Relationship between the changes in blood flow and volume in the finger during a Braille character discrimination task.

PURPOSE: We hypothesized that skin blood flow (SBF) of fingers are modulated during concentrated finger perception and that the changes in SBF reflect fluctuations in finger volume (FV). The aim of this study, therefore, was examine the relationship between the changes in SBF and FV during Braille reading. METHODS: We measured SBF of the finger, cutaneous vascular conductance (CVC), FV, and arterial blood pressure during Braille reading performed under blind conditions in thirty healthy subjects. The subjects were instructed to read a flat plate with raised letters (Braille reading) for 15 seconds using their forefinger, and to touch a blank plate as a control for the Braille discrimination procedure. RESULTS: Arterial blood pressure slightly increased during Braille reading but remained unchanged during the touching of the blank plate. SBF, CVC, and FV were reduced during Braille reading (decreased by -26%, -29%, and -0.3 mL/100 mL respectively). Furthermore, a significant relationship was observed between the changes in SBF and FV (r=.613) during Braille reading. CONCLUSION: These results suggested that SBF of fingers is modulated during concentrated finger perception, and that the variability of blood flow reflects the response in FV.

The constitutional t(11;22): implications for a novel mechanism responsible for gross chromosomal rearrangements.

The constitutional t(11;22)(q23;q11) is the most common recurrent non-Robertsonian translocation in humans. The breakpoint sequences of both chromosomes are characterized by several hundred base pairs of palindromic AT-rich repeats (PATRRs). Similar PATRRs have also been identified at the breakpoints of other nonrecurrent translocations, suggesting that PATRR-mediated chromosomal translocation represents one of the universal pathways for gross chromosomal rearrangement in the human genome. We propose that PATRRs have the potential to form cruciform structures through intrastrand-base pairing in single-stranded DNA, creating a source of genomic instability and leading to translocations. Indeed, de novo examples of the t(11;22) are detected at a high frequency in sperm from normal healthy males. This review synthesizes recent data illustrating a novel paradigm for an apparent spermatogenesis-specific translocation mechanism. This observation has important implications pertaining to the predominantly paternal origin of de novo gross chromosomal rearrangements in humans.

Overproduction of the follistatin-related gene protein in the placenta and maternal serum of women with pre-eclampsia.

OBJECTIVE: To characterise the follistatin-related gene (FLRG) in pre-eclampsia, one of the differentially expressed genes in pre-eclamptic placenta. DESIGN AND METHODS: We examined and compared the messenger RNA (mRNA) and protein levels of FLRG in placentas and maternal sera from women with uncomplicated pregnancy, and those with pre-eclampsia using real-time reverse transcription polymerase chain reaction, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assay. SETTING: Antenatal clinics in a teaching hospital. POPULATION: Women with uncomplicated pregnancy (n = 21) and those with pre-eclampsia (n = 21). RESULTS: FLRG mRNA is overexpressed in pre-eclamptic placental tissues (P < 0.01). Upregulated FLRG protein consists of both an immature 28-kDa cellular product and a mature 33-kDa secretory form, which are differentially glycosylated. FLRG is normally produced at its highest levels in endothelial cells and at moderate amounts in syncytiotrophoblast cells, but in pre-eclampsia, the syncytiotrophoblast FLRG levels are dramatically increased. We also determined the maternal serum concentrations of FLRG in our uncomplicated pregnancy subjects and in our pre-eclamptic groups, and found that they are significantly elevated in pre-eclampsia in a similar manner to activin A and inhibin A. However, the increase in FLRG in these cases is independent of activin A or inhibin A, and is associated with low-birthweight outcomes. CONCLUSION: Our current data show the placental and secretory changes of FLRG protein in pre-eclampsia, and also indicate the potential usefulness of FLRG as an additional diagnostic marker for pre-eclampsia.

Vascular endothelial growth factor increases messenger RNAs encoding cyclooxygenase-II and membrane-associated prostaglandin E synthase in rat luteal cells.

