Cooperative Interception of Neuronal Apoptosis by BCL-2 and BAG-1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species

Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N -acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert -butyl-α-phenylnitrone, and the antioxidant, N -acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.     

Does endothelin-1 participate in the exercise-induced changes of blood flow distribution of muscles in humans?

Maeda, Seiji, Takashi Miyauchi, Michiko Sakane, MakotoSaito, Shinichi Maki, Katsutoshi Goto, and Mitsuo Matsuda. Does endothelin-1 participate in the exercise-induced changes of blood flowdistribution of muscles in humans? J. Appl.Physiol. 82(4): 1107-1111, 1997.Endothelin-1(ET-1) is an endothelium-derived potent vasoconstrictor peptide thatpotentiates contractions to norepinephrine in human vessels. Wepreviously reported that the circulating plasma concentration of ET-1is significantly increased after exercise (S. Maeda, T. Miyauchi, K. Goto, and M. Matsuda. J. Appl.Physiol. 77: 1399-1402, 1994). Tostudy the roles of ET-1 during and after exercise, we investigatedwhether endurance exercise affects the production of ET-1 in thecirculation of working muscles and nonworking muscles. Male athletesperformed one-leg cycle ergometer exercise of 30-min duration atintensity of 110% of their individual ventilatory threshold. Plasmaconcentrations of ET-1 in both sides of femoral veins (veins in theworking leg and nonworking leg) and in the femoral artery (artery inthe nonworking leg) were measured before and afterexercise. The plasma ET-1 concentration in the femoralvein in the nonworking leg was significantly increased after exercise,whereas that in femoral vein in the working leg was not changed. Thearteriovenous difference in ET-1 concentration was significantlyincreased after exercise in the circulation of the nonworking leg butnot of the working leg, which suggests that the production of ET-1 wasincreased in the circulation of the nonworking leg by exercise. Thepresent study also demonstrated that the plasma norepinephrineconcentrations were elevated by exercise in the femoral veins of boththe working and nonworking legs, suggesting that the sympathetic nerveactivity was augmented in both legs during exercise. Therefore, thepresent study demonstrates the possibility that the increase inproduction of ET-1 in nonworking muscles may cause vasoconstriction andhence decrease blood flow in nonworking muscles through its directvasoconstrictive action or through an indirect effect of ET-1 toenhance vasoconstrictions to norepinephrine and that these responsesmay be helpful in increasing blood flow in workingmuscles. We propose that endogenous ET-1 contributes tothe exercise-induced redistribution of blood flow in muscles.

     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Isolation of Cytopathic Small Round Virus (Aichi Virus) from Pakistani Children and Japanese Travelers from Southeast Asia

Aichi virus was isolated in Vero cells from 5 (2.3%) of 222 Pakistani children with gastroenteritis but none was found in 91 healthy children. Aichi virus was also isolated from 5 (0.7%) of 722 Japanese travelers returned from tours to Southeast Asian countries and complained of gastrointestinal symptoms at the quarantine station of Nagoya International Airport in Japan. Of 5 Japanese travelers, 3 were returning from Indonesia, and 2 from Thailand or Malaysia. These results indicate that Aichi virus or a similar agent is endemic in Southeast Asian countries and is a cause of gastrointestinal symptoms in children in these areas or in Japanese travelers who visit there.     

Changes in cytokeratin expression in epidermal keratinocytes during wound healing

In order to investigate the re-epithelialization process during wound healing, the hair on the back of guinea pigs was shaved and then excisional wounds were made through the entire thickness of the skin. Histological changes were observed and changes in the expression of different cytokeratin polypeptides were examined using an immunohistochemical technique. Immunohisto chemical study revealed that the proliferating and migrating keratinocytes expressed the same cytokeratins as the basal cells of normal epidermis. In addition, the entire epidermis of fairly remote areas from the edges of the wound where no thickening was observed showed a temporarily abnormal staining pattern. The suprabasal cells in the regenerating epidermis temporarily expressed cytokeratins not only specific for suprabasal cells but also specific for basal cells. The cytokeratins expressed in normal basal keratinocytes were also present in the thickened granular layers. These data indicate that the expression of cytokeratins in the epidermal keratinocytes (even in fairly remote areas from the wound edges) changes during wound healing, that the origin of the migrating keratinocytes from the remaining epidermis seems to be the basal cells in the epidermis, and that the appearance of keratohyalin granules is not related to changes in cytokeratin expression.     

Effect of auxin on changes in the oriented structure of wall polysaccharides in response to mechanical extension in oat coleoptile cell walls

The increase in the dichroism of the ester-C=O and COO^–bands in response to mechanical extension in oat coleoptilecell walls was much enhanced by IAA pretreatment while the decreasein that of the C-O-C band was not enhanced or even suppressed.The results indicate the importance of the ultrastructure ofnon-cellulosic polysaccharides in controlling auxin-inducedcell elongation. ¹ Present adress: Department of Agricultural Chemistry, KyotoPrefectural University, Shimogamo, Kyoto 606, Japan. (Received May 16, 1978; )     

Microbial Production of Ursodeoxycholic Acid from Lithocholic Acid by Fusarium equiseti M41

A fungus identified as Fusarium equiseti was isolated from soil and found to carry out 7β-hydroxylation of lithocholic acid to ursodeoxycholic acid (35% yield; 350 mg/liter) in 112 h.     

Localization of laminin in nephritic glomeruli as revealed by a quick-freezing and deep-etching method with immunohistochemistry

Summary The three-dimensional localization of laminin in rat glomeruli at the chronic phase of Masugi nephritis was investigated by a quick-freezing and deep-etching method combined with immunohistochemistry. Light-microscopically, laminin was localized in increased mesangial matrix and thickened glomerular basement membrane. The quick-freezing and deep-etching method revealed that the increased mesangial matrix, which was newly formed in axial portions and areas of mesangial interposition, was composed of fine fibrillar networks. They were revealed with the 3,3-diaminobenzidine tetrahydrochloride (DAB) reaction products of peroxidase-labelled secondary antibody following anti-laminin antibody. However, these reaction products were not uniformly distributed in the newly formed matrix. Although the fibrils organizing lamina densa were also immunostained with anti-laminin antibody, the fibrils connected to mesangial cells, podocytes and endothelial cells had smaller amounts of DAB reaction products for laminin. These results indicate that one of the components of fibrils in the mesangial matrix and lamina densa is laminin, which is heterogeneously distributed in the newly formed matrix.     

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.     

Uridine Metabolism in the Goldfish Retina During Optic Nerve Regeneration: Whole Retina Studies

Accumulation of radioactivity from [3H]uridine in incubations of whole goldfish retinas is increased in the ipsilateral retina during a period of regeneration that follows unilateral optic nerve crush. Brief incubations to investigate the nature of enhanced labeling of the acid-soluble fraction showed a peak uptake 4 days following crush, with a gradual decrease to control levels by 21 days following crush. That nucleoside uptake may not mediate the effect is supported by the observation that the rate of uptake of 5'-deoxyadenosine, a nonmetabolizable nucleoside analog, is the same in post-crush (PC) and normal (N) retinal incubations. Following brief incubations of PC and N retinas with [3H]uridine, there is enhanced labeling in PC retinas relative to N retinas of recovered UMP, UDP, UTP, and uridine nucleotide sugars, whereas recovery of labeled uridine itself is slightly decreased. The results suggest that the increased accumulation of radioactivity in PC retinas following incubation with uridine reflects an increase in the activities of retinal uridine kinase and uridine nucleotide kinases.