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In the present study we used both in vivo and in vitro murine models to investigate the effects of cocaine on the release of cytokines (IL-1 alpha, IL-6, and TNF-alpha by peritoneal macrophages and splenocytes, IL-2, IL-4, IL-5, IL-10, and IFN-gamma produced by splenocytes. In vitro cocaine (0.1, 1, 10, 100 micrograms/ml) exposure inhibited all cytokines produced by ConA-stimulated spleen cells and LPS-stimulated macrophages in a concentration dependent manner. Different effects of cocaine administration on cytokine production were observed when female C57BL/6 mice were injected intraperitoneally with cocaine (40 mg/kg body weight for six weeks). Secretion of IL-2 by splenocytes was significantly enhanced by cocaine administration, whereas IFN-gamma was not affected. Secretion of IL-4 and IL-10 by splenocytes was significantly inhibited by cocaine administration, while secretion of IL-5 by splenocytes was significantly enhanced. Secretion of IL-6 and TNF-alpha by splenocytes was significantly enhanced by cocaine administration. Secretion of IL-6 by peritoneal macrophages was also significantly enhanced by the cocaine, while production of IL-1 alpha was not affected. However, release of TNF-alpha by peritoneal macrophages was significantly reduced by the cocaine administration. Therefore use of cocaine may alter the balance of cytokine production, and thereby adversely affects immune response and host defense.

Severe infections in intravenous drug abusers could be the consequence of morphine-induced damage on the immune system. To evaluate the long-term effect of in vivo morphine administration on the immune system we developed an experimental model where we studied the combined effects of morphine treatment and protein malnutrition. We treated protein-undernourished mice daily for 11 weeks with increasing doses of morphine. Morphine treatment produced a decrease in body weight and spleen cell number. The changes observed were partially independent of the nutritional status of the host. Saline-injected mice showed a decrease in the percentage of Thy 1+ cells in the spleen. Morphine treatment induced a decrease in the total number of cells and therefore in the absolute number of T-(Thy 1, CD4, CD8), B- and Mac 1+ (macrophages) cells in protein-undernourished mice. Saline-injected mice showed a decrease in the percentage of Thy 1+ cells and an increase in the percentage of B- and Ia(+)-cells in the spleen. We conclude that morphine altered the immune system by down-regulating splenocyte proliferation. We also studied the effects of i.p. administered morphine on expression of thymocyte phenotype in well-nourished and protein-undernourished mice. In well-nourished mice, morphine treatment reduced the number of Thy 1+, CD4+ and CD8+ cells per thymus to 30% of that found in untreated mice and to 40% of the cells in those saline-treated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

A murine acquired immunodeficiency syndrome (MAIDS) is induced in genetically susceptible strains of mice inoculated with LP-BM5 murine leukemia virus. It is characterized by progressive lymphoproliferation, profound immunodeficiency, and the subsequent loss of resistance to opportunistic pathogens, including intestinal pathogens. Cellular and/or humoral immunity of gut-associated lymphoid tissues may play a key role in the elimination of these pathogens. We have previously demonstrated reductions in the number of mucosal T and B cells in MAIDS. In this study, the cytokine production by mesenteric lymph nodes (MLN) cells and their proliferative response to mitogens during MAIDS were investigated. Alterations were observed in the kinetics of MLN cell proliferation and cytokine secretion by in vitro mitogen-stimulated MLN cells during the retrovirus infection. Cytokine production was abnormally changed, with a gradual decrease in interleukin-2 (IL-2) production as well as an increase in IL-5 and IL-6 secretion. Interferon-gamma production was increased during the progression to MAIDS. The dysregulated release of cytokines by MLN cells due to retrovirus infection could lead to immune dysfunction. These data indicate that dysregulated cytokine secretion by MLN cells may be responsible for impaired mucosal immunity in AIDS, explaining the dramatic increase of opportunistic intestinal pathogens in individuals with AIDS.

