NMDA Receptors are Expressed in Lymphocytes Activated Both In Vitro and In Vivo

There is increasing evidence showing that the interplay between neuronal and immune systems may be regulated by neuromediators. However, little is known about the involvement of glutamatergic system in such neuro-immune relations. In the present study, we have shown that some intact lymphocytes express N-methyl-d-aspartate activated receptors (NMDA receptors), an important constituent of glutamatergic system. The activation of lymphocytes with phytohemagglutinin (PHA) induces a time-dependent increase in the amount of NMDA receptor presenting cells, and NMDA stimulates this process. Immune response of such lymphocytes is suppressed and the amount of cells producing interferon γ (IFN-γ) in vitro is decreased to the level corresponding to intact (non-activated) cells. Furthermore, lymphocytes in the region of inflammation, induced by spinal cord injury (SCI), are also NMDA-positive. We suggest that expression of NMDA receptors in lymphocytes is regulated by central nervous system, which controls the inflammation process.     

Requirements for Sod Mimics Operating In Vitro to Work Also In Vivo

When an efficient SOD mimic operating in vitro is introduced into cells, the following requirements are needed in order that this compound will catalyze O^?₂ dismutation efficiently: it should be non toxic, stable, has a long metabolic half life, does not form ternary complexes with the cell components, its reduced form reacts slowly with molecular oxygen. should be able to cross cell membranes and also to reach lipophilic or hydrophobic regions. Thus, it seems that finding an efficient compound that has high SOD-like activity in vivo will not be easily achieved.     

Selective Pressures on RNA Hairpins In Vivo and In Vitro

Comparison of the most stable potential hairpins in the sequences of natural ribozymes with those in the randomized sequences has revealed that the hairpin loop energies are lower than expected by chance. Although these hairpins are not necessarily parts of functional structures, there is a selective pressure to diminish the destabilizing free energies of the hairpin loops. In contrast, no significant bias is observed in the stacking values of the most stable stems. In the ribozymes isolated in vitro the loops of potential hairpins are closer to random values, which can result in less efficient folding rates. Furthermore, the effects of kinetic traps seem to be more significant in the folding pathways of the in vitro isolates due to a potential to form stable stacks incompatible with the functional folds. Similarly to natural ribozyme sequences, the untranslated regions of viral RNAs also form hairpins with relatively low loop free energies. These evolutionary trends suggest ways for efficient engineering of improved RNA constructs on the basis of analysis of in vitro isolates and approaches for the search of regions coding for functional RNA structures in large genome sequences. Received: 12 January 2001 / Accepted: 21 May 2001     

In Vivo and In Vitro Action of Norethindrone on Staphylococci

Norethindrone has been examined in vitro for antibacterial activity against 10 microorganisms. Turbidimetric techniques were used to assay the antibacterial activity of norethindrone. The organisms tested included Staphylococcus aureus, S. epidermidis, Micrococcus conglomeratus, Listeria monocytogenes, Streptococcus faecalis, Salmonella typhosa, Shigella flexnerii, Klebsiella pneumoniae, Escherichia coli, and Proteus vulgaris. Bacteriostatic action was shown only against the gram-positive microorganisms when they were grown anaerobically in Tryptic Soy Broth containing 10 to 50 mug of norethindrone per ml. The bacteriostatic action of norethindrone was exerted primarily during the first 8 hr of incubation and it was reduced by the presence of oxygen. Mestranol at a concentration of 1 to 10 mug/ml failed to exert any significant action on S. aureus. However, incorporation of 5 mug of mestranol per ml in the culture medium enhanced the bacteriostatic action of norethindrone on staphylococci. Enhancement of the bacteriostatic action of norethindrone could not be obtained by the addition of a concentration of 5 mug/ml of testosterone, 17alpha-estradiol, and 17beta-estradiol. Progesterone and 4-pregnen-20beta-ol-3-one under similar conditions showed an additive bacteriostatic effect when they were incorporated into the culture medium containing norethindrone. In vivo studies indicated that female, adult New Zealand rabbits, injected subcutaneously with two injections of 10 to 20 mug of norethindrone, 24 hr apart, and challenged intradermally with S. aureus 4 hr after the second injection, had fewer lesions with smaller areas of swelling and erythema as compared to control, nontreated rabbits. The protective effect of norethindrone on the development of staphylococcal lesion seemed related to hormone concentration. Thus, it was demonstrated with doses of 20, 15, and 10 mug, but not with doses of 1 and 5 mug. When the lesions were excised 48 to 92 hr after infection and when viable cell counts were made, rabbits treated with norethindrone showed significantly lower staphylococcal counts than the control rabbits. During the 1st day after infection with S. aureus, leukocytic counts of the norethindrone-treated rabbits remained normal, whereas control animals showed elevated leukocytic counts.     

In Vitro and In Vivo Characterization of the Alkaloid Nuciferine

Rationale

The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.

Methods

Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.

Results

Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT_2A, 5-HT_2C, and 5-HT_2B, an inverse agonist at 5-HT₇, a partial agonist at D_2, D₅ and 5-HT₆, an agonist at 5-HT_1A and D₄ receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT_2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.

