Presence of nucleotide substitutions in the ABO promoter in individuals with phenotypes A3 and B3.

Recently, the involvement of mutation and deletion of transcription regulatory elements in the Bm , Am , A3 and B3 phenotypes has been reported. In the present study, we carried out genetic analysis of individuals with A3 and B3 using peptide nucleic acid-clamping PCR to exclude amplification of O alleles. Two single-point mutations, -76G>C and -68G>T, were found in the ABO promoter on the A-allele in three A3 individuals and on the B allele in a B3 individual, respectively. Transient transfection of luciferase reporter plasmids carrying the same mutations into K562 cells revealed decreased luciferase activity in comparison with that carrying the wild-type promoter. These observations suggest that the mutations downregulate the promoter activity, leading to reduction in A- or B-antigen expression on red blood cells in individuals with the A3 and B3 phenotypes.

Histone deacetylase 9 as a negative regulator for choline acetyltransferase gene in NG108-15 neuronal cells.

The biological function of histone deacetylases (HDACs), namely, repression of gene expression by removing an acetyl group from a histone N-terminal tail, plays an important role in numerous biological processes such as cell cycle, differentiation, and apoptosis in the development of individual tissues, including the brain. We previously showed the possible role of HDAC activity in the regulation of gene expression of choline acetyltransferase (ChAT), a specific marker for cholinergic neurons and their function, in NG108-15 neuronal cells as an in vitro model of cholinergic neurons. The objectives of the present study were to specify key HDACs and investigate the essential role of HDACs in ChAT gene regulation in NG108-15 cells. The experiments using different types of HDAC inhibitors indicated that class IIa HDACs substantially participate in the regulation of ChAT gene expression. In addition, HDAC9, a class IIa enzyme, was dramatically decreased at the protein levels, and dissociated from the promoter region of ChAT gene during neuronal differentiation. Furthermore, knockdown of HDAC9 by siRNA increased ChAT gene expression in undifferentiated cells. These findings demonstrate that HDAC9 is responsible for repressing ChAT gene expression in NG108-15 neuronal cells, and thus plays an important role in cholinergic differentiation.

Effect of estradiol and FBS on PRL cells, GH cells, and PRL/GH cells in primary cultures of pituitary cells from prenatal rats.

The effects of estradiol (E2) treatment on prolactin (PRL) cells, GH cells, and PRL/GH cells in immature pituitary cells were determined using primary cultures from prenatal rats and immunocytochemistry with fluorescent antibodies. Anterior pituitaries obtained from fetuses on day 22 of pregnancy were monodispersed and cultured in chemically defined medium or medium containing 10% fetal bovine serum (FBS). After pre-incubation for 24 hr, E2 (final concentrations were 0 M, 10(-8) M, 10(-7) M, and 10(-6) M) was added into each medium. After 72 hr of incubation, cells were subjected to immunocytochemistry. E2 stimulated the increase of PRL cells in a dose-dependent manner, and the PRL cell percentage cultured with FBS in all groups was significantly higher than that cultured in chemically-defined medium. PRL/GH cells also responded to E2 in the same manner as PRL cells. E2 was not effective in proliferating GH cells, and GH cell percentage significantly decreased with the addition of FBS into the medium. These results suggest that E2 is dose-dependently capable of increasing immature PRL cells and/or PRL/GH cells in vitro. Moreover, there is a factor(s) in FBS that regulates the increase of these cells.

Developmental profile of prolactin cells in the anterior pituitary of postnatal rats during the lactational stage.