Vascular endothelial growth factor (VEGF) is known to be necessary for the vascularization of the developing corpus luteum. Our recent data suggested that cyclooxygenase-II (COX-II) may play a role in the formation of vascular plexuses in developing corpora lutea of the rat. Here we examined the relationship between VEGF and the expression of prostaglandin (PG)- metabolizing enzymes in rat ovarian luteal cells. VEGF treatment caused a dose-dependent increase in the expression of COX-II and membrane-associated PGE synthase (mPGES) mRNA in cultured rat luteal cells. However, pretreatment of the luteal cells with a selective COX-II inhibitor, NS-398, abolished the VEGF-enhanced mPGES mRNA expression. VEGF also increased PGE2 secretion. Conversely, PGE2 dose-dependently stimulated VEGF mRNA expression. Furthermore, VEGF induced VEGF mRNA expression, but this effect was abolished by NS-398 pretreatment. These findings suggest that VEGF enhances PGE2 production by stimulating COX-II and mPGES expression in rat corpus luteum and that the effect of VEGF on luteal cells may be partially mediated by this stimulation of PGE2 production.

In vitro organogenesis of gut-like structures from mouse embryonic stem cells.

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues, including representatives of all three germ layers in the embryo. We have reported previously that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e. epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neurone and ICC development.

Differential localization of decidual stathmin during pregnancy in rats.

The present study was undertaken to determine the precise localization of stathmin, a protein associated with microtubule dynamics, during decidualization in rat uterus and to compare it with that of cyclin D3. Immunohistochemical analysis revealed that stathmin is exclusively localized in decidual cells, especially in the primary decidual zone surrounding the embryo, on days 7 and 9 of pregnancy. The intensity of staining was much higher on day 9 than day 7. On day 14, when the endometrial stromal cells had completely differentiated into decidual cells, the staining of decidual cells was faint. Cyclin D3 was expressed in decidual cells of the secondary but not the primary decidual zone on days 7 and 9. On day 14, cyclin D3 levels were low in decidua. Proliferating cell nuclear antigen (PCNA) was broadly detected in the uterus on days 7 and 9, and in the placenta and fetus on day 14. In an artificial decidualization model, cyclin D3 expression was stimulated as deciduoma was formed after an artificial stimulus. Stathmin mRNA levels also increased within 24 h and peaked at 48 h. The specific spatio-temporal uterine expression of stathmin and cyclin D3 suggest that they have a specific role in decidualization in rats.

Effects of carbamazepine on the first ovulation in gonadotropin-primed immature female rats.

1. The effects of carbamazepine (CBZ) and its possible mechanisms on the first ovulation were investigated in immature female rats. The first ovulation was induced by administration of equine chorionic gonadotropin (eCG) at 0800 h at 26 days of age. 2. A single s.c. injection of 360 mg x kg(-1) CBZ at 1300 h on the first pro-oestrus (day 28) completely inhibited the first ovulation on the morning of day 29. A marked elevation in 13, 14-dihydro-prostaglandin F2alpha (13, 14H2-PGF2alpha) forming capacity, a sensitive indicator of luteinizing hormone (LH) surge, was not detected in the CBZ-treated group at 0800 h on day 29 (72 h after eCG treatment). The elevation in serum LH levels at 57 h after eCG treatment was not observed in the CBZ-treated group, either. The blocking of the first ovulation and 13, 14H2-PGF2alpha forming capacity were recovered by an i.p. injection of human CG on day 28 in all animals. 3. However, the first ovulation was not blocked by repeated injections of 360 mg x kg(-1) CBZ at 1300 h once daily for 3 days (days 26 - 28). The repeated injections of CBZ caused a great fall (64% decrease) in the serum levels of CBZ at 4 h after the final CBZ injection as compared with the case of a single injection of CBZ and resulted in a delay for 5 h the occurrence of LH surge, which is normally observed around 57 h after eCG injection. 4. A significant increase in the activity of microsomal CBZ catabolism by the repeated injections of CBZ was quantitatively verified by the HPLC analysis. But, the activity of CBZ metabolism in the single injected-animals showed almost similar levels to that in the control. 5. The present results demonstrated that a single injection of CBZ blocks the ovulation by inhibiting LH surge but that the failure of the inhibition of ovulation by repeated injections of CBZ is due to a decrease in serum CBZ levels mediated through CBZ-induced hepatic enzyme induction.

Effects of phytoestrogens on acetylcholine- and isoprenaline-induced vasodilation in rat aorta.

