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We investigated whether peripheral combination treatment of a sodium–glucose cotransporter 2 (SGLT2) inhibitor and leptin improves glucose metabolism in insulin-dependent diabetes mellitus (IDDM) model mice. Twelve-week-old male C57BL6 mice were intraperitoneally administered a high dose of streptozotocin to produce IDDM. IDDM mice were then divided into five groups: SGLT2 inhibitor treatment alone, leptin treatment alone, leptin and SGLT2 inhibitor co-treatment, untreated IDDM mice, and healthy mice groups. The blood glucose (BG) level at the end of the dark cycle was measured, and a glucose tolerance test (GTT) was performed and compared between the five groups. Leptin was peripherally administered at 20 μg/day using an osmotic pump, and an SGLT2 inhibitor, ipragliflozin, was orally administered at 3 mg/kg/day. Monotherapy with SGLT2 inhibitor or leptin significantly improved glucose metabolism in mice as evaluated by BG and GTT compared with the untreated group, whereas the co-treatment group with SGLT2 inhibitor and leptin further improved glucose metabolism as compared with the monotherapy group. Notably, glucose metabolism in the co-treatment group improved to the same level as that in the healthy mice group. Thus, peripheral combination treatment with leptin and SGLT2 inhibitor improved glucose metabolism in IDDM mice without the use of insulin.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.

The reward system, which consists of dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and caudate-putamen in the striatum, has an important role in the pathogenesis of not only drug addiction but also diet-induced obesity. In the present study, we examined whether signaling through glucocorticoid receptors (GRs) in the reward system affects the rewarding value of a high-fat diet (HFD). To do so, we generated mice that lack functional GRs specifically in dopaminergic neurons (D-KO mice) or corticostriatal neurons (CS-KO mice), subjected the mice to caloric restriction stress conditions, and evaluated the rewarding value of a HFD by conditioned place preference (CPP) test. Caloric restriction induced increases in serum corticosterone to similar levels in all genotypes. While CS-KO as well as WT mice exhibited a significant preference for HFD in the CPP test, D-KO mice exhibited no such preference. There were no differences between WT and D-KO mice in consumption of HFD after fasting or cognitive function evaluated by a novel object recognition test. These data suggest that glucocorticoid signaling in the VTA increases the rewarding value of a HFD under restricted caloric stress.

Evidence has accumulated that higher consumption of high-fat diets (HFDs) during the juvenile/adolescent period induces altered hippocampal function and morphology; however, the mechanism behind this phenomenon remains elusive. Using high-resolution structural imaging combined with molecular and functional interrogation, a murine model of obesity treated with HFDs for 12 weeks after weaning mice was shown to change in the glutamate-mediated intracellular calcium signaling and activity, including further selective reduction of gray matter volume in the hippocampus associated with memory recall disturbance. Dysregulation of intracellular calcium concentrations was restored by a non-competitive α-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) antagonist, followed by normalization of hippocampal volume and memory recall ability, indicating that AMPARs may serve as an attractive therapeutic target for obesity-associated cognitive decline.

Previous studies suggest that signaling by the gamma-aminobutyric acid (GABA) type B receptor (GABABR) is involved in the regulation of binge eating, a disorder which might contribute to the development of obesity. Here, we show that intermittent access to a high fat diet (HFD) induced binge-like eating behavior with activation of dopamine receptor d1 (drd1)-expressing neurons in the caudate putamen (CPu) and nucleus accumbens (NAc) in wild-type (WT) mice. The activation of drd1-expressing neurons during binge-like eating was substantially increased in the CPu, but not in the NAc, in corticostriatal neuron-specific GABABR-deficient knockout (KO) mice compared to WT mice. Treatment with the GABABR agonist, baclofen, suppressed binge-like eating behavior in WT mice, but not in KO mice, as reported previously. Baclofen also suppressed the activation of drd1-expressing neurons in the CPu, but not in the NAc, during binge-like eating in WT mice. Thus, our data suggest that GABABR signaling in CPu neurons expressing drd1 suppresses binge-like consumption during a HFD in mice.

Binge eating is a characteristic symptom observed in obese individuals that is related to dysfunction of dopaminergic neurons (DNs). Intermittent administration of a high-fat diet (HFD) is reported to induce binge-like eating, but the underlying mechanisms remain unclear. We generated dopaminergic neuron specific IKKβ deficient mice (KO) to examine the effects of inflammation in DNs on binge-like eating under inflammatory conditions associated with HFD. After administration of HFD for 4 weeks, mice were fasted for 24 h, and then the consumption of HFD was measured for 2 h. We also evaluated that the mRNA expressions of inflammatory cytokines, glial markers, and dopamine signaling-related genes in the ventral tegmental area (VTA) and striatum. Moreover, insulin was administered intraventricularly to assess downstream signaling. The consumption of HFD was significantly reduced, and the phosphorylation of AKT in the VTA was significantly increased in female KO compared to wild-type (WT) mice. Analyses of mRNA expressions revealed that DNs activity and inflammation in the VTA were significantly decreased in female KO mice. Thus, our data suggest that HFD-induced inflammation with glial cell activation in the VTA affects DNs function and causes abnormal eating behaviors accompanied by insulin resistance in the VTA of female mice.

Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax^+ residual cells in the maturation phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax^+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax^+ early hypothalamic progenitor cells. Therefore, Rax^+ residual cells in the maturation phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax^+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y^+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax^+ residual cells were multipotent neural stem/progenitor cells. Rax^+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax^+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool., ファイル公開日: 2020/07/01

In 1997, the seminal manuscript by Asahara, Murohara, Isner et al outlined the evidence for the existence of circulating, bone marrow-derived cells capable of stimulating and contributing to the formation of new blood vessels. Consistent with the paradigm shift that this work represented, it triggered much scientific debate and controversy, some of which persists 2 decades later. In contrast, the clinical application of autologous CD34 cell therapy has been marked by a track record of consistent safety and clinical benefit in multiple ischemic conditions. In this review, we summarize the preclinical and clinical evidence from over 700 patients in clinical trials of CD34 cell therapy.

