Immunoelectron microscopic observations of hypothalamic TRH-containing neurons in rats

Summary Immunoreactive TRH-containing neurons and their synaptic associations were studied electron microscopically in the paraventricular nucleus (PVN) and dorsomedial nucleus (DMH) of the rat hypothalamus. In propylthiouracil (PTU)-treated rats, the immunoreactive cell bodies in the PVN appeared to be activated, showing a hypertrophic perikaryon, well developed Golgi bodies and numerous secretory granules. No such alterations were evident in the TRH neurons in the DMH. These findings suggest that the PVN-TRH neurons are involved in the hypothalamic-hypophysial-thyroid axis. Further, it was shown that unlabeled nerve terminals containing small and large clear vesicles make synaptic contacts with the TRH perikarya in the PVN. Thus it is likely that PVN-TRH neurons are regulated both by thyroid hormones and by other neuronal signals. In the DMH, unlabeled nerve terminals containing small and large clear vesicles, and immunoreactive terminals form synapses with TRH neurons. Thus the DMH-TRH neurons may be under dual neuronal control. It was further noted that in the DMH and PVN, TRH nerve terminals make synaptic contacts with other unlabeled neurons. It is evident that TRH acts as a neurotransmitter or neuromodulator, although the origin of TRH terminals should be elucidated.     

Directed differentiation of telencephalic precursors from embryonic stem cells

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.     

Bone-particle-impregnated bone cement: an in vivo weight-bearing study

To evaluate an experimental inorganic-bone-particle-impregnated bone cement, canine hip prostheses were implanted in dogs using a regular bone cement on one side and the experimental bone cement on the other. In a preliminary feasibility study, bone ingrowth into the resorbed bone-particle spaces was established 3 months after implantation in three dogs. In a more detailed study, twenty-eight (28) dogs were divided in four groups to delineate the effects of time on the phenomena of bony ingrowth. One month after implantation, active bone ingrowth into the bone cement was obvious. By 3 months postimplantation, the ingrowth appeared to have traversed the thickness of the bone-particle-impregnated cement. By the fifth month, most of the interconnected inorganic bone particles were replaced by new bone. At the end of a year, the ingrown bone was mature and negligible new bone activity was present. Biomechanical pushout tests closely corroborated the histologic observations. The maximum shear strength of the cement/bone interface of the experimental side reached 3.6 times that of the control side at 5 months postimplantation. No further improvements were seen at 12 months postimplantation. A viable bone/cement interface may result in a better orthopedic implant fixation system by combining the advantages of both cement for immediate rigidity and biological ingrowth for longterm stability.     

Involvement of cAMP response element-binding protein activation in salivary secretion.

OBJECTIVE: Saliva secretion is mediated by cAMP and the calcium signaling pathway in salivary acinar cells. The PKA signaling pathway plays an important role in protein secretion through the activation of cAMP, in fluid secretion through the elevation of intracellular calcium and in the activation of cAMP response element-binding protein (CREB), which is involved in these signaling cascades. In this study, we investigated whether the activation of CREB plays a part in the salivary secretion in mice. METHODS: We examined CREB activation by assessing phosphorylation at the serine-133 position using Western blotting. RESULTS: Carbachol (a muscarinic acetylcholine agonist) and isoproterenol (a beta-adrenergic agonist) markedly activated CREB in parotid acinar cells. Carbachol and isoproterenol-induced CREB phosphorylation was blocked by atropine (a muscarinic acetylcholine antagonist) and propranolol (a beta-adrenergic antagonist), respectively. The PKA inhibitor H89 inhibited CREB activation, but the PLC inhibitor U73122 did not. Moreover, carbachol- and isoproterenol-stimulated amylase secretion from parotid acinar cells was inhibited by H89 and adenoviral dominant-negative CREB. CONCLUSION: These results indicate that the muscarinic and beta-adrenergic activation of CREB was mediated through the PKA pathway and that CREB is involved in protein secretion from parotid acinar cells.     