Changes in prolactin (PRL)-, growth hormone (GH)- and PRL/GH-containing cells in the anterior pituitary of pre- and postnatal male and female rats were determined using immunocytochemistry with double fluorescent antibodies. The pituitary glands from a fetus on Day 20 of gestation and pups on Days 0, 1, 4, 7, 12, and 20 of the postnatal period with sex distinction were monodispersed and subjected to immunocytochemistry. Following immunostaining, the three types of cells described above were counted (i.e., only PRL-, only GH-, and both PRL and GH-positive cells in a visual field of microscope). Anterior pituitaries and blood were obtained from pups on Days 0, 1, 4, 7, 12, and 20 after birth and pooled with sex distinction, and bioactive PRL levels in the anterior pituitary and serum were measured using an Nb2 rat lymphoma cell bioassay. Double fluorescent-labeled immunocytochemistry was able to distinguish reliably between PRL-, GH-, and PRL/GH-containing cells. The PRL cells increased rapidly after parturition to 4 days of age and after 12 days of age in both genders. The latter increase was involved in the remarkable increase of PRL/GH-containing cells, which were first found at term and remained at low levels until 12 days of age. The weight and PRL level of the anterior pituitary increased in both sexes throughout the lactational stage, but the levels in female rats at 20 days of age were significantly greater than those of male rats. Serum PRL concentration remained at low levels except on Day 0. The present results demonstrate that the development of PRL-containing cells in postnatal rats during the lactational period is activated with two distinct stages, at term to Day 4 and after Day 12, and the development in the latter stage is involved with sexual dimorphism.

Exogenous oxytocin attenuates suckling-induced prolactin release but not maternal or infant behavior in lactating rats.

The influence of exogenous oxytocin (OT) on maternal and infant behavior over 60 min of suckling, which followed 6 h of isolation, was investigated on Day 12 of lactation in rats. Mothers administered 1 IU of OT or saline through an indwelling atrial catheter and their litters indicated a similar nursing and suckling pattern, which was estimated by the crouching time of the mothers and the number of stretch reactions performed by the litters during a suckling period. To assess the alteration of the suckling intensity by OT administration, the plasma prolactin (PRL) level was determined by an Nb2 lymphoma cell bioassay. In the control group, the plasma PRL level increased and reached a peak at 45 min after the onset of suckling in 60% of the animals. The suckling-induced PRL release was completely inhibited and/or markedly delayed by OT administration. The difference in body weight of the litters before and after a suckling period was estimated as an index of the amount of milk suckled by the litters. There was no difference in the amount of milk between the control and OT-treated groups during a 60-min suckling period. However, it was significantly greater in the OT-treated group during the first 20 min of the suckling period. These results indicate that a dose of OT is a factor in the attenuation of the intensity of suckling done by the pups, whereas the nursing and suckling behavior is not influenced by OT administration.

Effects of discrimination learning on the rat striatal dopaminergic activity: a microdialysis study.

The prefrontal cortex (PFC) has anatomical and functional relationships with the striatum. In a previous study we showed that dopamine (DA) turnover in the PFC of rats is enhanced during the performance of a discrimination task. In the present study, we used an in vivo microdialysis method to examine whether DAergic activity in the striatum could also be altered by the discrimination task. The results showed a substantial and sustained suppression of DAergic activity during and after the discriminative behaviour. The fact that the discriminative performance induced opposite changes in DAergic activity in the striatum and the PFC is consistent with the results of biochemical studies, suggesting that the suppressed DA turnover in the striatum may be induced by the enhanced DAergic activity in the PFC during the discrimination task.

Changes in prolactin bioactivity of milk following isolation from litter and oxytocin administration in rats.

