Iron-binding activity of FutA1 subunit of an ABC-type iron transporter in the cyanobacterium Synechocystis sp. Strain PCC 6803.

The futA1 (slr1295) and futA2 (slr0513) genes encode periplasmic binding proteins of an ATP-binding cassette (ABC)-type iron transporter in Synechocystis sp. PCC 6803. FutA1 was expressed in Escherichia coli as a GST-tagged recombinant protein (rFutA1). Solution containing purified rFutA1 and ferric chloride showed an absorption spectrum with a peak at 453 nm. The absorbance at this wavelength rose linearly as the amount of iron bound to rFutA1 increased to reach a plateau when the molar ratio of iron to rFutA1 became unity. The association constant of rFutA1 for iron in vitro was about 1 x 10(19). These results demonstrate that the FutA1 binds the ferric ion with high affinity. The activity of iron uptake in the Delta futA1 and Delta futA2 mutants was 37 and 84%, respectively, of that in the wild-type and the activity was less than 5% in the Delta futA1/Delta futA2 double mutant, suggesting their redundant role for binding iron. High concentrations of citrate inhibited ferric iron uptake. These results suggest that the natural iron source transported by the Fut system is not ferric citrate.

Genes essential to iron transport in the cyanobacterium Synechocystis sp. strain PCC 6803.

Genes encoding polypeptides of an ATP binding cassette (ABC)-type ferric iron transporter that plays a major role in iron acquisition in Synechocystis sp. strain PCC 6803 were identified. These genes are slr1295, slr0513, slr0327, and recently reported sll1878 (Katoh et al., J. Bacteriol. 182:6523-6524, 2000) and were designated futA1, futA2, futB, and futC, respectively, for their involvement in ferric iron uptake. Inactivation of these genes individually or futA1 and futA2 together greatly reduced the activity of ferric iron uptake in cells grown in complete medium or iron-deprived medium. All the fut genes are expressed in cells grown in complete medium, and expression was enhanced by iron starvation. The futA1 and futA2 genes appear to encode periplasmic proteins that play a redundant role in iron binding. The deduced products of futB and futC genes contain nucleotide-binding motifs and belong to the ABC transporter family of inner-membrane-bound and membrane-associated proteins, respectively. These results and sequence similarities among the four genes suggest that the Fut system is related to the Sfu/Fbp family of iron transporters. Inactivation of slr1392, a homologue of feoB in Escherichia coli, greatly reduced the activity of ferrous iron transport. This system is induced by intracellular low iron concentrations that are achieved in cells exposed to iron-free medium or in the fut-less mutants grown in complete medium.

A gene of Synechocystis sp. Strain PCC 6803 encoding a novel iron transporter.

A mutant of Synechocystis sp. strain PCC 6803 disrupted for sll1878 exhibited greatly reduced Fe(3+) transport activity. The K(m) value of sll1878-dependent Fe(3+) transport in cells grown in iron-replete medium was 0.5 microM. Both the maximal rate and K(m) value were increased in iron-starved cells.

The effect of maimendongtang on airway clearance and secretion.

We investigated the effect of Maimendongtang on airway clearance and secretion in anaesthetized quails. The oral application of 1 g/kg of Maimendongtang significantly increased tracheal mucociliary transport velocity (MCTV), whereas 100 or 300 mg/kg of Maimendongtang failed to do so. Moreover, 300 mg/kg or 1 g/kg of Maimendongtang markedly attenuated human neutrophil elastase (HNE) or DNA-induced decrease in MCTV although 100 mg/kg of Maimendongtang had little effect. Furthermore, we found that Maimendongtang significantly restored HNE-induced increases in DNA, fucose and protein contents of airway surface fluid (ASF), whereas only Maimendongtang itself significantly decreased the protein content. These results indicate that Maimendongtang increases MCTV and the increase may be, at least in part, ascribed to the amelioration of ASF in the trachea.

Biological effects of prolonged exposure to ELF electromagnetic fields in rats: III. 50 Hz electromagnetic fields.

