Effect of prior chronic aerobic exercise on overload-induced skeletal muscle hypertrophy in mice.

This study aimed to examine how regular aerobic training can affect the muscle hypertrophy induced by overloading. Male C57BL/6J mice were randomly divided into three groups: rest group, low-intensity aerobic exercise group, and high-intensity aerobic exercise group. Mice in the exercise groups were assigned to run at a speed of 10 m/min (low-intensity) or 25 m/min (high-intensity) for 30 min/day, five days/week, for four weeks. Then, the right hind leg gastrocnemius muscles were surgically removed to overload the plantaris and soleus muscles, while the left hind leg was subjected to a sham-operation. Both the plantaris and soleus muscles grew larger in the overloaded legs than those in the sham-operated legs. Muscle growth increased in the plantaris muscles in the low-intensity exercise group compared to that in the rest or high-intensity exercise groups at one and two weeks after overloading. This enhancement was not observed in the soleus muscles. Consistently, we observed changes in the expression of proteins involved in anabolic intracellular signaling, including Akt, mechanistic target of rapamycin (mTOR), and p70S6K, in the plantaris muscles. Our data showed for the first time that chronic low-intensity aerobic exercise precipitates overload-induced muscle growth.

X-ray crystal structure determination and molecular dynamics simulation of prophospholipase A2 inhibited by amide-type substrate analogues.

X-ray crystal structures of bovine pancreas prophospholipase A2 (proPLA2) inhibited by two amide-type inhibitors, [(R)-2-dodecanoyl-amino-1-hexanolphosphocholine (DAHPc) and (R)-2-dodecanoylamino-1-hexanolphosphoglycol (DAHPg)], were determined to R = 0.208 and 0.215 using reflections with up to 2.1 A resolution, respectively. Both complex crystals lacked defined electron densities for the prosequence of the N-terminal and for a loop region consisting of residues 65-70, retaining the disordered feature observed in free proPLA2 despite stabilization due to complex formation. The polar and nonpolar moieties of the amide-type inhibitors were located in the calcium-binding pocket and in the N-terminal alpha-helical hydrophobic region of the enzyme, respectively. As for the amide group of the inhibitor, which is lacking in the true substrate, a strong hydrogen bond was formed between the NH of the inhibitor and the unprotonated N(delta1) atom of His-48, resulting in the tight binding of the inhibitor to proPLA2, as well as to PLA2. The 20-30 times more potent inhibitory activity of DAHPg than DAHPc toward PLA2 could be explained by hydrogen bond formation between the glycol OH of DAHPg and the carbonyl O of Asp-49. The seven residues of the N-terminal prosequence of proPLA2, though disordered, block the access of a water molecule to Ala-1 of PLA2 or change the hydrogen-bonding property of Ala-1 alpha-amino group, resulting in breakage of the water-mediated hydrogen-bond network which is commonly formed in PLA2. The results of molecular dynamics (MD) calculation in an aqueous solution at 300 K indicate that this, rather than the close contact between the prosequence and the residues 65-70 loop region, is the main reason why the latter region becomes flexible in proPLA2, compared with in PLA2.

Novel ligands lacking a positive charge for the delta- and mu-opioid receptors.

Recently we reported using minilibraries to replace Lys(9) [somatostatin (SRIF) numbering] of the potent somatostatin agonist L-363,301 (c[-Pro-Phe-D-Trp-Lys-Thr-Phe-]) to generate the potent neurokinin receptor (NK-1) antagonist c[-Pro-Phe-D-Trp-p-F-Phe-Thr-Phe-]. This novel cyclic hexapeptide did not bind the SRIF receptor. Thus, a single mutation converted L-363,301, a SRIF agonist with potency ca. 2-8 times the potency of SRIF in laboratory animals,(24) into a selective NK-1 receptor antagonist with an IC(50) of 2 nM in vitro. During the screening of the same libraries for ligands of the delta-opioid receptor, we identified four compounds (1-4) which represent a new class of delta-opioid antagonists, some of which were also NK-1 receptor antagonists. The most potent delta-opioid antagonist, c[-Pro-1-Nal-D-Trp-Tyr-Thr-Phe-] (2), showed a K(e) value of 128 nM in the mouse vas deferens assay and a delta-receptor binding affinity constant of 152 nM in the rat brain membrane binding assay. These results are of interest because they represent a novel class of delta-opioid antagonists and, like two previously reported delta-opioid antagonists, they lack a positive charge. To examine further the requirement for a positive charge in the delta-opioid ligands, we prepared two analogues of the beta-casomorphin-derived mixed mu-agonist/delta-antagonist, H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-] (7), in which we eliminated the positive charge either through formylation of the primary amino group (5) or by the deletion of this N-terminal amino group (6). These latter compounds proved to be delta-opioid antagonists with K(e) values in the 16-120 nM range, as well as fairly potent mu-opioid antagonists (K(e) approximately 200 nM). These six compounds provide the most convincing evidence to date that there is no requirement for a positive charge in mu- and delta-opioid receptor antagonists. In addition, cyclic hexapeptide 4 lacks a phenolic hydroxyl group. Taken together, these data suggest that the prevailing assumptions about delta- and mu-opioid receptor binding need revision and that the receptors for these opioid ligands have much in common with the NK-1 and somatostatin receptors.