Changes in prolactin (PRL)-like bioactivity in rat milk after a longer isolation of litters and the effects of oxytocin (OT) were determined by Nb2 lymphoma cell bioassay and compared with the immunoreactivity using an enzyme immunoassay. First, we confirmed the appropriate dilution rates of rat milk which could neutralize the influence of an antimitogenic factor(s) in milk to Nb2 lymphoma cell proliferation. To prolong the litter removal, i.e. 6 h to 20 h, resulted in the increase in an antimitogenic factor(s) for Nb2 lymphoma cells in milk and in the significant decrease in milk PRL concentration measured by both assays, and plasma bioactive PRL level increased. The bioactivity/immunoreactivity ratio of PRL in milk accumulated in the mammary gland was 0.65 in rats after a 20-h isolation, but 1.18 after a 6-h isolation. The milk PRL concentration in rats isolated for 6 h decreased significantly within 45 min after the administration of OT and the plasma PRL concentration increased only slightly, but OT had no effect in rats after isolation for 20 h. The present findings indicate that milk PRL might transfer to plasma with the excessive engorgement of milk in the mammary gland and with OT administration. Moreover, PRL trapped in milk for a long period loses its biological activity more rapidly than its immunological activity, since bioactivity/immunoreactivity ratio in the 20-h isolation group was significantly lower than in the 6-h isolation group.

Acetylcholine in the hippocampus during the discrimination learning performance in a rat model of chronic cerebral ischaemia.

Acetylcholine (ACh) release in the hippocampus of Wistar strain rats with permanent bilateral occlusion of the common carotid arteries was examined during a discrimination learning task using a microdialysis method. Such occlusion resulted in obvious impairment of the discrimination performance. The state, the basal value and released patterns, of ACh in the hippocampus differed in the sham-operated control and the experimental group, while ACh release was elevated during the dialysis experiment in both groups. These findings suggested that the bilateral occlusion produced persistent learning deficits from an early stage after the operation and that the impaired discrimination learning performance might be related to the diffusion of ACh in the hippocampus.

Differences in electrophysiological properties of angiotensinergic pathways from the subfornical organ to the median preoptic nucleus between normotensive Wistar-Kyoto and spontaneously hypertensive rats.

Electrophysiological properties of angiotensinergic pathways from the subfornical organ (SFO) to the median preoptic nucleus (MnPO) were investigated in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) under urethane anesthesia. The activity of SFO neurons that were antidromically activated by electrical stimulation of the MnPO was compared between WKY (n = 28) and SHR (n = 27). No significant differences were observed between WKY and SHR in the latency, conduction velocity, or threshold of antidromic activation. The firing rate was significantly shorter in SHR. The activity of MnPO neurons was tested for a response to microiontophoretic application of angiotensin II (ANG II) and electrical stimulation of the SFO. Sixteen of 46 MnPO neurons tested in WKY and 15 of 47 MnPO neurons tested in SHR were excited by both ANG II applied iontophoretically and SFO stimulation, and the excitatory responses were prevented by iontophoretically applied saralasin, a specific ANG II antagonist. In these MnPO neurons that demonstrated the excitation to both SFO stimulation and ANG II, the firing rate was significantly higher and the threshold current required to evoke the SFO stimulus-induced excitation was significantly lower in SHR. The sensitivity to SFO stimulation was much greater in SHR than in WKY. These results provide evidence that there are marked alterations in the physiological properties of the angiotensinergic circuit from the SFO to the MnPO between WKY and SHR and imply that a disorder in the neural circuit may contribute, in part, to hypertension.

Septo-hippocampal cholinergic system under the discrimination learning task in the rat: a microdialysis study with the dual-probe approach.

To investigate regulation of the septo-hippocampal cholinergic system by dopaminergic inputs to the septum in rats which performed a discrimination learning task, an in vivo microdialysis method with the dual-probe approach was used. Rats were trained to discriminate between lamp-on and -off states under an operant-type learning procedure. After stable discriminative behavior was established, dialysis probes were implanted into the hippocampus and the lateral septum area of each rat. The concentration of dopamine (DA) in the septum rapidly increased within 20 min after the beginning of a learning session. However, another group of rats trained on a similar but non-discriminative task showed no such increase. The concentration of acetylcholine (ACh) in the hippocampus was significantly enhanced during the learning session and rapidly returned to the basal value after the session, but showed a delayed and diminished increase in the non-discrimination group. These results suggest that DAergic inputs to the septum may be involved in control of the septo-hippocampal cholinergic system which is of importance for discrimination learning behavior.

