Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro.

Galpha-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for Galpha subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIP-enriched vesicles could accelerate the GTPase activity of Galphai proteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5x) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant Galphai3 and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant Galphai3 protein increased the GTPase activity. We conclude that CCVs possess GAP activity for Galphai3 and that membrane-associated GAIP is capable of interacting with Galphai3. The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.     

Daphnetin: a novel antimalarial agent with in vitro and in vivo activity.

Daphnetin is a dihydroxycoumarin that is being used in China for the treatment of coagulation disorders. It is also a chelator and an antioxidant. In vitro, daphnetin causes a 50% inhibition (IC50) of 3H-hypoxanthine incorporation by Plasmodium falciparum at concentrations between 25 and 40 microM. Several related compounds, such as scopoletin, 2, 3-dihydroxybenzoic acid and 3, 4-dihydroxybenzoic acid show no inhibitory activity. The antimalarial activity of daphnetin is inhibited by the addition of iron. Daphnetin does not appear to be an oxidant drug, since it does not spontaneously generate superoxide in vitro. However, it does alkylate bovine serum albumin when incubated in the presence of iron. In vivo, daphnetin significantly prolongs survival of P. yoelli-infected mice.     

Hemolysin BL from novel Bacillus toyonensis BV-17 induces antitumor activity both in vitro and in vivo

ABSTRACT

The gut microbiota plays an important role in cancer development and immunotherapy. Bacterial toxins have enormous antitumor potential due to their cytotoxicity and ability to activate the immune system. Using 16S rRNA gene sequencing, we compared the gut microbiota composition of fecal samples from healthy individuals and patients with colorectal cancer (CRC) and observed that the genus Bacillus was common in the healthy donors but was absent in the CRC patients. Further, we isolated a novel Bacillus toyonensis BV-17 from the fecal samples of the healthy individuals. Our results showed that the supernatant of the Bacillus toyonensis BV-17 cultures could quickly kill various tumor cell lines within minutes in vitro, by causing cell membrane disruption, blebbing, and leakage of cytoplasmic content. Fast protein liquid chromatography (FPLC) and mass spectrometry analysis identified hemolysin BL (HBL) as the effector molecule, which exhibits a different cytotoxicity mechanism compared to previous studies. Intra-tumor injection of low dose HBL inhibited the growth of both treated and untreated tumors in mice. The outcomes of this pioneer study suggest that HBL exhibits antitumor activity and is a potential chemotherapeutic agent that could be engineered to target only tumor cells in future.     

Antibacterial activity of Co-trimazine in vitro and in vivo

Summary Co-trimazine is a new drug combination especially designed for the treatment of urinary tract infections. It consists of trimethoprim (90 mg) and sulphadiazine (410 mg). When combined in vitro, the components show high activity and a high frequency of synergy against urinary tract pathogens. After oral absorption sulphadiazine has a serum half-life similar to that of trimethoprim and is excreted in active form into the urine to a much higher degree than sulphamethoxazole. The ratio of the concentrations of trimethoprim and sulphadiazine in the urine following co-trimazine is favourable for a strong synergistic action between the compounds. In cross-over studies in volunteers receiving repeated daily doses of co-trimazine, either 500 mg twice daily or 1000 mg once daily, it was found that the antibacterial activity in the urine was at least as high as that provided by co-trimoxazole (2 × 960 mg) and considerably higher and more uniform than that given by nitrofurantion (3 × 50 mg).

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Die antibakterielle Aktivität in vitro und in vivo von Co-trimazine

Zusammenfassung Co-trimazine ist eine neue Kombination aus Trimethoprim (90 mg) und Sulfadiazin (410 mg), die besonders für die Behandlung von Harnwegsinfektionen entwickelt worden ist. Kombinationen von Trimethoprim und Sulfadiazin zeigen in vitro eine hohe Aktivität und in hohem Ausmaße synergistische Wirkung gegen Erreger der Harnwegsinfektionen. Nach oraler Gabe zeigt Sulfadiazin praktisch dieselbe Halbwertszeit in Serum wie Trimethoprim und wird in viel höherem Ausmaß als Sulfametoxazol in aktiver Form in den Harn ausgeschieden. Das Konzentrationsverhältnis Trimethoprim zu Sulfadiazin, das in Harn von Co-trimazine gegeben wird, begünstigt eine synergistische Wirkung zwischen den beiden Komponenten. In Cross-over-Versuchen bei Freiwilligen mit wiederholter Gabe zeigte Co-trimazine (2 × 500 mg und 1 × 1000 mg) eine antibakterielle Aktivität im Harn, die mindestens so hoch war wie die von Co-trimoxazole (2 × 960 mg) und deutlich höher und gleichmäßiger als die von Nitrofurantoin (3 × 50 mg).