Combined hepatocellular-cholangiocarcinoma with variable sarcomatous transformation.

An adult case of combined hepatocellular-cholangiocarcinoma with variable sarcomatous changes is presented. Histologically, the tumor was composed of hepatocellular carcinoma, cholangiocarcinoma, and sarcomatous portions, including spindle-shaped, pleomorphic, and osteoplastic varieties. There was a transitional cell form between the carcinoma and sarcomatous cells. These tumor elements showed both independent and concurrent metastases. Immunohistochemical examination for keratin revealed positive staining in the tumor cells except for osteoplastic immature cells, whereas vimentin had positive results only in some sarcomatous cells. On the basis of these findings, the possibility of sarcomatous transformation of combined hepatocellular-cholangiocarcinoma was discussed.

Crystallization and preliminary X-ray study of Agkistrodon halys blomhoffii phospholipase A2 complexed with a specific inhibitor.

Phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been crystallized as a complex with a specific inhibitor, (S)-2-dodecanoyl-amino-3-hexanol-1-phosphoglycol. The complex crystals belong to the hexagonal space group, P6(1)22 (or P6(5)22), with cell dimensions of a = b = 61.13 A, and c = 173.15 A. The diffraction extends to at least 2.3 A resolution.

Binding mode of phospholipase A2 with a new type of phospholipid analog having an oxazolidinone ring.

Inhibition of phospholipases A2 (PLA2s) by a new type of monodispersed phospholipid analog, 3-dodecanoyl-4-phosphatidylcholinohydroxymethyl-2-oxazolidinone (oxazolidinone-PC), was investigated by the pH stat assay method using monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC) as the substrate. The PLA2s used were those from bovine pancreas and cobra (Naja naja atra) venom (Group I) and from Japanese mamushi (Agkistrodon halys blomhoffii) venom (Group II). This new-type substrate analog was shown to inhibit competitively both types of venom and bovine pancreatic enzymes by binding to the active site in a similar manner to the carboxamide-type analog 2-dodecanoyl-amino-1-hexanol-phosphocholine (amide-PC). The binding of a stereoisomer, (R)-amide-PC, to N. naja atra (Group I) and A. halys blomhoffii (Group II) PLA2s was facilitated by the binding of Ca2+ to the enzymes. On the other hand, the binding of (R)-oxazolidinone-PC to the N. naja atra (Group I) enzyme was found to be independent of Ca2+ binding, while its binding to the A. halys blomhoffii (Group II) enzyme was markedly facilitated by the binding of Ca2+ to the enzyme. The binding of (R)-amide-PC to N. naja atra PLA2 (Group I) was markedly influenced by the ionization state of the catalytic residue His 48, whereas the binding of (R)-oxazolidinone-PC was found to be practically independent of the ionization state of this residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Ca2+ in the binding of phospholipase A2 with a monomeric substrate and with its amide-type analog.

Effects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by Group I phospholipases A2 (PLA2s) from Pseudechis australis, Naja naja atra, and bovine pancreas and by Group II enzymes from Vipera russelli russelli, Agkistrodon halys blomhoffii, and Trimeresurus flavoviridis, were studied by the pH-stat assay method at 25 degrees C, pH 7.5-8.2, and an ionic strength of 0.1 or 0.2 in the absence or presence of an amide-type substrate analog, 2-dodecanoyl-amino-1-hexanol-phosphoglycol. The binding of genuine substrate to the Group II enzymes and that of its analog to the Groups I and II enzymes were markedly facilitated by the binding of Ca2+ to the enzymes. On the other hand, the binding of genuine substrate to the Group I enzymes was found to be independent of the Ca2+ binding. The former result suggests that the structures of the Group II enzyme-genuine substrate complexes and both types of enzyme-analog complexes are generally stabilized by the Ca2+ binding, whereas the latter indicates that the structures of the Group I enzyme-genuine substrate complexes are already similar to those of their Ca2+ complexes and that, therefore, these enzyme-substrate interactions are independent of the Ca2+ binding.

Roles of asp126 and asp156 in the enzyme function of sphingomyelinase from Bacillus cereus.

