Cow's milk increases the activities of human nuclear receptors peroxisome proliferator-activated receptors alpha and delta and retinoid X receptor alpha involved in the regulation of energy homeostasis, obesity, and inflammation.

The nuclear peroxisome proliferator-activated receptors (PPAR) have been shown to play crucial roles in regulating energy homeostasis including lipid and carbohydrate metabolism, inflammatory responses, and cell proliferation, differentiation, and survival. Because PPAR agonists have the potential to prevent or ameliorate diseases such as hyperlipidemia, diabetes, atherosclerosis, and obesity, we have explored new natural agonists for PPAR. For this purpose, cow's milk was tested for agonistic activity toward human PPAR subtypes using a reporter gene assay. Milk increased human PPARalpha activity in a dose-dependent manner with a 3.2-fold increase at 0.5% (vol/vol). It also enhanced human PPARdelta activity in a dose-dependent manner with an 11.5-fold increase at 0.5%. However, it only slightly affected human PPARgamma activity. Ice cream, butter, and yogurt also increased the activities of PPARalpha and PPARdelta, whereas vegetable cream affected activity of PPARdelta but not PPARalpha. Skim milk enhanced the activity of PPAR to a lesser degree than regular milk. Milk and fresh cream increased the activity of human retinoid X receptor (RXR)alpha as well as PPARalpha and PPARdelta, whereas neither affected vitamin D3 receptor, estrogen receptors alpha and beta, or thyroid receptors alpha and beta. Both milk and fresh cream were shown by quantitative real-time PCR to increase the quantity of mRNA for uncoupling protein 2 (UCP2), an energy expenditure gene, in a dose-dependent manner. The increase in UCP2 mRNA was found to be reduced by treatment with PPARdelta-short interfering (si)RNA. This study unambiguously clarified at the cellular level that cow's milk increased the activities of human PPARalpha, PPARdelta, and RXRalpha. The possible role in enhancing the activities of PPARalpha, PPARdelta, and RXRalpha, and the health benefits of cow's milk were discussed.

Structure and expression of cDNA for D-amino acid oxidase active against cephalosporin C from Fusarium solani.

D-Amino acid oxidase (DAO) was extracted and purified from cultured mycelia of Fusarium solani M-0718 (FERM P-2688). The enzyme was able to oxidatively deaminate cephalosporin C to 7-beta-(5-carboxy-5-oxopentanamido)cephalosporanic acid. Ninety-eight amino acid residues of the F. solani DAO were determined by sequence analysis of 9 peptides derived from Acromobacter protease I digests of the protein. Complementary DNAs encoding F. solani DAO were isolated from the F. solani cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of the clones revealed a 1,186-nucleotide sequence with a 5'-terminal untranslated region of 41 nucleotides, an open reading frame of 1,083 nucleotides that encoded 361 amino acids, and a 3'-terminal untranslated region of 62 nucleotides. The amino acid sequence of F. solani DAO had 25% homology to that of porcine kidney DAO [EC 1.4.3.3] and 37% homology to that of Trigonopsis variabilis DAO. The constructed plasmid overproduced F. solani DAO in Escherichia coli. The recombinant DAO had almost the same molecular activity as the native DAO against cephalosporin C.

FK143, a novel nonsteroidal inhibitor of steroid 5 alpha-reductase: (1) In vitro effects on human and animal prostatic enzymes.

Steroid 5 alpha-reductase is an enzyme which converts testosterone into 5 alpha-dihydrotestosterone (DHT) and is implicated in the pathogenesis of benign prostatic hyperplasia (BPH) in men. We studied in vitro effects of FK143, a nonsteroidal new compound, on 5 alpha-reductase in human and animal prostates. Prostates were obtained from Wistar rats, Beagle dogs, and Cynomolgus monkeys as well as prostatic tissue from BPH patients obtained by the prostatectomy. Nuclear membrane fraction of prostates showed pH dependent 5 alpha-reductase activities, and inhibitory effects of drugs were assayed at pH 6.5. FK143 inhibited human prostatic 5 alpha-reductase in a dose-dependent manner with an IC50 of 1.9 nM and also inhibited animal 5 alpha-reductases with similar IC50 values. FK143 inhibited human and rat 5 alpha-reductases in a noncompetitive fashion while finasteride, a steroidal 5 alpha-reductase inhibitor, showed competitive inhibition. The affinities of FK143 for the human 5 alpha-reductase is constant at pH 5 and 6.5. No inhibitory effects were shown to other oxidoreductases. These results indicate that FK143 is a new type of potent and selective 5 alpha-reductase inhibitor.

