Preparation of artificial 2-, 3-, 4- and 8-domain myoglobins and comparison of their autoxidation rates

Although most hemoglobins and myoglobins consist of 15-kDa single-domain subunits, structurally unusual hemoglobins, such as Artemia 9-domain and Barbatia 2-domain hemoglobins, occur naturally in several invertebrates. These hemoglobins appear to be the result of gene duplication and fusion. Using cDNA coding for the open reading frame of Aplysia kurodai myoglobin, artificial cDNA inserts corresponding to contiguous dimer, trimer, tetramer and octamer myoglobins (2-, 3-, 4- and 8-domain myoglobins) were prepared and cloned into pMAL or pQE plasmids. These artificial myoglobins and wild-type single-domain myoglobins were successfully expressed in Escherichia coli in the heme-attached, oxygenated form. Myoglobin was purified partially by ammonium sulfate fractionation and gel filtration, and autoxidation rates were examined. The autoxidation rates of recombinant wild-type myoglobins with MBP or hexameric His tag were comparable to those of native myoglobin, suggesting that the recombinant proteins appear to be properly folded and that the N-terminal MBP or His tag does not have an affect on the rate. On the other hand, the rates were significantly decreased in the 2- and 3-domain myoglobins (50% and 30% of the single-domain myoglobins, respectively). The rates for 4- and 8-domain myoglobins were similar to those for 3-domain myoglobin. These results indicate that the artificial poly-domain structure of myoglobin is more stable than the usual single-domain myoglobin from the viewpoint of storage of bound dioxygen.     

Reduction in photostability by the esterification of beta-cryptoxanthin

Liposomes, in which beta-carotene, beta-cryptoxanthin, zeaxanthin, beta-cryptoxanthin palmitate or beta-cryptoxanthin acetate had been embedded, were irradiated by UVA, and the rate of degradation of each carotenoid was measured. There was no significant difference in the degradation rate between beta-carotene, beta-cryptoxanthin and zeaxanthin. The degradation rates of beta-cryptoxanthin palmitate and beta-cryptoxanthin acetate were faster than that of beta-cryptoxanthin, and the degradation rate of beta-cryptoxanthin palmitate was faster than that of beta-cryptoxanthin acetate.     

Effects of nitric oxide synthase inhibitor on decrease in peripheral arterial stiffness with acute low-intensity aerobic exercise

We previously reported that even low-intensity, short-duration acute aerobic exercise decreases arterial stiffness. We aimed to test the hypothesis that the exercise-induced decrease in arterial stiffness is caused by the increased production of NO in vascular endothelium with exercise. Nine healthy men (age: approximately 22-28 yr) performed a 5-min single-leg cycling exercise (30 W) in the supine position under an intravenous infusion of NG-monomethyl-L-arginine (L-NMMA; 3 mg/kg during the initial 5 min and subsequent continuous infusion of 50 mug.kg(-1).min(-1) in saline) or vehicle (saline) in random order on separate days. The pulse wave velocity (PWV) from the femoral to posterior tibial artery was measured on both legs before and after the infusion at rest and 2 min after exercise. Under the control condition, exercised leg PWV significantly decreased after exercise (P <0.05), whereas nonexercised leg PWV did not show a significant change throughout the experiment. Under L-NMMA administration, exercised leg PWV was increased significantly by the infusion (P <0.05) but decreased significantly after the exercise (P <0.05). Nonexercised leg PWV increased with L-NMMA administration and maintained a significantly higher level during the administration compared with baseline (before the infusion, all P <0.05). The NO synthase blockade x time interaction on exercised leg PWV was not significant (P=0.706). These results suggest that increased production of NO is not a major factor in the decrease of regional arterial stiffness with low-intensity, short-duration aerobic exercise.     

Molecular epidemiology of human T-lymphotropic virus type II infection in Amerindian and urban populations of the Amazon region of Brazil

Molecular characterization of human T-cell lymphotropic virus II (HTLV-II) isolates in North America and Europe has shown the existence of two principal subtypes of the virus, HTLV-IIa and HTLV-IIb. Subsequent studies on HTLV-II isolates from Brazil have suggested the existence of a unique variant phylogenetically related to HTLV-IIa but phenotypically similar to HTLV-IIb with respect to the transactivatory protein, Tax. This variant has been designated HTLV-IIc. To better clarify the variability and distribution of HTLV-II in Brazil, the viruses present in two population groups from the Amazon region were tested for the presence of HTLV-II using serological and molecular assays. The groups consisted of blood donors from three Amerindian communities and of HIV-1/HTLV-II coinfected patients residing in Belém, an urban area. Nucleotide sequences and phylogenetic analysis confirmed the presence of HTLV-IIc subtype among Amerindian populations and, for the first time, the presence of the same virus among urban groups in Belém. The isolated occurrence of the HTLV-IIc subtype among Amerindian populations in the Amazon region could be attributed to (1) the different migratory pathways and founder effect, or (2) the local origin of a proto-HTLV-II carried by Amerindian ancestors who migrated to the Amazon circa 11,000 to 13,000 years ago. These results suggest that not only is HTLV-IIc unique to this region, but that its presence in urban areas of Brazil has resulted from admixture processes during the colonization of the country.     