Groups of adult male Sprague Dawley rats (64 rats each) were exposed for 8 months to electromagnetic fields (EMF) of two different field strength combinations: 5microT - 1kV/m and 100microT - 5kV/m. A third group was sham exposed. Field exposure was 8 hrs/day for 5 days/week. Blood samples were collected for hematology determinations before the onset of exposure and at 12 week intervals. At sacrifice, liver, heart, mesenteric lymph nodes, bone marrow, and testes were collected for morphology and histology assessments, while the pineal gland and brain were collected for biochemical determinations. At both field strength combinations, no pathological changes were observed in animal growth rate, in morphology and histology of the collected tissue specimens (liver, heart, mesenteric lymph nodes, testes, bone marrow), and in serum chemistry. An increase in norepinephrine levels occurred in the pineal gland of rats exposed to the higher field strength. The major changes in the brain involved the opioid system in frontal cortex, parietal cortex, and hippocampus. From the present findings it may be hypothesized that EMF may cause alteration of some brain functions.

Evaluation of histopathologic changes in an animal model of mechanical corpus callosum impingement as seen in hydrocephalus.

RATIONALE AND OBJECTIVES: We evaluated histologic changes associated with chronic impingement of the corpus callosum. Similar callosal impingement has been postulated to be responsible for some of the symptoms in people who have hydrocephalus. METHODS: Eight rats with callosal impingement produced by surgical implantation of a blunt blade in the interhemispheric fissure and four control animals with no callosal impingement were evaluated by magnetic resonance (MR) imaging and by direct histologic evaluation after autopsy. The histologic evaluations occurred 1 month after surgery in half the animals and 6 months after surgery in the other half. RESULTS: MR imaging results showed that the implanted blade was in a good position in all animals. Histologically, the corpus callosum appeared normal 1 month after implantation of the impingement blade. Six months after surgery, the experimental group demonstrated decreased callosal thickness and a loss of axonal fibers in the corpus callosum both near and remote to the blade. CONCLUSION: Chronic impingement of the corpus callosum was associated with callosal thinning and by loss of callosal axons. Further research will be required to investigate the possible relation of these histologic findings to the clinical findings in normal-pressure hydrocephalus.

[Changes in monoamine concentrations in developing female rat brains under continuous light].

Norepinephrine (NE), dopamine (DA) and serotonin (5-HT) concentrations in the hypothalamus were measured by HPLC on days 10, 20, 30 and 40 of age in Sprague-Dawley albino female rats after continuously exposed to light (LL). When rats were housed under a lighting schedule (14hr light and 10hr dark: LD), the NE concentration increased linearly. The DA concentration remained lower on days 10, 20 and 30 of age, but a sudden increase in the DA concentration appeared on day 40 of age. An increase in the 5-HT concentration occurred at day 20 of age, and remained at the same level thereafter. LL exerts profound effects on DA and 5-HT concentrations in the hypothalamus; an increase in the 5-HT concentration appeared at day 10 of age, and the peak concentration of DA was reached at day 30 of age. The inference is then that LL exerts effects on the metabolism of 5-HT and DA and promotes the increase in 5-HT and DA concentrations in the hypothalamus, and that the advanced appearance of 5-HT and DA in the hypothalamus seems to correspond with the advancement of puberty onset. Indeed, LL exerted its effects on puberty onset and advanced it.

Long-term estrogen replacement therapy in female patients with dementia of the Alzheimer type: 7 case reports.

Seven female patients with mild to moderate dementia of the Alzheimer type (DAT) were treated with long-term, low-dose estrogen replacement therapy (ERT) over a period of 5-45 months. Five of the 7 patients were cases who had responded well to short-term ERT with 1.25 mg/day of conjugated equine estrogens (CEE) for 6 weeks. The 7 patients from 56 to 77 years of age received 0.625 mg/day of CEE for 21 days, followed by a pause of 7 days. A 28-day cycle of low-dose ERT was performed repeatedly. In 4 cases, these patients received 5 mg/day of medroxyprogesterone acetate (MPA) during the last 10-12 days of estrogen treatment. Therapeutic efficacy of estrogen was evaluated by psychometric assessments such as the Mini-Mental State Examination (MMSE) and the Hasegawa Dementia Scale (HDS) and a behavior rating scale of the Gottfries-Bråne-Steen geriatric rating scale (GBS). The MMSE and HDS evaluations were performed principally once in 2-4 weeks. In 4 out of the 7 patients, the MMSE and HDS scores were elevated above the pretreatment levels during ERT. The termination of ERT resulted in a decrease in both scores. Furthermore, the GBS scores and daily activities of the same 4 patients were improved during ERT. In these 4 patients cognitive functions were markedly improved throughout the treatment period, while the other 2 patients responded moderately well and another patient did not respond at all. These observations suggest that long-term, low-dose ERT improves cognitive functions, dementia symptoms and daily activities in women with mild to moderate DAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of estrogen treatment in female patients with dementia of the Alzheimer type.