FK143, a novel nonsteroidal inhibitor of steroid 5 alpha-reductase: (2) In vivo effects on rat and dog prostates.

FK143 is a nonsteroidal new inhibitor of steroid 5 alpha-reductase, an enzyme which converts testosterone into 5 alpha-dihydrotestosterone (DHT). We studied in vivo effects of FK143 on rat and dog prostates. FK143 was orally administered to mature male rats for 14 days. At doses above 1 mg/kg, FK143 significantly reduced the wet weights of the ventral prostate and seminal vesicle, but showed no effects on those of the epididymis, testis, and adrenal. Growth of ventral prostate and seminal vesicle was induced by the subcutaneous injection of testosterone propionate (TP) in the castrated young rats and was reduced by FK143 administration at doses above 3.2 mg/kg, while growth induced by 5 alpha-dihydrotestosterone propionate (DHTP) was not affected. FK143 had no binding affinity for the rat androgen receptor. FK143 showed neither estrogenic and antiestrogenic effects on the rat uterus nor androgenic effect on the rat prostate. Concentration of testosterone and DHT in the rat and dog prostates were measured by GC-MS, and administration of 10 mg/kg of FK143 significantly reduced the intraprostatic concentration of DHT. These results indicate that FK143 reduced the prostate growth by inhibiting 5 alpha-reductase activities in the prostates.

Subtype- and species-selectivity of a tachykinin receptor antagonist, FK888, for cloned rat and human tachykinin receptors.

We investigated the receptor-binding properties and potencies of FK888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-prolyl]-N- phenylmethyl-3-(2-naphthyl)-L-alaninamide), a tachykinin receptor antagonist, for the rat and human tachykinin receptor subtypes (NK1, NK2 and NK3) expressed in transfected mammalian cells. In displacement analyses, using membrane preparations derived from monkey kidney COS-7 cells transiently expressing tachykinin receptor subtypes, FK888 showed a subtype selectivity for NK1 receptor and its affinity for the human NK1 receptor was 320-fold higher than that for the rat NK1 receptor, demonstrating species difference in its binding affinity. This was in marked contrast to FK224 (N-[N2-[N-[N-[N-[2,3-didehydro-N-methyl-N-[N-[3-(2-pentylphenyl )- propionyl]-L-threonyl]tyrosyl-L-leucynyl]-D-phenylalanyl]-L- allothreonyl]-L-asparaginyl]-L-serine-n-lactone) that was selective for NK1 and NK2 receptors with similar affinities for the rat and human receptors. In Chinese hamster ovary cells permanently expressing the human NK1 receptor, FK888 inhibited the substance P-induced phosphatidylinositol hydrolysis and produced a parallel shift in the dose-response curve for substance P. Schild analysis of the antagonism of phosphatidylinositol hydrolysis by FK888 yielded a pA2 value of 8.9 and a slope of 0.97 of the regression line. FK888 itself showed no stimulatory effect on phosphatidylinositol hydrolysis in Chinese hamster ovary cells expressing the human NK1 receptor. Thus, FK888 is a potent, competitive and selective antagonist for human NK1 receptor.

Responsiveness of cerebral vessels to changes of blood pressure and partial pressure of carbon dioxide after a transient period of cardiac arrest in dogs.

