8-Cl-adenosine-induced inhibition of colorectal cancer growth in vitro and in vivo.

The cAMP analogue 8-Cl-cAMP induces apoptosis and inhibits proliferation of a wide variety of malignancies in vitro and in vivo with relatively little toxicity. The antitumor effects of this compound are thought to involve its ability to modulate type I protein kinase A (PKAI). However, a nontoxic metabolite of 8-Cl-cAMP, 8-Cl-adenosine, with no known activity against PKAI, exerts growth inhibitory effects in breast, ovary, pancreas, and colorectal cancer cells in vitro and accumulates in xenografted tumors after 8-Cl-cAMP treatment in vivo. To characterize further the antitumor effects of 8-Cl-adenosine in colorectal cancer, we examined its effects on cell growth in vitro (cell number, 3H-thymidine incorporation, and soft agar colony formation) using the isogenically matched colorectal cancer cell lines HCT116, HCT116-E6 (p53-depleted), and 80S14 (p21WAF1/Cip1-null). 8-Cladenosine inhibited cell growth by 89%, 74%, and 79%, respectively in HCT116, HCT116-E6, and 80S14 cells after a 72-hour exposure. Growth inhibition coincided with DNA endoreduplication and subsequent apoptosis. Furthermore, nontoxic doses of 8-Cl-adenosine administered i.p. twice weekly for 4 weeks to athymic mice suppressed growth of HCT116-derived xenografts by 50%. These results show that 8-Cl-adenosine exerts antitumor activity against colorectal cancer independent of p53 and p21WAF1/Cip1.

Protein kinase A regulatory subunits in colon cancer.

The protein kinase A (PKA) is classified as type I or II depending on the association of the catalytic subunit with either the R(I) or R(II) regulatory subunits. Alterations in the levels of these regulatory subunits and PKA activity itself appear to affect cellular proliferation and tumorigenesis. We examined colorectal tumor specimens from 45 patients to investigate the potential role of cAMP-related signaling molecules in regulating tumorigenesis. Western blot analysis (PKA subunit protein levels) and in vitro kemptide phosphorylation assays (PKA catalytic subunit activity) were performed on human colorectal tumor tissue homogenates. R(I)beta protein levels were decreased 200% in ascending and 50% in descending colonic tumors compared to adjacent mucosa. R(II) protein levels were decreased 77% in descending colonic tumors but no change was observed in ascending colonic tumors compared to adjacent mucosa. PKA activity and the absolute amount of catalytic subunit protein in ascending and descending tumors were unchanged compared to adjacent mucosa. Differences in cAMP-related signaling molecules exist between neoplastic and normal colorectal tissues. These differences may not only serve as potential therapeutic targets for chemotherapeutic agents, but also lead to the identification of novel regulatory mechanisms involved in cellular proliferation and tumorigenesis.

Lidocaine elimination: effects of metoprolol and of propranolol.

The effects of administration of metoprolol and propranolol on lidocaine elimination were studied in six healthy young men who did not smoke. Each received three single intravenous doses of lidocaine (2.5 to 3.0 mg/kg injected over 10 min): one alone, one after 1 day pretreatment with propranolol (40 mg orally every 6 hr), and one after 1 day pretreatment with metoprolol (50 mg orally every 6 hr). Lidocaine clearance was 0.88 +/- 0.28 l X hr-1 X kg-1 before beta blockade, 0.61 +/- 0.20 l X hr-1 X kg-1 during metoprolol dosing, and 0.47 +/- 0.16 l X hr-1 X kg-1 during propranolol dosing. There was no correlation between the change in lidocaine elimination and the steady-state concentrations of metoprolol or propranolol, nor between the change in lidocaine clearance and the change in resting heart rate produced by either beta blocker. Metoprolol and propranolol reduce lidocaine elimination significantly.

Putting the Web to work.

Can home care agencies use the Internet to help them succeed in today's highly volatile, highly competitive market? You bet they can. The potential of Internet technology is limited only by an organization's own vision. The Internet is new ground, however, in an industry that for the most part has limited experience with even traditional communications media.

The high-level tetraplegic: psychological survival and adjustment.

From the date of injury tetraplegics with spinal cord lesions of C4 and above are faced with unparalleled social and psychological adjustment. The individual will be involved in a process of re-establishing his identity by learning to develop and adapt his only remaining resources, his mind and his ability to communicate. This patient population is absolutely dependent in all aspects of meeting basic needs during the initial stages of the injury. If we attempt to return the patient to an active role in society, the rehabilitation team must be aware of the dynamics by which the patient achieves control of both himself and his environment. He will undergo a marked regression of his physical and emotional being and will face the turmoil of realigning ego parameters. The focal point of patient, family and staff interaction is the months of rehabilitation, where the team deals with the problem of social reintegration. Family involvement is essential from the beginning and reaches a high point at discharge and home planning. The scope of the paper will be to trace the psycho-social development of the patient with cervical lesion of C4 and above through the initial, rehabilitation and discharge phases of the injury. Our aim is to develop sufficient insight to the problems facing our patient population that we may aid in their search for meaningful lives.

