Effect of aqueous extract from Scutellaria baicalensis Georgi roots on CD4+ and CD8+ T cell responses during experimental infection with Trichinella spiralis in mice.

The aim of this study was to investigate the effect of aqueous extract from Scutellaria baicalensis Georgi roots (SB) on blood parameters and immune response during an experimental trichinellosis. A total of 60 mice infected with 200 Trichinella spiralis larvae were assigned into two groups. One of them served as a control and the second received SB extract orally from day 5 before infection to day 28 after infection (dpi). Blood was sampled at 7, 14, 21 dpi. Lymphocytes obtained from the spleen and mesenteric lymph nodes (MLN) at 7, 14, 21, and 28 dpi were counted, CD4+ and CD8+ subpopulations were analyzed by flow cytometry, and lymphocyte proliferation was estimated with colorimetric (MTT) assay. The intensity of intestinal and muscle invasion was also studied. SB caused a remarkable elevation of banded neutrophils in the blood at 7 dpi. SB increased ConA-stimulated splenocyte proliferation and CD4+ and CD8+ splenocyte subsets at 14 and 21 dpi, whereas MLN lymphocyte subset stimulation involved only CD4+ at 14 dpi. After administration of SB a downward trend in the number of T. spiralis larvae in the muscle was observed. These results suggest that Scutellaria baicalensis root extract stimulates murine cellular immune response during intestinal phase of T. spiralis infection.

Modulation of lymphocyte subsets and humoral immune response by florfenicol administered to sheep red blood cell-immunized broiler chickens.

Florfenicol is a broad-spectrum bacteriostatic antibiotic commonly used for the treatment of systemic infections in farm animals. The aim of this study was to determine the effect of florfenicol on the percentage of T lymphocytes (CD3+, CD4+, CD8+, TCRgd+ cells) and B lymphocytes (Bu-1+ cells) and on total serum anti - sheep red blood cell (SRBC) haemagglutinin titer in the peripheral blood of SRBC-immunized broiler chickens. The study included three groups of broiler chickens differentiated by weight (0.5, 1.2, 2.4 kg). Florfenicol was administered orally at a dose of 30 mg/kg. The drug was administered eight times at 24 h intervals. The chickens were immunized with SRBC 24 h after administration of the third dose of florfenicol. Florfenicol increased the percentage of CD3+ blood lymphocytes with a corresponding decrease in the percentage of B lymphocytes in birds weighing 0.5 and 2.4 kg. Florfenicol reduced the production of total anti SRBC-haemagglutinins on day 5 after antigen injection in all three body weight groups of the broiler chickens. In conclusion, florfenicol exerted a modulating effect on the immune response of the birds and this should be taken into consideration when using this antibiotic for certain indications.

Influence of betulinic acid on lymphocyte subsets and humoral immune response in mice.

Betulinic acid is a pentacyclic triterpene found in many plant species, among others, in the bark of white birch Betula alba. Betulinic acid was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial, anticancer and anti-inflammatory activities. The effects of betulinic acid (50, 5, 0.5 mg/kg) administered orally five times at 24 hours intervals to non-immunized and red blood cells (SRBC)-immunized mice were determined. The present study examined the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes, and the percentage of subsets of T cells (CD4+CD8+, CD4CD8, CD4+, CD8+) in thymus,T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes, as well as white blood cell (WBC) and differential leukocyte counts in non-immunized mice, and humoral immune response in SRBC-immunized mice. SRBC was injected 24 hours after administration of the last dose of betulinic acid. It was found that betulinic acid administered orally five times at the dose of 0.5 mg/kg increased the total number of thymocytes, splenocytes, lymphocytes of mesenteric lymph node cells, and the weight ratio of the spleen and mesenteric lymph nodes in non-immunized mice. Betulinic acid also changed the percentage of T cell subsets in the thymus and T and B lymphocytes in peripheral lymphatic organs. The effects of betulinic acid on T and B cell subpopulations depended on the dose applied. The strongest stimulating effect of betulinic acid was observed when the drug was administered at the dose of 0.5 mg/kg. Five exposures to betulinic acid (0.5 mg/kg) decreased the percentage of immature CD4+CD8+ thymic cells with corresponding increases in the percentage and absolute count of mature, single-positive CD4+ thymocytes and decreased the percentage and total count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Multiple administration of betulinic acid at the investigated doses augmented the percentage and absolute count of CD19+ cells in the peripheral lymphatic organs. Moreover, betulinic acid at the dose of 5 mg/kg administered prior to SRBC immunization increased the number of plaque forming cells (PFC) but decreased the production of anti-SRBC antibodies on day 4 after priming. Thus, betulinic acid is a potential biological response modifier and may strengthen the immune response of its host.