     

SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo

FLT3 (fms-related tyrosine kinase/Flk2/Stk-2) is a receptor tyrosine kinase (RTK) primarily expressed on hematopoietic cells. In blasts from acute myelogenous leukemia (AML) patients, 2 classes of FLT3 activating mutations have been identified: internal tandem duplication (ITD) mutations in the juxtamembrane domain (25%-30% of patients) and point mutations in the kinase domain activation loop (7%-8% of patients). FLT3-ITD mutations are the most common molecular defect identified in AML and have been shown to be an independent prognostic factor for decreased survival. FLT3-ITD is therefore an attractive molecular target for therapy. SU11248 is a recently described selective inhibitor with selectivity for split kinase domain RTKs, including platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and KIT. We show that SU11248 also has potent activity against wild-type FLT3 (FLT3-WT), FLT3-ITD, and FLT3 activation loop (FLT3-Asp835) mutants in phosphorylation assays. SU11248 inhibits FLT3-driven phosphorylation and induces apoptosis in vitro. In addition, SU11248 inhibits FLT3-induced VEGF production. The in vivo efficacy of SU11248 was investigated in 2 FLT3-ITD models: a subcutaneous tumor xenograft model and a bone marrow engraftment model. We show that SU11248 (20 mg/kg/d) dramatically regresses FLT3-ITD tumors in the subcutaneous tumor xenograft model and prolongs survival in the bone marrow engraftment model. Pharmacokinetic and pharmacodynamic analysis in subcutaneous tumors showed that a single administration of an efficacious drug dose potently inhibits FLT3-ITD phosphorylation for up to 16 hours following a single dose. These results suggest that further exploration of SU11248 activity in AML patients is warranted.     

The microfilaricidal activity of ivermectin in vitro and in vivo

Ivermectin has been tested against the microfilariae of Onchocerca lienalis, Brugia pahangi and Dirofilaria immitis in vitro and in vivo. All in vitro tests were performed on larvae incubated for 48 hours at 37 degrees C in Hepes buffered medium 199 containing 20% serum, benzylpenicillin and streptomycin. In vivo tests were performed on larvae in female BALB/C mice dosed with ivermectin, 5 mg/kg, orally. The microfilariae of B. pahangi in vitro were insensitive to ivermectin at concentrations to 30 ng/ml. In vivo, an 87% reduction in the level of microfilaraemia was obtained by 24 hours after drugging but no reduction was observed in the numbers of peritoneal microfilariae. O. lienalis microfilariae in vitro were killed by ivermectin at 3 ng/ml and the larvae of this species within the subcutaneous and cutaneous tissues of the mouse were also eliminated by ivermectin at 5 mg/kg. D. immitis larvae within the bloodstream of the mouse were also sensitive to ivermectin at the dosage employed but were unaffected by ivermectin in vitro at concentrations up to 30 ng/ml.     

In vitro and in vivo antileishmanial efficacy of a combination therapy of diminazene and artesunate against Leishmania donovani in BALB/c mice

The in vitro and in vivo activity of diminazene (Dim), artesunate (Art) and combination of Dim and Art (Dim-Art) against Leishmania donovani was compared to reference drug; amphotericin B. IC50 of Dim-Art was found to be 2.28 ± 0.24 μg/mL while those of Dim and Art were 9.16 ± 0.3 μg/mL and 4.64 ± 0.48 μg/mL respectively. The IC50 for Amphot B was 0.16 ± 0.32 μg/mL against stationary-phase promastigotes. In vivo evaluation in the L. donovani BALB/c mice model indicated that treatments with the combined drug therapy at doses of 12.5 mg/kg for 28 consecutive days significantly (p < 0.001) reduced parasite burden in the spleen as compared to the single drug treatments given at the same dosages. Although parasite burden was slightly lower (p < 0.05) in the Amphot B group than in the Dim-Art treatment group, the present study demonstrates the positive advantage and the potential use of the combined therapy of Dim-Art over the constituent drugs, Dim or Art when used alone. Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.     