Supersensitivity of sigma receptors after repeated administration of cocaine.

We investigated the role of sigma receptors in the expression of behavioral sensitization induced by cocaine. Rats received intraperitoneal injections of either 20 mg/kg cocaine or saline once daily for 14 consecutive days. Cocaine-treated rats became sensitized. After a 5-day abstinence period, a challenge dose of (+)-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine ((+)-3-PPP), a sigma receptor agonist, was administered. (+)-3-PPP at doses of 12 and 24 mg/kg induced significantly more frequent rearing and more potent stereotypy consisting of repetitive head movement and sniffing in cocaine-sensitized rats than in saline-pretreated rats. These enhanced responses to (+)-3-PPP lasted for at least a month. The enhanced responses to (+)-3-PPP were attenuated by 30 mg/kg BMY 14802, a putative sigma antagonist, and also attenuated by 100 mg/kg (+/-)-sulpiride, a D2 dopamine antagonist. These findings show that repeated administration of cocaine produces lasting supersensitivity of simga receptors, which may induce subsequent activation of dopaminergic transmission.     

Angiotensin II type 1 receptor interaction is an important regulator for the development of pancreatic fibrosis in mice

The renin-angiotensin system (RAS) plays important roles in various pathophysiological processes. However, the role of the RAS in pancreatic fibrosis has not been established. We investigated the role of angiotensin II (ANG II)-ANG II type 1 (AT(1)) receptor pathway in the development of pancreatic fibrosis with AT(1a) receptor-deficient [AT(1a)(-/-)] mice. To induce pancreatic fibrosis, AT(1a)(-/-) and wild-type (WT) mice were submitted to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 microg/kg body wt cerulein at hourly intervals, per week, for four consecutive weeks. Pancreatic fibrosis was assessed by histology and hydroxyproline content. Pancreatic stellate cell (PSC) activation and the localization of AT(1) receptors were assessed by Western blot analysis for alpha-smooth muscle actin and immunostaining. Transforming growth factor-beta(1) (TGF-beta(1)) mRNA expression in the pancreas was assessed by RT-PCR. Six intraperitoneal injections of cerulein induced acute pancreatitis in both AT(1a)(-/-) and WT mice. There were no significant differences between two groups with regard to serum amylase and histological changes. Pancreatic fibrosis induced by repeated episodes of acute pancreatitis was significantly attenuated in AT(1a)(-/-) mice compared with that in WT mice. This finding was accompanied by a reduction of activated PSCs. Dual-immunofluorescence staining in WT mice revealed that activated PSCs express AT(1) receptors. The level of TGF-beta(1) mRNA was lower in AT(1a)(-/-) mice than in WT mice. Our results demonstrate that the ANG II-AT(1) receptor pathway is not essential for the local pancreatic injury in acute pancreatitis but plays an important role in the development of pancreatic fibrosis through PSC activation and proliferation.     

Recognition of N-glycolylneuraminic acid linked to galactose by the alpha2,3 linkage is associated with intestinal replication of influenza A virus in ducks

The hemagglutinin (HA) of H3 human influenza viruses does not support viral replication in duck intestine despite its avian origin. A Leu-to-Gln mutation at position 226 and a Ser-to-Gly mutation at position 228 in the HA of human A/Udorn/307/72 (H3N2) permit a reassortant virus [human Udorn HA, with all other genes from A/mallard/New York/6750/78 (H2N2)] to replicate in ducks. To understand the molecular basis of this change in host range restriction, we investigated the receptor specificity of duck influenza viruses as well as of human-duck virus reassortants. The results indicate that the recognition of a glycoconjugate moiety possessing N-glycolneuramic acid (NeuGc) linked to galactose by the alpha2,3 linkage (NeuGcalpha2,3Gal) is associated with viral replication in duck intestine. Immunofluorescence assays with NeuGcalpha2,3Gal-specific antiserum detected this moiety primarily on the crypt epithelial cells of duck colon. Such recognition, together with biochemical evidence of NeuGc in crypt cells, correlated exactly with the ability of the virus to replicate in duck colon. These results suggest that recognition of the NeuGcalpha2,3-Gal moiety plays an important role in the enterotropism of avian influenza viruses.     

Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH(2)-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (alpha PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of alpha PIX (DeltaCH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated alphaPIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted alphaPIX (Delta SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase-negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, overexpression of alphaPIX (Delta CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas alphaPIX (Delta SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the alphaPIX (Delta CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that alphaPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases.