The impact of stratospheric ozone recovery on the Southern Hemisphere westerly jet.

In the past several decades, the tropospheric westerly winds in the Southern Hemisphere have been observed to accelerate on the poleward side of the surface wind maximum. This has been attributed to the combined anthropogenic effects of increasing greenhouse gases and decreasing stratospheric ozone and is predicted to continue by the Intergovernmental Panel on Climate Change/Fourth Assessment Report (IPCC/AR4) models. In this paper, the predictions of the Chemistry-Climate Model Validation (CCMVal) models are examined: Unlike the AR4 models, the CCMVal models have a fully interactive stratospheric chemistry. Owing to the expected disappearance of the ozone hole in the first half of the 21st century, the CCMVal models predict that the tropospheric westerlies in Southern Hemisphere summer will be decelerated, on the poleward side, in contrast with the prediction of most IPCC/AR4 models.

Effect of bovine lactoferrin on the immune responses of captive bottlenosed dolphins (Tursiops truncatus) being transported over long distances.

Bovine lactoferrin was administered orally, in feed, to six bottlenosed dolphins (Tursiops truncatus) before they were transported for approximately six hours; their stress responses were compared with those of five untreated dolphins. During the journey the dolphins had an increased plasma concentration of cortisol, and lymphopenia, eosinopenia and mild neutrophilia, indicating a stress response. The administration of lactoferrin did not affect the function of the dolphins' polymorphonuclear leucocytes, but affected their leucogram by maintaining the number of circulating eosinophils.

Production of nitric oxide from endothelial cells by 31-amino-acid-length endothelin-1, a novel vasoconstrictive product by human chymase.

Human chymase selectively converts big endothelin (ET)-1 to 31-amino-acid-length ET-1 [ET-1(1-31)]. In this study we examined effect of ET-1(1-31) on endothelial function. ET-1(1-31) evoked contraction in a concentration-dependent manner at > 10(-8) M, which was about 10 times weaker than that of conventional ET-1 [ET-1(1-21)]. BQ485, an ETA receptor antagonist, completely abolished ET-1(1-31)-induced contraction, but BQ788, an ETB receptor antagonist, slightly enhanced it, suggesting that ET-1(1-31) relaxes artery via endothelium. On endothelial cells, ET-1(1-21) and ET-1(1-31) increased [Ca2+]i and produced NO, both of which were significantly inhibited by BQ788 and not by BQ485. These results indicate that ET-1(1-31) increased [Ca2+]i and produced NO in endothelial cells through ETB receptor similarly with ET-1(1-21), although slight difference in effect on smooth muscle cells.

A novel mutation of KCNQ3 (c.925T-->C) in a Japanese family with benign familial neonatal convulsions.

At present, only one mutation of KCNQ3, a KCNQ potassium channel gene, has been identified as a cause of benign familial neonatal convulsions type 2 (BFNC2). We found a T to C substitution (c.925T-C) on one allele of affected individuals in a Japanese family with BFNC but not on 200 alleles from healthy subjects. c.925T-->C replaced Trp309, a conserved residue within the P-loop of the KCNQ potassium channel family that holds the channel pore open, with an Arg (W309R). We report c.925T-->C as the second mutation of KCNQ3 responsible for BFNC2.

Effect of protein kinase C on glucose-mediated insulin secretion in HIT-T15 cells.

OBJECTIVE: To clarify the regulation of protein kinase C on glucose-mediated insulin secretion. MAIN OUTCOME MEASURES: We examined the effect of protein kinase C on the cytosolic free Ca(2+) concentration ([Ca(2+)]i) and the activity of Ca(2+)-activated K(+) channels (K(Ca)-channel) in the insulinoma cell line, HIT-T15. RESULTS: Glucose at a concentration of 10 mmol/L increased the secretion of insulin. This increase was partly inhibited by 1 nmol/L staurosporine, a protein kinase C inhibitor. Staurosporine (1 nmol/L) also attenuated the glucose-induced elevations in [Ca(2+)]i. On the contrary, glibenclamide (100 nmol/L) specifically blocked ATP-sensitive K(+) channels, and increased both [Ca(2+)]i and insulin secretion, but staurosporine had no effect on them. Patch clamp studies showed that 10 mmol/L glucose almost completely blocked K(Ca) channel activity, an effect that was reversed by 1 nmol/L staurosporine. Phorbol 12-myristate 13-acetate (1 mmol/L), a protein kinase C activator, also decreased K(Ca) channel activity. CONCLUSIONS: These results indicate that the activation of protein kinase C is involved in the glucose-induced release of insulin by modulating K(+) channel function in HIT-T15 cells.

Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus.

An amphotericin B (AmB)-resistant mutant was isolated from a wild-type AmB-susceptible strain of Aspergillus flavus by serial transfer of conidia on agar plates containing stepwise increased concentrations of AmB up to 100 microg ml-1. The acquired resistance of mycelia was specific for polyene-antibiotics AmB, nystatin and trichomycin. Spheroplasts derived from the resistant mycelia were as susceptible to AmB as the wild-type. Chemical analysis of the cell wall revealed that levels of alkali-soluble and -insoluble glucans were significantly higher in the resistant mycelia as compared to those in the wild-type. When resistant mycelia were treated with SDS, they adsorbed as much AmB as wild-type mycelia. These results suggest that alterations in the cell wall components of mycelia, especially 1,3-alpha-glucan and protein complex in the outermost wall layer, lead to AmB resistance in A. flavus.

A novel mutation of CHRNA4 responsible for autosomal dominant nocturnal frontal lobe epilepsy.

OBJECTIVE: To identify the mutation responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in a nonwhite family. BACKGROUND: ADNFLE is newly recognized as an entity of idiopathic partial epilepsy. Recently, two different mutations of the neuronal nicotinic acetylcholine receptor alpha4 subunit (CHRNA4) gene were identified in a white family as a cause of ADNFLE. METHODS: Four affected and three unaffected individuals in three generations of a Japanese family with ADNFLE, and 100 unrelated healthy Japanese volunteers were studied. Clinical features and EEG findings in affected individuals were consistent with those of ADNFLE reported in white families with ADNFLE. Mutations within the CHRNA4 gene were screened for using single-strand conformation polymorphism analysis (SSCA) and were determined by direct sequencing. The mutation identified was sought in volunteers by the amplification refractory mutation system. RESULTS: A C-to-T exchange (C755T) was found in exon 5 of the CHRNA4 gene on one allele of affected individuals. C755T segregated in affected individuals and was not found in 200 alleles obtained from the volunteers. C755T replaced serine 252 (Ser252) in the second membrane-spanning domain (M2) of CHRNA4 with a leucine. Ser252 is conserved characteristically in the alpha-subunit of acetylcholine receptor and is considered to play an important role in channel function. CONCLUSION: C755T is a novel missense mutation of the CHRNA4 gene causing autosomal dominant nocturnal frontal lobe epilepsy in this Japanese family.

Hepatoprotective action of adenovirus-transferred HNF-3gamma gene in acute liver injury caused by CCl(4).

Hepatocyte nuclear factor-3gamma (HNF-3gamma) is an important regulator of liver-specific genes and the expression of this factor is reduced in the liver injured by carbon tetrachloride (CCl(4)). Wistar rats were infected with a recombinant adenovirus carrying the cDNA for HNF-3gamma (AxCAHNF3gamma) via the tail vein and were treated with CCl(4) by intraperitoneal injection. Liver damage, such as swelling of the hepatocytes and increases in serum marker enzymes were markedly alleviated by AxCAHNF3gamma infection. Interestingly, hepatocyte growth factor (HGF) was strongly induced in the AxCAHNF3gamma-infected liver. Likewise, HNF-1alpha and HNF-1beta levels were increased, but HNF-3alpha and HNF-3beta levels were depressed in the liver. Our results suggest that the transduced HNF-3gamma gene leads to a hepatoprotective effect via the induction of HGF by the combined actions of liver-enriched transcription factors.

Lactate stimulates insulin secretion without blocking the K+ channels in HIT-T15 insulinoma cells.