Post-ischemic cerebral hypoperfusion supposedly due to constriction of cerebral vessels is considered to be one of the most important factors limiting the recovery of the brain after cerebral ischemia. An experimental study on dogs was carried out to determine the changes in the responsiveness of cerebral vessel to the dilating effects of increase of arterial pressure (AP) and of CO2 inhalation after 3-6 min of cardiac arrest. Responsiveness was measured by the ratio of change in intracranial pressure (ICP) to change in AP (delta ICP/delta AP) and to change in PCO2 (delta ICP/delta PCO2), since in a bony cranium the changes in cerebral vessel diameter are reflected by instantaneous ICP change. delta ICP/delta AP following the administration of intravenous epinephrine was 33%, 43%, 36%, 37% and 16% of pre-ischemic value 1 h, 2 h, 3 h, 4 h and 5 h after cerebral ischemia, respectively. delta ICP/delta PCO2 following 10% CO2 inhalation was 13%, 32%, 55%, 50%, 70% and 75% of pre-ischemic value 1/2 h, 1 h, 2 h, 3 h, 4 h and 5 h after cerebral ischemia, respectively. Normal delta ICP/central venous pressure (delta CVP) was observed during the post-ischemic period, although statistical analysis was not done. From this we concluded: After 3-6 min of cardiac arrest, cerebral arteries constricted for more than 5 h during post-ischemic period. These arteries did not respond well to the dilating effects of increased arterial pressure or CO2 inhalation, but after 3 h their responses to CO2 inhalation returned to their pre-ischemic levels. The intracranial pressure became more or less dependent on CVP during post-ischemic period.

A novel method for evaluating the outer membrane permeability to beta-lactamase-stable beta-lactam antibiotics.

A novel method is described which allows estimation of the outer membrane permeability to beta-lactamase-stable beta-lactams by determining antibiotic concentrations in the periplasm. The method is based on measurement of the inhibiting activity of beta-lactamase-stable beta-lactams on the hydrolysis of a substrate by periplasmic beta-lactamase. Application of the method to carbenicillin revealed that the high level of resistance to carbenicillin of an Escherichia coli strain acquiring the plasmid encoding ampicillin resistance resulted from the poor ability of carbenicillin to penetrate the outer membrane of E. coli.

Enterobacter cloacae outer membrane permeability to ceftizoxime (FK 749) and five other new cephalosporin derivatives.

The ability of ceftizoxime to penetrate the outer membrane was compared with those of five other new cephalosporins: cefotiam, cefuroxime, cefotaxime, cefmetazole and cefoxitin, using a clinical isolate of Enterobacter cloacae as a test strain. Estimation of permeability was performed by a method utilizing the inhibitory activities of the cephalosporins against beta-lactamase located in the periplasm. Of the cephalosporins tested, both ceftizoxime and cefmetazole gave remarkably high concentrations in the periplasm, several times higher than those of cefotaxime and cefoxitin and ten or more times higher than those of cefuroxime and cefotiam. The approximate permeability coefficient of ceftizoxime was also several times higher than those of cefotiam and cefmetazole and over ten times higher than those of cefoxitin, cefuroxime and cefotaxime.

FR-31564, a new phosphonic acid antibiotic: bacterial resistance and membrane permeability.

Mutants which acquired resistance to FR-31564 were also resistant to fosfomycin and vice versa. Some exceptions to cross-resistance were found among clinical isolates of certain species. FR-31564 was found to be incorporated into bacterial cells more efficiently than fosfomycin although the extent of incorporation varied among species. In particular, the uptake rate of FR-31564 by a strain of Pseudomonas aeruginosa was ten times that of fosfomycin. The uptake rate of FR-31564 by both FR-31564- and fosfomycin-resistant mutants was less than one-tenth of that by the parent strain. FR-31564 was scarcely inactivated in the culture broths of FR-31564-resistant strains. All of the FR-31564-resistant mutants of P. aeruginosa came under the classification of strains lacking an L-alpha-glycerophosphate transport system.

Nocardicin A, a new monocyclic beta-lactam antibiotic IV. Factors influencing the in vitro activity of Nocardicin A.

Factors influencing the in vitro antimicrobial activity of nocardicin A against Pseudomonas aeruginosa and Proteus mirabilis were investigated. Sodium chloride was identified as a major inhibitor. Some of the amino acids, sugars and divalent cations were found to be minor inhibitors. The presence of potassium phosphates enhanced nocardicin A activity against P. aeruginosa, but antagonized the activity against P. mirabilis.

Nocardicin A, a new monocyclic beta-lactam antibiotic III. In vitro evaluation.

