Brugia pahangi: Feeding and nutrient uptake in vitro and in vivo

Incubation in vitro of adult Brugia pahangi in an apparatus which permitted the separate exposure of the anterior, middle, or posterior region of the worms to medium-containing radioactively labeled d-glucose, l-leucine, and adenosine has provided evidence that these materials are taken up in physiologically significant amounts by a transcuticular route. No evidence for an oral ingestion of materials has been obtained from worms in vitro, but in vivo an oral uptake of Trypan blue has been demonstrated. The ultrastructure and cytochemical staining reactions for nonspecific esterase, acid phosphatase (EC-3.1.3.2), and leucine naphthylamidase of the gut and body wall are described.     

In vivo calcium regulation in diabetic skeletal muscle

In skeletal muscle, dysfunctional contractile activity has been linked to impaired intracellular Ca²⁺ concentration ([Ca²⁺]_i) regulation. Muscle force production is impaired and fatigability and muscle fragility deteriorate with diabetes. Use of a novel in vivo model permits investigation of [Ca²⁺]_i homeostasis in diabetic skeletal muscle. Within this in vivo environment we have shown that diabetes perturbs the Ca²⁺ regulatory system such that resting [Ca²⁺]_i homeostasis following muscle contractions is compromised and elevations of [Ca²⁺]_i are exacerbated. This review considers the impact of diabetes on the capacity of skeletal muscle to regulate [Ca²⁺]_i, following muscle contractions and, in particular, the relationship between muscle fatigue and elevated [Ca²⁺]_i in a highly ecologically relevant circulation-intact environment. Importantly, the role of mitochondria in calcium sequestration and the possibility that diabetes impacts this process is explored. Given the profound microcirculatory dysfunction in diabetes this preparation offers the unique opportunity to study the interrelationships among microvascular function, blood-myocyte oxygen flux and [Ca²⁺]_i as they relate to enhanced muscle fatigability and exercise intolerance.     

Antibacterial performance of nanoscaled visible-light responsive platinum-containing titania photocatalyst in vitro and in vivo

Background

Traditional antibacterial photocatalysts are primarily induced by ultraviolet light to elicit antibacterial reactive oxygen species. New generation visible-light responsive photocatalysts were discovered, offering greater opportunity to use photocatalysts as disinfectants in our living environment. Recently, we found that visible-light responsive platinum-containing titania (TiO₂–Pt) exerted high performance antibacterial property against soil-borne pathogens even in soil highly contaminated water. However, its physical and photocatalytic properties, and the application in vivo have not been well-characterized.

Methods

Transmission electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, ultraviolet–visible absorption spectrum and the removal rate of nitrogen oxides were therefore analyzed. The antibacterial performance under in vitro and in vivo conditions was evaluated.

Results

The apparent quantum efficiency for visible light illuminated TiO₂–Pt is relatively higher than several other titania photocatalysts. The killing effect achieved approximately 2 log reductions of pathogenic bacteria in vitro. Illumination of injected TiO₂–Pt successfully ameliorated the subcutaneous infection in mice.

Conclusions

This is the first demonstration of in vivo antibacterial use of TiO₂–Pt nanoparticles. When compared to nanoparticles of some other visible-light responsive photocatalysts, TiO₂–Pt nanoparticles induced less adverse effects such as exacerbated platelet clearance and hepatic cytotoxicity in vivo.

General significance

These findings suggest that the TiO₂–Pt may have potential application on the development of an antibacterial material in both in vitro and in vivo settings.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O₂ and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O₂ exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

Antihypertensive effect of insect cells: In vitro and in vivo evaluation

In this study, we investigated the in vitro ACE inhibitory and in vivo antihypertensive effect of insect cell extracts. The IC₅₀ of three insect cell lines from different type and insect species origin: S2 (embryo, Drosophila melanogaster), Sf21 (ovary, Spodoptera frugiperda) and Bm5 (ovary, Bombyx mori), were evaluated. Most interesting results were that the IC₅₀ values ranged between 0.4 and 0.9 mg/ml, and that an extra hydrolysis with gastrointestinal enzymes did not increase the ACE inhibitory activity conspicuously. Finally, a single oral administration with a gavage of 150 mg cell extract/kg BW to spontaneous hypertensive rats (SHR) significantly decreased (p < 0.05) their systolic blood pressure (SBP) with 5-6% (9-12 mm Hg) compared to the controls at 6 h post-administration. Here the undigested and digested insect S2 cell extracts were equal in activity to lower the SBP. To the best of our knowledge, this is the first report of in vivo antihypertensive activity of insect cell extracts and this without an extra digestion requirement.     

Oesophagostomum radiatum: Glucose metabolism of larvae grown in vitro and adults grown in vivo

Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO₂ and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO₂, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N₂:5% CO₂) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0–3%, respectively).     