In vitro and in vivo antitumor activity of melatonin receptor agonists

Abstract: Melatonin has been shown to inhibit the proliferation of estrogen receptor α (ERα)‐positive human breast cancer cells in vitro and suppress the growth of carcinogen‐induced mammary tumors in rats. Melatonin’s antiproliferative effect is mediated, at least in part, through the MT1 melatonin receptor and mechanisms involving modulation of the estrogen‐signaling pathway. To develop melatonin analogs with greater therapeutic effects, we have examined the in vitro and in vivo antimitotic activity of two MT1/MT2 melatonin receptor agonists, S23219‐1 and S23478‐1. In our studies, both agonists are quite effective at suppressing the growth of MCF‐7 human breast cancer cells. At a concentration of 10^?6 m , S23219‐1 and S23478‐1 inhibited the growth of MCF‐7 cells by 60% and 73%, respectively. However, S23478‐1 is more effective than melatonin and S23219‐1 at repressing the expression and transactivation of the ERα, and modulating the expression of pancreatic spasmolytic polypeptide (pS2), an estrogen‐regulated gene. The melatonin agonist S23478‐1 exhibited enhanced antitumor potency in the subsequent studies in our animal model. At a dosage of 25 mg/kg/day, S23478‐1 is more efficacious than melatonin at inducing regression of the established N‐nitroso‐N‐methyl‐urea‐induced rat mammary tumors. This dose of S23478‐1 (25 mg/kg/day) generated a significant (P < 0.05) overall regression response of 52%. Furthermore, at this dosage, S23478‐1 is more effective than melatonin at suppressing the estrogen‐signaling pathway and promoting tumor cell apoptosis, significantly increasing the expression of the pro‐apoptotic protein Bax, while decreasing the expression of ERα and the anti‐apoptotic protein Bcl‐2.     

Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs

Development of new therapeutic approaches for leishmaniasis treatment requires new high throughput screening methodologies for the antileishmanial activity of the new compounds both in vitro and in vivo. Reporter genes as the GFP have become one of the most promissory and widely used tools for drug screening in several models, since it offers live imaging, high sensibility, specificity and flexibility; additionally, the use of GFP as a reporter gene in screening assays eliminates all the drawbacks presented in conventional assays and also those technical problems found using other reporter genes. The utility of the GFP as a reporter gene in drug screening assays with Leishmania parasites depends on the homogeneity and stability of the GFP transfected strains. Stable expression of the GFP in the Old World Leishmania species has been demonstrated using integration vectors; however, no reports exist yet about the success of this methodology in the New World species. Here we report the generation of New World Leishmania strains expressing the GFP protein from an integration vector, which replaces one copy of the 18S RNA in the chromosome with the GFP coding sequence by homologous recombination. We also prove that the expression of the integrated GFP is stable and homogeneous in the transfected parasites after months in culture without selective pressure or during its use in hamster infection assays. The fluorescent strains are useful for in vitro, ex vivo and in vivo drug screening assays since no considerable variations in virulence or infectivity where seen attributable to the genetic manipulation during both in vitro and in vivo infection experiments. The platform described here for drug testing assays based on the use of stable fluorescent Leishmania strains coupled to flow cytometry and fluorescent microscopy is more sensitive, more specific and faster than conventional assays used normally for the evaluation of compounds with potential antileishmanial activity.     

Epidermal growth factor enhances guanylate cyclase activity in vivo and in vitro

Epidermal growth factor (EGF) increases DNA synthesis and cell division both in vivo and in vitro. The mechanism by which EGF increases growth and DNA synthesis is unknown. Since the intracellular messenger cGMP stimulates DNA synthesis, the present investigation was designed to determine if EGF might have part of its mechanism of action through activating guanylate cyclase [EC 4.6.1.2], the enzyme that catalyzes the formation of cGMP. EGF enhanced soluble and particulate guanylate cyclase activities as well as cGMP levels 2- to 3-fold in hypophysectomized and nonhypophysectomized tissues both in vivo and in vitro. EGF increased guanylate cyclase activity 0.5 h after ip injection in mice, and this increased activity was still present 12 h later. Guanylate cyclase activity was increased to a greater extent secondary to EGF in hypophysectomized cecum compared to nonhypophysectomized cecum. Dose-response curves revealed that maximal stimulation of guanylate cyclase by EGF occurred at 1 nM. There was no augmented guanylate cyclase activity when the concentration of EGF was decreased to 0.01 nM. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of EGF.     

Sphingosine 1-phosphate induces antimicrobial activity both in vitro and in vivo

Sphingosine 1-phosphate (S1P), a polar sphingolipid metabolite, is involved in a wide spectrum of biological processes, including Ca(++) mobilization, cell growth, differentiation, motility, and cytoskeleton organization. Here, we show a novel role of S1P in the induction of antimicrobial activity in human macrophages that leads to the intracellular killing of nonpathogenic Mycobacterium smegmatis and pathogenic M. tuberculosis. Such activity is mediated by host phospholipase D, which favors the acidification of mycobacteria-containing phagosomes. Moreover, when it was intravenously injected in mycobacteria-infected mice, S1P reduced mycobacterial growth and pulmonary tissue damage. These results identify S1P as a novel regulator of the host antimicrobial effector pathways.     