Intravenous heroin abusers suffer a great variety of infections, including AIDS (acquired immune deficiency syndrome). We developed an experimental mouse model to evaluate the long-term effect of in vivo morphine administration during retrovirus-induced immune dysfunction. Mice were treated daily for 11 weeks with increasing doses of morphine. Morphine treatment produced a decrease in body weight and spleen cell number. Murine retrovirus infection provoked an increase in body weight due to enlargement of lymphoid organs, and an increase in the percentage and absolute number of CD4+ and Mac 1+ cells. Interestingly, retrovirus-infected mice that were also morphine-treated did not show the increase in the relative proportion of Mac 1+ cells. Moreover, under the experimental conditions of protein-malnutrition and morphine treatment potentiation of immune dysfunction by murine retrovirus infection was investigated. Retrovirus infection-induced splenocyte proliferation was partially regulated by morphine treatment. Splenocytes from retrovirus-infected mice presented a higher percentage of IL-2R+ cells and, lower levels of sIL-2R in splenocyte supernatants. Mitogen-stimulated splenocytes had a lower production of interferon-gamma as well as an increase in the secretion of tumor necrosis factor-alpha. Thus morphine altered the immune system by down-regulating splenocyte proliferation, because retrovirus infection-induced splenocyte proliferation was partially regulated by morphine treatment. We also evaluated the effects of joint murine retrovirus infection and protein undernutrition on the thymus cell subsets. Retrovirus infection was associated with a decrease in the absolute number of Thy 1+, CD4+ and CD8+ cells per thymus with the CD8+ cell subset being the most affected. Moreover, retrovirus-infected mice presented a dramatic decrease in the percentage of double-positive (CD4+ CD8+) cells in the thymus as well as changes in its immunoarchitecture. While protein undernutrition alone did not produce further differences between infected versus non-infected, protein-undernourished, morphine treatment induced a greater decrease in thymocyte number than that seen in retrovirus- or morphine-treated animals alone.

Alcoholics have frequent bronchial and intestinal bacterial-associated infections compromising the mucosal-associated lymphoid tissues. Alcoholics also suffer undernutrition due to changes in their lifestyles. Thus, we studied the intestinal mucosa-associated lymphoid tissues using two types of diets in young and mature mice. The objective was to elucidate if ethanol consumption alters the T and B cell populations in the intestine, and if the alteration could be associated with the age and/or the nutritional status of the host. There was an increase in the number of CD8+ cells per field in the intestinal lamina propria (ILP) of young mice consuming ethanol, with no change in CD4+ and IgA+ cells in the ILP and CD4+ and CD8+ cells in the intestinal intraepithelium (IE). There was also an increase in the number of CD4+ and CD8+ cells in the ILP of well-nourished mature mice consuming ethanol, and an increase in the number of CD4+ cells in the ILP of undernourished mice. An increase in the number of CD8+ cells in the IE was also detected in the well-nourished group consuming ethanol. Therefore, we conclude that ethanol administration alters the intestinal mucosa-associated lymphoid tissue.

Ethanol (ETOH) consumption has been associated with general suppression of the immune response, resulting in increased susceptibility to infection. Chronic dietary ETOH consumption may be one of the cofactors accelerating development of human acquired immune deficiency syndrome (AIDS) after retrovirus infection. Chronic dietary ETOH [5% (v/v)] in the Lieber-DeCarli liquid diet was fed female C57BL/6 mice inoculated with LP-BM5 retrovirus causing murine AIDS for 11 weeks. Because cytokines are key regulators of humoral and cellular immunity, their production by concanavalin A (ConA) and lipopolysaccharide (LPS)-induced splenocytes was measured by ELISA methods. Decreased levels of interleukin (IL)-2 caused by retrovirus infection remained unchanged. Elevated levels of IL-5 and IL-6 produced in vitro by ConA-stimulated spleen cells during retrovirus infection were significantly further increased by dietary ETOH. Elevated IL-4 due to retroviral infection were not affected by dietary ETOH. Increased production of IL-10 induced by retrovirus infection, however, was significantly reduced by dietary ETOH, whereas decreased release of interferon-tau induced by retrovirus infection was significantly enhanced. Elevated levels of tumor necrosis factor-alpha produced by LPS-stimulated splenocytes from retrovirus infected mice were significantly further increased by dietary ETOH, whereas levels of IL-6 by LPS-stimulated splenocytes were not affected. Suppressed T-cell proliferation caused by retrovirus infection was significantly reduced further by dietary ETOH. However, no effect of dietary ETOH was observed on decreased B-cell proliferation by retrovirus infection. These results suggest that dietary ETOH aggravates progression of immune dysfunction leading to AIDS, because dietary ETOH modifies production of immunological regulatory cytokines.