Conclusions

The molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions.     

Effects of Lead In Vivo and In Vitro on GABAergic Neurochemistry

Abstract: Alterations in aspects of neurotransmission utilizing -γ-aminobutyric acid (GABA) are associated with in vivo exposure of rats to lead at doses that do not produce convulsions, but sensitize animals to convulsant agents. These effects are observed regionally and include: decreased GABA levels in cerebellum; increased activity of glutamate decarboxylase (GAD) in caudate; and decreased GABA release (both resting and K⁺-stimulated) in cortex, caudate, cerebellum and substantia nigra. Sodium-dependent uptake of GABA by synaptosomes of cerebellum, substantia nigra and caudate was also affected: in these regions, affinity (K_m) was increased and maximal velocity (V_max) was reduced. Sodium-independent binding of GABA to synaptic membranes was increased in cerebellum, but was observed only when tissue was Tritonized and prepared without freezing and washing. No effects on GAD or on GABA uptake, release, or binding were observed when lead was added to brain tissue in vitro in concentrations as high as 100 μM. The results suggest that lead may produce chronic inhibition of presynaptic GABAergic function, notably in the cerebellum, which is associated with supersensitivity of postsynaptic GABA receptors. Failure of lead to affect GABAergic function in vitro may indicate that these effects are secondary to another neurotoxic action of lead in the CNS or are consequent to a nonneuronal metabolic action of lead.     

Antithrombotic Activities of Luteolin In Vitro and In Vivo

The objectives of present study were to investigate whether luteolin affects procoagulant proteinase activity and fibrin clot formation and influences thrombosis and coagulation in Sprague–Dawle rats. Luteolin significantly inhibited the enzymatic activity of thrombin and FXa activity by 29.1% and 16.2%. Luteolin also inhibited fibrin polymer formation in turbidity and microscopic analysis using fluorescent conjugate. Coagulation assay of luteolin was found to prolong activated partial thromboplastin time and prothrombin time. Moreover, luteolin protected the development of oxidative stress induced thrombosis in the FeCl₃‐induced carotid arterial thrombus model. This study demonstrated that luteolin may be useful by reducing or preventing thrombotic challenge and can help us better understand the antithrombotic action of luteolin.     

In Vitro and In Vivo Observations on a Murine C-Type Virus

From 40 discrete mouse tissue culture cell lines examined by electron microscopy or complement fixation, or both, for the presence of detectable virus, one (NCTC 4705), initiated and maintained on chemically defined medium, was chosen for a more extensive study. Virus-like particles (100 to 110 mμ), morphologically similar to previously reported immature and mature C-type leukemia virus particles, were found budding from the plasma membrane and free in the intracellular spaces of cells in tissue culture and in fibrosarcomas resulting from intramuscular implants of these tissue cultures. Complement-fixation tests for group reactive murine leukemia antigens were positive, with titers consistently higher to a broadly reactive anti-serum than to anti-Friend, anti-Moloney, or anti-Rauscher sera. The 4705 virus was neutralized by Gross antiserum, but not by the F-M-R antisera. When injected into DD, BALB/c, or C3H/He newborn mice, the virus thus far has manifested no leukemogenicity, though virus from tumor extracts and tissue culture medium has been shown to be capable of infecting C3H and Swiss mouse embryo tissue cultures and successfully replicating in them. The role of the virus in accelerating or inducing neoplastic transformation in NCTC 4705 is still not known. When it was introduced into NCTC [ill], a non-neoplastic cell line in other respects similar to NCTC 4705, 4823 manifested no signs of neoplastic transformation after harboring the virus more than 300 days in vitro.     

Cytoglobin Exhibits Anti-Fibrosis Activity on Liver In Vivo and In Vitro

Cytoglobin, generated using genetic engineering method, is a kind of recombinant human stellate cell activation-associated protein. We speculate that it could influence the development of hepatic fibrosis like Sellate cell activation-associated protein which was discovered by Kawada et al. Therefore, we investigated its anti-fibrosis effect on liver both in vivo and in vitro. During our research, we found that cytoglobin showed obvious effect compared with the control group on Thioacetamide-induced liver fibrosis in SD rats, including significantly decrease in aspartate aminotransferase, Hyaluronic acid, laminin and collagen I(Col I) levels in serum and hydroxyproline in livers, which are the important indices reflecting the degree of hepatic fibrosis. Meanwhile, the viability of rat hepatic stellate cell line T6 (HSC-T6) cells was inhibited by cytoglobin and the apoptosis induced by cytoglobin in HSC-T6 cells was detected by Annexin V/PI double staining. Activation of the caspase cascade including caspase-3 for the intrinsic pathways was demonstrated. The results also showed that the expression of Bcl-2 protein decreased whereas that of Bax protein increased, leading to an increase of the Bax/Bcl-2 ratio. Our results demonstrated that cytoglobin exhibited anti-fibrosis activity on livers in vivo and in vitro, involving apoptosis induction.     