Influence of bestatin, an inhibitor of aminopeptidases, on T and B lymphocyte subsets in mice.

Bestatin, a low-molecular weight dipeptide, is a potent inhibitor of aminopeptidase N which has been demonstrated to have antitumor and immunomodulatory effects. The effects of bestatin (10, 1 and 0.1 mg/kg) administered intraperitoneally once, five or ten times to mice on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes were studied. It has been found that bestatin administered ten times at doses of 10, 1 and 0.1 mg/kg increased the total number of thymocytes, splenocytes and lymphocytes of mesenteric lymph nodes. Bestatin also changed the percentage and the absolute number of T cell subsets in the thymus and T and B lymphocytes in the peripheral lymphatic organs. Five and ten exposures to bestatin (10, 1 and 0.1 mg/kg) increased the absolute count of both immature CD4+CD8+ and CD4-CD8- thymic cells. Moreover, both a single and multiple administration of bestatin (1 and 0.1 mg/kg) decreased the percentage and absolute count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Both a single and multiple administration of bestatin at all the doses under investigation augmented the percentage and the absolute count of CD19+ (B lymphocytes) in the peripheral lymphatic organs. The results of the study show that there is a relationship between the effect induced by bestatin and the dose of the drug as well as the number of doses applied. The strongest effect on the T and B lymphocyte subsets was noted after five injections of bestatin at doses of 1 and 0.1 mg/kg.

The effects of florfenicol on lymphocyte subsets and humoral immune response in mice.

Florfenicol is a broad-spectrum bacteriostatic antibiotic used in domestic animals. The aim of the study was to determine the effect of florfenicol on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the peripheral lymphatic organs in non-immunized mice and humoral immune response in sheep red blood cells (SRBC)-immunized mice. Florfenicol was administered orally at a dose of 30 mg/kg six times at 24 h intervals to non-immunized mice and four or seven times at 24 h intervals to SRBC-immunized mice. SRBC was injected 2 hours prior to the first dose of the drug. Florfenicol increased the percentage of CD4CD8- thymocytes and the absolute number of CD4+ and CD8+ thymocytes on day 7. The increased percentage and absolute number of CD3+, CD4+ and CD8+ lymphocytes in mesenteric lymph nodes and decreased percentage of lymphocytes B were also observed 24 hours from the last administration of florfenicol. Florfenicol administered after SRBC immunization reduced the number of plaque forming cells (PFC) and the production of anti-SRBC antibodies on days 4 and 7 after priming.

The effect of immunomodulating drugs on the percentage of apoptotic and necrotic lymphocytes in inflammatory infiltrations in the muscle tissue of mice infected with Trichinella spiralis.

In order to determine the effect of apoptosis and necrosis on the intensity of the muscular phase of infection by Trichinella spiralis, male CFW mice were orally infected with T. spiralis larvae and treated with some immunomodulating drugs: calf thymus extract (TFX), lipopolysaccharide from Escherichia coli (LPS), and dexametasone (DEX). Treatment with TFX increased the proportion of apoptotic lymphocytes and decreased the proportion of necrotic lymphocytes from 14 to 60 days after infection in mice infected with T. spiralis. Treatment with LPS increased proportions of both apoptotic and necrotic lymphocytes from 21 to 60 days after infection, especially at 28 days after infection. Treatment with DEX increased the proportion of apoptotic lymphocytes only at 28 days after infection, and significantly increased the proportion of necrotic lymphocytes at 21 days after infection. Parasite load in the affected muscle tissue was significantly lower than the control in mice treated with TFX, not significantly different from the control in mice treated with LPS, and significantly higher than the control in mice treated with DEX. The results of the study suggest that the parasite made an effort to reduce the effectivity of the host immune response in order to ensure its own survival.