Wolfram syndrome (WS) is mainly caused by mutations in the WFS1 gene and characterized by diabetes mellitus, optic atrophy, hearing loss, and central diabetes insipidus (CDI). WFS1 is an endoplasmic reticulum (ER)-resident transmembrane protein, and Wfs1 knockout (Wfs1−/−) mice, which have been used as a mouse model for WS, reportedly manifested impairment of glucose tolerance due to pancreatic β-cell loss. In the present study, we examined water balance, arginine vasopressin (AVP) secretion, and ER stress in AVP neurons of the hypothalamus in Wfs1−/− mice. There were no differences in urine volumes between Wfs1−/− and wild-type mice with free access to water. Conversely, when mice were subjected to intermittent water deprivation (WD) for 20 weeks, during which water was unavailable for 2 days a week, urine volumes were larger in Wfs1−/− mice, accompanied by lower urine AVP concentrations and urine osmolality, compared to wild-type mice. The mRNA expression of immunoglobulin heavy chain binding protein, a marker of ER stress, was significantly increased in the supraoptic nucleus and paraventricular nuclei in Wfs1−/− mice compared to wild-type mice after WD. Our results thus showed that Wfs1 knockout leads to a decrease in AVP secretion during dehydration, which could explain in part the mechanisms by which Wfs1 mutations cause CDI in humans.

Excessive sodium salt (NaCl) or fat intake is associated with a variety of increased health risks. However, whether excessive NaCl intake accompanied by a high-fat diet (HFD) affects glucose metabolism has not been elucidated. In this study, C57BL/6J male mice were fed a normal chow diet (NCD), a NCD plus high-NaCl diet (NCD plus NaCl), a HFD, or a HFD plus high-NaCl diet (HFD plus NaCl) for 30 weeks. No significant differences in body weight gain, insulin sensitivity, and glucose tolerance were observed between NCD-fed and NCD plus NaCl-fed mice. In contrast, body and liver weights were decreased, but the weight of epididymal white adipose tissue was increased in HFD plus NaCl-fed compared to HFD-fed mice. HFD plus NaCl-fed mice had lower plasma glucose levels in an insulin tolerance test, and showed higher plasma glucose and lower plasma insulin levels in an intraperitoneal glucose tolerance test compared to HFD-fed mice. The β-cell area and number of islets were decreased in HFD plus NaCl-fed compared to HFD-fed mice. Increased Ki67-positive β-cells, and increased expression levels of Ki67, CyclinB1, and CyclinD1 mRNA in islets were observed in HFD-fed but not HFD plus NaCl-fed mice when compared to NCD-fed mice. Our data suggest that excessive NaCl intake accompanied by a HFD exacerbates glucose intolerance, with impairment in insulin secretion caused by the attenuation of expansion of β-cell mass in the pancreas.

The immunoglobulin heavy chain binding protein (BiP), also referred to as 78-kDa glucose-regulated protein (GRP78), is a pivotal endoplasmic reticulum (ER) chaperone which modulates the unfolded protein response under ER stress. Our previous studies showed that BiP is expressed in arginine vasopressin (AVP) neurons under non-stress conditions and that BiP expression is upregulated in proportion to the increased AVP expression under dehydration. To clarify the role of BiP in AVP neurons, we used a viral approach in combination with shRNA interference for BiP knockdown in mouse AVP neurons. Injection of a recombinant adeno-associated virus equipped with a mouse AVP promoter and BiP shRNA cassette provided specific BiP knockdown in AVP neurons of the supraoptic (SON) and paraventricular nuclei (PVN) in mice. AVP neuron-specific BiP knockdown led to ER stress and AVP neuronal loss in the SON and PVN, resulting in increased urine volume due to lack of AVP secretion. Immunoelectron microscopy of AVP neurons revealed that autophagy was activated through the process of AVP neuronal loss, whereas no obvious features characteristic of apoptosis were observed. Pharmacological inhibition of autophagy by chloroquine exacerbated the AVP neuronal loss due to BiP knockdown, indicating a protective role of autophagy in AVP neurons under ER stress. In summary, our results demonstrate that BiP is essential for the AVP neuron system.

The reward system plays an important role in the pathogenesis of not only drug addiction, but also diet-induced obesity. Recent studies have shown that insulin and leptin receptor signaling in the ventral tegmental area (VTA) regulate energy homeostasis and that their dysregulation is responsible for obesity and altered food preferences. Although a high-fat diet (HFD) induces inflammation that leads to insulin and leptin resistance in the brain, it remains unclear whether HFD induces inflammation in the VTA. In the present study, we placed male mice on a chow diet or HFD for 3, 7, and 28 days and evaluated the mRNA expression of inflammatory cytokines and microglial activation markers in the VTA. The HFD group showed significantly elevated mRNA expressions of IL1β at 3 days; tumor necrosis factor-alpha (TNFα), IL1β, IL6, Iba1, and CD11b at 7 days; and TNFα, IL1β, Iba1, and CD11b at 28 days. The changes in TNFα were also confirmed in immunohistochemical analysis. Next, after administration of chow or HFD for 7 days, we selected mice with equal weights in both groups. In experiments using these mice, Akt phosphorylation in the VTA was significantly decreased after intracerebroventricular injection of insulin, whereas no change in STAT3 phosphorylation was found with leptin. Taken together, these results suggest that HFD induces inflammation at least partly associated with microglial activation in the VTA leading to insulin resistance, independently of the energy balance. Our data provide new insight into the pathophysiology of obesity caused by a dysfunctional reward system under HFD conditions.

Immune-related adverse events induced by anti-programmed cell death-1 antibodies (PD-1-Ab), including destructive thyroiditis (thyroid-irAE), are thought to be caused by activated T cells. However, the T cell subsets that are directly responsible for damaging self-organs remain unclear. To clarify which T cell subsets are involved in the development of thyroid-irAE, a mouse model of thyroid-irAE was analyzed. PD-1-Ab administration 2.5 months after immunization with thyroglobulin caused destructive thyroiditis. Thyroiditis was completely prevented by previous depletion of CD4