Effects of exogenous interferon on L cells persistently infected with Sendai virus

Summary A carrier culture of L cells persistently infected with Sendai virus (steady state) designated as L-Sendai_ts cells was established with a temperature-sensitive strain of the virus. When interferon was added to culture fluids from the start of the cultures at permissive (35° C) or non-permissive temperature (38° C), cell-associated infectivity was unaffected at 35° C, while it was unexpectedly enhanced at 38° C, although the cell-associated infectivity was titrated after further incubation at 32° C for 2 days. The titer of cell-associated infectivity was increased by subculturing in the continuous presence of interferon at 38° C. The effect of interferon on the paradoxical enhancement of cell-associated infectivity was shown to be dose dependent. When L-Sendai_ts cells were successively subcultured 6 times at 38° C in the continuous presence or absence of interferon, more than 95 per cent of the cells contained a detectable amount of nucleocapsid (NP) antigen in the presence of interferon, whereas the antigen could be detected in only 30–40 per cent of the cells subcultured in the absence of interferon. Only when the cells subcultured at 38° C in the presence of interferon were transferred to permissive temperature, could the distinct hemadsorbing and cell-associated hemagglutinating activities and the release of virus particles, as measured by hemagglutinating activity in the culture fluids, be detected. Cells subcultured in the presence of interferon accumulated more virus polypeptides than in the absence of interferon. Accumulation of virus specific RNA in the cells subcultured in the presence of interferon was about twice as much as that in the absence of interferon. Larger sized RNA (probably 50S) was the major species and two smaller RNAs could be detected in both the treated and untreated cells.When L-Sendai_ts cells were cultured at 38° C in the presence of interferon, their multiplication was clearly inhibited. However, the cells which were subcultured twice at 38° C in the continouos presence of interferon acquired resistance to the anti-cell proliferative action of interferon. Interestingly, the conversion of the sensitive state to resistant state of the cells was reversible.With 3 Figures     

IL-8 as an important chemoattractant for neutrophils in ulcerative colitis and Crohn's disease.

IL-8 is generating increasing interest as a powerful neutrophil chemoattractant and activator. To elucidate the mechanisms of neutrophil infiltration in inflammatory bowel disease, we examined 33 patients with ulcerative colitis (UC), 18 with Crohn's disease (CD), eight with some other type of colitis, and 18 normal control subjects for measurement of IL-8 in homogenates of colonic biopsy specimens. The affected colonic mucosa was found to contain significantly more IL-8 in patients with active inflammatory bowel disease than in patients with inactive disease (UC, P < 0.001; CD, P < 0.001), in patients with other types of colitis (UC, P < 0.05; CD, P < 0.01), or in normal control subjects (UC, P < 0.001; CD, P < 0.001). Colonic IL-8 levels correlated significantly with the macroscopic grade of local inflammation, especially in patients with UC (P < 0.001). Colonic IL-8 levels also correlated well with the neutrophil numbers in mucosal tissue (UC, r = 0.950, P < 0.001; CD, r = 0.940, P < 0.001), and with colonic IL-1 beta (r = 0.911, P < 0.001) and tumour necrosis factor-alpha (TNF-alpha) levels (r = 0.604, P < 0.001) in patients with these two conditions. These data suggest a potential role for IL-8 and its regulatory cytokines IL-1 and TNF-alpha in mediating neutrophil infiltration of the gut wall in inflammatory bowel disease.     

A complex composed of USF1 and USF2 activates the human FcepsilonRI alpha chain expression via a CAGCTG element in the first intron

The high-affinity IgE receptor, FcepsilonRI, is a key regulatory molecule in the allergic reaction. During the course of studies to find cis-acting elements for FcepsilonRI alpha chain gene expression, a CAGCTG sequence located in the first intron was revealed to serve as a crucial enhancer element. Electromobility shift assays using antibodies and in vitro translation products showed that the CAGCTG element was recognized by the USF1/USF2 complex. As was the case for other intronic cis-elements, the CAGCTG element regulated the promoter in an orientation- and position-dependent manner. Overexpression of USF2 antisense repressed the FcepsilonRI alpha chain gene promoter and decreased the amount of alpha chain mRNA in mast cell lines. All these results indicated that the USF1/USF2 complex activates the human FcepsilonRI alpha chain gene expression via the CAGCTG element in the first intron.     

