 1 light chain gene, in addition to an amyloid precursor protein homolog (0.7%). In conclusion, we performed a molecular biological quantification of transcripts in mesangial cells. Fibronectin was the most abundantly expressed, followed by calcyclin, gamma-actin, calpactin 1 light chain, and an amyloid precursor protein homolog. We also discovered some candidate genes specific for human mesangial cells. The expression profile of the transcripts serves as an important tool in understanding the biological properties of mesangial cells.

[Chinese herbs nephropathy in the Kansai area: a warning report].

In 1993, Vanherweghem and his associates reported cases of rapidly progressive renal interstitial fibrosis in young women who were administered a slimming regimen including Chinese herbs. Subsequently, similar cases have been reported. In Japan, especially in the Kansai area, several cases of Chinese herbs nephropathy have already been reported. We experienced a patient suffering from Chinese herbs nephropathy (CHN), and further detected aristolochic acids from the Chinese herbs taken by the patient. Aristolochic acids are known to be causative agents of CHN. The danger of CHN should be noted as soon as possible and drugs containing aristolochic acids should be prohibited.