Persistent changes in extracellular lactate dynamics following synaptic potentiation.

A diverse array of neurometabolic coupling mechanisms exist within the brain to ensure that sufficient metabolite availability is present to meet both acute and chronic energetic demands. Excitatory synaptic activity, which produces the majority of the brain's energetic demands, triggers a rapid metabolic response including a characteristic shift towards aerobic glycolysis. Herein, astrocytically derived lactate appears to serve as an important metabolite to meet the extensive metabolic needs of activated neurons. Despite a wealth of literature characterizing lactate's role in mediating these acute metabolic needs, the extent to which lactate supports chronic energetic demands of neurons remains unclear. We hypothesized that synaptic potentiation, a ubiquitous brain phenomenon that can produce chronic alterations in synaptic activity, could necessitate persistent alterations in brain energetics. In freely-behaving rats, we induced long-term potentiation (LTP) of synapses within the dentate gyrus through high-frequency electrical stimulation (HFS) of the medial perforant pathway. Before, during, and after LTP induction, we continuously recorded extracellular lactate concentrations within the dentate gyrus to assess how changes in synaptic strength alter local glycolytic activity. Synaptic potentiation 1) altered the acute response of extracellular lactate to transient neuronal activation as evident by a larger initial dip and subsequent overshoot and 2) chronically increased local lactate availability. Although synapses were potentiated immediately following HFS, observed changes in lactate dynamics were only evident beginning ~24 h later. Once observed, however, both synaptic potentiation and altered lactate dynamics persisted for the duration of the experiment (~72 h). Persistent alterations in synaptic strength, therefore, appear to be associated with metabolic plasticity in the form of persistent augmentation of glycolytic activity.

Crystallization and a preliminary X-ray diffraction study of macrophage migration inhibitory factor from human lymphocytes.

Two crystal forms of the macrophage migration inhibitory factor (MIF) from human lymphocytes have been obtained by the hanging drop method of vapor diffusion from ammonium sulfate solution. A trigonal crystal form belongs to the space group P3(1)21 (P3(2)21), with unit cell dimensions of a = b = 96.4 A and c = 105.5 A. Assuming that the asymmetric unit contains two or three dimers, the Vm value is calculated as 2.9 or 1.9, respectively. A second crystal form is orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions a = 68.4 A, b = 68.8 A and c = 86.8 A. The Vm value is 2.1 for two dimers in the asymmetric unit. Both crystals diffract to at least 1.9 A and are suitable for X-ray crystallographic structure determination at a high resolution.

Follistatin and activin in bone: expression and localization during endochondral bone development.

The involvement of activin and follistatin, an activin-binding protein, in endochondral bone development was examined by sc implantation of demineralized bone matrix in rats. Immunoreactive follistatin was localized in proliferating chondrocytes and round osteoblasts, whereas it was not detected in hypertrophic chondrocytes and osteoblasts surrounding bone marrow. Western blot analysis also revealed that immunoreactive follistatin was higher during the initial stages of chondrogenesis (day 5) and osteogenesis (days 11 and 14) and lower during the conversion from cartilage to bone (day 9). These results suggest that follistatin is produced by proliferating cells, and the expression decreases with differentiation of the cells. Implants injected with follistatin on days 9 and 10 contained lower calcium levels on day 14 than those injected with rat albumin. Furthermore, the follistatin-injected implants were still mainly composed of cartilage, suggesting that the disappearance of follistatin is necessary for the conversion of cartilage to bone. In contrast, immunoreactive activin beta A (55-60 kDa) was continuously detected in implants on days 7-14. The content of C propeptide of type II procollagen was increased and cartilageous area was enlarged on day 7 by activin A injections on days 5 and 6, suggesting a chondrogenic effect of activin in the initial stage of cartilage formation. These results indicate that proliferating chondrocytes and round osteoblasts produce follistatin, and that the activity of activin is regulated by changes in the expression of follistatin at the stages of chondrogenesis and transition from cartilage to bone.

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo.

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer-graphic modeling based on the stereo-structure. The common fragment, N-(L-trans-carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethyla mide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10(-7) M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10(-7) M in vitro.

Oxytocin receptor gene expression in rat uterus: regulation by ovarian steroids.

The present study was designed to investigate a possible role for ovarian steroids in the regulation of rat uterine oxytocin receptor (OTR) mRNA expression before labour. By using a competitive RT-PCR system, we have previously reported that parturition was associated with high levels of uterine OTR mRNA in all the animals examined. On the other hand, near term, some rats showed high OTR mRNA levels while others did not. We therefore examined the changes in OTR mRNA expression before and during prostaglandin F(2)(alpha) (PGF(2)(alpha))-induced parturition; a paradigm adopted to reduce the variation in the onset of parturition. Injection of PGF(2)(alpha) on day 18 of pregnancy significantly increased OTR mRNA expression in all the rats within 24 h of treatment, suggesting that the variation in OTR mRNA levels during spontaneous parturition may be due to the difference in the timing of the onset of parturition. The increase in OTR mRNA was significantly abolished by injection of the anti-oestrogen compound, tamoxifen. The stimulatory action of oestrogen on OTR mRNA expression was then examined in the presence or absence of ovarian factors. Pregnant rats were ovariectomized (OVX) or sham-operated on day 18 of pregnancy and either oestrogen or vehicle was administered 6 h after the surgical operation. Oestrogen increased OTR mRNA significantly in OVX rats 18 h after administration compared with sham-operated animals. Moreover, ovariectomy alone on day 18 of pregnancy increased OTR mRNA expression to a level which reached statistical significance 24 h after the operation. In addition, oestrogen treatment increased OTR mRNA levels in OVX virgin rats in which progesterone tubes were implanted for 1 week and removed 6 h before oestrogen injection. The stimulatory effect of oestrogen was not observed in rats in which the progesterone tubes were implanted for 1 week and not removed. These results suggest that the decline of progesterone is necessary for the expression of the stimulatory effects of oestrogen on uterine OTR mRNA.

Immunolocalization of type I or type II activin receptors in the rat brain.

We have studied immunolocalization of activin receptors in the central nervous system using polyclonal antibodies (IgG) to type I (50-55 kDa, ActRI), type II (70-75 kDa, ActRII) or a subtype of type II known as type IIB (ActRIIB) receptors of activin. A total of 7 antisera to rat activin receptors was generated, i.e. 3 kinds of antisera to the extracellular domain (ActRI(81-89), ActRII(91-100), or ActRIIB(90-99)) and 4 antisera to the kinase domain (ActRI(323-333), ActRII(307-319), ActRII(407-420) or ActRIIB(306-319)). The region of aa 407-420 of ActRII is identical with that of ActRIIB. At first, we characterized these antibodies by Western blot analysis using ovarian proteins fractionated by preparative SDS-PAGE. All antibodies to ActRII and ActRIIB specifically reacted with 75 kDa-proteins which could also bind to activin-A. Anti-ActRII(91-100) antibody also reacted with 62 kDa-proteins which were capable of binding with activin-A. Although no positive reactions to anti-ActRI(81-89) antibody were seen in ovarian proteins, a positive reaction was detected at 52 kDa only when the proteins were deglycosylated. By use of these antibodies, immunolocalization of activin receptors was examined in the rat brain. The patterns of expression of activin type I and type II receptors were different. Positive reactions to anti-ActRII(91-100) antibody were detected in neurons of the cerebral cortex, hippocampus, medial amygdala and thalamus. In the hypothalamus, some neurons of the supraoptic nucleus were weakly stained, and widely scattered neurons of the lateral hypothalamic area were moderately stained. On the contrary, the most intense reactions to anti-ActRI(81-89) antibody were detected in neurons of the lateral hypothalamic area. In addition, many neurons of the cerebral cortex were also stained, but neurons of the hippocampus and the amygdala were not stained. These results suggest that activin may have physiological roles not only for hypothalamic neuroendocrinological and feeding-related systems as suggested previously but may also have functions in cortical and limbic pathways as a neuromodulator or for maintenance of neurons.