The effects of inoculation of LP-BM5 murine leukemia retrovirus and chronic ethanol (5% v/v) ingestion on immunomodulation and Cryptosporidium parvum infection in C57BL/6 female mice were evaluated. The intestinal mucosae of retrovirally immunosuppressed animals were heavily colonized by Cryptosporidium parasites, and oocysts shedding in the feces persisted throughout the duration of the study. Mortality was exacerbated by murine retrovirus infection alone and exacerbated with concomitant chronic alcohol feeding (42.8 and 69.4%). Chronic ethanol ingestion decreased production of interferon-gamma and soluble interleukin-2 receptor released in supernatants of splenocytes when stimulated with concanavalin A, compared with the control group. Decreased production of interferon-gamma and interleukin-2 receptor was further exacerbated due to retrovirus infection. Tumor necrosis factor production by splenocytes stimulated with lipopolysaccharide, however, was significantly increased because of retrovirus infection. LP-BM5 retrovirus infection alone as well as with concomitant ethanol feeding altered cytokine production, which might have led to immunodeficiency. These changes may help explain the enhanced persistence of Cryptosporidiosis.

The therapeutic efficacy of pooled bovine colostrum for the control of cryptosporidiosis was investigated during murine acquired immunodeficiency syndrome in female C57Bl/6 mice. Mice were infected with LP-BM5 murine leukemia retrovirus for four months and then inoculated with Cryptosporidium parvum oocysts. Persistent cryptosporidiosis was established in all retrovirus immunosuppressed mice, while control mice were refractory to infection. Parasite colonization of intestinal villi was significantly (P < 0.05) reduced in immunosuppressed animals that received dietary supplemental pooled bovine colostrum compared with to those that did not receive colostrum treatment. Similarly, shedding of oocysts in the feces of immunosuppressed animals that received dietary pooled bovine colostrum was significantly (P < 0.05) reduced compared with those that did not at 26 days post-parasite challenge. Since the nonimmune bovine colostrum contained no anti-Cryptosporidium antibodies, this suggests that passively transferred antibodies alone are unlikely to have provided the improved resistance shown in this study.

Female C57BL/6 mice were infected with LP-BM5 retrovirus, causing murine AIDS, which is functionally similar to human AIDS. Retrovirus infection targeted the thymus, producing altered T cell differentiation via the dysregulation of thymocyte cytokine production. Human AIDS causes vitamin deficiencies, therefore the effects of dietary vitamin E supplementation were determined on the kinetics of cytokine production by concanavalin A-stimulated thymocytes in uninfected normal mice and mice with murine AIDS. Dietary supplementation, with a 15-fold increase in vitamin E (160 IU/l) in the liquid diet (National Research Council), modulated interleukin-2 (IL) production in both uninfected mice and retrovirus-infected mice. Vitamin E significantly reduced the level of IL-4 secretion in the uninfected mice at 4 and 8 weeks, but not at 12 and 16 weeks. It also significantly reduced IL-4 production, elevated by retrovirus infection. Vitamin E significantly reduced IL-6, and interferon-gamma production increased in murine AIDS. The effects of dietary vitamin E on concanavalin A-induced proliferation of thymocytes were consistent with the finding of changes in IL-2 secretion. No effects of dietary vitamin E on thymus weight were observed in uninfected or retrovirus-infected mice, whereas vitamin E significantly increased serum and thymic vitamin E concentration, which had been reduced by retrovirus infection. These data indicate that dietary vitamin E supplementation can modulate cytokine production by thymocytes, affecting T cell differentiation, especially during retrovirus-induced immune dysfunction.

The effect of cocaine and LP-BM5 murine leukemia retrovirus infection on tumor necrosis factor (TNF) production were investigated. Three types of macrophages were used 1) peritoneal macrophage (PM), 2) thioglycollate induced peritoneal macrophage (TPM), and 3) alveolar macrophage (AM). Cells were cultured with and without cocaine during in vitro stimulation by lipopolysaccharide (LPS) and gamma-interferon (IFN). Retroviral infection enhanced the TNF production by PM and AM, but not by TPM. Intraperitoneal cocaine injection reduced TNF production by PM, but increased TNF production by AM and TPM. TNF production by AM from cocaine injected mice was stimulated by cocaine applied in vitro. In contrast, 100 micrograms/ml of cocaine in vitro significantly inhibited the TNF production by TPM from uninfected and retrovirally infected mice. Thus, TNF production by macrophages is modulated by murine retroviral infection and cocaine treatment. This could play an important role in host defense.