The influence of the phytoestrogen, isoflavones, on vasodilating responses of the thoracic aorta precontracted with norepinephrine, together with the stimulatory effect on uterine weight (uterotrophic effect), was investigated in ovariectomized rats. In comparison with intact rats, acetylcholine (ACh)-induced vasodilation showed a tendency to be decreased by ovariectomy. On the other hand, isoprenaline (ISO)-induced vasodilation was significantly increased by ovariectomy. Estrogen replacement (17beta-estradiol dipropionate, 300 microg/kg per week, for 1 month) completely restored the impaired ACh- and ISO-induced vasodilation caused by ovariectomy. Dietary isoflavone aglycones (containing 52% genistein, 42% daidzein and 6% glycitein) of 157 mg/kg per day (not 67 mg/kg per day) for 1 month, in addition to the effects of estrogen replacement, completely restored the impaired vasodilation caused by ovariectomy. However, the uterotrophic effect of dietary isoflavones of 157 mg/kg per day was incomplete as compared with that by estrogen replacement. These results indicate that phytoestrogen, isoflavones, certainly possess estrogenic actions on the vasodilating responses caused by ACh and ISO, as well as a weaker uterotrophic effect.

Possible mechanisms for the suppressing action of 17beta-estradiol on beta-adrenoceptor-mediated vasorelaxation in rat aorta.

The mode of action of estrogen on beta-adrenoceptor-mediated relaxation was investigated by using isolated ring preparations of thoracic aorta from ovariectomized rats. Administration of 17beta-estradiol to ovariectomized rats significantly suppressed isoprenaline-induced relaxation of aortic rings. There was no alteration in the beta-adrenoceptor binding characteristics. The suppressing action of 17beta-estradiol on the N(G)-nitro-L-arginine and indomethacin-resistant relaxation induced by isoprenaline disappeared after pretreatment with N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525A), an inhibitor of cytochrome P450 (CYP). The levels of CYP2C11 expression were the highest of the CYP mRNAs examined in rat aorta. 17beta-Estradiol replacement increased the expression of CYP2C11 mRNA in the aorta, compared with that in ovariectomized rats. These results suggest that estrogen suppresses beta-adrenoceptor-mediated vasorelaxation, and that the mechanisms may be associated with alterations in CYP2C11 metabolites.

Dexamethasone increases inhibin and estradiol secretion mediated by endogenous FSH in equine chorionic gonadotropin (eCG)-primed immature female rats.

In the present study, we have examined whether the effects of dexamethasone on follicle stimulating hormone (FSH) secretion were mediated by hypophysiotropic factors, and whether the increased levels of FSH induced by dexamethasone can stimulate ovarian functions in equine chorionic gonadotropin (eCG)-primed immature female rats. Dexamethasone (500 microg) significantly increased serum concentrations of FSH in hypophysectomized rats implanted with pituitary under the kidney capsule, as well as in intact rats. Serum concentrations of inhibin and estradiol in eCG (2.5, 5 i.u.)-primed rats were significantly increased by simultaneous treatment with dexamethasone (500 microg) and eCG. These simultaneous effects were not confirmed in hypophysectomized rats. The results had shown that hypophysiotropic factors do not mediate the selective increase of FSH secretion caused by dexamethasone. Dexamethasone induces the excess amount of FSH secretion from anterior pituitary and this FSH can stimulate inhibin and estradiol secretion in eCG-primed immature female rat.

Interleukin-6 inhibits the expression of luteinizing hormone receptor mRNA during the maturation of cultured rat granulosa cells.

Interleukin-6 (IL-6) and its receptor components have been shown to be present in rat follicular granulosa cells. The present study was designed to examine the effect of this cytokine on changes in expression of the luteinizing hormone receptor (LHR) messenger RNA and of the steroidogenic enzyme, CYP11A1 (cytochrome P450 scc) in an in vitro model of granulosa cell maturation. Ovarian granulosa cells harvested from immature rats 2 days after treatment with equine chorionic gonadotropin were cultured for 48 h in media containing 10% fetal bovine serum. They were then transferred to a chemically defined serum-free medium and cultured for an additional 72 h. Within 24 h of transfer, the expressions of LHR and CYP11A1 mRNA increased significantly and remained increased for 72 h. The cells responded to exposure to FSH, but not LH, by an increase in production of cAMP before the additional 72 h of culture. The cAMP response to LH was attained within 24 h and persisted for 72 h, whereas the response to FSH decreased continuously with time. Inclusion of IL-6 in the culture medium caused a dose-dependent decrease in expression of LHR mRNA, in addition to a decrease in the cAMP response to LH. Immunoneutralization of endogenous granulosa cell IL-6 resulted in an increase in expression of LHR mRNA, but not CYP11A1 mRNA. The results are consistent with the view that IL-6 may have a physiological role in the maturation of ovarian follicles by modulating the attainment of the LHR in granulosa cells.