To elucidate the roles of conserved Asp residues of Bacillus cereus sphingomyelinase (SMase) in the kinetic and binding properties of the enzyme toward various substrates and Mg2+, the kinetic data on mutant SMases (D126G and D156G) were compared with those of wild type (WT) enzyme. The stereoselectivity of the enzyme in the hydrolysis of monodispersed short-chain sphingomyelin (SM) analogs and the binding of Mg2+ to the enzyme were not affected by the replacement of Asp126 or Asp156. The pH-dependence curves of kinetic parameters (1/Km and kcat) for D156G-catalyzed hydrolysis of micellar SM mixed with Triton X-100 (1:10) and of micellar 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP) were similar in shape to those for WT enzyme-catalyzed hydrolysis. On the other hand, the curves for D126G lacked the transition observed for D156G and WT enzymes. Comparison of the values and the shape of pH-dependence curves of kinetic parameters indicated that Asp126 of WT SMase enhances the enzyme's catalytic activity toward both substrates and its binding of HNP but not SM. The deprotonation of Asp126 enhances the substrate binding and slightly suppresses the catalytic activity toward both substrates. Asp156 of WT SMase acts to decrease the binding of both substrates and the catalytic activity to HNP but not SM. From the present study and the predicted three-dimensional structure of B. cereus SMase, Asp126 was thought to be located close to the active site, and its ionization was shown to affect the catalytic activity and substrate binding.

Stereocontrolled synthesis of a sphingomyelin methylene analogue as a sphingomyelinase inhibitor.

[reaction: see text]Efficient synthesis of a sphingomyelin methylene analogue, which was designed as a sphingomyelinase inhibitor, was stereoselectively achieved. The Hofmann rearrangement of the alpha-hydroxyethyl-beta-hydroxy amide 4 followed by the intramolecular oxazolidinone ring formation was one of the key steps.

A randomized trial of intrahepatic arterial infusion of 4'-epidoxorubicin with Lipiodol versus 4'-epidoxorubicin alone in the treatment of hepatocellular carcinoma.

We conducted a prospective randomized trial to evaluate the efficacy of Lipiodol in intrahepatic arterial infusion chemotherapy for patients with hepatocellular carcinoma (HCC). A total of 38 patients with unresectable HCCs and underlying cirrhosis were entered in this trial, and 36 of them were evaluable. Every 4 weeks, 17 patients received 70 mg of 4'-epidoxorubicin (epirubicin) alone (group A), whereas 19 patients received a Lipiodol emulsion containing the same dose of epirubicin (group B) through the hepatic artery. A tumor response (CR+PR) was observed in 12% of group A patients and in 42% of group B patients. The group B patients showed a significantly higher response rate than the group A patients. There was a tendency for an increased duration of survival (P = 0.09) in the group B patients. These results suggested that the infusion of the Lipiodol emulsion with epirubicin was more effective than epirubicin alone for the treatment of these patients with HCC.

Liver-specific F antigen in the serum of patients with liver diseases and the detection of early stage hepatocellular carcinoma.

Liver-specific F antigen was detected in serum using human-specific guinea pig antiserum, in 75 out of 121 patients with various liver diseases. The antigen was detectable in most of the patients (86.7%) with hepatocellular carcinoma, and the levels of F antigen were often high in these patients. There was no correlation between the levels of F antigen and alpha-fetoprotein. Temporal changes of serum F antigen level were also studied in patients with hepatocellular carcinoma. In 8 out of 13 patients with hepatocellular carcinoma, serum F antigen level remained continuously high, and in these patients, it had been significantly high for several months to one year before hepatocellular carcinoma became clinically detectable. Extracts of hepatocellular carcinomas obtained by necropsy was examined for the presence of F antigen, and it was found that most of hepatocellular carcinomas contained the human-specific determinant of F antigen, but lacked the species non-specific determinant of the antigen. The use of human-specific anti-F antiserum may facilitate early detection of hepatocellular carcinoma in some patients.

[Therapeutic effect of midecamycin on Mycoplasma pneumoniae pneumonia in adults (author's transl)].

The clinical efficacy of a macrolide antibiotic, midecamycin, was studied in 12 adult cases with Mycoplasma pneumoniae pneumonia. The therapeutic effects were excellent or good in 9 cases and fair in 2 cases. On defervescence and disappearance of shadows on chest X-ray the therapeutic effect was satisfactory, but on disappearance of cough therapeutic effect was not clear in some cases. Taking into consideration the antimicrobial activity of midecamycin against Mycoplasma pneumoniae, serum concentration and side effects of midecamycin, this antibiotic is expected to be effective in the treatment of Mycoplasma pneumoniae pneumonia.

[A case of rupture of a posterior pancreaticoduodenal artery aneurysm].

A 75 year old man complaining of right upper abdominal pain was admitted to our hospital. He became shocked with hypotension and cold sweat, and immediately underwent operation. Laparotomy revealed massive hemorrhage in the retroperitoneal region, particularly in the right upper space. It was derived from the rupture of the posterior pancreaticoduodenal artery aneurysm. The arterial trunk was ligated above and below the aneurysm and the aneurysmectomy was carried out. The patient recovered and was discharged from the hospital 56 days after operation. There was no other visceral artery aneurysm and the etiology of this aneurysm was unknown. Four cases of pancreaticoduodenal artery aneurysm have been reported in Japan and three of them were operated, but this is the first surgically resected case for the rupture of pancreaticoduodenal artery aneurysm with success.