Glial cells can mediate hypothalamic inflammatory processes induced in response to a high-fat diet (HFD). We used magnetic-activated cell sorting (MACS) to isolate microglia and astrocytes from hypothalamus of mice fed HFD and examined changes in expression of inflammation-related cytokines and markers related to glial cell activation status. Hypothalamus from male C57BL6 mice fed a chow diet (chow) or HFD for 1, 3, or 28 days were collected and microglia and astrocytes were isolated by MACS. After confirming cell viability by fluorescence activated cell sorting, mRNA expression levels of inflammation-related cytokines and markers of glial cell activation status were examined by qRT-PCR, which revealed that both glial cell types isolated by MACS retained specificity. On day 3 of HFD, both CD86 and TNFα mRNA expression was significantly increased in microglia relative to the chow group. In astrocytes, TNFα mRNA expression levels were similar between the chow and HFD groups on day 3, but anti-inflammatory cytokine IL-10 levels were significantly increased. On day 7 of HFD, TNFα expression in microglia decreased to levels comparable to the chow group while that in astrocytes remained unchanged. On day 28 of HFD, TNFα levels were significantly increased in both microglia and astrocytes, which had increased mRNA expression of CD86 and MAO-B, respectively. For both glial cell types, results for TNFα expression assessed by RT-PCR and immunohistochemical analysis were similar. These results indicate that the role of microglia and astrocytes in hypothalamic inflammation under HFD conditions changed with time and these changes were accompanied by changes in the activation status of glial cells. Our data suggest that early after initiating HFD, hypothalamic astrocytes suppress diet-induced inflammation at least in part by secreting IL-10, whereas continued HFD feeding impairs this suppressive function such that both microglia and astrocytes promote hypothalamic inflammation.

Arginine vasopressin (AVP) synthesized in the magnocellular neurons of the hypothalamus is transported through their axons and released from the posterior pituitary into the systemic circulation to act as an antidiuretic hormone. AVP synthesis and release are precisely regulated by changes in plasma osmolality. Magnocellular AVP neurons receive innervation from osmosensory and sodium-sensing neurons, but previous studies showed that AVP neurons per se are osmosensitive as well. In the current study, we made AVP-Venus reporter mice and showed that Venus was expressed exclusively in AVP neurons and was upregulated under water deprivation. In hypothalamic organotypic cultures from the AVP-Venus mice, Venus-labeled AVP neurons in the supraoptic and paraventricular nuclei survived for 1 month, and Venus expression was upregulated by forskolin. Furthermore, in dissociated Venus-labeled magnocellular neurons, treatment with NaCl, but not with mannitol, decreased Venus fluorescence in the soma of the AVP neurons. Thus, Venus expression in AVP-Venus transgenic mice, as well as in primary cultures, faithfully showed the properties of intrinsic AVP expression. These findings indicate that AVP-Venus mice as well as the primary hypothalamic cultures could be useful for studying magnocellular AVP neurons.

Background Periostin (POSTN) is a protein that binds to integrins to support adhesion and migration of epithelial cells. Mice lacking this gene exhibit cardiac valve disease as well as skeletal and dental defects. Recent studies indicated that periostin is involved in the pathogenesis and progression of knee osteoarthritis (OA). We investigated the influence of periostin and matrix metalloproteinases (MMPs) on OA synoviocytes. Materials and methods OA patients were classified according to the Kellgren-Lawrence system and the levels of periostin, interleukin (IL)-4, IL-13 and transforming growth factor-β (TGFβ) in the synovial fluid were measured. MMPs or tissue inhibitor of MMPs (TIMPs) with periostin in cultured cells were measured when periostin was added to OA-associated synovial cells. Dexamethasone, a steroid medication which shows immunosuppressive effects, was used to investigate the influence of the downstream cascade. Results Periostin and IL-13 levels were up-regulated during the progression of OA. MMP-2 and MMP-3 levels increased in a periostin concentration-dependent manner. Increase in MMP-2 and MMP-3 levels was inhibited by dexamethasone treatment. Conclusion In vivo results herein indicate that IL-13 may induce periostin production in OA. Furthermore, periostin may facilitate MMP production in OA-associated synovial cells.

Ricinoleic acid (RA) is the main fatty acid component of castor oil and was found to inhibit Ca2+ -signal transduction pathway-mediated cell cycle regulation in a yeast-based drug screening assay. RA is expected to have antidiabetic, antiallergy, and/or anticancer properties but its target molecule is unknown. To identify a novel pharmacological effect of RA, we investigated its target molecule in the Ca2+ -signal transduction pathway. RA inhibition of calcineurin (CN) was examined in a yeast-based CN inhibitor screening assay using the rsp5A401E mutant and in a phosphatase assay using recombinant human CN. RA showed growth-restoration activity at 5 μg/spot in the CN inhibitor screening assay with the rsp5A401E yeast strain. Furthermore, it directly inhibited CN without immunophilins at Ki = 33.7 μM in a substrate-competitive manner. The effects of RA on CN in mammalian cells were further evaluated by measuring β-hexosaminidase (β-HEX) release in RBL-2H3 cells. RA at 50 μM suppressed the release of β-HEX from RBL-2H3 cells. Moreover, this compound was found to inhibit glycogen synthase kinase-3β (GSK-3β), as determined by a kinase assay using recombinant human GSK-3β. RA inhibited GSK-3β at Ki = 1.43 μM in a peptide substrate-competitive manner. The inhibition of GSK-3β by this molecule was further assessed in mammalian cells by measuring the inhibition of glucose production in H4IIE rat hepatoma cells. RA at 25 μM suppressed glucose production in these cells. These findings indicate that RA and/or castor oil could be a useful functional fatty acid to treat allergy or type 2 diabetes.

High differentiation efficiency is one of the most important factors in developing an in vitro model from pluripotent stem cells. In this report, we improved the handling technique applied to mouse-induced pluripotent stem (iPS) cells, resulting in better differentiation into hypothalamic vasopressin (AVP) neurons. We modified the culture procedure to make the maintenance of iPS cells in an undifferentiated state much easier. Three-dimensional floating culture was demonstrated to be effective for mouse iPS cells. We also improved the differentiation method with regards to embryology, resulting in a greater number of bigger colonies of AVP neurons differentiating from mouse iPS cells. Fgf8, which was not used in the original differentiation method, increased iPS differentiation into AVP neurons. These refinements will be useful as a valuable tool for the modeling of degenerative disease in AVP neurons in vitro using disease-specific iPS cells in future studies. Keywords: AVP neuron, Hypothalamus, Mouse iPS cells, Differentiation, Maintenance

Giant presynaptic terminal brain slice preparations have allowed intracellular recording of electrical signals and molecular loading, elucidating cellular and molecular mechanisms underlying neurotransmission and modulation. However, molecular genetic manipulation or optical imaging in these preparations is hampered by factors, such as tissue longevity and background fluorescence. To overcome these difficulties, we developed a giant presynaptic terminal culture preparation, which allows genetic manipulation and enables optical measurements of synaptic vesicle dynamics, simultaneously with presynaptic electrical signal recordings. This giant synapse reconstructed from dissociated mouse brainstem neurons resembles the development of native calyceal giant synapses in several respects. Thus, this novel preparation constitutes a powerful tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development.SIGNIFICANCE STATEMENTWe have developed a novel culture preparation of giant mammalian synapses. These presynaptic terminals make it possible to perform optical imaging simultaneously with presynaptic electrophysiological recording. We demonstrate that this enables one to dissect endocytic and acidification times of synaptic vesicles. In addition, developmental elimination and functional maturation in this cultured preparation provide a useful model for studying presynaptic development. Because this giant synapse preparation allows molecular genetic manipulations, it constitutes a powerful new tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development.

Familial neurohypophysial diabetes insipidus (FNDI), characterized by progressive polyuria and loss of arginine vasopressin (AVP) neurons, is an autosomal dominant disorder caused by AVP gene mutations. Our previous studies with FNDI model mice demonstrated that mutant proteins accumulated in the endoplasmic reticulum (ER) of AVP neurons. Here, we examined therapeutic effects of the chemical chaperone 4-phenylbutylate (4-PBA) in FNDI mice. Treatment with 4-PBA reduced mutant protein accumulation in the ER of FNDI mice and increased AVP release, leading to reduced urine volumes. Furthermore, AVP neuron loss under salt loading was attenuated by 4-PBA treatment. These data suggest that 4-PBA ameliorated mutant protein accumulation in the ER of AVP neurons and thereby prevented FNDI phenotype progression.

Isolated adrenocorticotropin deficiency (IAD) is characterized by low or absent adrenocorticotropic hormone (ACTH) production. IAD is presumed to be caused in part by an autoimmune mechanism, and several lines of evidence have suggested the presence of anti-pituitary antibodies in IAD. However, the exact autoantigens remain unknown. The present study was designed to identify the autoantigen(s) in IAD using chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Rat anterior pituitary lysate was subjected to SDS-PAGE, and immunoblotting was performed using the sera from two patients with IAD and from a healthy subject. The bands detected by the patient serum samples, but not by the healthy subject sample, were excised, in-gel digested using trypsin, and subjected to LC-MS/MS analysis. On immunoblots, a 51-kDa band in the insoluble pellet was detected by the sera from the IAD patients but not from the healthy subject. Mass spectrometric analysis revealed the 51-kDa band contained Rab guanine nucleotide dissociation inhibitor (GDI) alpha. Consistent with the mass spectrometric analysis, a recombinant full-length human Rab GDI alpha was recognized by the two IAD patient samples but not by the healthy subject sample using immunoblotting. In total, anti-Rab GDI alpha antibodies were detected in serum samples from three of five patients with IAD (60%) but were absent in 5 healthy subjects. In addition, Rab GDI alpha was expressed in the anterior pituitary. In conclusion, it appears that Rab GDI alpha is a candidate autoantigen involved in IAD, and that anti-Rab GDI alpha antibodies are present predominantly in patients with IAD.

Overly rapid correction of chronic hyponatremia can cause osmotic demyelination syndrome (ODS). Minocycline protects ODS associated with overly rapid correction of chronic hyponatremia with hypertonic saline infusion in rats. In clinical practice, inadvertent rapid correction frequently occurs due to water diuresis, when vasopressin action suddenly ceases. In addition, vasopressin receptor antagonists have been applied to treat hyponatremia. Here the susceptibility to and pathology of ODS were evaluated using rat models developed to represent rapid correction of chronic hyponatremia in the clinical setting. The protective effect of minocycline against ODS was assessed. Chronic hyponatremia was rapidly corrected by 1 (T1) or 10 mg/kg (T10) of tolvaptan, removal of desmopressin infusion pumps (RP), or administration of hypertonic saline. The severity of neurological impairment in the T1 group was significantly milder than in other groups and brain hemorrhage was found only in the T10 and desmopressin infusion removal groups. Minocycline inhibited demyelination in the T1 group. Further, immunohistochemistry showed loss of aquaporin-4 (AQP4) in astrocytes before demyelination developed. Interestingly, serum AQP4 levels were associated with neurological impairments. Thus, minocycline can prevent ODS caused by overly rapid correction of hyponatremia due to water diuresis associated with vasopressin action suppression. Increased serum AQP4 levels may be a predictive marker for ODS.

Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxin-binding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons.

SummaryAt presynaptic terminals vesicular membranes are fused into plasma membrane upon exocytosis and retrieved by endocytosis. During a sustained high-frequency transmission, exoendocytic coupling is critical for the maintenance of synaptic transmission. Here, we show that this homeostatic coupling is supported by cGMP-dependent protein kinase (PKG) at the calyx of Held. This mechanism starts to operate after hearing onset during the second postnatal week, when PKG expression becomes upregulated in the brainstem. Pharmacological tests with capacitance measurements revealed that presynaptic PKG activity is supported by a retrograde signal cascade mediated by NO that is released by activation of postsynaptic NMDA receptors. Activation of PKG also upregulates phosphatidylinositol-4,5-bisphosphate, thereby accelerating endocytosis. Furthermore, presynaptic PKG activity upregulates synaptic fidelity during high-frequency transmission. We conclude that maturation of the PKG-dependent retrograde signal cascade strengthens the homeostatic plasticity for the maintenance of high-frequency synaptic transmission at the fast glutamatergic synapse.

Fumitremorgin C, a diketopiperazine mycotoxin produced by Aspergillus fumigatus, is a potent and specific inhibitor of breast cancer resistance protein (BCRP). Elucidation of the fumitremorgin C biosynthetic pathway provides a strategy for new drug design. A structure-activity relationship study based on metabolites related to the ftm gene cluster revealed that the process most crucial for inhibitory activity against BCRP was cyclization to form fumitremorgin C. To determine the gene involved in the cyclization reaction, targeted gene inactivation was performed with candidate genes in the ftm cluster. Analysis of the gene disruptants allowed us to identify ftmE, one of the cytochrome P450 genes in the cluster, as the gene responsible for the key step in fumitremorgin biosynthesis. Additionally, we demonstrated that the other two cytochrome P450 genes, ftmC and ftmG, were involved in hydroxylation of the indole ring and successive hydroxylation of fumitremorgin C, respectively.