C57BL/6 female mice were fed a liquid ethanol (ETOH) diet (27% of calories derived from ETOH) for 5 months as an animal model of chronic alcohol use. A isocaloric liquid diet supplemented with maltose dextran was fed to controls. Splenocytes from ETOH-treated and control mice, and purified macrophages from normal mice and retrovirus infected mice were exposed in vitro to various concentrations of ETOH (0.1-1.0% v/v). The effect of chronic ETOH exposure in vivo, and of in vitro treatment with ETOH on tumor necrosis factor (TNF) and gamma-interferon (IFN) production by cultured murine splenocytes and purified macrophages was investigated. Dietary ethanol did not significantly affect in vitro TNF or IFN production. However, TNF and IFN production by splenocytes from mice fed either the ETOH or control diet decreased significantly when the cells were cultured with ETOH and stimulated with LPS or Con A in vitro. Thus ETOH in vitro directly down regulates TNF and IFN secretion by LPS- or Con A-stimulated spleen cells. ETOH treatment in vitro did not significantly change TNF production by purified peritoneal macrophage (PM) and thioglycollate-induced peritoneal macrophage (TPM) from normal mice, but increased TNF production by alveolar macrophage (AM). Although murine retrovirus infection per se increased TNF production it did not change the responsive pattern in TNF production of PM and TPM to ETOH in vitro and reduced the TNF production of AM.

Retrovirus infection, cocaine administration, and nutritional deficiencies are known to individually produce impairment of the immune system. Therefore, we developed a murine model to study the effect of daily cocaine administration, protein malnutrition, and retrovirus infection causing murine AIDS on the lymphoid cell populations of the thymus. C57BL/6 female mice fed a diet containing 4% protein were studied following chronic cocaine administration and LP-BM5 murine leukemia virus (MuLV) infection. Cocaine administration reduced body and thymus weight. Cocaine partially prevented thymus enlargement due to lymphoid cell proliferation induced by murine retrovirus infection. Cocaine treatment affected dramatically the thymus of protein-malnourished mice where the absolute number of Thy 1.2+, CD4+ and CD8+ cells represented only 10% of the control values. Daily saline injection also induced a significant decrease in the number of Thy 1.2+, CD4+ and CD8+ cells per thymus. These results suggest that the thymus glands of mice fed a low protein diet were susceptible to stress. Retrovirus infection provoked a decrease in the percentage and absolute number of Thy 1.2+, CD4+ and CD8+ cells in the thymus. This effect was potentiated by cocaine treatment. Therefore, cocaine was able to potentiate the impairment of the immune system caused by MuLV infection. We consider that cocaine could alter the immune system by altering the expression of T cell differentiation markers after direct interaction with thymocytes or through the neuroendocrine-thymus axis. Moreover, this effect was more dramatic and severe during protein malnutrition.

LP-BM5 murine leukemia virus (MuLV) infection and cocaine administration are known to impair the murine immune system. We have developed a murine model to study the effect of daily cocaine administration and retrovirus infection on the lymphoid cell populations of the thymus. C57BL/6 female mice were studied following chronic cocaine administration for 11 weeks with simultaneous LP-BM5 MuLV infection. Cocaine administration reduced body and thymus weight, significantly reduced the number of CD8+ cells in the thymus, and partially prevented thymus enlargement due to lymphoid cell proliferation induced by LP-BM5 MuLV infection. Retrovirus infection was associated with a decrease in the percentage and absolute number of Thy 1.2+, CD4+, and CD8+ cells in the thymus, an effect potentiated by cocaine administration. Therefore cocaine impairs thymic function by altering the number of cells expressing T cell differentiation markers in MAIDS.

