Induced expression of p16(INK4a) inhibits both CDK4- and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes.

To investigate the mode of action of the p16(INK4a) tumor suppressor protein, we have established U2-OS cells in which the expression of p16(INK4a) can be regulated by addition or removal of isopropyl-beta-D-thiogalactopyranoside. As expected, induction of p16(INK4a) results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16(INK4a) also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27(KIP1). Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16(INK4a), this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16(INK4a). Sequestration of CDK4 by p16(INK4a) allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16(INK4a), p27(KIP1) appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27(KIP1) and p21(CIP1). Significantly, p16(INK4a) itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.

Therapeutic efficacy of cefpiramide-ciprofloxacin combination in experimental Pseudomonas infections in neutropenic mice.

The therapeutic efficacy of cefpiramide and ciprofloxacin alone and in combination was investigated and compared with that of ticarcillin plus tobramycin against pseudomonal infections in mice made neutropenic by administration of cyclosphosphamide. Therapy with cefpiramide plus ciprofloxacin was significantly more effective than that by either antibiotic alone. These results were consistent with in-vitro synergistic effects. At a higher dose of ciprofloxacin (4 mg/kg) plus cefpiramide (50 mg/kg), the combination therapy protected all neutropenic mice from fatal bacteraemia, and was more protective than ticarcillin (200 mg/kg) plus tobramycin (1 mg/kg). The peak serum concentration of cefpiramide in infected neutropenic mice was 51 mg/l when they were given 50 mg/kg subcutaneously. Ciprofloxacin attained a peak serum concentration of 1.2 mg/l and a serum half-life of 34 min.

Therapeutic efficacy and pharmacokinetic properties of ciprofloxacin in intra-abdominal abscesses caused by Bacteroides fragilis and Escherichia coli.

Experimental intra-abdominal abscesses were produced in mice by intraperitoneal injections of Bacteroides fragilis and Escherichia coli. The therapeutic efficacy of ciprofloxacin was investigated in this mixed intra-abdominal abscess model and was compared with that of rifampicin. Treatment with ciprofloxacin at 0.2 to 20 mg/kg or rifampicin at 20 mg/kg prevented all mice from death, as compared to the 60% mortality rate observed in the vehicle-treated controls. Rifampicin concentrations at 10 and 20 mg/kg were effective in preventing abscess formation and eradicated bacterial abscess. Ciprofloxacin at all the levels tested neither reduced the incidence of abscess nor eradicated Bact. fragilis from abscesses. However, ciprofloxacin at levels of 20, 10, 5, and 1 mg/kg reduced significantly the number of E. coli cells in the abscess. The peak serum level of ciprofloxacin at the oral dose of 20 mg/kg was 0.43 mg/l which was well above the MIC values for E. coli but not for Bact. fragilis.

Interaction of clavulanic acid, sulbactam and cephamycin antibiotics with beta-lactamases.

The inhibitory effects of clavulanic acid, sulbactam and cephamycin antibiotics on chromosomally-mediated or plasmid-mediated beta-lactamases were investigated. The inhibition constants were determined by a non-linear regression analysis. Clavulanic acid and sulbactam had high affinities for the purified plasmid-mediated beta-lactamases such as SHV-1, TEM-1 and PSE-4, and were potent inhibitors as shown by their low Ki values. Except for Bacteroides beta-lactamase, which is sensitive to inhibition by cephamycin antibiotics, clavulanic acid and sulbactam were found not to be as effective against chromosomally-mediated beta-lactamases. The cephamycin antibiotics were better inhibitors of chromosomally-mediated beta-lactamases than those that are plasmid mediated. Except for P99 beta-lactamase, against which sulbactam and clavulanic acid were inactive, the cephamycin antibiotics were less effective inhibitors than sulbactam and clavulanic acid.

Purification and properties of a beta-lactamase from Proteus penneri.

A cephalosporin-hydrolyzing enzyme from strains of Proteus penneri resistant to beta-lactam antibiotics was purified and characterized. The enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 30,000. This cephalosporinase has an isoelectric point of 6.8, a pH optimum of 6.5 and a temperature optimum of 45 degrees C. The enzyme hydrolyzed cephaloridine, cephalothin, cefuroxime, and cefotaxime more rapidly than penicillins. The relative rate, with cephaloridine as 100, were: cephalothin, 50; cefuroxime, 93; cefotaxime, 48; ceftriaxone, 23; cefoperazone, 11; benzylpenicillin, 3; ampicillin, 9; and carbenicillin, less than 1. Cephamycins had low affinities for the enzyme. However, clavulanic acid and sulbactam, with high affinities for the enzyme, were inhibitors of this enzyme.