Effect of exogenous oxytocin on the leakage of lactose from the mammary gland and on suckling-induced plasma prolactin in the lactating rat.

The leakage of lactose from the mammary gland into plasma and the increase in the suckling-induced concentration of prolactin in the plasma of oxytocin-injected lactating rats suckling different numbers of pups were investigated. Rats nursing eight pups were isolated from their litter for 6 h and injected i.v. with either 1 iu oxytocin or saline and allowed to resuckle either two, eight or 12 pups for 1 h. The concentration of lactose in plasma increased significantly in rats injected with oxytocin and allowed to resuckle two or eight pups; however, there was no difference in rats allowed to resuckle 12 pups. When compared within oxytocin-injected groups, the concentration of lactose was significantly lower in rats allowed to resuckle eight or 12 pups than in rats allowed to resuckle only two pups. Plasma prolactin concentrations increased during the suckling period in mothers suckling either eight or 12 pups, and oxytocin diminished the increase in prolactin concentration in both groups. In rats suckling two pups, prolactin release was not observed. The second objective of this study was to determine the relationship between the leakage of lactose from the mammary gland by oxytocin injection and suckling. Mothers that had the three left nipples of their mammary glands occluded were isolated from their litter of eight pups for 6 h and were allowed to resuckle with eight pups for 1 h. At the start of resuckling, 0.01, 0.1 or 1 iu oxytocin was injected i.v.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between learning performance and dopamine in the prefrontal cortex of the rat.

To clarify the relationship between dopamine (DA) release in the prefrontal cortex and learning performance, extracellular DA was measured by an in vivo microdialysis method during a discrimination learning task. Rats were trained to discriminate between lamp-on and -off states under an operant-type learning procedure. After stable discriminative behavior was established, a microdialysis experiment was conducted. Inverse relationships were seen between the relative ratio of DA after the beginning of a learning session to the basal level and the responses (total, correct and error responses) during a session. The results indicate that the degree of DA release in the prefrontal cortex is involve in the inhibitory mechanism of the response during discrimination learning performance.

Changes with age of mammary glands in male and female soft-furred rat, Millardia meltada, in relation to prolactin and testosterone.

The soft-furred rat, millardia (Millardia meltada), is characterized by the development of androgen-dependent mammary tumours only in males. The age-related changes of the activities of thymidylate synthetase (TS) and thymidine kinase (TK), which contribute to DNA synthesis through de novo and salvage pathways, respectively, and structure in the mammary glands were studied in both males and females of this species between 5-28 months of age. While TK activity had no relation to age, TS activity decreased with age in males. In the females, TK activity increased with age, but not TS activity. These enzyme activities were generally higher in females than in males. The mammary glands of both sexes consisted of fine ducts with small end-buds and the glands of males contained mostly black pigments at any age examined. In either males or females, serum levels of prolactin and testosterone related little with age, DNA synthesizing enzyme activities or structure of the mammary glands. Furthermore, elevation by pituitary grafting of circulating prolactin affected neither DNA synthesizing enzyme activities nor structure of mammary glands in both sexes. The histological structures of adrenal, testis, ovary, ventral prostate and uterus of millardia were essentially similar to those of mice or rats.

Responses of subfornical organ neurons projecting to the hypothalamic paraventricular nucleus to hemorrhage.

The activity of subfornical organ (SFO) neurons that were antidromically identified by electrical stimulation of the rat hypothalamic paraventricular nucleus (PVN) was tested for a response to microiontophoretic application of angiotensin II (ANG II) or hemorrhage (10 ml/kg b.w.t.). Microiontophoretically (MIPh) applied ANG II caused an increased excitability in 24 out of 28 neurons tested and the excitation was blocked by MIPh-applied saralasin (Sar), a specific ANG II antagonist. Of these neurons that responded to ANG II, 14 displayed an increase in neuronal firing in response to hemorrhage, while 10 were unresponsive. The excitatory response to hemorrhage in 5 out of 14 neurons tested was prevented by MIPh-applied Sar, whereas the response of the remaining neurons was not affected. These results show that part of SFO neurons projecting to the PVN may receive neural inputs from the peripheral baroreceptors, and suggest that the inputs may be partially attributable to the involvement of central angiotensinergic circuits.