Hyponatremia is the most common clinical electrolyte disorder. Once thought to be asymptomatic in response to adaptation by the brain, recent evidence suggests that chronic hyponatremia may be linked to attention deficits, gait disturbances, risk of falls, and cognitive impairments. Such neurologic defects are associated with a reduction in quality of life and may be a significant cause of mortality. However, because underlying diseases such as adrenal insufficiency, heart failure, liver cirrhosis, and cancer may also affect brain function, the contribution of hyponatremia alone to neurologic manifestations and the underlying mechanisms remain unclear. Using a syndrome of inappropriate secretion of antidiuretic hormone rat model, we show here that sustained reduction of serum sodium ion concentration induced gait disturbances; facilitated the extinction of a contextual fear memory; caused cognitive impairment in a novel object recognition test; and impaired long-term potentiation at hippocampal CA3-CA1 synapses. In vivo microdialysis revealed an elevated extracellular glutamate concentration in the hippocampus of chronically hyponatremic rats. A sustained low extracellular sodium ion concentration also decreased glutamate uptake by primary astrocyte cultures, suggesting an underlying mechanism of impaired long-term potentiation. Furthermore, gait and memory performances of corrected hyponatremic rats were equivalent to those of control rats. Thus, these results suggest chronic hyponatremia in humans may cause gait disturbance and cognitive impairment, but these abnormalities are reversible and careful correction of this condition may improve quality of life and reduce mortality.

Context:Central diabetes insipidus (CDI) can be caused by several diseases, but in about half of the patients the etiological diagnosis remains unknown. Lymphocytic infundibulo-neurohypophysitis (LINH) is an increasingly recognized entity among cases of idiopathic CDI; however, the differential diagnosis from other pituitary diseases including tumors can be difficult because of similar clinical and radiological manifestations. The definite diagnosis of LINH requires invasive pituitary biopsy.Objective:The study was designed to identify the autoantigen(s) in LINH and thus develop a diagnostic test based on serum autoantibodies.Design:Rat posterior pituitary lysate was immunoprecipitated with IgGs purified from the sera of patients with LINH or control subjects. The immunoprecipitates were subjected to liquid chromatography-tandem mass spectrometry to screen for pituitary autoantigens of LINH. Subsequently, we made recombinant proteins of candidate autoantigens and analyzed autoantibodies in serum by Western blotting.Results:Rabphilin-3A proved to be the most diagnostically useful autoantigen. Anti-rabphilin-3A antibodies were detected in 22 of the 29 (76%) patients (including 4 of the 4 biopsy-proven samples) with LINH and 2 of 18 (11.1%) patients with biopsy-proven lymphocytic adeno-hypophysitis. In contrast, these antibodies were absent in patients with biopsy-proven sellar/suprasellar masses without lymphocytic hypophysitis (n = 34), including 18 patients with CDI. Rabphilin-3A was expressed in posterior pituitary and hypothalamic vasopressin neurons but not anterior pituitary.Conclusions:These results suggest that rabphilin-3A is a major autoantigen in LINH. Autoantibodies to rabphilin-3A may serve as a biomarker for the diagnosis of LINH and be useful for the differential diagnosis in patients with CDI.

In the field of neuroscience, low-invasive in vivo imaging would be a very useful method of monitoring the morphological dynamics of intact neurons in living animals. At present, there are two widely used in vivo imaging methods; one is the two-photon microscope method, and the other is the fiber optics method. However, these methods are not suitable for the in vivo imaging of deeper subcortical structures. In our study, we have developed a novel method for the in vivo imaging of pyramidal neurons in layer V of the cerebral cortex, utilizing a MicroLSM system and a stick-type objective lens that can be directly inserted into the target tissue. By using this method, we succeeded in obtaining clear images of pyramidal neurons in layer V of the cerebral cortex under a low-invasive condition. The MicroLSM system is a useful and versatile in vivo imaging system that will be applicable not only to the brain but also to other organs.

Background. Costimulatory molecules such as CD28 and B7 are essential for T cell activation, as well as playing a role in the T cell receptor and major histocompatibility complex pathway. It is well known that rejection in allotransplantation is diminished by treatment with CTLA4Ig, but whether a similar effect occurs in xenotransplantation remains to be determined. Methods. In this study, we investigated whether adenovirus-mediated gene transfer with CTLA4Ig cDNA by intravenous injection to the recipient is effective in suppression in hamster-to-rat cardiac xenotransplantation. Results. With CTLA4Ig gene transfer, the duration of gene expression was clearly prolonged, based on reduced production of antiadenovirus antibody and shrinkage of the spleen. The survival of cardiac xenografts was significantly prolonged with CTLA4Ig gene transfer compared to the control graft, and survival with combination use of FK506 and CTLA4Ig gene transfer in xenotransplantation was also significantly prolonged compared to that with CTLA4Ig gene transfer alone. Cessation of the cardiac graft in the combination treatment occurred in parallel with the elevation of antihamster IgM antibodies in rat sera. Conclusions. These results suggest that adenovirus-mediated CTLA4Ig gene transfer is effective for immunosuppression in hamster-to-rat xenotransplantation.

Sericin is the silk protein enveloping fibroin fibers in cocoons. Sericin hydrolysate protects cultured Sf9 insect cells from death caused by serum deprivation; the activity depends on the repeats of 38 amino acids. A partial peptide from the 38 residues, SGGSSTYGYS, inhibited serum-deprivation death as well. Cell viabilities in the presence of 10% (v/v) foetal calf serum, no additives and 1 mM: SGGSSTYGYS were 96, 12 and 31% on the third day after inoculation, respectively. Aromatic residues seemed to be important because SGGSSTWGWS had the same activity as SGGSSTYGYS but SGGSSTAGAS had no activity.

As a result of screening for terpenoids produced by Actinomycetes, naphterpins B and C, two new congeners of naphterpin were isolated from Streptomyces sp. CL190 and their structures were determined by NMR spectral analysis.