This study was designed to investigate the therapeutic efficacy of estrogen in female patients with dementia of the Alzheimer type (DAT). Fifteen DAT patients with a mean age of (mean +/- SE) 71.9 +/- 2.4 years were treated with 0.625 mg of conjugated equine estrogens orally twice a day for 6 weeks. Of the 15 DAT patients, 4 were diagnosed as mild, 7 as moderate and 4 as severe. The effects of estrogen on DAT patients were evaluated by psychometric assessments, behavior rating scales, regional cerebral blood flow (rCBF) measurement and quantitative EEG analysis. Psychometric assessments consisted of Mini-Mental State Examination (MMSE) and Hasegawa Dementia Scale (HDS). Dementia syndromes were evaluated by the GBS-Scale (GBSS) and Hamilton Depression Rating Scale (HDRS). During estrogen replacement therapy (ERT), the mean MMSE score (mean +/- SE) increased significantly from 11.6 +/- 1.9 to 13.2 +/- 2.0 at 3 weeks (P < 0.01) and 13.8 +/- 2.0 at 6 weeks (P < 0.001). The mean HDS score increased significantly from 8.6 +/- 2.1 to 11.5 +/- 2.3 at 3 weeks (P < 0.001) and 11.6 +/- 2.6 at 6 weeks (P < 0.01). Significant improvements in the mean scores of the GBSS and HDRS were also observed in the estrogen-treated group, but not in the untreated control group with a mean age of 71.2 +/- 2.5 years (n = 15). The rCBF was measured by single photon emission computed tomography (SPECT). ERT increased the mean rCBF significantly in the lower frontal region (P < 0.01) and primary motor area (P < 0.02) of the right hemisphere. The mean absolute power delta band values in both left and right frontal EEG (Fp1 and Fp2) (P < 0.01) and theta, band values in Fp2 (P < 0.05) decreased significantly during ERT. It is inferred that ERT significantly improves cognitive functions, dementia symptoms, regional cerebral blood flow and EEG activity in female patients with DAT.

Estrogen in cognetive behaviors--clinical application of estrogen for dementia of Alzheimer type.

We may assume the presence of specific neuron elements in the region between the optic chiasma and the paraventricular nuclei, which are sensitive to estrogen and may be inhibited (or excited) by the estrogen level of the blood and in turn regulate (increase or decrease) the secretion of gonadotropin.

In vivo studies of the effect of magnetic field exposure on ontogeny of choline acetyltransferase in the rat brain.

Developmental increases of the activity of choline acetyltransferase (ChAT) were examined in the brains of fetuses and offspring from parent rats continuously exposed to a 500 mG, 60 Hz circularly polarized (CP) magnetic field (MF) prior to pregnancy, and further, during pregnancy and lactation. In developing rats between 12 days and 20 days of embryogenesis that were housed in a control unit, i.e., nonexposed to MF, the specific activity of ChAT in whole brain specimens increased from 2.4% to 6.9% of adult activity, while specific activity of ChAT in rat brain specimens between 12 days of embryogenesis and 10 days of postpartum increased from 2.4% to 21.6% of adult activity. On the other hand, the specific activity of ChAT in whole brain specimens from rats under housed MF exposure conditions was found to increase from 2.6% to 6.7% of adult activity between 12 days and 20 days of embryogenesis and from 2.6% to 21.6% of adult activity between 12 days of embryogenesis and 10 days postpartum. Furthermore, the effect of the same test magnetic environment on the specific activity of ChAT in the brains of parental rats was examined in order to determine whether magnetic field exposure of parental rats might reflect onto the development of fetal brain. It was observed that continuous exposure of parental rats to a 500 mG, 60 Hz CP MF did not show any significant changes in the specific activity of ChAT in the septodiagonal band complex, dorsal and ventral hippocampus, striatum, and frontoparietal cerebral cortex, as compared with the same brain regions of control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

An animal model of corpus callosum impingement as seen in patients with normal pressure hydrocephalus.