Synergistic activity of apalcillin and gentamicin in a combination therapy in experimental Pseudomonas bacteraemia of neutropenic mice.

The therapeutic activity of a combination of apalcillin and gentamicin was evaluated in experimental Pseudomonas aeruginosa infection in neutropenic mice. Mice made neutropenic by administration of cyclophosphamide were more susceptible to P. aeruginosa infection than normal mice. At both challenge levels of 2LD50 and 20LD50, therapy with gentamicin alone was more effective than that with apalcillin or piperacillin. However, therapy with apalcillin-gentamicin combinations was significantly more effective than that by either component alone, and was as active as that with piperacillin-gentamicin. These in-vivo findings correlated with those of in-vitro studies, thus establishing a synergistic effect when apalcillin and gentamicin were combined. The results show that apalcillin when combined with gentamicin is effective in treating serious P. aeruginosa bacteraemia in neutropenic mice.

Therapeutic efficacy of cefpiramide and cefoperazone alone and in combination with gentamicin against pseudomonal infections in neutropenic mice.

Cefpiramide and cefoperazone alone and in combination with gentamicin were compared for therapeutic efficacy against pseudomonal infections in normal mice and in mice made neutropenic by administration of cyclophosphamide. Neutropenic mice were more susceptible to infection with Pseudomonas aeruginosa than normal mice. At all challenge doses, combination therapy with either cefpiramide-gentamicin or cefoperazone-gentamicin was more effective than that with a single agent. Therapy with the cefpiramide-gentamicin combination was significantly more active than that with the cefoperazone-gentamicin combination in protecting mice from fatal bacteremia. Pharmacokinetic studies in mice showed that cefpiramide attained a peak serum concentration of 12 micrograms/ml and a serum half-life of 40 min, which are higher than attained by cefoperazone with values of 4 micrograms/ml and 18 min. These factors may have caused the combined cefpiramide-gentamicin therapy to result in significantly improved survival rates in mice as well as in higher bactericidal titers than the cefoperazone-gentamicin combination. The results show that cefpiramide when combined with gentamicin is effective in treating serious infections with P. aeruginosa in neutropenic mice.

Discrepancy between the antibacterial activities and the inhibitory effects on Micrococcus luteus DNA gyrase of 13 quinolones.

Thirteen quinolone antibacterial agents were investigated as to their ability to inhibit Micrococcus luteus DNA gyrase and cell growth, and compared to those of novobiocin and coumermycin. Among the quinolones tested, the most active were found to be CI-934 and ciprofloxacin, which inhibited gyrase full supercoiling activity at concentrations of 100 and 200 micrograms/ml, respectively, while inhibiting cell growth at a concentration of 1 microgram/ml. However, both novobiocin and coumermycin inhibited gyrase full supercoiling activity at concentrations of 0.5 and 1.0 microgram/ml, respectively, which were comparable to those concentrations causing inhibition of cell growth.

Experimental efficacy and pharmacokinetic properties of cefpiramide, a new cephalosporin.

The in vivo therapeutic efficacy of cefpiramide was investigated and compared with that of cefoperazone. Cefpiramide was more potent than cefoperazone against infections produced by both beta-lactamase-producing and non-beta-lactamase-producing S. aureus. The protective activity of cefpiramide against experimental infections with selected members of Enterobacteriaceae was lower than that of cefoperazone. Against carbenicillin-resistant P. aeruginosa infections, cefpiramide was as active as gentamicin and aztreonam and three times more potent than cefoperazone, cefotaxime and piperacillin. The pharmacokinetic properties of cefpiramide in mice and rats were superior to those of cefotaxime and cefoperazone. The peak serum concentrations of cefpiramide, administered subcutaneously at a dose of 50 mg/kg, were 76 micrograms/ml in mice and 174 micrograms/ml in rats and the corresponding serum half-lives of cefpiramide were 87 min and 49 min in mice and rats respectively.

Inhibitory activity of cefpiramide on beta-lactamase.