Effect of alcohols and selected solvents on serum osmolality measurements.

The method by which serum osmolality is measured can significantly affect the result if certain volatiles or solvents are present in the specimen. Commonly available solvents and alcohols were added to aliquots of pooled human serum to produce toxicologically relevant concentrations. Increasing concentrations of carbon tetrachloride, chloroform, mono-n-butyl ether (butyl cellosolve), 1, 1,1 trichloroethylene, toluene, and xylene did not change vapor pressure (VP) or freezing point depression (FPD) osmolality. Acetone, ethanol, isopropanol, and methanol in increasing concentrations produced a linear increase in FPD osmolality, but no change in VP osmolality. Only ethylene glycol produced a linear increase in VP and FPD osmolality across the range of concentrations studied. Despite the excellent correlation between osmolality and ethanol concentration in prepared serum samples, this relationship could not accurately predict patient ethanol concentrations from FPD osmolality. The osmolal gap, "delta" osmolality, (measured FPD minus calculated osmolality) did not correlate with the difference between measured FPD and VP osmolalities. Patient ethanol levels could not be predicted with accuracy using an equation based on the osmolal gap or "delta" osmolality.

Amitriptyline plasma protein binding: effect of plasma pH and relevance to clinical overdose.

Reversing ventricular ectopy with plasma alkalinization following acute tricyclic antidepressant overdose is a recognized mode of therapy. The mechanism responsible for this effect is unclear. Changes in plasma protein binding of free drug, effects of the sodium ion on the myocardium, and alterations of plasma concentrations of alpha-1-acid glycoprotein may all interact to alter toxicity of tricyclics in overdose. An in vitro investigation using equilibrium dialysis was designed to examine the effect of altering plasma pH on percentage of free amitriptyline at clinical overdose plasma concentrations. A 1973 report on this effect lacked adequate controls and was faulty in experimental protocol. The current investigation used plasma concentrations typically present in amitriptyline overdose, a sensitive gas liquid chromatographic assay to detect total and free drug, and adequate control of plasma pH. The results of two separate experiments demonstrated a significant decrease in percentage of free amitriptyline of 20% over a pH range of 7.0-7.4 (P less than 0.05) and 42% over a pH range of 7.4-7.8 (P less than 0.05). The rate of change in slope in both experiments was not significantly different (P less than 0.01) indicating similar effects of pH change on plasma protein binding of amitriptyline within the two groups.

8-Cl-adenosine induces differentiation in LS174T cells.

8-Cl-adenosine represents a novel nontoxic chemotherapeutic agent shown to inhibit growth of a number of colorectal cancer cell lines. We have utilized the mucin-secreting colorectal cancer cell line, LS174T, to assess the growth inhibitory properties of 8-Cl-adenosine independent of its parental compound, 8-Cl-cAMP. Conversion of 8-Cl-cAMP to 8-Cl-adenosine is required for growth inhibition in LS174T cells. 8-Cl-Adenosine inhibited growth by inducing a G1 cell cycle arrest that was associated with large (eightfold) increases in p21WAF1/Cip1 and p53 protein levels and a decrease in the phosphorylation status of the retinoblastoma protein. LS174T cells did not undergo apoptosis. In addition, 8-Cl-adenosine also induced some degree of enterocytic differentiation. Both villin protein levels as well as alkaline phosphatase activity rose (2- and 3.5-fold, respectively) in response to treatment with 8-Cl-adenosine. Our results suggest that in LS174T cells, 8-Cl-adenosine not only serves as a growth inhibitory agent but also as an inducer of enterocytic differentiation.

Effects of human serum on cathepsin D activity.

The proteolytic activity of bovine splenic cathepsin D was evaluated by using the digestion of tritiated hemoglobin. Acid-denatured human serum was an enhancer of cathepsin D activity. Concentrated serum sources showed decreased activity because of competition of serum albumin with tritiated hemoglobin for cathepsin D. The enhancing activity of serum did not separate with any isolated protein as assessed by Sephadex G-50-80 chromatography or protein electrophoresis. The extraction of serum with ethanol-ether and chloroform-methanol showed that the enhancing activity separated with the phospholipid fraction, which had 2.6 times as much activity as the total lipid fraction. The activity was stable to heat at 56 degrees C for 30 minutes and to freeze-thawing, and was not dependent on metal ions.

Radiosensitivity of thymic interleukin-7 production and thymopoiesis after bone marrow transplantation.