Nocardicin A, a new monocyclic beta-lactam antibiotic, exerts a comparatively potent antimicrobial activity against gram-negative organisms, especially Pseudomonas aeruginosa, the indole-positive and indole-negative Proteus groups (except Pr. morganii), Serratia marcescens and the Neisseria groups. The in vitro antimicrobial activity of nocardicin A against clinical isolates of Ps. aeruginosa was about twice that of carbenicillin. The mean MICs of nocardicin A for Pr. mirabilis, Pr. rettgeri and Pr. inconstans ranged from 3.13 to 12.5 microgram/ml and were 25 similar to 50 microgram/ml for Pr. vulgaris. Nocardicin A in concentrations of 12.5 similar to 50 microgram/ml inhibited 30 strains (48 percent) of S. marcescens usually resistant to beta-lactam antibiotics. However, nocardicin A had no significant in vitro activity against Staphylococci and Escherichia coli. No cross-resistance was seen between nocardicin A and other beta-lactam antibiotics. This antibiotic was stable to beta-lactamase. The in vitro activity of nocardicin A against Ps. aeruginosa and Pr. mirabilis was greatly influenced by the assay media used. Nocardicin A was bactericidal and appeared to act synergistically with serum bactericidal factors against Ps. aeruginosa and with polymorphonuclear leukocytes against Ps. aeruginosa, E. coli and Pr. vulgaris. The bactericidal activity of nocardicin A against the above 3 organisms, therefore, increased markedly in the presence of fresh serum and polymorphonuclear leukocytes.

Role of the cephalosporinase gene in the resistance of the clinically isolated cephem-resistant Escherichia coli.

A small number of highly cephem-resistant strains was found in extensive susceptibility testing of clinical isolates of Escherichia coli to the new cephalosporin derivatives. The cephem-resistance of these clinical isolates appeared to be due to the increased cephalosporinase activities. To clarify the mechanism of the resistance, we cloned the cephalosporinase genes from two typical cephem-resistant clinical isolates as well as from an E. coli K-12 strain. The following two lines of evidence indicated that the cephem-resistance resulted from hyper production of the cephalosporinase due to the up-mutation of the regulatory sequence of the cephalosporinase gene. 1) Reciprocal exchange of the regulatory sequence including a short segment of N-terminal coding sequence and the rest of the coding sequence between the cephalosporinase genes from E. coli K-12 and the cephem-resistant clinical isolate showed that the higher cephalosporinase activity was accompanied by the regulatory sequence of the cephalosporinase gene from the clinical isolate. 2) The promoter activities of the cephalosporinase genes were determined by cloning the regulatory sequences into a promoter analysis vector. The promoter activities of the cephalosporinase genes from the clinical isolates were 23-33-fold higher than that of the cephalosporinase gene from E. coli K-12.

Mechanism of action of the new orally active cephalosporin FK027.

The mechanism of action of a new orally active cephalosporin, FK027, was compared to that of cephalexin and cefaclor to elucidate its excellent antibacterial activity against Gram-negative bacteria. FK027 showed very high affinity for the penicillin-binding proteins (PBPs) 3, 1a and 1bs of Escherichia coli whereas cephalexin showed fairly high affinity for PBPs 1a, 4 and 3. The ability of FK027 to penetrate the outer membranes of E. coli and Enterobacter cloacae was less than that of cephalexin and cefaclor. However, FK027 was extremely stable to both plasmid-mediated penicillinases and chromosomal beta-lactamases except the Bacteroides fragilis enzyme and its stability was superior to that of cephalexin and cefaclor. These results indicate that the potent antibacterial activity of FK027 is based on its enhanced affinity for the target enzymes and its high stability to beta-lactamases.

Nocardicin A, a new monocyclic beta-lactam antibiotic VI. Absorption, excretion and tissue distribution in animals.