Quantitative assessment of neurochemical changes in a rat model of long-term alcohol consumption as detected by in vivo and ex vivo proton nuclear magnetic resonance spectroscopy

The aim of present study was to quantitatively investigate the neurochemical profile of the frontal cortex region in a rat model of long-term alcohol consumption, by using in vivo proton magnetic resonance spectroscopy (¹H-MRS) at 4.7 T and ex vivo¹H high-resolution magic angle spinning (HR-MAS) technique at 11.7 T. Twenty male rats were divided into two groups and fed a liquid diet for 10 weeks. After 10 weeks, in vivo¹H MRS spectra were acquired from the frontal cortex brain region. After in vivo¹H MRS experiments, all animals were sacrificed and 20 frontal cortex tissue samples were harvested. All tissue examinations were performed with the 11.7 T HR-MAS spectrometer and high-resolution spectra were acquired. The in vivo and ex vivo spectra were quantified as absolute metabolite concentrations and normalized ratios of total signal-intensity (i.e., metabolites_Norm), respectively. The absolute quantifications of in vivo spectra showed significantly higher glycerophosphocholine plus phosphocholine (GPC + PCh) and lower myo-inositol (mIns) concentrations in ethanol-treated rats compared to controls. The quantifications of ex vivo spectra showed significantly higher PCh_Norm, Cho_Norm and tCho_Norm, and lower GPC_Norm and mIns_Norm ratio levels in ethanol-treated rats compared to controls. Our findings suggest that reduced mIns concentrations caused by the long-term alcohol consumption may lead to hypo-osmolarity syndrome and astrocyte hyponatremia. In addition, increased choline-containing compound concentrations may reflect an increased cell turnover rate of phosphatidylcholine and other phospholipids, indicating an adaptive mechanism. Therefore, these results might be utilized as key markers in chronic alcohol intoxication metabolism.     

Pseudomonas aeruginosa overexpression system of nitric oxide reductase for in vivo and in vitro mutational analyses

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N₂O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO₂^?) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies.     

Ornithine decarboxylase in Phycomyces: In vitro and in vivo properties

The properties of ornithine decarboxylase from Phycomyces blakesleeanus were examined. Enzyme from mycelial cultures was extracted and purified approximately 70-fold. The apparent molecular weight is 96K. The Michaelis constants with respect to ornithine and pyridoxal 5′-phosphate are 90 and 0.37 μm, respectively. Putrescine is a potent competitive inhibitor with a K_i of 75 μm. Exposure of ornithine decarboxylase to sulfhydryl-modifying reagents resulted in a rapid inhibition of activity. In vivo addition of putrescine produced characteristic decreases in cellular ornithine decarboxylase activity. Light stimulation of dark-adapted mycelial cultures also decreased cellular ornithine decarboxylase activity.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

The importance of transamination and decarboxylation in phenylalanine metabolism in vivo in the rat

The extent of hydroxylation, transamination, and decarboxylation in the metabolism of excess phenylalanine in vivo has been examined by measuring the amount of radioactivity from [¹⁴C]phenylalanine that is converted to ¹⁴CO₂ and urinary metabolites. Transamination and direct decarboxylation represent only 6% of total phenylalanine metabolism. The major aromatic metabolites in the urine after phenylalanine loading are phenylacetylglycine, phenylacetic acid, phenylpyruvate, and phenylalanine. A small but significant portion (1.5%) of phenylalanine is degraded to nonaromatic compounds. The maximum phenylalanine oxidation in vivo is approximately $\text{75\%}\text{24}$ h at saturating concentrations of phenylalanine; thus, the major route of degradation of phenylalanine in the rat, even when intake is high, is via formation of acetoacetic acid and fumaric acid.     

Schistosoma mansoni: Tyrosine,a putative in vivo substrate of phenol oxidase

l-Tyrosine, l-[3,4]dihydroxyphenylalanine (l-DOPA), and dopamine are known to be in vitro substrates for Schistosoma mansoni phenol oxidase. Since all three compounds are present in the female schistosome, it is not clear which one serves as the substrate for phenol oxidase in intact S. mansoni. However, the concentration of l-tyrosine in the female schistosome (252 ng/mg worm) is 4-fold higher than the K_m of phenol oxidase for this amino acid while the concentrations of l-DOPA and dopamine (0.954 and 0.790 ng/mg worm, respectively) are 100- and 500-fold lower than the K_m of these substrates. Tri-l-tyrosine methyl ester is oxidized at less than 3% of the rate of l-tyrosine methyl ester. A tyrosine:lysine peptide and chymotrypsinogen are not oxidized. Female S. mansoni do not incorporate l-tyrosine into proteins to a significantly greater extent than l-leucine. The results suggest that free l-tyrosine is the substrate for S. mansoni phenol oxidase in vivo.     

In vivo Biosynthesis of isopentenylacetophenones in Eupatorium rugosum

The biosynthesis of dehydrotremetone in Eupatorium rugosum has been investigated by feeding radioactive precursors to intact plants. The carbon atoms of acetate-[1-¹⁴C] and acetate-[2-¹⁴C] were identified in dehydrotremetone by degradation of the molecule. From the pattern of labeling it was concluded that the acetophenone moiety was derived from acetate via the polyacetate pathway. From the incorporation of mevalonate it appeared that the furan ring and its side chain were formed from an isoprenoid compound. Potential aromatic intermediates were chemically synthesized and also fed to plants but only tremetone was found to be efficiently incorporated into dehydrotremetone. Neither 4-hydroxyacetophenone nor 4-hydroxy-3[isopenten-(2)-yl]-acetophenone were efficiently incorporated into dehydrotremetone.     

