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.

Three types of gonadotrophin-releasing hormone neurones and steroid-sensitive sexually dimorphic kisspeptin neurones in teleosts.

In general, the gonadotrophin-releasing hormone (GnRH) neuronal systems of vertebrates consist of one group of hypothalamic neuroendocrine and one or two group(s) of extrahypothalamic neuromodulatory GnRH neurones. By taking advantage of the brains of dwarf gourami and GnRH-green fluorescent protein transgenic medaka, the spontaneous electrical activities of all three different types of GnRH neurones have now been characterised. The hypophysiotrophic preoptic area-gnrh1 neurones show irregular and episodic spontaneous electrical activities, whereas extrahypothalamic midbrain gnrh2 and terminal nerve-gnrh3 neurones show regular pacemaker potentials. It is suggested that these spontaneous electrical activities are related to their different functions as neuroendocrine hormones (gnrh1 neurones) and neuromodulators (gnrh2 and gnrh3 neurones). On the other hand, recent evidence strongly suggests that the GnRH neurones are regulated by another class of peptidergic neurones, the kisspeptin neurones. The gene encoding kisspeptin (kiss1 gene) has been cloned, and the anatomical distribution of kiss1 mRNA expressing neurones (kiss1 neurones) has recently been studied in brains of several fish species. In medaka, two kiss1 neuronal populations in hypothalamic areas, called the nucleus ventral tuberis (NVT) and nucleus posterioris periventricularis (NPPv), have been identified. The NVT kiss1 neurones are sexually dimorphic in number (male >> female) under breeding conditions and are sensitive to ovarian oestrogens, whereas the NPPv kiss1 neurones are neither sexually dimorphic, nor sensitive to steroids. The steroid-sensitive changes in kiss1 mRNA expression in the NVT occur physiologically, closely linked to the reproductive state. As in the mammalian counterpart, the medaka kiss1 neuronal system is suggested to be involved in the central regulation of reproductive functions. Interestingly, kiss2, another gene paralogous to kiss1, has been cloned in some fish species. The possible regulation of reproduction and other unknown functions by kiss1 and kiss2 neurones may be the focus of future studies.

Imaging postsynaptic activities of teleost thalamic neurons at single cell resolution using a voltage-sensitive dye.

Optical recording of neuronal activities using voltage-sensitive dyes (VSDs) is a useful method for simultaneous multi-site recording. However, it has been rather difficult to distinguish optical signals from individual, identified cells. We applied the optical recording technique using a high-speed charge coupled device (CCD) imaging system to a teleost thalamic nucleus, corpus glomerulosum (CG) which has a well-defined histological organization and large postsynaptic dendrites. Patch-like dye (di-4-ANEPPS) signals were observed in the dendritic layer of the CG in response to afferent nerve stimulations. These responses were completely blocked by an alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA) receptor antagonist, did not propagate, and the size of the patches were close to that of a single dendritic tip of the 'large cell'. Thus, we found that these patch-like VSD signals most likely represent postsynaptic potentials at individual dendritic tips of the large cells.

Effects of characteristic dendritic tip geometry on the electrical properties of teleost thalamic neurons.

Of the factors that characterize the properties and functions of neurons, dendritic geometry is one of the most critical. We used simulations employing the multi-compartment model to study the effects of dendritic tip geometry on the electrical properties of the "large cell" in a teleost thalamic nucleus from the corpus glomerulosum. We demonstrated a dramatic geometrical "boosting" effect; passive propagation of the synaptic inputs from the dendritic tip to the soma through the dendritic stalk is less attenuated in cells with larger tips.

Cloning of cDNAs encoding two isoforms of 68-kDa type I phosphatidylinositol-4-phosphate 5-kinase.

Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell. Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinositol- 4-phosphate 5-kinase (PI4P5K) have been identified as cytosolic components required for ATP-dependent, Ca2+-activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic beta-cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic beta-cell line MIN6. Two novel cDNAs, designated PI4P5K-Ialpha and PI4P5K-Ibeta, were identified, which contained complete coding sequences encoding 539- or 546-amino acid proteins, respectively. These cDNAs were expressed in mammalian cells with an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phosphatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoforms. While these isoforms share a marked amino acid sequence homology in their central portion, the amino- and carboxyl-terminal regions differ significantly. Northern blot analysis depicted that tissue distributions differed between the two isoforms. Molecular identification of type I PI4P5K isoforms in insulin-secreting cells should provide insights into the role of phosphatidylinositol metabolism in regulated exocytosis of insulin-containing large dense core vesicles.

[Study of components in crude drugs by headspace gas chromatography. II. Components of atractylodes].

Determination of volatile components in essential oils from Atractylodis plants was studied by headspace gas chromatography. The crude drug of 0.20 g with 1.0 ml of water in a capped vial was heated at 130 degrees C for 45 min. Then 0.5 ml of vaporized components were collected by gas tightsillinge, and were applied into the injection port of GC or GC/MS. Consequently we could analyze and confirm the following components; hinesol and beta-eudesmol were found to be contained in Atractylodis Lanceae Rhizoma and atractylon in Atractylodis Rhizoma. beta-Eudesmol was analyzed by headspace gas chromatography, and the obtained calibration curve showed good linearity over the range from 2.5 micrograms to 10.0 mg. The result agreed with those obtained using numerical analyses by the steam distillation method. Atractylodis was found to have a wide variety of components depending on the available sources and on the stored conditions. This method was, therefore, more rapid and simpler determination of essential oils in crude drugs using headspace gas chromatography than those used previously. The method was useful means of the analyses of components in crude drugs such as Atractylodis plants and quality control.

Effect of Lactobacillus acidophilus on iron bioavailability in rats.