Interleukin-7 (IL-7) is the major thymopoietic cytokine. Injections of IL-7 after murine bone marrow transplantation (BMT) correct defects in thymic differentiation, including thymic hypocellularity, abnormal differentiation of CD3- CD4- CD8- (triple-negative [TN]) thymocytes into CD4+ CD8+ (double-positive [DP]) cells, and antigen-specific mature T-lymphocyte proliferation. To determine whether IL-7 production is decreased in BMT recipients, BMT was performed with congenic murine donor-recipient strains and escalating doses of pre-BMT conditioning. Increasing doses of radiation resulted in decreased thymic cellularity and maturation from the TN to the DP stage. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that intrathymic production of IL-7 was significantly decreased in irradiated mice than in nonirradiated controls. Decline in IL-7 transcript levels was correlated with the dose of radiation administered. Analyses of the numbers of CD45- major histocompatibility complex class II+ thymic stromal cells suggested that the mechanism for the decreased IL-7 production was loss of IL-7-producing thymic stromal cells. Experiments indicated that pre-BMT conditioning with radiation led to decreased stromal production of IL-7 and consequent blocks in the maturation of thymocytes. They provided a mechanism for both the abnormal thymopoiesis observed after BMT and the previously observed beneficial effects of IL-7 administration in murine models. Impaired production of IL-7 by thymic stroma may be a general model for the clinically observed adverse effects of cytotoxic therapy on thymopoiesis.

Activity of ampicillin/sulbactam, ticarcillin/clavulanate, clarithromycin, and eleven other antimicrobial agents against anaerobic bacteria isolated from infections in children.

The activity of 14 antimicrobial agents against 253 clinical isolates of anaerobic bacteria from pediatric infections was assessed by the agar dilution method. Fifty-eight percent of the isolates were from intraabdominal sites. The drugs tested were ampicillin/sulbactam, ticarcillin/clavulanate, ampicillin, sulbactam, piperacillin, cefoxitin, cefotaxime, cefoperazone/sulbactam, clarithromycin, azithromycin, erythromycin, clindamycin, metronidazole, and chloramphenicol. Ticarcillin/clavulanate was active against all isolates. Clarithromycin was the most active macrolide; combination of this agent with its 14-hydroxy metabolite did not result in synergy. Sixty-two percent of Bacteroides fragilis group isolates, 13% of B. fragilis isolates, and 22% of peptostreptococcal isolates were resistant to clindamycin at a concentration of 4 micrograms/mL. The distribution of these strains in clinical specimens and the patterns of antimicrobial susceptibility documented were different from the findings for isolates from adults in the Los Angeles area.

Activities of the benzoxazinorifamycin KRM 1648 and ethambutol against Mycobacterium avium complex in vitro and in macrophages.

KRM 1648 is a 4-aminobenzoxazine derivative of rifamycin S with potent in vitro activity against the Mycobacterium avium complex (MAC); the MIC for 90% of 24 MAC isolates from AIDS patients was 0.25 microgram/ml as determined by a radiometric broth macrodilution assay. KRM 1648 was bactericidal for MAC isolates in Middlebrook 7H9 broth, with a reduction in viability of 1 to 4 orders of magnitude over 72 h. In human macrophages, KRM 1648 also was bactericidal, with a reduction of 3 to 4 orders of magnitude in CFU per ml of macrophage lysate at a concentration of 1 microgram/ml; however, the bactericidal activity varied approximately 10-fold among the three MAC serovars tested. In growth medium, ethambutol potentiated the effect of KRM 1648, but this potentiation was modest when tested against MAC in macrophages and also varied between MAC strains. KRM 1648 has potential as an antimycobacterial agent for MAC disease, perhaps in combination with other agents so that the use of lower dosages of KRM 1648 than are needed with other rifamycins may be possible.

Activities of bay Y 3118, levofloxacin, and ofloxacin alone or in combination with ethambutol against Mycobacterium avium complex in vitro, in human macrophages, and in beige mice.

Levofloxacin, ofloxacin, and Bay Y 3118 are new fluoroquinolones with variable in vitro bacteriostatic and bactericidal activities against the Mycobacterium avium complex (MAC). The potential therapeutic activities of these agents both alone and combined with ethambutol were evaluated in a human macrophage test system and in the beige mouse animal test system with MAC strain 101. Bay Y 3118 at a human-equivalent dose of 30 mg/kg/day for 4 weeks caused a significant reduction in mortality compared with that in untreated controls (P = 0.02). Bay Y 3118 also caused significant reductions in the number of MAC organisms in the blood, liver tissue, and spleen tissue compared with those in untreated controls. Levofloxacin at a human-equivalent dose of 200 mg/kg/day was associated with a significant reduction in mortality (10 versus 39%); however, treatment with either levofloxacin or ofloxacin (200 mg/kg/day) did not result in significant reductions in the numbers of MAC organisms in blood, liver, and spleen compared with those in untreated controls. When Bay Y 3118 was combined with ethambutol, there was no enhancement in therapeutic activity except in the spleen in terms of CFU per gram (reductions of 89% compared with the untreated control, 63% compared with Bay Y 3118 alone, and 72.5% compared with ethambutol alone). Levofloxacin in combination with ethambutol was more active than either drug alone in the reduction of organisms in blood, liver, and spleen. Bay Y 3118 was the most active fluoroquinolone for monotherapy of MAC infection in beige mice, and the combination of ethambutol plus either levofloxacin or ofloxacin was at least additive. In summary, this study demonstrates that quinolones, although active, are inhibitory against MAC in vivo and that there is little correlation between the activity of quinolones in vitro and the activity in mice.

The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase.

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.