Immunological localization and ontogenetic development of inhibin alpha subunit in rat brain.

This study examined the immunolocalization and ontogeny of the inhibin-specific alpha subunit in the brain of male rats. Immunohistochemistry using antiserum directed against the mature region of porcine inhibin alpha (1-19, Tyr20) revealed positive reactions in process-bearing cells resembling astroglia in several regions, especially in the dorsal region of the third ventricle, medial and ventral arcuate nucleus, hippocampal dentate gyrus, and layers 1-3 of the cerebral cortex. Generally, inhibin alpha-positive cells in the limbic cortex had larger cell bodies and longer processes than those in the hypothalamus. These inhibin alpha-positive cells were verified to be positive for glial fibrillary acidic protein (GFAP), a differentiated astroglial marker, by double immunolabelling. The expression of inhibin alpha mRNA was higher in the brains of neonatal rats than in those of adult rats, as revealed by reverse transcription-competitive polymerase chain reaction, although the similar changes of immunoreactive inhibin alpha subunit in the brain was not observed. Orchiectomy did not affect expression of inhibin alpha mRNA in the hypothalamic area. This study suggests that inhibin-related peptide is produced by differentiated astrocytes, especially in the hypothalamic arcuate nucleus, the hippocampal dentate gyrus, and the cerebral cortex, and that the expression of inhibin alpha is regulated during brain development.

Unique recognition of activin and inhibin by polyclonal antibodies to inhibin subunits.

Inhibin-A is a glycoprotein composed of an alpha subunit containing a glycosylation site and a beta A subunit, whereas activin-A is a homodimer of two inhibin beta A subunits. We examined the recognition of activin-A and inhibin-A by several antisera to the alpha or beta A subunit, and factors affecting the recognition. A total of six polyclonal antibodies to inhibin subunits, i.e., two antisera to a peptide fragment of the alpha subunit [alpha (1-19) and alpha (1-26)], and four antisera to the beta A subunit [beta A (1-10), beta A (70-79), beta A (87-99), and beta A (94-105)], was generated. On Western blot analysis, the anti-beta A (87-99) and beta A (94-105) sera recognized recombinant human activin-A but not inhibin-A under non-reducing conditions. When inhibin-A was deglycosylated with N-glycosidase-F, inhibin-A could be recognized by the anti-beta A (87-99) and beta A (94-105) sera. In addition, when activin-A bound to a nitrocellulose membrane was pre-incubated with recombinant human follistatin, the recognition of activin-A by the anti- beta A (87-99) and beta A (94-105) sera was decreased. These results suggested that the lower affinity of follistatin to inhibin-A than to activin-A might be likely explained as reflecting a site associated with the glycosylation of inhibin-A. However, the exposure of amino acids 87-105 of the inhibin beta A subunit on the molecular surface through deglycosylation did not increase the affinity of inhibin-A for follistatin but rather resulted in poor binding with follistatin. The present data suggest that (1) amino acids 87-105 of the inhibin/activin beta A subunit are located on the molecular surface, although this region of inhibin-A is concealed by the carbohydrate chain of the alpha subunit, (2) the region responsible for follistatin binding within the activin beta A subunit is spanned by amino acids 87-105, and (3) the mode of binding of inhibin-A to follistatin is quite different from that of activin-A to follistatin, and the former may be influenced by glycosylation.

Immuno-histochemical expression of alpha1, alpha2 and alpha3 integrin subunits during angiogenesis in vitro.