Bisphenol A inhibits testicular functions and increases luteinizing hormone secretion in adult male rats.

Effects of a xenobiotic estrogen, bisphenol A (BPA), on reproductive functions were investigated using adult male rats. BPA was dissolved into sesame oil and injected s.c. every day (1 mg/rat) for 14 days. Animals were killed by decapitation after the final administration of BPA, and the trunk blood, pituitary, and testes were collected. Plasma concentrations of prolactin were dramatically increased and pituitary contents of prolactin were slightly increased in the BPA group compared to the control group. Plasma concentrations of testosterone were decreased and plasma concentrations of LH were increased in BPA-treated rats compared to control rats. Testicular contents of inhibin were decreased in BPA-treated rats compared to control rats, although plasma concentrations of inhibin were not changed after administration of BPA. The testicular response to hCG for progesterone and testosterone release was decreased in BPA-treated rats. Administration of BPA did not change the pituitary response to luteinizing hormone-releasing hormone (LH-RH) in castrated male rats treated with testosterone. Male sexual behavior also was not changed as a result of BPA treatment. These results suggest that BPA directly inhibits testicular functions and the increased level of plasma LH is probably due to a reduction in the negative feedback regulation by testosterone. The testis is probably a more sensitive site for BPA action than the hypothalamus-pituitary axis.

Short term hypercholesterolemia alters N(G)-nitro-L-arginine- and indomethacin-resistant endothelium-dependent relaxation by acetylcholine in rabbit renal artery.

The tension of isolated rings was measured isometrically to compare the N(G)-nitro-L-arginine- and indomethacin-resistant relaxation by acetylcholine (ACh) in the renal artery from normal rabbits and short term hypercholesterolemia rabbits (0.5% cholesterol chow for 5 weeks). ACh-induced relaxation in the renal artery precontracted with phenylephrine was not influenced by cholesterol-enriched chow. However, in comparison with artery from normal rabbits, the N(G)-nitro-L-arginine- and indomethacin-resistant endothelium-dependent relaxation by ACh was significantly enhanced by the chow. The resistant part of ACh-induced relaxation was significantly inhibited when the artery was treated with tetraethylammonium or SKF 525a. Results suggest that short term hypercholesterolemia modulates endothelium-derived hyperpolarizing factor-mediated relaxation in rabbit renal artery.

Caveolin-2 is targeted to lipid droplets, a new "membrane domain" in the cell.

Caveolin-1 and -2 constitute a framework of caveolae in nonmuscle cells. In the present study, we showed that caveolin-2, especially its beta isoform, is targeted to the surface of lipid droplets (LD) by immunofluorescence and immunoelectron microscopy, and by subcellular fractionation. Brefeldin A treatment induced further accumulation of caveolin-2 along with caveolin-1 in LD. Analysis of mouse caveolin-2 deletion mutants revealed that the central hydrophobic domain (residues 87-119) and the NH(2)-terminal (residues 70-86) and COOH-terminal (residues 120-150) hydrophilic domains are all necessary for the localization in LD. The NH(2)- and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein. In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain. It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.

Antagonist of pituitary adenylate cyclase activating polypeptide suppresses prolactin secretion without changing the activity of dopamine neurons in lactating rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a relatively new neuropeptide, and it has a potent stimulatory effect on adenylate cyclase activity in rat pituitary cells. However, the role of PACAP in the physiological control of prolactin (PRL) secretion is still unclear. In the present study, we investigated the physiological significance of endogenous PACAP on PRL secretion in lactating rats. On lactation days 7-8, pups were separated from their mother rats for 5 h before the onset of suckling and PACAP6-38 (16 microg), a receptor antagonist, was injected through the lateral ventricle cannula just after the removal of pups. The effects of PACAP6-38 on PRL and oxytocin secretion, and on the activity of tyrosine hydroxylase (TH), were examined after the onset of suckling. Administration of PACAP6-38 inhibited PRL levels in response to suckling, but it did not affect the activity of TH, as measured by DOPA accumulation at 15 min after administration of NSD 1015 (25.0 mg/kg), an L-aromatic amino acid decarboxylase inhibitor, or the plasma concentrations of oxytocin in lactating rats. Injection of alpha-methyl-p-tyrosine (alpha-MT; 50 mg/kg), an inhibitor of dopamine synthesis, increased PRL levels, and suckling caused a further increase in the plasma concentrations of PRL. An injection of PACAP6-38 (i.c.v.) also inhibited the PRL response to suckling under dopamine depletion. These results suggest that endogenous PACAP acts as a neurotransmitter or neuromodulator within the hypothalamus and plays an important role for PRL secretion in lactating rats. Endogenous PACAP may regulate PRL secretion, possibly mediated by PRL-releasing factors such as vasoactive intestinal polypeptide or vasopressin.