The beta-lactamase stability and inhibitory activity of cefpiramide were investigated. Cefpiramide was found to be stable to hydrolysis and inhibited beta-lactamase produced by Citrobacter freundii, Enterobacter cloacae, Morganella morganii, and Escherichia coli. Kinetic studies showed that cefpiramide is a competitive inhibitor of cephaloridine hydrolysis by E. cloacae beta-lactamase.

Immunomodulating activity of 3-(p-chlorophenyl)thiazolo-[3,2-a] benzimidazole-2-acetic acid (Wy-18,251, NSC 310633) in mice implanted with Lewis lung tumor. Effect of Wy-18,251 on lymphocyte proliferation and phagocytosis.

Proliferation of B lymphocytes is depressed in Lewis lung tumor bearing mice. Treatment of these mice with Wy-18,251 (5 mg/kg) significantly increased the responsiveness of their splenic T cells to concanavalin A in cell culture. Levamisole (5 mg/kg) acted more weakly than Wy-18,251. Neither Wy-18,251 nor levamisole elevated the depressed B cell mitogenesis. Wy-18,251 significantly increased macrophage phagocytosis against 51chromium labeled opsonized chicken red blood cells. Levamisole behaved differently: it either had no effect on phagocytosis, or depressed it.

Immunomodulating and antimetastatic activity of 3-(p-chlorophenyl) thiazolo[3,2-a]benzimidazole-2-acetic acid (Wy-18,251, NSC 310633).

The antimetastatic activity of a thiazolobenzimidazole, Wy-18,251 was investigated using various dosage regimens in mice with implanted Lewis lung tumors. The low doses, 1 and 5 mg/kg (i.p.) given 24 hours after implantation with two subsequent doses at 1 week intervals were most effective in reducing lung metastases. Delayed hypersensitivity reactions in guinea pigs were significantly increased by oral doses of 50 and 150 mg/kg. In normal rats, peripheral blood lymphocytes determined by rosette assay, were significantly increased by oral doses of 50 to 150 mg/kg of Wy-18,251 and in a more sensitive assay using anti-theta serum the lymphocyte levels were increased by doses of 7.5 and 10 mg/kg. When cultured T lymphocytes from CBA/J mouse spleens were incubated with a suboptimal concentration of Concanavalin A, [3H]thymidine uptake was significantly increased in the presence of 0.05 to 1.0 microgram per culture. These results suggest that Wy-18,251 may have potential therapeutic value as an antimetastatic agent through its stimulation of the cellular immune system.

Anti-tumour and anti-metastatic activity of 3-(P-Chlorophenyl)-2,3-Dihydro-3-Hydroxythiazolo (3,2-A)-Benzimidazole-2-acetic acid (WY-13,876).

Extensive investigation of 3-(p-chlorophenyl)-2,3-dihydro-3-hydroxythiazolo(3,2-alpha)-benzimidazole-2-acetic acid (Wy-13,876) in BDF1 mice implanted with Lewis lung tumour has shown that it is an effective anti-tumour and anti-metastatic agent. In vitro examination using HEp-2 human epidermal tumour cells has indicated that Wy-13,876 is not cytotoxic. When mice implanted with Lewis lung tumour and treated with Wy-13,876 are also injected with anti-thymocyte serum, an increase in lung metastases is observed suggesting that thymocyte activity is involved in the drug's mechanism of action. An increase in peripheral T lymphocytes observed in rats 18 h after a single oral dose of Wy-13,876 further supports this possibility. When Wy-13,876 is given to tumour -bearing mice in combination with low, ineffective doses of 5-fluorouracil or cyclophosphamide, further reduction of primary tumour growth is observed.

Antitubercular activity of substituted 5-oxo-1-thiocarbamoyl-3-pyrazoline-4-alkanoic acid derivatives.

Several novel pyrazolin-5-ones prepared by the cyclization of variously substituted thiosemicarbazone derivatives of ethyl formylsuccinate, ethyl acetylsuccinate, and ethyl acetylglutarate were tested for antitubercular activity against Mycobacterium tuberculosis, human type, strain H37Rv, by a tube dilution technique. Minimum inhibitory concentrations (MIC) for these derivatives ranged from 0.05 to 100 mug/ml. The most active compound was ethyl 3-methyl-1-methylthiocarbamoyl-5-oxo-3-pyrazoline-4-acetate (MIC=0.05-0.1 mug/ml).