We have developed an experimental mouse model to study the effect of daily cocaine administration on the immune system during an acquired immune deficiency syndrome (AIDS). Mice were infected with LP-BM5 murine leukemia virus, a retrovirus which causes immunosuppression with the development of functional murine AIDS. Increasing doses of cocaine given by daily intraperitoneal injection for 11 weeks reduced body weight. A daily cocaine injection in some mice as well as a saline injection in others showed a decrease in the percentage of Thy 1.2+, CD4+ and CD8+ cells, while both treatments increased the percentage and absolute numbers of B-cells per spleen. Saline and cocaine treatment induced an increase in gamma-IFN and TNF-alpha production by splenocytes. Cocaine treatment favored a decrease in sIL-2R secretion. Saline and cocaine treatment had slightly different effects on the splenocytes of protein-malnourished mice. Cocaine treatment induced an increase in the percentage of CD8+ cells. Saline and cocaine treatments decreased the number of Mac 1+ cells in the spleen. Moreover, saline- and cocaine-treated protein-malnourished mice splenocytes did not present the increase in gamma-IFN production as well-nourished mice splenocytes showed. Retrovirus-infected mice showed a decrease in the percentage of Thy 1.2+ and CD8+ cells and an increase in the percentage and absolute numbers of CD4+, IL-2R+, Mac 1+ and B-cells. Cocaine partially prevented the enlargement of lymphoid organs due to lymphoid cell proliferation induced by murine retrovirus infection, but had little effect on the elevated percentage of CD4+ cells or B-cells or the depressed numbers of CD8+ cells associated with virus infection. However, cocaine did reduce the number of activated IL-2R+ cells and macrophages (Mac 1+) in addition to reducing the total number of cells per spleen in all subsets in retrovirus-infected mice, but not in uninfected controls. Cocaine treatment and retrovirus infection alone or in combination suppressed the release of sIL-2R into supernatant fluid during in vitro culture of splenocytes. These data illustrate that cocaine treatment modulates cell proliferation in retrovirus-infected mice and thus modifies the absolute number of cells in those subsets already altered by retrovirus infection. Retrovirus-infected and retrovirus-infected cocaine-treated protein-malnourished mice showed similar results.

The antitumorigenic effects of carotenoids, in addition to their immuno-enhancing effects, may occur by their direct action on growing tumor cells. To test this hypothesis the direct inhibitory effect of various concentrations of canthaxanthin (CX; 4,4'-diketo-beta-carotene), a non-provitamin A carotenoid, was tested on the in vitro growth of JB/MS, B16F10 melanomas and PYB6 fibrosarcoma and murine non-transformed NIH-3T3 (ATCC CRL 1658) cells. At concentrations of 1 x 10(-8) M up to 1 x 10(-4) M, CX significantly reduced the overall number of tumor cells. The greatest inhibition was observed at a CX concentration of 1 x 10(-4) M after 72 h and 96 h of incubation. However, CX had no inhibitory effect on the growth of the non-transformed NIH-3T3 cell line; rather it significantly enhanced growth of this cell line (P less than 0.05) after 96 h of incubation. Thus, the inhibitory action of CX on growing tumor cells appears to be due to its direct actions on tumor cells and not via its conversion to vitamin A or its immuno-enhancing effects.

LP-BM5 murine leukemia virus (MuLV) infection induces an immunodeficient state in susceptible strains of mice. It has been previously characterized at the level of spleen and peripheral lymph nodes. We recently demonstrated that LP-BM5 MuLV-infected mice lost intestinal host resistance to common opportunistic pathogens. In this article we investigated how murine retroviral infection alters the differentiation of IgA B cell precursors in Peyer's patches (PP), mesenteric lymph nodes (MLN), and the intestinal lamina propria (ILP). After 4 months of LP-BM5 MuLV infection, there was a significant decrease in the absolute numbers of Thy1+, CD4+, and CD8+ cells in PP with a concomitant decrease in the percentage and in the absolute numbers of surface IgA+--(sIgA+) and surface IgM+--bearing (sIgM+) cells. Infection also produced an enlarged MLN with a six-fold increase in cell numbers and a decrease in the relative percentage of sIgA+, cytoplasmic IgA+, and cytoplasmic IgM+ cells. However, murine retrovirus infection caused no significant changes in the percentages of Thyl+, CD4+, CD8+, and CD5+ cells in the MLN. After 4 months of murine retrovirus infection cIgA+ cells from MLN were not able to populate the intestinal lamina propria as the number of IgA plasma cells was significantly decreased. Moreover, there was a concomitant decrease in the number of CD4+ cells per field in the ILP. These results suggest that murine retrovirus infection favors the expansion of IgA B cell precursors at the level of MLN, while simultaneously interfering with the terminal differentiation step and thus preventing IgA plasma cell precursors from seeding the ILP.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar macrophages (Am phi s), resident peritoneal macrophages (RPm phi s), and thioglycolate-elicited peritoneal macrophages (TGPm phi s) were isolated from C57BL/6 mice and incubated with lipopolysaccharide (LPS), stimulated cell supernatant, or recombinant interferon gamma (IFN-gamma) for 24 h. Tumor necrosis factor (TNF) in cell-free supernatants was measured by enzyme-linked immunosorbent assay. Amo phi s incubated with 10(3) ng/ml LPS produced 50 times more TNF than RPm phi s and 5 times more than TGPm phi s, and LPS alone induced maximum TNF production by Am phi s. Stimulated cell supernatant or recombinant IFN-gamma alone did not induce TNF production. A combination of LPS with stimulated cell supernatant or IFN-gamma had only a limited synergistic effect on TNF production by Am phi s. However, both LPS and stimulated cell supernatant or recombinant IFN-gamma induced maximum TNF production by RPm phi s and TGPm phi s. TGPm phi s showed greater sensitivity to LPS and stimulated cell supernatant or IFN-gamma with regard to TNF production than the other macrophage populations investigated.