Isoforms of caveolin-1 and caveolar structure.

The relationship between caveolin-1 isoforms alpha and beta and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alphabeta) were not labeled by another antibody specific for the alpha isoform (anti-alpha). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-alpha labeled deep caveolae preferentially over shallow ones, whereas anti-alphabeta labeled both forms with an equivalent frequency and intensity. The presence of the beta isoform in deep caveolae was confirmed by labeling epitope-tagged beta-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.

Phenytoin inhibits both the first ovulation and uterine development in gonadotropin-primed immature rats.

This study was planned to determine the effects and possible mechanism of action of phenytoin on development of the reproductive tract and first ovulation in immature rats. Rats were injected s.c. with 5 IU of equine chorionic gonadotrophin (equine CG) on day 26 to induce ovarian and uterine development. Treatment with phenytoin (140 mg/kg) at 1200 h on day 28, which induces serum levels approximately twice those reached with the clinical dose as anticorvulsant drug for humans, was effective for inhibiting the first ovulation and normal secretion of serum follicle - stimulating hormone and luteinizing hormone (LH) on day 29 as well as the preovulatory gonadotrophin surge on day 28. The block of ovulation was overcome by administration of human chorionic gonadotrophin or LH-releasing hormone on day 28. Simultaneous treatment with equine CG and phenytoin at 0800 h on day 26 did not affect either ovarian weight or ovarian hormones secretion, whereas phenytoin clearly inhibited the normal increase in uterine weight on day 27. Furthermore, phenytoin suppressed uterine growth after 17beta-oestradiol injection. These results indicate that phenytoin inhibits the first ovulation by inhibiting the gonadotrophin surge and further, that the drug impairs the stimulatory effects of oestrogen on uterine proliferation in the gonadotropin-induced ovulation model.

Concentration of caveolin-1 in the cleavage furrow as revealed by time-lapse analysis.

Caveolin-1 is a major component of caveolae. Recent studies have suggested a possible role of caveolin-1 in cell transformation and normal cell proliferation. To observe the behavior of caveolin-1 in living mitotic cells, we prepared cDNA constructs encoding the chimeric protein of alpha- or beta-caveolin-1 and green fluorescent protein (GFP) and transfected culture cells with them. Correct targeting of the chimera to the caveolae was confirmed by colocalization with the caveolar inositol 1,4,5-trisphosphate receptor-like protein. By time-lapse observation of mitotic MDCKII cells, the GFP-caveolin-1 chimeras were seen throughout the plasma membrane before cell division, but became markedly concentrated at the cleavage furrow during cytokinesis. Accumulation around the spindle poles was also observed at late telophase. The result showed that caveolin-1 undergoes a drastic distributional change during cell division and suggested that the protein may be involved in the cytokinetic process.

Caveolin-1 isoforms are encoded by distinct mRNAs. Identification Of mouse caveolin-1 mRNA variants caused by alternative transcription initiation and splicing.

By searching the EST database with the known cDNA sequence encoding alpha-caveolin-1 (full-length: FL), we found a variant having a hitherto unknown sequence in place of the first exon (5'-end variant: 5'V). The expression level of 5'V mRNA was equivalent to that of FL mRNA. The entire sequences of FL and 5'V mRNA were determined by 3'- and 5'-RACE analysis; their sizes were 2484 bp and 2533 bp, respectively, and the sequences were identical except for the region of the first exon. By Northern blotting, FL and 5'V mRNAs showed the same tissue distribution, and were intensely expressed in the lung, heart, and skeletal muscle. Analyzing the protein production from these mRNAs using green fluorescent protein as a tag, we found FL mRNA to produce the alpha-isoform predominantly, but to form little beta-isoform. The production of the beta-isoform from 5'V mRNA was also demonstrated. By sequence analysis of the first intron of the caveolin-1 gene, a TATA box was found at 28 bp upstream of the transcription initiation site for 5'V mRNA. This is the first demonstration of caveolin-1 mRNA variants generated by alternative transcription initiation, and it indicates that the two isoforms of caveolin-1 are produced from two distinct mRNAs.