Effects of some natural products as components of Chinese herbal medicines on mammary gland growth and function in mice.

As a possible step to evaluate the role in the mammary glands of the scales of Guanshanjia, the fruits of Lulutong and the seeds of Wangbuliouxing, which are the natural products used widely as the components of herbal medicine in China for the improvement of lactation and the therapy of breast disorders, the effects of these agents on mammary gland growth and function were studied in female mice. The chronic administration of each agent in drinking water improved several parameters; however, the mode of action differed markedly among agents. The results indicate the necessity of acquiring enough knowledge of the characteristics of each agent for the efficient prescription of herbal medicine as well as the single use of the agent.

Rat milk inhibits the mitogenic response of Nb2 lymphoma cells to prolactin.

Prolactin-like bioactivity in the rat milk was observed using the lactogen specific Nb2 lymphoma assay. The water soluble fraction (infranatant) of pooled milk samples obtained on days 2, 12 or 22 postpartum stimulated Nb2 cell growth in the range of 0.08-2.5 microliters/well. Higher concentrations of day-12 and day-22 (but not day-2) milk infranatant, however, decreased Nb2 proliferation in a dose-dependent fashion. [3H]-thymidine incorporation used as an indicator of cell growth was decreased by 21%, 49% and 83% at the doses of 5, 10, 20 microliters/well concentrations of day-22 milk infranatant, respectively. Milk infranatant did not reduce cell viability as assessed by Erythrosin B exclusion test. Addition of exogenous rat PRL (NIH B-6) at concentrations of ED50-ED90 did not restore the Nb2 proliferation rate decreased by milk infranatant. Saturating doses of PRL (ED100-ED400) resulted in maximal cell growth, but failed to counteract the inhibitory effect of milk infranatant. The relative molecular weight of the putative Nb2 cell inhibitor of rat milk is between 10 kDa and 30 kDa as determined by ultrafiltration and dialysis. The inhibitory activity of milk infranatant is stable at physiological pH, but is destroyed upon acidification. Thirty min of incubation at 37 degrees C enhanced but 30 min of incubation at 100 degrees C only slightly decreased the calculated total inhibitory effect of milk infranatant. These initial results indicate the presence of a water-soluble antimitogenic factor in rat milk (rMAF) which inhibits the Nb2 lymphoma cell response to prolactin in a non-competitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular heterogeneity of prolactin in lactating rats and their pups: biological and immunological activities in pituitary gland, serum and milk.