RATIONALE AND OBJECTIVES: Callosal impingement has been postulated to cause the symptoms associated with normal pressure hydrocephalus. The authors developed an animal model for the investigation of corpus callosum impingement by the falx cerebri. METHODS: The corpus callosum was compressed from above by a plastic blade and surgically placed in the interhemispheric fissure in adult Sprague-Dawley rats. Magnetic resonance imaging (MRI) was performed both preoperatively and postoperatively. The brains of the rats also were examined grossly at the time of autopsy. RESULTS: All rats survived the surgical procedure. MRI proved to be a suitable method to image the corpus callosum, to confirm the location of the blade, and to demonstrate the corpus callosum impingement. MRI correlated well with necroscopy sections. CONCLUSIONS: It was possible to surgically produce callosal impingement in rats, and this impingement could be confirmed by MRI. In the future, this rat model of callosal impingement will be used to search for evidence of changes in metabolism, neuroelectrical activity, behavior, and neuronal anatomy which are known or are thought to be associated with hydrocephalus.

Ontogenesis of the GnRH neuron system in the rat. A quantitative immunohistochemical study with special reference to the extra cerebral GnRH-positive cells and the occupation of intracerebral termination fields.

The distribution of GnRH immunopositive cells in the rat was studied from day 13 of fetal life to day 10 of postnatal life with the aid of a GnRH antibody (H-16) generated against the decapeptide p-Glu1-LHRH in the rabbit and following a PAP immunohistochemical staining protocol on 70 um serial Vibratome sections. The first appearance of GnRH cells was observed simultaneously in both intra- and extra-cerebral regions on day 16 of fetal life. At all stages of development, at least 10 times more GnRH cells were found in the brain than in extracerebral areas. The population of GnRH cells was maximum in the nasal mucosa on day 20, and in intracranial, extracerebral regions (along the anterior cerebral artery and the terminal nerve) on day 22 of fetal life: it declined thereafter. The population of GnRH cells in the brain continued to increase until the day of birth and remained unchanged thereafter. Our findings are in accordance with the theory of nasal placodal origin of the GnRH neuronal system, but also suggest that in the rat, shifting of the cells from the nasal placode to the brain is somewhat preceding the onset of GnRH synthesis. Our other field of endeavor was to examine the GnRH axon terminal fields in the course of development. On day 18 of fetal life, the vascular organ of the lamina terminalis, the median eminence and the medial habenular nuclei received their GnRH axon terminals. On day 19 the subfornical organ, mammillary nuclei and the central nucleus of the amygdala, and on day 21 the periaqueductal gray and the interpeduncular nucleus became invaded by GnRH axons. These data serve the better understanding of the onset of the different functional activities of the GnRH system in the rat.

[Experience of multimodal therapy for advanced neuroblastoma].

The prognosis of advanced neuroblastoma is extremely poor. We treated 5 patients with advanced neuroblastoma, older than 3 years, with multimodal therapy including intraoperative irradiation and autologous bone marrow transplantation. Elevated serum NSE and ferritin level and unfavorable histology according to the Shimadas histological classification, all of which are indicators of poor prognosis, were found in all of them. N-myc oncogene was amplified in 3 cases. After preoperative intensive induction chemotherapy, delayed primary operation and intraoperative irradiation (10-15 Gy) were performed. The postoperative lethal dose chemotherapy and total body irradiation (33 Gy x 3 days) were followed by autologous bone marrow transplantation. Tumor cells were purged using immunomagnetic beads method. Two cases showed recurrence (brain; 1, bone and bone marrow; 1) and a metastatic brain tumor was extirpated completely. All of them are alive during the follow up period from 6mo. to 4y8mo. (mean; 2y5mo.) with no evidence of disease except one. It may be concluded that our multimodal therapy is effective in achieving better results for advanced neuroblastoma.

Radioimmunoassay of 3 alpha-hydroxy-5 alpha-pregnan-20-one in rat and human plasma.