Campylobacter jejuni is a major cause of human enteritis which mimics the inflammatory bowel disease (IBD). In this study, microstructural changes on the surfaces of the murine gastrointestinal tract persistently colonized by Campylobacter jejuni, strain GJ-S131, were investigated by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results revealed that the appearance of the gastrointestinal mucosa in both BALB/C and KM mice resembled that in human with inflammatory bowel disease. Under SEM, the mucosa of the jejunum and ileum, with broken or distorted villi had a "worm eaten" look; crypts were irregular in shape and size, and the mucosa showed atrophy, especially in the colon. Epithelial junctions demonstrated furrows, clefts or deep crevasses, with exudates containing a large number of leukocytes. Cytologic appearances were characterized by microvilli dysplasia and/or atrophy, patchy erosions or necrosis and pelade-like appearance due to absence of microvilli, which were similar to the findings under TEM.

An experimental model which resembles human drug addiction was developed to study the effect of chronic drug (cocaine or morphine) administration on the immune system. As malnutrition has been associated with drug use, a low protein diet has been evaluated for its contribution to the impairment of the immune system during cocaine/morphine addiction. Female C57BL/6 mice that received a 20% or 4% casein diet were studied. Both drugs were administered intraperitoneally daily for 11 weeks and drugs were administered in increasing daily doses, beginning after 3 weeks of diet consumption. Doses of cocaine began with 5 mg/kg body weight and reached the maximum dose of 40 mg/kg/day at the fourth week. Doses of morphine gradually increased from 10 mg/kg to 75 mg/kg body weight with the maximum dose reached after 5 weeks of treatment. Cocaine administration reduced body weight, particularly in the low protein diet group, and spleen weight in protein malnourished mice. Cocaine as well as saline injected mice showed a decrease in the percentage of CD4+ CD8+ and Mac-1+ cells and an increase in B cells in the spleens of well nourished mice. Morphine-treated mice showed similar results to those observed in cocaine or saline treated mice. These results suggest that cocaine, morphine or saline injection can alter the percentage of cells that express a defined phenotype independently of the nutritional status of the subject. Moreover, the effect appears dependent on a stress mediated process.