Effects of Propyzamide on Tobacco Cell Microtubules In Vivo and In Vitro

Treatment with propyzamide at 2 ? 10^-6 M or at higher concentrationsarrested the cell cycleat metaphase in tobacco BY-2 cells. Metaphasecells having disorganized spindle microtubulesand scatteredchromosomes began to appear within several minutes of the additionof propyzamide. Within 30 min, disrupted spindle microtubulesand dispersed chromosomes were seenin all metaphase cells. Propyzamideat 2 ? 10^-6 M or at higher concentrations also disrupted corticalmicrotubules, but disruption of cortical microtubules requiredmore time than disruption of spindle microtubules. The effectof propyzamide on microtubules was found to be readily reversible.The cells arrested at metaphase by 2 ? 10^-6 M propyzamide resumedmitosis within 2 h from the termination of treatment with propyzamide.Spindle microtubules reappeared within 15 min from the terminationof treatment with propyzamide, and the cortical microtubuleswithin 1 h. Tubulin was isolated from tobacco BY-2 cells bycolumn chromatography on ethyl Nphenylcarbamate-Sepharose 4B.On incubation with EGTA, Mg²⁺ and DMSO, the purified tobaccotubulin polymerized into microtubules. Propyzamide at 1 ? 10^-4M completely inhibitedthe polymerization of tobacco tubulin,but did not inhibit polymerization of bovine braintubulin. Tobaccotubulin was adsorbed onto a column of propyzamide-analogue-linkedSepharose 4B and then purified by chromatography on this column. (Received February 15, 1988; Accepted June 29, 1988)     

In Vitro and in Vivo Effects of Nitrofurantoin on Experimental Toxoplasmosis

Toxoplasma gondii is an important opportunistic pathogen that causes toxoplasmosis, which has very few therapeutic treatment options. The most effective therapy is a combination of pyrimethamine and sulfadiazine; however, their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound, (Z)-1-[(5-nitrofuran-2-yl)methyleneamino]-imidazolidine-2,4-dione (nitrofurantoin), showed anti-T. gondii effects in vitro and in vivo. In HeLa cells, the selectivity of nitrofurantoin was 2.3, which was greater than that of pyrimethamine (0.9). In T. gondii-infected female ICR mice, the inhibition rate of T. gondii growth in the peritoneal cavity was 44.7% compared to the negative control group after 4-day treatment with 100 mg/kg of nitrofurantoin. In addition, hematology indicators showed that T. gondii infection-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, biochemical parameters involved in liver injury, were reduced by nitrofurantoin significantly. Moreover, nitrofurantoin exerted significant effects on the index of antioxidant status, i.e., malondialdehyde (MDA) and glutathione (GSH). The nitrofurantoin-treated group inhibited the T. gondii-induced MDA levels while alleviating the decrease in GSH levels. Thus, nitrofurantoin is a potential anti-T. gondii candidate for clinical application.     

Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose

The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods

The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results

In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion

This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.     

Dysmyelination of Shiverer Mutant Mice In Vivo and In Vitro

Demyelination in the CNS of shiverer mutant mice was studied in vivo and in vitro. By immunohistochemical reaction with glial fibrillary acidic protein antibody, hypertrophy of the fibrous astrocytes was observed in the white matter of shiverer cerebella. The cerebella of shiverer mice in primary culture from the day of birth showed very poor myelination under optical microscopy. Axons of Purkinje cells are thought to be the main myelinated axons in the primary culture of the cerebellum. Purkinje cells from shiverer appeared normal with regard to Bodian silver impregnation, hematoxylin and eosin staining, and P400 protein characterization of Purkinje cells. Addition of the conditioned culture medium of shiverer to the control culture did not interfere with myelination. We concluded that the demyelination in the CNS of shiverer could be caused by an intrinsic defect of the oligodendrocyte rather than by hypertrophy of the astrocytes or by diffusible factors.     

In Vivo and In Vitro Development of the Protist Helicosporidium sp.

We describe the discovery and developmental features of a Helicosporidium sp. isolated from the black fly Simulium jonesi. Morphologically, the helicosporidia are characterized by a distinct cyst stage that encloses three ovoid cells and a single elongate filamentous cell. Bioassays have demonstrated that the cysts of this isolate infect various insect species, including the lepidopterans, Helicoverpa zea, Galleria mellonella, and Manduca sexta, and the dipterans, Musca domestica, Aedes taeniorhynchus, Anopheles albimanus, and An. quadrimaculatus. The cysts attach to the insect peritrophic matrix prior to dehiscence, which releases the filamentous cell and the three ovoid cells. The ovoid cells are short-lived in the insect gut with infection mediated by the penetration of the filamentous cell into the host. Furthermore, these filamentous cells are covered with projections that anchor them to the midgut lining. Unlike most entomopathogenic protozoa, this Helicosporidium sp. can be propagated in simple nutritional media under defined in vitro conditions, providing a system to conduct detailed analysis of the developmental biology of this poorly known taxon. The morphology and development of the in vitro produced cells are similar to that reported for the achorophyllic algae belonging to the genus Prototheca.     

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.