A radioimmunoassay for measuring 3 alpha-hydroxy-5 alpha-pregnan-20-one in plasma has been developed. Polyclonal antibodies were raised in rabbits against 3 alpha-hydroxy-20-oxo-5 alpha-pregnan-11 alpha-yl carboxymethyl ether coupled to bovine serum albumin. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was purified from either extracts of plasma by high-performance liquid chromatography. These antibodies were then used for the radioimmunoassay of this centrally active progesterone metabolite in rat and human plasma. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was detected in plasma from female rats on the day of estrus (2.0 to 9.3 ng/ml) and in the plasma of women during the luteal phase of the menstrual cycle at levels ranging from 0.25 to 2.5 ng/ml. The latter was highly correlated with plasma progesterone levels.

Direct ovulatory response with human menopausal gonadotropin of FSH/LH ratio 4 to 1.

The experiment was designed to examine if human menopausal gonadotropin (Gonadoryl, GNR, Mochida, Tokyo) influences the ovary and induces ovulation. When fifteen I.U. of GNR 4 (the ratio of FSH to LH is 4 to 1) was given on 28 days of age to immature female rats, ovulation was observed within 24 hours. Neither administration of GNR 1.6 (FSH to LH ratio is 1.6 to 1) nor GNR 3.1 (FSH to LH ratio is 3.1 to 1) induced ovulation. Administration of sodium pentobarbital (Nembutal) on the afternoon in GNR 4 treated rats did not block ovulation. It infers that GNR 4 acts on the ovary directly and induces ovulation within 24 hours.

Neuroendocrine effect of toki-shakuyaku-san on ovulation in rats.

In this study, the experiments were designed to examine if Toki-Shakuyaku-San (TSS) has a neuroendocrine effect on ovulation. TSS (500 mg/kg Bwt in drinking water) was given to Sprague Dawley immature female rats on 25 days of age and during experimental sessions. Fifteen IU of the hMG (Gonadoryl 4; GNR 4) was injected intraperitoneally on the morning of 28 days of age, and ovulation occurred in 70% of rats on the following day. In TSS drinking immature female rats, administration of GNR 4 on 28 days of age results in ovulation twice: once on 29 days of age and again on 31 days of age. Administration of sodium pentobarbital (Nembutal) at 12:30 p.m. on 28 days of age did not block the first ovulation on 29 days of age, but administration at 12:30 p.m. on 30 days of age blocked the second ovulation on 31 days of age. However, delayed ovulation was observed on 32 days of age. This evidence indicates that GNR 4 acts on the ovary directly and induces first ovulation in TSS treated rats. However, TSS treatment modulates the chain of events in the neuroendocrine control of ovulation and results in the second ovulation.

Effect of toki-shakuyaku-san on ovulation induced by human menopausal gonadotropin in rats.

One hundred and thirty-five immature female rats were treated with Toki-Shakuyaku-San (TSS:500 mg/kg, Bwt in drinking water) beginning at 25 days of age and continuing during experimental sessions. Fifteen IU of the human menopausal gonadotropin (hMG:Gonadoryl:GNR:GNR 4 contains 4 to 1 ratio of FSH and LH) was injected intraperitoneally on the morning of 28 days of age, and ovulation (presence of tubal ova) was examined on the morning of 29, 30 and 31 days of age. GNR 4 alone induced ovulation at 29 days of age (70%) and at 30 days of age (35%); however, no ovulation was observed at 31 days of age. TSS treatment alone causes 30% to ovulate at 31 days of age. When GNR 4 was combined with TSS treatment, animals demonstrated ovulation at 31 days of age, that is, they ovulated at 29 days of age (93%), at 30 days of age (30%) and at 31 days of age (90%). These results infer that TSS treatment may accelerate the chain of events in the neuroendocrine control on ovulation, thus causing more frequent ovulation in rats.

Effect of exogenous dopamine on hypothalamic dopamine and norepinephrine concentrations in the neonatal brain in rats.

Hypothalamic dopamine (DA) and norepinephrine (NE) concentrations were studied in the neonatal rats after acute (postnatal day 4) or chronic (postnatal days 1-10) DA injections (0.5 mg in 5% dextrose in 0.45% saline). Acute injection of DA twice on postnatal day 4 resulted in an increase of hypothalamic DA and NE concentrations 16 hr later. Chronic treatment with the DA (twice in a day) for 10 days resulted in a reduction of NE concentration in the hypothalamus. The results of these studies suggest that the amount and duration of exposure to exogenous DA during postnatal development may result in divergent effects on hypothalamic catecholamine concentration.