Methane monooxygenase catalyzes the oxygenation of 1,1-dimethylcyclopropane in the presence of O2 and NADH to (1-methylcyclopropyl)methanol (81%), 3-methyl-3-buten-1-ol (6%), and 1-methyl-cyclobutanol (13%). Oxygenation by 18O2 using the purified enzyme proceeds with incorporation of 18O into the products. Inasmuch as methane monooxygenase catalyzes the insertion of O from O2 into a carbon-hydrogen bond of alkanes, (1-methylcyclopropyl)methanol appears to be a conventional oxygenation product. 3-Methyl-3-buten-1-ol is a rearrangement product that can be rationalized on the basis that enzymatic oxygenation of 1,1-dimethylcyclopropane proceeds via the (1-methylcyclopropyl)carbinyl radical, which is expected to undergo rearrangement with ring opening to the homoallylic 3-methyl-3-buten-1-yl radical in competition with conventional oxygenation. Oxygenation of the latter radical gives 3-methyl-3-buten-1-ol. 1-Methylcyclobutanol is a ring-expansion product, whose formation is best explained on the basis that the 1-methylcyclobutyl tertiary carbocation is an oxygenation intermediate. This cation would result from rearrangements of carbocations derived by one-electron oxidation of either radical intermediate. The fact that both 3-methyl-3-buten-1-ol and 1-methylcyclobutanol are produced suggests that the oxygenation mechanism involves both radical and carbocationic intermediates. Radicals and carbocations can both be intermediates if they are connected by an electron-transfer step. A reasonable reaction sequence is one in which the cofactor (mu-oxo)diiron reacts with O2 and two electrons to generate a hydrogen atom abstracting species and an oxidizing agent. The hydrogen-abstracting species might be the enzymic radical or another species generated by the iron complex and O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Described are some pharmacokinetic parameters for 5-fluorouridine, a potentially useful intermediate metabolite of 5-fluorouracil (5-FU), following subconjunctival and intravitreal injection in the pigmented rabbit. Immediately following a 500 microgram intravitreal injection of 3H-5-fluorouridine, a peak ipsilateral vitreous concentration of 308 micrograms/ml was measured. This decreased to 199 micrograms/ml at 2 hours, 33 micrograms/ml at 12 hours and 4.4 micrograms/ml at 24 hours, indicating that 5-fluorouridine is cleared from the vitreous at a rate similar to 5-fluorouracil. Aqueous concentrations in the ipsilateral eye reached a maximum at 6 hours (9.6 micrograms/ml) and decreased to 0.8 micrograms/ml at 24 hours. Following subconjunctival injection of 5 mg 3H-5-fluorouridine (0.5 ml), peak ipsilateral anterior chamber concentration of 10.7 micrograms/ml was measured 1 hour after injection and decreased to 2.4 micrograms/ml after 12 hours. Maximum vitreal concentrations of 1.7 micrograms/ml were measured 12 hours after injection.

In this study the mitogenic effect of demineralized bone powder (DBP) on cells in tissue culture was evaluated. The cells studied have osteoblastic properties. At a DBP concentration of 100 micrograms/well, rat osteosarcoma cells (ROS) 17/2.8 cells, ROS 24/1 cells, and periosteal fibroblasts (POF) all showed a significant increase in proliferation in comparison to controls. The peak effect was on day two for ROS 17/2.8 (125% of control), on day three for ROS 24/1 (139% of control), and on day four for POF (145% of control). The results indicate that this in vitro system may be useful for evaluating the osteoinductive activity of DBP preparations.

The reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol 1,2-dioxygenase and catechol 2,3-dioxygenase from Pseudomonas putida were examined. Both 3-substituted catechols are oxidized by catechol 2,3-dioxygenase at approximately 30% of the rate observed for catechol oxidation by this enzyme. Analysis of the products of the reactions showed that ring cleavage occurs in a normal fashion between carbons 2 and 3 of the alternate substrates. 3-Ethylcatechol is oxidized by catechol 1,2-dioxygenase at about 6% of the rate of catechol oxidation; ring cleavage occurs between carbons 1 and 2 to give 2-ethyl-cis,cis-muconic acid. However, 3-(methylthio)catechol is a very poor substrate for catechol 1,2-dioxygenase (0.8% of the rate of catechol), but it is a potent competitive inhibitor (Ki = 0.6 microM). The effects of 3-(methylthio)catechol and 3-ethylcatechol on the visible and EPR spectra of catechol 1,2-dioxygenase are also reported.

A variety of analogues of stearic acid in which one of the methylene groups was replaced by a sulfur atom were examined as inhibitors of growth and fatty acid biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The 8-, 9-, 10-, and 11-thiastearic acids were found to suppress the synthesis of the cyclopropane-containing fatty acid dihydrosterculic acid (9,10-methyleneoctadecanoic acid) at micromolar concentrations in the growth medium, and all but the 9-thiastearate were found to inhibit the growth of the protozoa at concentrations. The most potent inhibitor, 8-thiastearic acid (I50 for growth = 0.8 microM; I50 dihydrosterculate synthesis = 0.4 microM), was also observed to inhibit the synthesis of gamma-linolenic acid at a similar concentration. The sulfoxide derivatives of the 9- and 10-thiastearates were found to have little effect on growth or fatty acid synthesis, and several long-chain amides of 3-amino-1,2-propanediol were found to have effects similar to those of the fatty acids from which they were derived.