To clarify the mechanism by which lactate affects insulin secretion, we investigated the effect of lactate on insulin secretion, cytosolic free Ca2+ ([Ca2+](i), the ATP sensitive K+ channel (K(ATP)) and the Ca2+-activated K+ channel (K(Ca)) in HIT-T15 cells, and the results were compared with those of glucose and glibenclamide. All three agents caused insulin secretion and increased [Ca2+](i), but the effects on the K+ channels were different. In cell-attached patch configurations, 10 mmol/l glucose blocked both the K(ATP) and KCa channels, while 100 nmol/l glibenclamide had no effect on KCa channels, but blocked K(ATP) channels. Lactate at a concentration of 10 mmol/l activated both the K(ATP) and KCa channels, not only in cell-attached, but also in inside-out patch configurations, indicating that the increase in [Ca2+](i) and secretion of insulin by lactate cannot be explained by the blocking of the K+ channels. Lactate, at concentrations of 10 mmol/l and 50 mmol/l decreased 45Ca2+ efflux, while glibenclamide increased the efflux. These results suggest that the lactate-induced Ca2+ increase is not due to the closing of K+ channels, but at least in part, to the suppression of Ca2+ efflux from HIT cells.

Centrilobular and perisinusoidal fibrosis in experimental congestive liver in the rat.

BACKGROUND/AIMS: The pathogenesis of congestive hepatic fibrosis is known to be a reaction of hepatic stromal cells following prolonged congestive heart failure or hepatic outflow obstruction. However, little is known about the fibrotic process itself. This study documents the hepatic morphology and ultrastructure of the fibrotic processes in an experimental model of congestive hepatic fibrosis in rats. METHODS: In this model we ligated the abdominal portion of the inferior vena cava in the space between the diaphragm and liver, and observed liver morphology 24 h, 1 week and 6 weeks after the operation. The cytoskeletal components of the hepatic stellate cells and myofibroblasts were identified by immunohistochemistry for glial fibrillary acidic protein (GFAP) and a-smooth muscle actin (a-SM actin). Extracellular matrices of reticulin fibers and fibronectin were localized using silver impregnation and immunohistochemical staining. RESULTS: Soon after ligation of the vena cava, foci of cells at variable stages of necrosis appeared in the centrilobular areas, the topographical localization of which was highly variable within the liver. The fibrotic processes were subclassified into three stages. In the first stage (24 h after ligation), abundant neutrophils, macrophages and GFAP-positive stellate cells appeared, but a-SM actin-positive cells were not detected in the necrotic areas. In the second stage (1 week after ligation), the GFAP-positive cells disappeared, but a-SM actin-positive myofibroblasts appeared. In the third stage (6 weeks after ligation), a large number of a-SM actin-positive myofibroblasts were observed, and there was heavy deposition of connective tissue proteins, such as reticulin fibers and fibronectin, in centrilobular areas. Two interesting observations were that: (1) the distribution of centrilobular necrosis was highly variable within the liver, and (2) the fibrosis was confined to focal centrilobular areas involving the perivenular sinusoidal area without periportal fibrosis. CONCLUSIONS: These findings suggest that GFAP-positive stellate cells are transformed into a-SM actin-positive myofibroblasts, and these myofibroblasts produce extracellular matrix proteins in centrilobular sinusoidal areas under congestive conditions.

Adenovirus-mediated gene expression in the septal cells of cirrhotic rat livers.