The present study was designed to elucidate the roles of dendritic voltage-gated K+ channels in Ca2+ influx mechanism of a rat Purkinje cell using a computer simulation program. First, we improved the channel descriptions and the maximum conductance in the Purkinje cell model to mimic both the kinetics of ion channels and the Ca2+ spikes, which had failed in previous studies. Our cell model is, therefore, much more authentic than those in previous studies. Second, synaptic inputs that mimic stimulation of parallel fibers and induce sub-threshold excitability were simultaneously applied to the spiny dendrites. As a result, transient Ca2+ responses were observed in the stimulation points and they decreased with the faster decay rate in the cell model including high-threshold Ca2+-dependent K+ channels than in those excluding these channels. Third, when a single synaptic input was applied into a spiny dendrite, Ca2+-dependent K+ channels suppressed Ca2+ increases at stimulation and recording points. Finally, Ca2+-dependent K+ channels were also found to suppress the time to peak Ca2+ values in the recording points. These results suggest that the opening of Ca2+-dependent K+ channels by Ca2+ influx through voltage-gated Ca2+ channels hyperpolarizes the membrane potentials and deactivates these Ca2+ channels in a negative feedback manner, resulting in local, weak Ca2+ responses in spiny dendrites of Purkinje cells.

In Bengalese finches, the maturation of distance calls and the stabilization of their song are obtained through developmental learning stages. In this study, whole cell patch clamp recordings were made from neurons in the robust nucleus of the archistriatum (RA), which is a motor control nucleus of male-specific vocalization in male birds of different developmental stages. The neurons in the RA were categorized into three types according to their electrophysiological properties under voltage clamp recordings. Distribution ratios of the neuron types changed during development. The firing pattern of these neurons was highly co-related with the expression of slow-inactivated (SI) type outward current. In this study, we first showed that the developmental change of the neural circuit in the RA was induced by the expression of the SI-type outward current.

The silk proteins, fibroin and sericin, are produced in the silk gland of Bombyx mori, and hydrophilic sericin envelops fibroin with successive sticky layers in the formation of a cocoon. To study the biological functions of sericin, we focused on the serine-rich sericin peptide consisting of 38 amino acids, which is a highly conserved and internally repetitive sequence of a sericin protein. The corresponding gene was chemically synthesized, and the PCR-amplified gene was ligated to oligomerize sericin peptide and fused at the amino terminus to a His-tagged and proteolytic cleavage sequence in an inducible expression vector. When the dimers of sericin peptides were overexpressed in Escherichia coli, the transformants showed a prominent increase in cell viability after freezing in medium. Further, the purified dimeric sericin peptide from E. coli was found to be effective in protecting lactate dehydrogenase from denaturation caused by freeze-thaw. Both of these protective effects against freezing stress in cells and proteins were also observed with sericin hydrolysate. These results indicate that this unique sericin peptide, like sericin, has a high cryoprotective activity and will be valuable as a new biomaterial for industrial use.

Various types of voltage-gated ion channels are distributed along the dendrites of neurons in the central nervous system. We have recently shown experimentally that the dendrites of cerebellar Purkinje neurons contain low-threshold voltage-gated Ca2+ channels and low-threshold voltage-gated K+ channels. Although we found that these channels are involved in regulating the onset of Ca2+-dependent action potentials in the dendrites, we were unable to identify which of the known types of low-threshold Ca2+ channels and K+ channels were responsible, since there was no reliable method of discriminating between them. Here, we have built a detailed compartmental model of a Purkinje neuron by incorporating two types of low-threshold Ca2+ channel (T-type and class-E, or R-type) and two types of low-threshold K+ channel (A-type and D-type), in addition to another eight voltage-gated channel types, using a compartmental model neuron simulator. The model reproduces the basic features of the depolarization-induced responses of Purkinje neurons, such as fast Na+ spikes in the soma, Ca2+ spikes in the dendrites, the slow onset of Ca2+ spikes, repetitive Ca2+ spikes in the presence of TTX, the marked shortening of Ca2+ spike onset in the presence of 4-aminopydridine, and the longer Ca2+ spike onset in the presence of Ni2+. Our model shows that the D-type K+ channel and the class-E Ca2+ channel regulate the onset of depolarization-induced Ca2+ spikes in Purkinje neurons. These channels might be involved in integrating synaptic inputs in Purkinje neurons.

Many different kinds of voltage-gated ion channels (Na+ channels, K+ channels, Ca2+ channels) exist at neuronal dendrites. Integration of dendritic electric signals (excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs) and action potentials) and/or non-electric signals (Ca2+ and second messengers) occurs in restricted dendritic compartments consisting of spines and adjacent fine dendrites. Voltage-gated ion channels at neuronal dendrites play crucial roles in the integration of dendritic signals. Dendritic signals, in turn, play important roles in the modulation of local dendritic physiological functions (e.g. input-specific synaptic plasticity, long-term potentiation (LTP) and long term depression (LTD)). A combined experimental and theoretical approach is a good way to clarify the biophysical behaviors of dendritic ion channels. Analyses of dendritic ion channels can open the door to a new wave of discoveries about EPSP integration at neuronal dendrites.

In situ splitting of cadaver livers has been reported to reduce cold ischemic damage, to avoid biliary complications, and to result in improved graft survival. In this study, which involved a wider application of split liver transplantation (SLT), we examined the effects of a technique combining both ex situ and in situ splittings in triple SLT in pigs and compared it to ex situ splitting alone. In the combination splitting group, the splitting between the right and left lobes was done in situ with perfusion of the left lobe with cold, lactated Ringer's solution; that between the lateral and medical right lobes was done ex situ in backtable surgery. The time required for in situ splitting was 28 ± 5 min. The time for backtable surgery and the total ischemia time were significantly shorter in the combination splitting group than that in the ex situ splitting group (P < 0.05). One day after triple SLT, the elevations in both serum AST and LDH in the ex situ splitting group were significantly greater than those in the combination splitting group (P < 0.05). We conclude that combination splitting may provide a technical improvement and have a beneficial effect on the clinical application of triple SLT.

We examined the roles of A- and D-type K channels in the integration of EPSPs, particularly EPSP reduction, in a model dendrite using a computer simulation program (NEURON), instead of conventional electrophysiological methods which present technical difficulties. We obtained two important results: (1) KD channels play a crucial role in every synaptic input event, and (2) KA channels affect reduction of the second EPSP only when the second synaptic input is applied with a short interval (≤100 ms). These results suggest that KD channels, which have not yet been analyzed experimentally, play a major role in synaptic integration in dendrites by altering their cable properties.

The donor source of vascular endothelial cells for hybrid blood vessels seeded with genetically engineered endothelial cells is generally considered to be autologous. The purpose of this study was to determine whether porcine endothelial cells transduced with double-expression retroviral vector using complement-resistant gene could be substituted for autologous endothelial cells. Decay-accelerating factor (DAF) and tissue plasminogen activator (tPA) cDNA were inserted into retroviral vector with homologous restriction factor 20 cDNA as a complement regulatory factor gene. Porcine aortic endothelial cells were transduced with these double-expression retroviral vectors, followed by the complement-dependent selection. Porcine endothelial cells transduced withdouble-expression retroviral vectors showed a high gene expression of both DAF and tPA. Complement-dependent cytotoxicity and adherence of U937 were significantly inhibited by the transduction of double-expression vectors with complement regulatory factor gene. Double-expression retroviral vector using complement regulatory factor gene was efficacious in substituting porcine endothelial cells for the autologous endothelial cells.

The distribution and function of voltage-gated Ca2+ channels in Purkinje neurons in rat cerebellar slices were studied using simultaneous Ca2+ imaging and whole-cell patch clamp recording techniques. Voltage-gated Ca2+ channels were activated by applying depolarizing voltage steps through the pipette attached at the soma in a voltage-clamp mode in the presence of tetrodotoxin. Poor space clamp due to extensive arborization of the dendrites allowed the dendrites to fire Ca2+ spikes. Ca2+ imaging with Fura-2 injected through the pipette, showed a steady [Ca2+]i increase at the soma and transient, spike-linked [Ca2+]i jumps in the dendrites. omega-Agatoxin-IVA (200 nM) abolished the depolarization-induced Ca2+ spikes, the spike-linked [Ca2+]i increase in the dendrites, and the steady [Ca2+]i increase at the soma. omega-Conotoxin-GVIA (5 microM) and nifedipine (3 microM) had no significant effect on the depolarization-induced responses. In the presence of 4-aminopyridine(2 mM) and omega-Agatoxin-IVA, transient [Ca2+]i increases remained in the dendrites. Low concentrations of Ni2+(100 microM) reversibly suppressed this [Ca2+]i increase. The voltage for half-maximal activation and inactivation of this component were lower than -50 mV and -31 mV, respectively. In normal conditions, low concentration of Ni2+ slowed the onset of the Ca2+ spike without changing the time course of the spikes or the amplitude of the accompanying [Ca2+]i increase. These results show that omega-Agatoxin-IVA-sensitive Ca2+ channels are distributed both in the soma and the dendrites, and are responsible for dendritic Ca2+ spikes, whereas low-voltage activated, Ni2+-sensitive Ca2+ channels are distributed in the whole dendrites including both thick and fine branches, and provide boosting current for spike generation.

Background and Objective: Peritoneal dissemination and hepatic metastasis commonly occur after patients with pancreatic cancer have undergone surgery. It is thought that specific adhesion molecules play corresponding roles in cancer metastasis. Study Design/Materials and Methods: We conducted in vitro and in vivo studies to assess the role of adhesion molecules in these processes, using SW1990 cells derived from human pancreatic cancer. Results SW1990 cells pronouncedly expressed sialyl Lewisa (s-Lea) and sialyl Lewisx antigens (s-Lex), CD44H, and β1 integrin. Also, SW1990 cells showed a strong binding activity to IL-1β activated human umbilical vein endothelial cells, cultured murine endothelial cells (F-2 cells), and human peritoneal mesothelial cells. Invasive ability of SW1990 cells to F-2 cells was also observed. The adhesion leading to implantation of cancer cells to endothelial cells were inhibited by treatment with the antibodies against s-Lea and against β1 integrin, respectively. Treatments with the antibodies against s-Lea and β1 integrin each inhibited the development of liver metastasis in nude mice with SW1990 cells. The adhesion of SW1990 cells to peritoneal mesothelial cells was markedly inhibited by antibodies each against CD44 or β1 integrin, but was completely blocked by using a combination of these two antibodies. These antibodies inhibited the dissemination of SW1990 cells in the peritoneal cavity of nude mice and prolonged their survival. Conclusion These findings suggest that s-Lea and integrin mediate the process from adhesion to implantation of SW1990 cells to endothelial cells, and CD44 and integrin play important roles in the initial attachment of SW1990 cells to mesothelial cells. It is thus speculated that compounds that interfere with the function of cell adhesion molecules may decrease the incidence of pancreatic cancer metastasis. J. Surg. Oncol. 1998;67:77–84. © 1998 Wiley-Liss, Inc.

Homologous restriction factor 20 (HRF20, CD59) is one of the complement regulatory factors. In this study, the complement-resistant retroviral vector, which possesses the HRF20 gene as a selection gene, was constructed and examined. The virus-producing cell, transduced with complement-resistant retroviral vector, was established after complement-dependent selection. NIH3T3 and PK15 cells transduced with this virus-producing cell were suc- cessfully selected by complement-dependent selection, which showed significant expression of the HRF20 antigen. In addition, these cells, transduced with tissue plasminogen activator (tPA) cDNA using complement-resistant retroviral vector, expressed tPA antigen after complement-dependent selection. These findings suggest that complement-resistant retroviral vector can be used for the double transduction of HRF20, as well as other genes.

The light and electron microscopic localizations of single stranded DNA (SSD) protein, a marker of apoptosis and programmed cell death, in the gerbil hippocampus were examined by immunocytochemistry after transient brain ischemia. SSD-immunoreactive (IR) cells appeared from post-operative day 1 (PO 1) to PO 7 after 5- or 10-min ischemia. Immunoreaction was recognized in the nucleus of the CA1 pyramidal neurons without remarkable morphological changes on PO 1. These findings suggest that SSD degradation can occur during delayed neuronal death in the CA1, preceding the appearance of double strand breaks, one of the characteristic features of apoptosis.