Suppression of interferon production in mouse spleen cells by cytochalasin D.

Cytochalasin D is thought to impair microfilament function. The present study has investigated its effects on four different systems in which interferon is formed, namely (1) mouse fibroblasts induced with virus (2) mouse spleen cells induced with virus, or (3) with endotoxin or (4) by allogeneic stimulation. Cytochalasin D did not suppress formation of interferon by fibroblasts (L cells) or spleen cells stimulated with either HVJ or NDV. However it did suppress production of interferon by spleen cells in response to endotoxin or an allogeneic stimulation; here its action was apparently not on the secretion of interferon, but on some earlier event. It also suppressed the production of interferon by mouse spleen cells induced with HVJ if this had been u.v. irradiated for more than 15 min: this suggests that cytochalasin D sensitive structures do play some role in interferon production by mouse spleen cells when stimulated with HVJ, as well as when they are stimulated with endotoxin or an allogeneic stimulus.     

Peliosis Hepatis in a Child with X-Linked Myotubular Myopathy Treated with Living-Donor Liver Transplant: A Case Report

BackgroundMyotubular myopathy is a rare disease sometimes accompanied by peliosis hepatis, a leading cause of fatal liver hemorrhage.Case ReportWe present a case of a 2-year-old boy with myotubular myopathy who developed liver hemorrhage because of peliosis hepatis and was successfully treated with living-donor liver transplant. The patient initially presented with fever, anemia, and liver dysfunction. A computed tomographic scan revealed hemorrhages in the liver, and the patient underwent hepatic artery embolization twice. After the second embolization, multiple peliosis hepatis cavities appeared in the left lobe of the liver that had increased in size. Therefore, the patient underwent ABO-incompatible living-donor liver transplant using a lateral segment graft from his father. The patient developed severe septic shock with an unknown focus on postoperative day 18, which resolved with antibiotic therapy. On postoperative day 62, he was discharged. Fourteen months after undergoing living-donor liver transplant, the patient showed no recurrence of peliosis hepatis.ConclusionsAlthough the long-term prognosis of peliosis hepatis due to myotubular myopathy after living-donor liver transplant remains unclear, liver transplant may be a curative treatment for patients with myotubular myopathy who have uncontrollable peliosis hepatis.     

The systemically administered competitive AMPA receptor antagonist, YM872, has analgesic effects on thermal or formalin-induced pain in rats

A new competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, (2,3-dioxo-7-[1H-imidazol-1-yl]-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl) acetic acid (YM872) has analgesic effects on acute thermal- and formalin-induced nociception by intrathecal administration. The purpose of this study was to determine the analgesic effects of systemically administered YM872 in both acute thermal- and irritant-induced pain. Sprague-Dawley rats were tested for tail withdrawal response by the tail flick test and for paw flinches by formalin injection after intraperitoneal administration of YM872. The tail flick latency increased dose-dependently with a 50% effective dose value of 156.3 microg. The number of flinches in both first and second phases of the formalin test decreased with increasing the dose of YM872. The 50% effective dose values were 1.0 microg in the first phase and 38.7 microg in the second phase. Transiently, intraperitoneal administration of 1 and 10 mg of YM872 induced motor disturbance and 10 mg induced loss of pinna reflex. We conclude that intraperitoneal administration of YM872 had analgesic effects on both acute thermal- and formalin-induced nociceptions in rats. Transient motor disturbance and loss of pinna reflex occurred only with large doses. IMPLICATIONS: Intraperitoneally administered YM872, a new alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, had analgesic effects on thermal- and formalin-induced pain in rats. Larger doses induced transient motor disturbance and loss of pinna reflex mediated in the brain.