Trichinella spiralis: Effect of thymus factor X on apoptosis and necrosis in mice.

The aim of the study was to determine the effect of thymus factor X (TFX-Jelfa) on the percentage of apoptotic and necrotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with 200 larvae of Trichinella spiralis. TFX was administered subcutaneously at a dose of 15mg/kg. On days 7, 14, 21, 28, 35, 42, and 60 after infection, apoptotic and necrotic cells were detected by flow cytometry after staining with the Annexin V-Fluos Staining Kit. TFX increased the percentage of apoptotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with T. spiralis. The effect of TFX on the percentage of necrotic lymphocytes was weaker and less clear. Parasite load was lower in infected mice treated with TFX than in the untreated control mice. The effect of TFX on the host immune response and the survival of parasite larvae was therefore probably affected by the extent of inflammatory infiltrates, and not by the percentage of lymphocytes undergoing apoptosis.

Effect of phytohaemagglutinin-P on apoptosis and necrosis in Trichinella spiralis infected mice.

Flow cytometry analyses were used to evaluate the contribution of apoptotic and necrotic lymphocytes in the selected organs of Trichinella spiralis infected mice treated with phytohaemagglutinin-P (PHA-P). The Tunnel method was used to examine apoptosis in a cryostat section from the jejunum and masseter muscle. CFW mice (Groups I and II) were infected with 200 larvae of T. spiralis. PHA-P was administered intravenously at a dose of 10mg/kg 24h prior to infection in Group II mice only. Group III mice were treated with PHA-P without T. spiralis infection, and Group IV mice were untreated controls. The lymphocytes obtained from the spleen, mesenteric lymph nodes (MLN) and muscular inflammatory infiltration on 7, 14, 21, 28, 35, 42 and 60 days post infection (DPI) were incubated with the Annexin-V-Fluos Staining Kit (Roche). The cryostat preparation made from the jejunum and masseter muscle was evaluated using a fluorescence microscope. PHA-P administration stimulated apoptosis in the jejunal mucosa and in the muscular inflammatory infiltration. In Group I mice, infected with T. spiralis only, the highest percentage of apoptotic cells was found on 7 DPI in the spleen and in MLN, and on 14 DPI among the cells of the muscular inflammatory infiltration. The peak of the necrotic lymphocytes was found on 7 DPI in the spleen, on 28 DPI in MLN, and on 21 DPI in the cells of muscular inflammatory infiltration. In Group II mice, infected with T. spiralis and treated with PHA-P, the peak in apoptotic cells occurred on 7 DPI in the spleen and in the muscular inflammatory infiltration. The highest level of necrotic lymphocytes was observed only on 7 DPI in the muscular inflammatory infiltration. Percentage of necrotic lymphocytes in the spleen was the same and in MLN it was lower than in Group I (T. spiralis only). Moreover, the number of muscle larvae in mice treated with PHA-P (Group II) was lower than in Group I (T. spiralis only).

Effect of calf thymus extract and zinc supplementation on the cellular response of mice exposed to restraint stress.