Prolactin (PRL)-like bioactivities (in Nb2 lymphoma assay), immunoreactivities (in RIA) and B/I ratios in rat milk, maternal and neonatal pituitary glands and sera were investigated. The PRL-like bioactivity in the water-soluble fraction of rat milk (infranatant prepared by ultracentrifugation) exceeded its immunoreactivity 3-7-fold. The elevated B/I ratio was in part due to the presence of a glycosalated PRL (G-rPRL)-like material, since 5-70% of the PRL-like bioactivity was recovered from the glycosylated fraction of rat milk infranatant prepared by concanavalin-A affinity chromatography. We were unable to detect PRL-like immunoreactivity in the glycosylated fraction of rat milk, and calculated that the maximal cross-reactivity of G-rPRL in the RIA is less than 3.8%. In day 12 milk, over 80% of the G-rPRL-like bioactivity eluted from a Sephadex G-100 column as a high apparent molecular weight (Mr) substance (approximately 50 kD), while the rest eluted as a monomeric G-rPRL (24-25 kD). The PRL-like bioactivity in the nonglycosylated fraction eluted in three peaks (Mr: 50, 24 and 16 kD), while two immunoreactive peaks occurred (Mr: 24 and 8 kD). The concentration of rPRL-like immunoreactivity in rat milk increased during the first days of lactation, remained high in midlactation, and declined by the end of lactation. The PRL-like bioactivity in the nonglycosylated fraction of rat milk displayed a similar timecourse. G-rPRL-like bioactivity in rat milk, however, changes inversely, i.e. decreased between days 2 and 18 postpartum then increased by day 22. The concentration of high Mr PRL-like bioactivity in rat milk was greatly reduced by day 22 from day 2 postpartum. No PRL-like bioactivity or immunoreactivity was recovered from the IgG fraction (prepared by protein A affinity chromatography) of rat milk. The B/I ratio in day 2 maternal pituitary glands was close to 1. In neonatal pituitaries and in maternal sera, however, the B/I ratio was slightly elevated (2-3). The B/I ratio in day 2 neonatal serum was between 6 and 22, while the B/I ratio of PRL secreted by day 2 neonatal pituitary glands in vitro was 1. The present results demonstrate that the concentrations of PRL in rat milk and neonatal serum have been grossly underestimated because levels were detected by RIA. The high B/I ratio reflects the presence of PRL variants. Milk appears to be the most likely source of PRL variants in the circulation of the neonate.(ABSTRACT TRUNCATED AT 400 WORDS)

Cause of pregnancy-dependent mammary tumour-induced suppression of lactation in GR/A mice.

To clarify the cause of pregnancy-dependent mammary tumour (PDMT)-induced suppression of lactation due to the retardation of mammary gland growth, we studied the following: (I) mammary gland growth at the end of pregnancy in mice with PDMT [PDMT(+)] in comparison with mice without PDMT [PDMT(-)] and (II) effects of PDMT removal at the end of pregnancy on mammary gland growth and function. In Experiment I, little difference was observed between PDMT(-) and PDMT(+) groups in mammary DNA content and plasma level of lactogenic hormone or progesterone at the end of pregnancy, indicating that the retardation of mammary gland growth by PDMT does not occur during pregnancy. In Experiment II, the surgical removal of PDMT 1 or 2 days before parturition resulted in a complete restoration of mammary gland growth and lactation. All results strongly suggest that PDMT has a deleterious effect on mammary gland growth during their abrupt regression after parturition, perhaps by secreting some mammary growth inhibitory factor(s).

Suppression of lactation by pregnancy-dependent mammary tumors in GR/A mice.

Pregnancy-dependent mammary tumors (PDMT) in GR/A mice appear during pregnancy, disappear soon after parturition, and appear again during subsequent pregnancies. The retardation of pup growth, an indication of the level of milk production, was also observed with the advance of lactation numbers in this strain. This study was performed to elucidate the relationship between PDMT and lactational performance. At the end of the second pregnancy, mice were divided into two groups according to the presence of PDMT [PDMT(-) and PDMT(+) groups]. Although all PDMT disappeared within a day after parturition, the weight and growth of pups on Day 12 of lactation were significantly less in the PDMT(+) group than in the PDMT(-) group. Associated with this, the DNA and RNA contents of the mammary glands were apparently lower in the former than in the latter, although the differences were not statistically significant. There was little difference in mammary RNA/DNA ratio between groups. No difference was also observed between groups in endocrine organ weights, mother body weights, morphology of the mammary glands, adrenals and ovaries and plasma prolactin and progesterone levels. These results suggest that PDMT suppression of lactation is principally due to the retardation of mammary gland growth. Furthermore, no significant correlations were obtained between the size of PDMT and the parameters for mammary gland function. The data suggest that the development of PDMT per se is important for the retarded mammary gland growth.