Aortic explants were obtained from mouse fetuses and cultured in collagen gels. Immuno-fluorescence microscopy, antibodies (anti alpha1, alpha2 and alpha3 integrin subunits) were used. Fibroblastic cells migrated from the aortic explant after one day of cultivation. The migrating cells located in the peripheral part of the aortic explant were positive for alpha1 and alpha2 integrin subunit antibodies. Immuno-fluorescence-positive staining for the alpha3 integrin subunit antibody was clearly seen in the migrating cells located near the aortic explant and surrounding tube-like structures. In an immuno-electron microscope study performed by pre-embedding immuno labeling, gold particles associated with the alpha3 integrin subunit were found to reside on the membranes of the cells surrounding the capillary-like tubes. Two synthetic peptides, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and KDGEA (Lys-Asp-Gly-Glu-Ala), were added to the growth medium to study their effects on cell migration. KDGEA, a compound containing the recognition sequence for alpha2beta1 integrin, decreased cell migration, while GRGDSP exhibited no effect. The migration of fibroblastic cells is an important phenomenon for tube formation. The present study suggested that the alpha1 and alpha2 integrin subunits are both involved in the cell migration, and more specifically, that the alpha2 integrin subunit participates in cell migration through the KDGEA sequence. The alpha3 integrin subunit played a role in tube formation.

Parturition upregulates nitric oxide synthase activity in the rat anterior pituitary gland.

Recent evidence suggests that endogenous nitric oxide (NO) contributes to the modulation of hormonal secretion from the anterior pituitary gland according to the physiological state of the animal. In this study, nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and specific neurochemical assay were used to asses possible changes of NO synthase (NOS) activity in the anterior pituitary during pregnancy and parturition in rats. The anterior pituitary showed (weak) NADPH-d activity throughout pregnancy. Parturition increased the number and intensity of NADPH-d-positive cells. The NADPH-d-positive cells co-localized with immunofluorescent LH-positive cells. No variation in NADPH-d activity was apparent during the various stages of the oestrous cycle. Furthermore, NOS activity during parturition increased significantly when compared with non-pregnant and pregnant rats. Increases in both specific activity and NADPH-d activity gradually decreased within 24 h post-partum, suggesting that NO may modulate anterior pituitary function during parturition.

Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction.

The possible localization of nitric oxide (NO) synthase (NOS) in proximity to the microvasculature was examined in the rat adenohypophysis using immunohistochemistry and nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. A population of NOS-positive cells was localized in very close contact with the sinusoidal capillaries. The pattern of this perivascular localization was either unicellular, bicellular or multicellular. These observations suggest that, at least, some actions of NO in the adenohypophysis can be accounted for by a local regulation of the glandular microvasculature.

Study of the functional share of lysosomal cathepsins by the development of specific inhibitors.

To analyze the functional share of individual cathepsins, we developed powerful and specific inhibitors for individual cathepsins using computer graphics of substrate binding pockets based on X-ray crystallography. These new inhibitors were named CLIK group. Epoxy succinate peptide derivatives, CLIK-066, 088, 112, 121, 148, 181, 185 and 187, are typical specific inhibitors for cathepsin L. Aldehyde derivatives CLIK-060 and CLIK-164 showed specific inhibition against cathepsin S and cathepsin K, respectively. We found that pyridoxal phosphate (PLP), a coenzyme form of vitamin B6, inhibits all cathepsins and also new artificially synthesized pyridoxal derivatives, CLIK-071 and -072, in which the phosphate esters of PLP were replaced by propionic acid, exhibited strong inhibition for cathepsins. Furthermore, CLIK-071 was easy to incorporate into cells and showed powerful inhibition for intracellular cathepsins. Using these selective inhibitors, the allotment of individual cathepsin functions in cells has been studied as follows. Cathepsin L and/or K participate in bone resorption based on bone type-1 collagen degradation and the L-type protease inhibitors suppressed the bone resorption. Cathepsins B and S participate in antigen presentations based on antigen processing and invariant chain degradation, respectively. Also cathepsin L participates in cell apoptosis mediated by caspase III activation.

Suppressed bone induction by follistatin in spontaneously hypercholesterolemic rat bone.