The studies were carried out on Balb/c mice exposed to restraint stress twice for 12 h at 24 h intervals. Prior to stress exposure, the mice were treated with calf thymus extract (TFX - Jelfa) i.p. at a dose of 10 mg/kg, ten times at 24 h intervals. TFX was used per se or with zinc ions interaction, by adding zinc ions (as sulfate salt) to drinking water at a dose of 72 microg/mouse per day. The results obtained show that restraint stress dramatically decreased the total number of thymocytes and splenocytes which is also accompanied by decreasing weight ratio of the thymus and spleen. The decreasing number of thymic and spleen cells corresponded to a diminishing percentage of immature, double-positive CD4+CD8+ thymocytes, mature single-positive CD4+ thymic cells and CD4+, CD8+ and CD19+ splenocytes. Changes in the number of thymic cells affect their activity, which is expressed as a decreased proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohaemagglutinin (PHA). Besides, exposure to the restraint stress decreased interleukin-1 (IL-1) production by murine intraperitoneal macrophages stimulated in vitro with lipopolisacharide (LPS) from E. coli. Previous treatment with TFX counteracted restraint stress-induced immunosuppression, which is expressed as partial normalisation of the total number of thymic and spleen cells, accelerated regeneration of these two lymphatic organs, shortned suppressive action of restraint stress on the percentage of immature CD4+CD8+ thymocytes and CD4+ splenocytes and in total normalisation of the CD4+ thymocytes and CD8+ splenocytes. TFX administered prior to restraint stress not only counteracted the suppresive effects of stress on the proliferative activity of thymic cells stimulated in vitro with Con A and PHA, but also augmented the proliferative response of these cells to two mitogens. The immunorestorative effect of TFX was augmented by zinc supplementation.

Comparative effects of fluoroquinolones on subsets of T lymphocytes in normothermic and hyperthermic mice.

The subsets of T lymphocytes in thymus, spleen and mesenteric lymph nodes were investigated in normothermic and hyperthermic mice treated with fluoroquinolones administered orally six times at 24 h intervals at doses of 15 or 75 mg/kg (flumequine, norfloxacin and ciprofloxacin) and 5 or 25 mg/kg (enrofloxacin). It has been found that fluoroquinolones can modulate CD3+, CD4+ and CD8+ marker expression on thymocytes, splenocytes and lymphocytes of mesenteric lymph nodes. Flumequine (15 mg/kg) decreased the percentage of immature CD4+CD8+ thymic cells and increased the percentage of mature CD4+ and CD8+. When the dose of flumequine was increased to 75 mg/kg a reduction in the maturation of thymocytes was observed. Administration of flumequine, norfloxacin and ciprofloxacin, irrespective of doses applied, increased the percentages of CD3+ splenocytes of CD4+ spleen cells. Exposure to enrofloxacin decreased the percentage of T helper-inducer cells. Flumequine and ciprofloxacin augmented the percentage of CD3+ mesenteric lymph node cells and increased the percentage of CD8+ cells. In contrast, norfloxacin and enrofloxacin decreased the percentage of CD3+ mesenteric lymph node cells and the percentage of CD4+ cells. Lipopolysaccharide (LPS) from E. coli (25 micro g/mouse) increased the percentage of single-positive CD4+ thymocytes, but did not affect the percentage of CD3+, CD4+ and CD8+ splenocytes and mesenteric lymph node cells. Flumequine and ciprofloxacin administered to mice pior to LPS potentiated its stimulant effect on the maturation of thymic cells ( increased percentage of mature CD4+ and CD8+ thymocytes). Pre-treatment with norfloxacin or enrofloxacin either reduced or did not modify the stimulant effect of LPS on maturation of thymic cells. Flumequine, norfloxacin, enrofloxacin and ciprofloxacin administered prior to LPS decreased the percentage of CD8+ splenocytes and increased the percentage of CD4+ spleen cells. Norfloxacin and ciprofloxacin at a dose of 75 mg/kg reduced the percentage of CD8+ mesenteric lymph node cells in hyperthermic mice. Pretreatment with norfloxacin at a dose of 15 mg/kg augmented the percentage of mesenteric lymph node cells. It was concluded that the modulating effects of fluoroquinolones depends on the chemial structure of drugs, dose administered as well as immunologic status.

Effects of thymomimetic drugs and zinc supplementation on the cellular immune response in hydrocortisone-suppressed mice.