“Bound” insulin: in vivo and in vitro biologic activity

Summary Partially purified preparations of human serum bound insulin were injected intraperitoneally or intravenously into intact fed, fasted or fasted-refed rats, or incubatedin vitro with their isolated epididymal adipose tissue. The biologic activity of bound insulin on muscle and adipose tissue,in vivo andin vitro, was compared with the activity of crystalline insulin standards. Bound and crystalline insulin injected intraperitoneally into rats stimulated the incorporation of glucose-u-¹⁴C into the glycogen of muscle and into the glycogen and fat of adipose tissue. The activities of both bound and crystalline insulin were affected by the nutritional state of the animals. Intravenous administration of partially purified bound insulin or crystalline insulin stimulated the incorporation of glucose-u-¹⁴C into the muscle glycogen of intact fed, fasted or fasted-refed rats and into the fat of the adipose tissue of fed or fasted-refed rats. Bound insulin, but not crystalline insulin stimulated fat synthesis in the adipose tissue of fasted rats. Bound or crystalline insulin, at the concentration used, failed to stimulate glycogen synthesis in adipose tissue. Bound insulin stimulated the oxidation of glucose into CO₂ in isolated rat adipose tissue and the incorporation of glucose-u-¹⁴C into the glycogen and fat of the adipose tissue. The effect of bound or crystalline insulin on isolated adipose tissue was also affected by the nutritional state of the animal.

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Zusammenfassung Teilweise gereinigte Präparate von gebundenem Insulin aus menschlichem Serum wurden normalen gefütterten, hungernden oder nach Hungern wiederaufgefütterten Ratten intraperitoneal oder intravenös injiziert oderin vitro mit dem isolierten epididymalen Fettgewebe dieser Ratten inkubiert. Die biologische Aktivität des gebundenen Insulins am Muskel und Fettgewebe,in vivo undin vitro, wurde mit der Aktivität von Standardlösungen kristallinen Insulins verglichen. Wurde gebundenes und kristallines Insulin Ratten intraperitoneal injiziert, kam es zu einer vermehrten Einlagerung von Glukose-U-¹⁴C in Muskelglykogen und in das Glykogen und Fett des Fettgewebes. Sowohl die Aktivität des gebundenen als auch des kristallinen Insulins wurden durch den Ernährungszustand der Tiere beeinflußt. Die intravenöse Verabreichung des teilweise gereinigten gebundenen Insulins oder des Kristallinsulins führte zu einer verstärkten Einlagerung von Glukose-U-¹⁴C in das Muskelglykogen der gefütterten, hungernden oder der nach Hungern wiederaufgefütterten Ratten und auch in das Fett des Fettgewebes der gefütterten und nach Hungern wiederaufgefütterten Ratten. Nur das gebundene Insulin, nicht das kristalline Insulin steigerte die Fettsynthese des Fettgewebes der hungernden Ratten. Weder das gebundene noch das kristalline Insulin führten bei den verwendeten Konzentrationen zu einer Steigerung der Glykogensynthese des Fettgewebes. Das gebundene Insulin stimulierte die Oxydation von Glukose zu CO₂ am isolierten Fettgewebe der Ratte und die Einlagerung von Glukose-U-¹⁴C in das Glykogen und Fett des Fettgewebes. Die Wirkung des gebundenen und des kristallinen Insulins auf das isolierte Fettgewebe wurde ebenfalls durch den Ernährungszustand der Tiere beeinflußt.

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Résumé Cette étude porte sur l'injection intrapéritonéale ou intraveineuse de préparations partiellement purifiées d'insuline liée de sérum humain. Des rats nourris ad libitum, mis à jeun, ou renourris après une période de jeûne, ont servi pour ces injections, et leur tissu adipeux épididymaire a servi à l'étude de l'effet in vitro de ces préparations. L'activité biologique de cette insuline liée a été comparée avec celle d'une insuline cristalline standard. L'insuline liée tout comme l'insuline cristalline stimule l'incorporation de glucose-u-C¹⁴ dans le glycogène du muscle et dansle glycogène et la graisse du tissu adipeux, à la suite de leur injection intrapéritonéale. L'état de nutrition des animaux injectés modifie les activités et de l'insuline liée et de l'insuline cristalline. L'injection intraveineuse d'insuline liée partiellement purifiée, ou d'insuline cristalline, stimule l'incorporation de glucoseu-C¹⁴ dans le glycogène musculaire de rats nourris ad libitum, à jeun, ou renourris après un jeûne prolongé. Elle stimule également l'incorporation du glucose dans la graisse du tissu adipeux de rats nourris ou renourris après jeûne. L'insuline liée seulement, non l'insuline cristalline, stimule la lipogénèse du tissu adipeux de rats à jeun. Tant l'insuline cristalline que l'insuline liée est restée sans effet, pour les dosages utilisés, sur la synthèse du glycogène dans le tissu adipeux. L'insuline liée stimule l'oxydation du glucose par le tissu adipeux isolé ainsi que son incorporation dans le glycogène et la graisse de ce tissu. L'action de l'insuline liée ou cristalline est également modifiée par l'état de nutrition de l'animal dont le tissu adipeux est utilisé in vitro.