Bone inducing activity in demineralized bone matrix (DBM) of young spontaneously hypercholesterolemic (SHC) rats has been shown to be lower than that of aged SHC rats. This study examined the involvement of bone follistatin, an activin-binding protein, in bone induction. Immunoreactive follistatin was higher in DBM from 10-week-old SHC rats (DBM-10wk) than in DBM from 6-month-old SHC rats (DBM-6mo). When DBM without follistatin supplement was implanted, the C-propeptide of type II procollagen and calcium contents on day 12 in implants of DBM-6mo were 68% and 40% higher than those of DBM-10wk, respectively. In contrast, follistatin supplement to DBM decreased C-propeptide of type II procollagen and calcium contents in implants of both DBM-10wk and DBM-6mo, and the levels of these parameters were comparable between DBM-10wk and DBM-6mo, indicating reduced formation of cartilage and bone. These findings suggest that 1) follistatin content in bone matrix decreases with advancing age in SHC rats, and 2) the follistatin interferes with endochondral bone formation. We demonstrate that the lower bone induction of DBM from young SHC rats was partly due to the abundance of follistatin in bone matrix.

Increased cartilage and bone formation in spontaneously hypercholesterolemic rats.

Spontaneously hypercholesterolemic (SHC) rats are known to exhibit accelerated bone resorption. We compared endochondral bone formation induced by implantation of demineralized bone matrix (DBM) to 4-week-old SHC rats with that of age-matched Sprague-Dawley (SD) rats. When DBM prepared from adult SD rats was implanted, the cartilageous area enlarged, and C-propeptide of type II procollagen content on day 7 was higher in SHC rats. Alkaline phosphatase activity and calcium content on day 12 and tartrate-resistant acid phosphatase activity on day 19 were higher in SHC rats. These results suggest active chondrogenesis, with a subsequent increase in osteogenesis, and stimulated osteoclastic bone resorption in SHC rats. When DBM from 10-week-old SHC rats was implanted into SD or SHC rats, the levels of bone forming parameters on day 12 were reduced to one-third, suggesting inhibiting factor(s) for bone induction in bone matrix of SHC rats. In contrast, when DBM from 6-month-old SHC rats was implanted, although bone forming parameters in SD rats were comparable to the case of implantation of DBM from SD rats, the accelerated bone formation detected in SHC rats was blocked, indicating resistance to systemic bone inducing factor(s) of SHC rats in aged bone matrix. These results suggest that age-related decrease in responses to some systemic bone inducing factor may lead to the bone loss with advancing age.

Effect of dietary vitamin B6 contents on antibody production.

When mice were placed on diets extreme deficient in vitamin B6, ovalbumin-dependent antibody productions (IgE, IgG1, IgG2a) were significantly suppressed, and alanine aminotransferase activity in the liver was also significantly decreased. In the case of pyridoxine excess (6 mg% = about ten times standard amount) in a 70% casein diet, ovalbumin-dependent antibody productions were also considerably suppressed. These responses were weaker in a low casein (5%) or normal casein (20%) diet than in a 70% casein diet. The administration of high doses of pyridoxine (6 mg%) resulted in the suppression of hepatic cathepsin B activity. Therefore, we conclude that ovalbumin-dependent antibody productions (IgG1, IgE) were suppressed by pyridoxine excess diet (6 mg%), because hepatic cathepsin B activity was suppressed by the excess pyridoxine in diet.

Inhibition of cathepsin activities by vitamin B6 derivatives.

We found that pridoxal phosphate (PLP), a coenzyme form of vitamin B6, inhibited the activity of cathepsins B, K, S and L in vitro. Cathepsins activities in cultured splenocytes were suppressed by the addition of pridoxal (PL) or pridoxine (PI) in to the culture medium. A newly synthesized artificial vitamin B6 derivative, a pridoxal propionate derivative, CLIK-164, showed selective inhibition of cathepsin O/K.

Morphology of capillary-like structures in a three-dimensional aorta/collagen gel culture.