The studies were carried out on Balb/c mice (5-6 weeks of age) exposed to immunosuppression by a single intraperitoneal dose (125 mg/kg) of hydrocortisone. Prior to hydrocortisone injection the mice were treated with diethyldithiocarbamate (DTC) intra-peritoneally at a dose of 20 mg/kg, five times at 48 h intervals or calf thymus extract (TFX) at a dose of 10 mg/kg, 10 times at 24 h intervals. The two drugs were used per se or in zinc ions interactions, by adding zinc ions (as sulphate salt) to drinking water at a dose of 72 microg/mouse per day. The results obtained in the study show that hydrocortisone injection drastically decreases the number of thymocytes and splenocytes, which is also accompanied by a decreasing weight ratio of the thymus and spleen. The decreasing number of thymic and spleen cells corresponds to a decreasing percentage of CD4+, CD8+ and CD19+ splenocytes and double positive CD4+CD8+ thymocytes. Changes in the number of thymic cells affect their activity, which is expressed in a decreased proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohaemagglutinin (PHA). It has also been found that a single hydrocortisone dose decreases interleukin (IL)-1 production by murine intraperitoneal macrophages stimulated in vitro with lipopolysaccharide (LPS) from Escherichia coli. TFX or DTC counteract hydrocortisone-induced immunosuppression, which is expressed in partial normalization of the total number of thymic and spleen cells, accelerated regeneration of the two lymphatic organs, shorter suppressive action of hydrocortisone on the percentage of CD4+, CD8+ splenocytes and double positive (CD4+CD8+) and CD4+ thymocytes. Furthermore, total counteraction against the suppressive action of hydrocortisone to proliferative activity of thymocytes stimulated in vitro with Con A and PHA was observed. TFX administered prior to hydrocortisone injection partially prevented the suppressive action of the drug on IL-1 production by intraperitoneal macrophages, but such an effect was not observed with DTC. The immunorestorative effect of TFX and DTC was augmented by zinc supplementation. The results obtained in the study show that neither TFX nor DTC administration per se and in interaction with zinc supplementation were able to change the suppressive effect of hydrocortisone on the percentage of B splenocytes (CD19+ cells).

Modulation of murine macrophages and T lymphocytes by lysozyme dimer.

The effects of lysozyme dimer (2 and 20 microg/kg) administered i.p. once and four times to mice on the phagocytic and killing ability of peritoneal macrophages, interleukin-1 (IL-1) production by murine macrophages stimulated in vitro with lipopolisaccharide of E. coli and expression of thymocyte, splenocyte and mesenteric lymphonode cell CD3+, CD4+ and CD8+ markers were studied. It was found that lysozyme dimer administered once or four times at doses of 2 microg/kg and 20 microg/kg augments the phagocytic and killing activity of peritoneal macrophages. The strongest stimulating effect was noted after four injections of lysozyme dimer at a dose of 20 microg/kg. Moreover, lysozyme dimer is able to modulate the production of IL-1 by murine macrophages stimulated in vitro with LPS. Exposure to four doses of lysozyme dimer (20 microg/kg) enhances the synthesis and release of IL-1, but this drug administered once (2 microg/kg and 20 microg/kg) or four times (2 microg/kg) decreases IL-1 production by peritoneal macrophages. It was also found that administration of lysozyme dimer at a dose of 20 microg/kg, irrespective of the number of doses applied, increases the percentage of CD4+ thymocytes and splenocytes. Moreover, exposure to four doses of lysozyme dimer (2 and 20 microg/kg) increases the percentage of CD4+ and CD8+ mesenteric lymphonode cells.

Modulatory effects of calf thymus extract on the subset of T lymphocytes in Trichinella spiralis-infected mice.