GH3 pituitary cells have high tendency to exhibit spontaneous Ca2+ action potentials and their frequency (Ca2+ APF) is increased by treatment with thyrotropin-releasing hormone (TRH). Although spontaneous Ca2+ firing was thought to be significant for the induction of oscillations in cytosolic Ca2+ concentration ([Ca2+]i), little attempt to elucidate the mechanism has been done so far. We demonstrate here that spontaneous Ca2+ APF in GH3 cells was increased 1.5-3 fold, comparable to that for TRH, by injection of guanosine 5'-0-3-thiotriphosphate (GTPgammaS), rab3A effector domain peptide, and phorbol-dibutyrate (PDBu), whereas guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), H-rab5 peptide, ras peptide, and 4 alpha-phorbol did not. The enhancement of Ca2+ firing by rab3A effector domain peptide was blocked by a protein kinase C (PKC) inhibitor, PKC(19-36). The present study suggests that the spontaneous Ca2+APF may be controlled by small G protein phosphorylated by PKC.

l -Tyrosyl- l -arginine (kyotorphin) is known as an endogenous analgesic neuropeptide. We examined whether kyotorphin and other arginine-containing neuropeptides were endogenous substrates for neuronal nitric oxide synthase (NOS) in the rat brain. Cytosol fractions of the rat cerebellum contained higher concentrations of neuronal NOS (nNOS) than endothelial NOS. In rat cerebellar cytosol, the binding activity of [ 3 H]N G -nitro- l -arginine (NNA) was inhibited equally by l -arginine ( l -Arg), kyotorphin, and l -leucyl- l -Arg (a kyotorphin receptor antagonist). Binding activities were inhibited to lesser degrees by fibronectin active fragments, bradykinin, and dynorphin A, but were not inhibited by l -tyrosyl- d -Arg or substance P. Interestingly, the inhibition of [ 3 H]NNA binding by kyotorphin was attenuated by inhibitors of kyotorphin-hydrolyzing peptidases (KTPases) such as bestatin and arphamenine B. These results suggest that kyotorphin is degraded to l -Arg by KTPases, which in turn may act as substrate for nNOS.

Portal venous flow (PVF) was serially monitored after pig liver transplantation (LTX) with the use of an implantable, miniature Doppler probe developed in our laboratory. Throughout the study period, the mean PVF in pigs that underwent LTX was significantly greater than that in pigs that were sham operated. For three animals with early graft failure secondary to primary nonfunction and for six that survived longer than 7 days, the mean PVF on postoperative day (POD) 1 was 18.7 +/- 3.8 cm/s and 41.7 +/- 11.2 cm/s, respectively (P0.05). For animals with acute cellular rejection (ACR), the mean PVF was 61.3 +/- 9.9 cm/s on POD 7 and 54.3 +/- 6.38 cm/s on POD 14. These values were significantly higher than those for animals without ACR (P0.05). Moreover, the increase in PVF correlated well with the degree of ACR. The actual PVF volume was measured by ex vivo perfusion, which showed a clear correlation with the PVF velocity obtained with the implanted, miniature Doppler probe. We feel that the liver graft requires increased PVF volume after transplantation to facilitate functional recovery from damage to hepatocytes due to preservation-reperfusion injury, and that ACR is also associated with an increased PVF. We conclude that monitoring the PVF in the early postoperative period after LTX is useful in the evaluation of graft function, particularly for predicting primary nonfunction and severity of ACR.

Antibodies to an alpha-galactosyl saccharide structure are mainly responsible for hyperacute rejection after pig-to-primate xenotransplantation. The beneficial effect of alpha-galactosyl oligosaccharides has been shown on the inhibition of anti-pig natural antibodies. We synthesized polyethylene glycol (PEG)-conjugates of alpha-galactosyl disaccharide (Di) and trisaccharide (Tri) as potential inhibitors of the rejection reaction. The half lives of Di, Tri, PEG-conjugated Di (Di-PEG) and PEG-conjugated Tri (Tri-PEG) were 18.1 +/- 2.3 min, 20.2 +/- 0.9 min, 38.7 +/- 2.8 min and 35.8 +/- 1.6 min, respectively. Furthermore, Di-PEG and Tri-PEG showed biphasic clearance, and their half lives at the second phase were longer than 10 hours. PEG-conjugated oligosaccharides (Di-PEG, Tri-PEG) markedly inhibited cytotoxic action of human sera to pig kidney cell line (PK15) compared to unconjugated oligosaccharides (Di, Tri). The binding of IgM antibodies to PK15 cells, however, was more strongly blocked by unconjugated oligosaccharides than PEG-conjugated oligosaccharides. This phenomenon can be explained by the finding that PEG has anti-complement activity and masks antigenic sites of oligosaccharides. In conclusion, conjugation of PEG to oligosaccharides provided two beneficial effects; prolonged intravascular retention time and anti-complement activity, upon systemic application of the oligosaccharides. The present findings opened a new approach to treatment of hyperacute rejection after xenotransplantation.

One cause of a false positive limulus test after surgery or hemodialysis has been identified as extrinsic (1--3)-beta-D-glucan which was derived from surgical gauze or cellulose dialyzer. However, there have been no investigations concerning intrinsic factors and the presence of (1--3)-beta-D-glucan in mammalian organs. In this study, (1--3)-beta-D-glucan in homogenate of various rat organs and stool was measured by a Gluspecy test (G test) using factor G, which specifically reacts with (1--3)-beta-D-glucan. In small intestine and lung, large amounts of factor G-activating substance were observed and identified as (1--3)-beta-D-glucan by a digestion study using end-(1--3)-beta-D-glucanase. However, only very small amounts of (1--3)-beta-D-glucan were found in the kidney, spleen, vena cava, aorta, thymus, heart and liver. In serum and plasma, no (1--3)-beta-D-glucan was observed. On the other hand, extremely large amounts of (1--3)-beta-D-glucan were found in stool. Minute amounts of (1--3)-beta-D-glucan were observed in a variety organs except for the small intestine and lung. High levels of (1--3)-beta-D-glucan found in the small intestine might be traced to contamination by stool in the small intestine, and such levels in the lung might derive from macrophages which have trapped (1--3)-beta-D-glucan in the air.

Information on genetic abnormalities in primary hyperparathyroidism has accumulated gradually. Genetic alterations responsible for tumorigenesis have been identified in multiple endocrine neoplasia types 1 and 2. Point mutations in a calcium-sensing receptor gene were recently found to be responsible for familial hypocalciuric hypercalcaemia and neonatal severe hyperparathyroidism. Evidence has been provided that abnormalities of cell cycle regulation participate at tumorigenesis in parathyroid adenoma and carcinoma. Clonal analysis has shown that in renal hyperparathyroidism the parathyroid glands initially grow diffusely and polyclonally, after which foci of nodular hyperplasia are transformed to monoclonal neoplasia. Somatic changes of specific genes have been suspected of being responsible for parathyroid tumorigenesis in renal hyperparathyroidism. However, the genetic loci responsible for the frequent monoclonality largely remain to be identified, and heterogeneous genetic abnormalities may contribute to the progression of secondary parathyroid hyperplasia.