The effect of Lactobacillus acidophilus on iron bioavailability in rats was examined by the hemoglobin regeneration method. For hemoglobin depletion, female Wistar rats were fed an iron-deficient diet at 3 weeks of age for 13 days. Rats were then assigned to one of four groups, such that average blood hemoglobin value and average body weight were similar among the groups. For hemoglobin regeneration, they were fed one of two ferrous sulfate-supplemented diets that contained the following iron levels (mg/kg): 13.7 for two groups; 21.7 for the other two groups. In the two groups fed the same diet, one group additionally received oral administration of 2 ml of skim milk fortified with 0.3% yeast extract once or twice a day for 7 days, and other rats were administered with skim milk fermented by L. acidophilus SBT 2062 in the same manner. Hemoglobin regeneration efficiency (HRE) was significantly higher in the fermented product-given rats than in the skim milk-supplied rats. There was no significant interaction in HRE between the dietary iron group and the oral administration group. These results indicate that L. acidophilus SBT 2062 is effective for increasing of iron bioavailability in rats.

[Study of components in crude drugs by head space gas chromatography. I. Components of Asiasari radix].

Determinations of volatile components of essential oil contained in Asiasari Radix were studied by head space gas chromatography. The head space gas, which was generated by heating 0.2 g of crude drug with 1.0 ml of water at 90 degrees C for 45 min, was analyzed directly by gas chromatography or gas chromatography-mass spectrometry. This method was rapid, and did not require tedious procedures and time consuming, and without contamination of the column.

[A case of remarkable regression of a colonic cancer involving metastasis to the liver as a result of FT-207 treatment].

In mid-August, 1986, a 50-year-old man underwent a detailed examination following the finding of a tumor in the upper abdominal region. The tumor was revealed to be a sigmoid colon cancer with multiple metastasis to both halves of the liver. Subsequent administration of FT-207 suppositories (750 mg X 2/day) resulted in a recognizable shrinkage of the metastatic lesions in the liver after one month, and a barium enema a month later indicated a decrease in the size of the original tumor. The pre-operative CEA value was 2,317 ng/ml, but this has reduced to 46.7 ng/ml to date March, 1987. In this report of single chemotherapy treatment with FT-207 of a sigmoid colon cancer involving metastasis to the liver, the prognosis obtained for the original and hepatic lesions has been good, and the patient's course to date healthy.

The cohering telomeres of Oxytricha.

We have studied the process by which purified Oxytricha macronuclear DNA associates with itself to form large aggregates. The various macronuclear DNA molecules all have the same terminal or telomeric DNA sequences that are shown below. 5' C4A4C4A4C4--mean length----G4T4G4T4G4T4G4T4G4 G4T4G4T4G4T4G4T4G4-----2.4 kb------C4A4C4A4C4. When incubated at high concentrations, these telomeric sequences cohere with one another to form an unusual structure--one that is quite different from any DNA structure so far described. The evidence for this is the following: 1) These sequences cohere albeit slowly, in the presence of relatively high concentrations of Na+, and no other cation tested. This contrasts with the rapid coherence of complementary single-chain terminals of normal DNA (sticky ends) which occurs in the presence of any cation tested. 2) If the cohered form is transferred into buffers containing a special cation, K+, it becomes much more resistant to dissociation by heating. We estimate that K+ increases the thermal stability by 25 degrees or more. The only precedent known (to us) for a cation-specific stabilization is that seen in the quadruplex structure formed by poly I. The thermal stability of double helical macronuclear DNA depends on the cation concentration, but not the cation type. Limited treatment with specific nucleases show that the 3' and 5'-ended strands are essential for the formation of the cohering structure. Once in the cohered form, the telomeric sequences are protected from the action of nucleases. Coherence is inhibited by specific, but not by non-specific, synthetic oligomers, and by short telomeric fragments with or without their terminal single chains. We conclude that the coherence occurs by the formation of a novel condensed structure that involves the terminal nucleotides in three or four chains.

Serum zinc and copper concentrations in low birth weight infants during first three months of life: correlation to birth weight and different feedings.

Serum zinc (Zn) and copper (Cu) concentrations were measured in infants weighing 740 g to 2,500 g at birth. They were divided into three groups depending on their birth weight; Group I ranged from 740 to 1,500 g (N = 35), Group II from 1,501 to 2,000 g (N = 26), and Group III from 2,001 to 2,500 g (N = 19). They were fed breast milk (N = 24), commercially available non-supplemented formula with low Zn and low Cu content (N = 42), or Zn- and Cu-supplemented formula (N = 14). Serum Zn levels of breast fed infants were apparently higher than those of the other two different formula fed infants, the difference being significant at 1 month of age in Group I. After the decline of serum Zn level during the first month of life, the level in Groups II and III remained unchanged, whereas the level in Group I further declined progressively until 3 months of age. An incidence of hypozincemia (0.65 micrograms/ml) was highest in Group I, medium in Group II, and lowest in Group III. The serum Cu level was elevated progressively after birth in all groups, but the mean level was consistently highest in Group III, medium in Group II and lowest in Group I. No difference in serum Cu level was found among the infants with different feedings.

Multiple lipomas of the ileum with volvulus.

We describe a patient with symptomatic multiple lipomas with volvulus. He complained of gradually increased abdominal pain and distention. Preoperative roentgenograms showed many rounded filling defects of the small intestine. Operation was performed to reduce intestinal obstruction and ileal resection was performed. The dilated ileum was rotated 720 degree clockwise and it contained 115 submucosal and 26 subserosal lipomas. Histologically, the lipomas were benign and consisted of well-developed fat tissue. The patient's postoperative course was satisfactory, and neither residual lipoma nor recurrence were found at barium enema examination two years after operation.