The immunotropic properties of calf thymus extract (TFX-Jelfa) is connected with the mimic action of the thymus to modulate the differentiation, maturation and function of prothymocytes and mature thymus dependent (T) cells. The studies were carried out on CFW male mice aged 3 months. The animals were infected per os with 200 larvae of Trichinella spiralis. TFX-Jelfa was administered i.p. at a dose of 10 mg/kg seven times at 24 hour intervals prior to infection. The percentage of CD4+ and CD8+ in suspension of splenocytes and mesenteric lymphonode cells by flow cytometry using monoclonal antibodies coupled with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) were determined. At the same time, cryostat preparations, made from jejunum and muscle samples, were examined by the direct immunofluorescence method using FITC-labeled antibody to mouse CD4+ and CD8+. It has been found that infection with T. spiralis in mice decreases the percentage of CD8+ splenocytes, while the percentage of CD8+ mesenteric lymphonode cells does not change. However, in infected mice the percentage of CD4+ spleen cells and mesenteric lymphonode cells is increased. It has been also found that during the course of infection an increase in the number of CD8+ and CD4+ cells in the basal lamina propria of the intestines was observed. In infected mice, CD4+ lymphocytes were visible in the inflammatory infiltrates of the muscle tissue on the 14th day, whereas CD8+ lymphocytes were first observed a week later. Pretreatment with TFX does not change the inhibitory effect of infection on the percentage of CD8+ splenocytes, but potentiates the percentage of CD4+ spleen cells and mesenteric lymphonode cells increased by infection. Furthermore, administration of TFX prior to infection also potentiates the stimulatory effect of T. spiralis on the number of CD8+ and CD4+ in the basal lamina propria of the jejunum, and on the number of CD8+ cells in the inflammatory infiltrates of the muscle tissue.

Dopamine production in the caudate putamen restores feeding in dopamine-deficient mice.

Dopamine-deficient (DD) mice cannot synthesize dopamine (DA) in dopaminergic neurons due to selective inactivation of the tyrosine hydroxylase gene in those neurons. These mice become hypoactive and hypophagic and die of starvation by 4 weeks of age. We used gene therapy to ascertain where DA replacement in the brain restores feeding and other behaviors in DD mice. Restoration of DA production within the caudate putamen restores feeding on regular chow and nest-building behavior, whereas restoration of DA production in the nucleus accumbens restores exploratory behavior. Replacement of DA to either region restores preference for sucrose or a palatable diet without fully rescuing coordination or initiation of movement. These data suggest that a fundamental difference exists between feeding for sustenance and the ability to prefer rewarding substances.

Dopamine-deficient mice are hypersensitive to dopamine receptor agonists.

Dopamine-deficient (DA-/-) mice were created by targeted inactivation of the tyrosine hydroxylase gene in dopaminergic neurons. The locomotor activity response of these mutants to dopamine D1 or D2 receptor agonists and l-3,4-dihydroxyphenylalanine (l-DOPA) was 3- to 13-fold greater than the response elicited from wild-type mice. The enhanced sensitivity of DA-/- mice to agonists was independent of changes in steady-state levels of dopamine receptors and the presynaptic dopamine transporter as measured by ligand binding. The acute behavioral response of DA-/- mice to a dopamine D1 receptor agonist was correlated with c-fos induction in the striatum, a brain nucleus that receives dense dopaminergic input. Chronic replacement of dopamine to DA-/- mice by repeated l-DOPA administration over 4 d relieved the hypersensitivity of DA-/- mutants in terms of induction of both locomotion and striatal c-fos expression. The results suggest that the chronic presence of dopaminergic neurotransmission is required to dampen the intracellular signaling response of striatal neurons.

Dopamine is required for hyperphagia in Lep(ob/ob) mice.

Feeding is a complex process responsive to sensory information related to sight and smell of food, previous feeding experiences, satiety signals elicited by ingestion and hormonal signals related to energy balance. Dopamine released in specific brain regions is associated with pleasurable and rewarding events and may reinforce positive aspects of feeding. Dopamine also influences initiation and coordination of motor activity and is required for sensorimotor functions. Thus, dopamine may facilitate integration of sensory cues related to hunger, initiating the search for food and its consumption. Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens, where they modulate movement and reward. There are projections from the nucleus accumbens to the lateral hypothalamus that regulate feeding. Dopamine-deficient mice (Dbh(Th/+), Th-/-; hereafter DD mice) cannot synthesize dopamine in dopaminergic neurons. They gradually become aphagic and die of starvation. Daily treatment of DD mice with L-3,4-dihydroxyphenylalanine (L-DOPA) transiently restores brain dopamine, locomotion and feeding. Leptin-null (Lep(ob/ob)) mice exhibit obesity, decreased energy expenditure and hyperphagia. As the hypothalamic leptin-melanocortin pathway appears to regulate appetite and metabolism, we generated mice lacking both dopamine and leptin (DD x Lep(ob/ob)) to determine if leptin deficiency overcomes the aphagia of DD mice. DD x Lep(ob/ob) mice became obese when treated daily with L-DOPA, but when L-DOPA treatment was terminated the double mutants were capable of movement, but did not feed. Our data show that dopamine is required for feeding in leptin-null mice.