BACKGROUND/AIMS: Liver cirrhosis is characterized by the formation of fibrous septa following hepatic necrosis and fibrosis, and finally progression to severe hepatic failure and/or hepatocellular carcinoma. To establish effective therapy for cirrhosis using a designed gene, we examined whether recombinant adenovirus vectors could transfer foreign genes into the septal cells of cirrhotic livers. METHODS: Rats with cirrhosis induced by 4-8 weeks treatment with carbon tetrachloride were intravenously infected with a recombinant adenovirus Adex1CALacZ bearing a bacterial lacZ gene. Expression of the transferred gene was determined by X-gal staining. The infectivity of the vectors in vitro was examined using slice cultures from the cirrhotic rat livers. RESULTS: In normal rat livers, almost all hepatocytes expressed beta-galactosidase from the recombinant adenovirus vectors. In rat liver fibrosis, the adenovirus-mediated gene transfer to hepatocytes is markedly reduced compared with normal rat liver. In cirrhosis, there is an even stronger reduction in the number of transduced hepatocytes. On the other hand, in vitro infection to the slice culture demonstrated that the cirrhotic hepatocytes still maintained adenovirus infectivity. Moreover, in the kidney, which is the second target organ of adenovirus, there was no difference in infectivity between normal and cirrhotic rats. CONCLUSION: The recombinant adenovirus intravenously transmitted the foreign gene to septal cells in extranodular fibrous septa, rather than to hepatocytes within small nodules. Therefore, septal cells in the cirrhotic liver should be targeted with the adenovirus vector for a successful in vivo therapeutic strategy.

A comparative histochemical and immunohistochemical study of aminergic, cholinergic and peptidergic innervation in rat, hamster, guinea pig, dog and human livers.

AIMS/BACKGROUND: The mammalian liver receives both sympathetic and parasympathetic nerves that contain aminergic, cholinergic and peptidergic components. The intrahepatic distribution of nerve fibers are highly species-dependent; and also, even within one species, there are notable variations. To reveal the pattern and type of hepatic innervation in different species, we examined the distribution and density of these nerve fibers. METHODS: The livers of rats, golden hamsters, guinea pigs, dogs and humans were used. Aminergic and peptidergic nerve fibers were identified by immunohistochemistry for tyrosine hydroxylase (TH), neuropeptide Y (NPY), substance P (SP), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), and galanin (GAL), and cholinergic fibers were identified by the acetylcholinesterase (AChE) neurohistochemistry method. RESULTS: AChE-, TH-, NPY-, CGRP-, VIP-, and SP-positive nerves were observed in the connective tissue of the portal region, and they were in close contact with hepatic arteries, portal veins and bile ducts in all five species. Within the parenchyma of guinea pig, dog and human livers, TH-, NPY- and SP-positive fibers were observed, but no AChE- and CGRP-positive fibers were observed. In rat and hamster livers, no parenchymal nerve fibers could be demonstrated, but CGRP-, NPY- and SP-positive fibers were observed in the border of periportal areas. The density of CGRP-positive nerve fibers were slightly higher around bile ducts than around hepatic arteries and portal veins. GAL-positive fibers were not detected in any animal. CONCLUSIONS: These data indicate that there were differences in the patterns of hepatic innervation among rats, golden hamsters, guinea pigs, dogs and humans. The data also show that: 1) in rat and hamster livers, hepatic functions may be regulated by both sympathetic and parasympathetic nerves in the portal region; 2) in guinea pig, dog and human livers they may be regulated by these fibers both in the interlobular region (parasympathetic and sympathetic systems) and in the intraparenchymal region (sympathetic system); and thus, 3) in the latter three species, hepatocytes and sinusoidal cells may be innervated by sympathetic nerves.

Mast cell, myofibroblast and nerve terminal complexes in carbon tetrachloride-induced cirrhotic rat livers.

BACKGROUND/AIMS: Extracellular matrices in liver fibrosis are known to be produced by myofibroblasts that are transformed from fat-storing cells. The development of the fibrotic process is thought to be mediated by various fibrogenic mediators. Recently, the involvement of mast cells and cholinergic neurotransmitters in fibrogenesis has been suggested. We have studied the distribution of these cells and cholinergic nerve fibers in normal rat livers and 6-week carbon tetrachloride-induced rat cirrhotic livers. METHODS: Mast cells and myofibroblasts were identified by immunohistochemistry for mast cell tryptase (AA1) and alpha-smooth muscle actin. Cholinergic nerve fibers and terminals were localized using the acetylcholinesterase neurohistochemistry method for light and transmission electron microscopy. RESULTS: In normal rat livers, a few nerve terminals were connected with fibroblasts near the vascular walls in the portal tracts. In contrast, in cirrhotic rat livers, numerous acetylcholinesterase-positive nerve fibers were observed in the fibrous septa, forming a network. Ultrastructurally, the nerve terminals were observed in close contact with mast cells and myofibroblasts in fibrous septa, forming characteristic mast cell/myofibroblast/nerve terminal complexes. In cirrhotic nodules, nerve terminals were situated in close contact with myofibroblasts in the periseptal sinusoids. These axon terminals contained numerous small clear vesicles, and acetylcholinesterase-positive products were noted in the space of the synaptic membranes. CONCLUSIONS: Our findings demonstrate that mast cell/ myofibroblast/cholinergic nerve terminal complexes may play a role in the development of liver fibrosis, probably because of the production of extracellular matrix components by myofibroblasts.