The absorption, excretion and tissue distribution of nocardicin A, a new monocyclic beta-lactam antibiotic, were studied in various animals. When nocardicin A was given intramuscularly in single doses of 20 mg/kg to rats, rabbits, and dogs, the peak serum levels of nocardicin A were about 1.6 similar to 2.8 times higher than those of carbenicillin in all animals though the levels varied among the species tested. The serum half-life of nocardicin A in these animals was about twice that of carbenicillin. The 24-hour urinary recovery rate of nocardicin A after intramuscular injection was 68.5 percent in rabbits and 77.0 percent in dogs, but was low in rats; i.e., 0.7 percent. When nocardicin A was given intravenously in single doses of 20 mg/kg to these animals, the peak serum levels varied widely among the test species; i.e. about 3 times higher than those of carbenicillin in rabbits and dogs, similar to those in rats. The peak serum and tissue levels of nocardicin A after intramuscular to intravenous injection were the highest in the kidneys, followed by the liver, serum, lungs, heart and spleen. The levels in the liver were prolonged. Nocardicin A, and traces of unknown substances less active than nocardicin A were observed as active substances in the urine recovered after injection of nocardicin A.

Nocardicin A, a new monocyclic beta-lactam antibiotic V. In vivo evaluation.

Nocardicin A is a new monocyclic beta-lactam antibiotic which provides a potent therapeutic effect in mice experimentally infected with gram-negative bacilli. When given subcutaneously to mice, the therapeutic effect of the drug was stronger than had been anticipated from in vitro studies. Nocardicin A was more potent in therapeutic effect than carbenicillin against infections due to Pseudomonas aeruginosa, Proteus mirabilis, Pr. vulgaris, Pr. rettgeri and Pr. inconstans, and was similar in effect to carbenicillin against infections due to Escherichia coli in mice. In addition, nocardicin A proved to be active against infections due to Serratia marcescens and other organisms resistant to beta-lactam antibiotics. When nocardicin A was given subcutaneously to mice, blood and hepatic levels of the drug were higher than those of carbenicillin.

Adult-type neuroblastoma originated in retroperitoneum beginning with obstructive jaundice.

Abdominal neuroblastoma in adults is a rare neoplasm and only 30 patients have been described in Japan since 1985. The patient was a 43-year-old woman with jaundice. The tumor originated from retroperitoneum. The enlarged gall bladder and dilatation of intrahepatic bile ducts were noted by ultrasonography and computed tomography. We report the first adult-type neuroblastoma with obstructive jaundice.

[The histological changes in the spinal cord following percutaneous cervical cordotomy (PCC) and correlation of these changes with the efficacy of PCC].

We observed the histological changes in the spinal cord following percutaneous cervical cordotomy (PCC) and correlation of these changes with the efficacy of PCC in 7 cases. A fine monopolar electrode which we used, measured 0.25 mm or 0.27 mm in diameter with exposed length of 2 mm. An oval or elliptical-shaped lesion, 1 to 3 mm in width and 3 to 4 mm in length, was observed in 6 of these cases. The main lesion was found in the anterolateral column in 5 cases. In 3 of these 5 cases, the whole anterolateral column had been destroyed, and in the other 2 cases dorsal one half to two thirds of it had been destroyed. In these 5 cases, pain sensation on the opposite side of PCC was lost for a long time and pain was relieved until death (21-239 days after PCC). In the other 2 cases the lesion in the anterolateral column was tenuous or unrecognizable, and the loss of pain sensation was temporary with pain recurring by the next day. This study showed that an oval or elliptical-shaped lesion was made in PCC and that in cases in which main lesion was located in the anterolateral column, pain relief was long lasting.

Construction of a 7-aminocephalosporanic acid (7ACA) biosynthetic operon and direct production of 7ACA in Acremonium chrysogenum.

We have used cDNA encoding D-amino acid oxidase, and genomic DNA encoding cephalosporin acylase from Fusarium solani and Pseudomonas diminuta, respectively, to construct a novel hybrid 7-aminocephalosporanic acid (7ACA) biosynthetic operon under the control of regulatory elements from the alkaline protease gene of Acremonium chrysogenum. Transformants of A. chrysogenum BC2116, a high cephalosporin-producing strain, containing this operon, synthesized and secreted low levels of 7ACA. Although the amounts are not yet commercially significant, this represents the first microbial production of 7ACA and demonstrates the feasibility of introducing new biosynthetic capabilities into industrial microorganisms by combining fungal and bacterial genes.