Feeding behavior in dopamine-deficient mice.

Mice that cannot make dopamine (DA), a condition caused by the selective inactivation of tyrosine hydroxylase in dopaminergic neurons, are born normal but gradually become hypoactive and hypophagic, and die at 3 weeks of age. We characterized the feeding and locomotor responses of these DA-deficient (DA-/-) mice to 3, 4-dihyroxy-L-phenylalanine (L-DOPA) to investigate the relationship between brain DA levels and these complex behaviors. Daily administration of L-DOPA to DA-/- mice stimulated locomotor activity that lasted 6 to 9 hr; during that time the mice consumed most of their daily food and water. The minimal dose of L-DOPA that was sufficient to elicit normal feeding behavior in the DA-/- mice also restored their striatal DA to 9.1% of that in the wild-type (WT) mice at 3 hr; then DA content declined to <1% of WT levels by 24 hr. This dose of L-DOPA induced locomotor activity that exceeded that of treated WT mice by 5- to 7-fold, suggesting that DA-/- mice are supersensitive to DA. Unexpectedly, DA-/- mice manifested a second wave of activity 24 to 48 hr after L-DOPA treatment that was equivalent in magnitude to that of WT mice and independent of DA receptor activation. The DA-/- mice approached, sniffed, and chewed food during this second period of activity, but they ate <10% of that required for sustenance. Therefore, DA-/- mice can execute behaviors necessary to seek and ingest food, but they do not eat enough to survive.

Viral gene delivery selectively restores feeding and prevents lethality of dopamine-deficient mice.

Dopamine-deficient mice (DA-/- ), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA-/- mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior.

Dopamine-stimulated sexual behavior is testosterone dependent in mice.

Mice unable to synthesize dopamine (DA) in dopaminergic neurons were generated by gene-targeting techniques (Q.-Y. Zhou & R. D. Palmiter, 1995). These dopamine-deficient (DA-/-) mice required daily administration of 3,4-dihydroxyphenylalanine (L-DOPA) for survival beyond 2 to 3 weeks of age. This treatment stimulated mounting and aggressive behavior of adult DA-/- males toward both male and female mice. Both a nonspecific DA agonist (apomorphine) and a specific D1 agonist (SKF81297) stimulated aggression and mounting behavior; however, a D2 agonist (quinpirole) was less effective. Castration of male DA-/- mice demonstrated that these L-DOPA-stimulated behaviors depend on testosterone. In addition, replacement of testosterone to castrated males showed that the testosterone-responsive pathways of DA-/- males were more sensitive to testosterone than wild-type mice.

A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathionato)cadmium.

The yeast cadmium factor (YCF1) gene encodes an MgATP-energized glutathione S-conjugate transporter responsible for the vacuolar sequestration of organic compounds after their S-conjugation with glutathione. However, while YCF1 was originally isolated according to its ability to confer resistance to cadmium salts, neither its mode of interaction with Cd2+ nor the relationship between this process and organic glutathione-conjugate transport are known. Here we show through direct comparisons between vacuolar membrane vesicles purified from Saccharomyces cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild-type strain DTY165 that YCF1 mediates the MgATP-energized vacuolar accumulation of Cd-glutathione complexes. The substrate requirements, kinetics and Cd2+/glutathione stoichiometry of cadmium uptake and the molecular weight of the transport-active complex demonstrate that YCF1 selectively catalyzes the transport of bis(glutathionato)cadmium (Cd x +GS2). On the basis of these results--the Cd2+ hypersensitivity of DTY167, versus DTY165, cells, the inducibility of YCF1-mediated transport, and the rapidity and spontaneity of Cd-GS2 formation--this new pathway is concluded to contribute substantially to Cd2+ detoxification.