Adenovirus-transferred HNF-3 gamma conserves some liver functions in primary cultured hepatocytes of adult rats.

Hepatocyte nuclear factor-3 (HNF-3) isoforms are key factors for regulation of gene expression and differentiation in hepatocytes. HNF-3gamma is abundantly expressed in the mature liver, but down-regulated in primary cultured hepatocytes, in which some other hepatic gene expressions are also decreased. In this study, the primary hepatocytes were infected with the recombinant adenovirus carrying HNF-3gamma gene (AxCAHNF3gamma), and this led to marked induction of the HNF-3gamma gene. As a result, the expressions of albumin, catalase, and ornithine transcarbamylase (OTC) genes were also recovered to significant levels in the AxCAHNF3gamma-infected hepatocytes. Moreover, hepatocyte proliferation stimulated by epidermal growth factor (EGF) and insulin was also inhibited by AxCAHNF3gamma infection. Our results demonstrate that the enforced expression of HNF-3gamma gene can lead to conservation of some original liver functions in the primary cultured hepatocytes accompanied by morphological differentiation and growth inhibition.

Effects of cholestatic agents on the structure and function of bile canaliculi in neonatal rat hepatocytes in primary culture.

The effects of cytochalasin B and colchicine on the structure and function of bile canaliculi were studied in neonatal rat hepatocytes in primary culture. Cellular contacts of neonatal hepatocytes were not as tight as those of adult hepatocytes. There was no remarkable difference in the ultrastructure of bile canaliculi between neonatal and adult hepatocytes. Neonatal hepatocytes treated with cytochalasin B were round in shape and aggregated in groups of several cells. Actin filaments stained by rhodamine-phalloidin were disrupted and condensed at the cell periphery or around dilated bile canaliculi. Markedly-dilated bile canaliculi with less microvilli were observed by transmission electron microscopy while the secretory function of horseradish peroxidase, which was used as a marker for uptake, transport and secretion into bile canaliculi, were maintained. The lumen of dilated bile canaliculi was found close to the undersurfaces of hepatocytes by scanning electron microscopy after turning over the cultured cells. By colchicine treatment, the filamentous structure of microtubules in neonatal hepatocytes disappeared. The ultrastructure of the bile canaliculi was not affected by the treatment, but transport and secretion of horseradish peroxidase into bile canaliculi were inhibited. The development of strict cellular polarity in neonatal hepatocytes may be suppressed in neonatal hepatocytes; however, cholestatic agents which rearrange the cytoskeleton caused the same morphological or functional changes of bile canaliculi as in adult hepatocytes.

[Addition of fentanyl to epidural lidocaine raises the toe temperature].

In order to investigate effects of addition of fentanyl epidurally on the onset of sympathectomy from epidural lidocaine, we have measured the toe temperature of 29 healthy patients undergoing elective lower extremity or lower abdominal surgeries. The latency of onset of the toe temperature was significantly shorter in patients receiving both epidural lidocaine and fentanyl compared with those receiving epidural lidocaine alone (258 +/- 135 vs 398 +/- 184 sec, P < 0.05 [mean +/- SD]). Osmolarity and pH of the epidural solutions were similar between the two groups. These results suggest, but do not indicate, that sympathectomy from epidural lidocaine is accelerated by the addition of fentanyl.