The morphology of capillary-like tubes was investigated by electron microscopy (TEM and SEM) using an in vitro model of capillarogenesis (aorta/collagen type I gel). This model allowed morphological comparisons with in vivo capillaries and an evaluation of the functional maturity of the endothelium to be made. The lumina developing in vitro were demarcated by endothelial cells of varying thickness (0.1-2 microns). Pericytes were resting on the outside. The endothelial cells were characterized by contacts of varying length with tight and gap junctions and occasional indentations. The inner surface exhibited areas both with pronounced and without any endocytotic activity. In addition to a large Golgi apparatus, a varying number of cell organelles occurred depending on the thickness of the endothelium. Bundles consisting of microfilaments were often located underneath the outer cell membrane and in the vicinity of contact areas. A lamina densa was in the process of formation. The capillaries grown in vitro closely resembled those in vivo and showed a high degree of differentiation. Hence, this in vitro model allows the study of a number of functions of endothelial cells.

Formation of new capillary-like tubes in a three-dimensional in vitro model (aorta/collagen gel).

Direct sprouting (angiogenesis) does not occur during the formation of capillary-like tubes in an aorta/ collagen gel in the in vitro model. However, emigration of cells which stretch, arrange themselves side by side, form contacts (unspecific, tight and gap junctions), develop a lumen and show differentiation of endothelial cells (including the formation of a lamina densa and the appearance of pericytes) have been observed, i.e. vasculogenesis occurs. The origin of long, stretched cells is not known with certainty. They possibly represent smooth muscle cells. In addition, other cell types have been found, such as fibrocyte-like and fibroblast-like cells, elastoblasts, fat cells, monocytes and macrophages. All these cells are able to produce factors that promote the formation of new capillaries. Hence, a knowledge of these cells appears to be important for the analysis of in vitro systems. Moreover, the occurrence of these cell types must be considered when assessing possible effects.

Unilateral anomalous left common carotid artery; a case report.

An anomaly of the left common carotid artery was observed in a Japanese male cadaver during an anatomy class at the Saitama Medical School in 1995. The superior thyroid, lingual and facial arteries arose from the common carotid artery, and the posterior auricular, maxillary and superficial temporal arteries arose from the common carotid artery by a common trunk. The occipital and ascending pharyngeal arteries arose from the internal carotid artery. The left carotid body (glomus caroticum) was observed to be slightly below the lingual artery, behind the common carotid artery, and it was located at the level of the intervertebral disk between C2 and C3 or at the same level as the right carotid body. The carotid body was richly innervated by a branch of the glossopharyngeal nerve and by a plexus of sympathetic fibers from the vagus and glossopharyngeal nerves. We assumed that the artery above the level of the carotid body was the internal carotid artery; there was no specific external carotid artery and all branches of the external carotid artery arose from the internal carotid artery.

Observation on the basal lamina of duodenal mesothelial cells during metamorphosis of Xenopus laevis.

Morphologic changes in the basal lamina of duodenal mesothelial cells during metamorphosis of Xenopus laevis were observed. In the prometamorphosis stage (Stage 56-59), the basal lamina was almost completely flat; the lamina densa of the basal lamina was a 50 nm layer of high electron density. In the early stages of metamorphic climax (Stage 60-62), the basal lamina showed occasional slight folding (stage 60), with the lapse of time, the folding became continuous and deeper. The development of an additional thin basal lamina was observed in areas where the folded basal lamina was separated from mesothelial cells, viz. on the side adjacent to the mesothelium. The lamina densa in this stage was approximately twice the thickness of the prometamorphosis stage and exhibited high electron density. In the later stages of metamorphic climax (stage 63-66), the basal lamina just under mesothelium became more apparent. The folded basal lamina shifted from the mesothelium into the subserosa and gradually disappeared, and the basal lamina became a single layer. The thickness of the lamina densa was almost the same as in the prometamorphosis stage. Since the timing of the folding of the basal lamina coincides with the shortening of the digestive tract and the marked narrowing of the lumen, we suggest that physical changes in the digestive tract during metamorphosis may play an important role in these morphologic changes of the basal lamina.