Leishmania major: In vitro and in vivo anti-leishmanial effect of cantharidin

Cantharidin is a natural poisonous compound secreted by male blister beetles. The effect of different doses of cantharidin on Leishmania major (MRHO/IR/75/ER) were investigated both in vitro (promastigote and amastigote viability) and in experimentally-infected BALB/c mice (skin lesions) using ointment or soluble cantharidin. In this study, cantharidin with concentrations of 0.5, 1, 2, 5, 10, 20 and 50 μg/ml inhibited the growth of L. major promastigotes after 24 h and the resultant inhibition levels were 39.22%, 41.95%, 49.88%, 54.78%, 58.01%, 68.30% and 80.04%, respectively. After 72 h, the mean number of amastigotes per macrophage in the culture using 2 μg/ml of cantharidin, (the 50% inhibitory concentration dose (IC₅₀)), was 1.2 while in the control group it was 2.7. In order to perform the inflammatory blister technique, 500 μg of cantharidin were solved in 25 μl of DMSO to show the formation of the blister which leads to treatment of cutaneous leishmaniasis. Using the blister technique, the small lesions (<5 mm) healed after one session. Two weeks of topical treatment with 0.1% cantharidin ointment was an effective method for treating cutaneous leishmaniasis in infected BALB/c mice.     

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H₂O₂ system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H₂O₂ was 86-times lower than that of HRP (K_m = 23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H₂O₂ as electron donor was 2838-times lower than that of HRP (k_cat/K_m = 15.8 and 44 833 mM⁻¹ min⁻¹, respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H₂O₂ concentration, and that it poorly protects recombinant Escherichia coli from H₂O₂ stress. These observations indicate that rice Hb1 inefficiently scavenges H₂O₂ as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.     

In vivo and in vitro preparation of divinyl-132,173-cyclopheophorbide-a enol

Divinyl-13²,17³-cyclopheophorbide-a enol was in vivo produced as a metabolite of divinyl-chlorophyll-a by protists and in vitro prepared by the intramolecular cyclization of methyl divinyl-pyropheophorbide-a, one of the divinyl-chlorophyll-a derivatives. The ¹H NMR spectra in CDCl₃ showed that the obtained product took exclusively its enol form in the solution. The intramolecular cyclization of chlorin π-system at the C13² and C17³ positions affected the optical properties of such chlorophyll derivatives including the non-fluorescent emission of the enol.     

The thylakoid proton motive force in vivo. Quantitative, non-invasive probes, energetics, and regulatory consequences of light-induced pmf

Endogenous probes of light-induced transthylakoid proton motive force (pmf), membrane potential (Δψ) and ΔpH were used in vivo to assess in Arabidopsis the lumen pH responses of regulatory components of photosynthesis. The accumulation of zeaxanthin and protonation of PsbS were found to have similar pK_a values, but quite distinct Hill coefficients, a feature allowing high antenna efficiency at low pmf and fine adjustment at higher pmf. The onset of “energy-dependent’ exciton quenching (q_E) occurred at higher lumen pH than slowing of plastoquinol oxidation at the cytochrome b₆f complex, presumably to prevent buildup of reduced electron carriers that can lead to photodamage. Quantitative comparison of intrinsic probes with the electrochromic shift signal in situ allowed quantitative estimates of pmf and lumen pH. Within a degree of uncertainly of ∼ 0.5 pH units, the lumen pH was estimated to range from ∼ 7.5 (under weak light at ambient CO₂) to ∼ 5.7 (under 50 ppm CO₂ and saturating light), consistent with a ‘moderate pH’ model, allowing antenna regulation but preventing acid-induced photodamage. The apparent pK_a values for accumulation of zeaxanthin and PsbS protonation were found to be ∼ 6.8, with Hill coefficients of about 4 and 1 respectively. The apparent shift between in vitro violaxanthin deepoxidase protonation and zeaxanthin accumulation in vivo is explained by steady-state competition between zeaxanthin formation and its subsequent epoxidation by zeaxanthin epoxidase. In contrast to tobacco, Arabidopsis showed substantial variations in the fraction of pmf (0.1-0.7) stored as Δψ, allowing a more sensitive qE response, possible as an adaptation to life at lower light levels.     

Mature interleukin-33 is produced by calpain-mediated cleavage in vivo

Interleukin (IL)-33 is a novel member of the IL-1 family. IL-33 is primarily synthesized as a 30-kDa precursor (pro-IL-33). Pro-IL-33 is cleaved by caspase-1 into an 18-kDa mature form (mature IL-33) in vitro. Recombinant mature IL-33 has been known to induce T-helper type-2 (Th2)-associated cytokines and inflammatory cytokines via its receptor, ST2L. However, processing of pro-IL-33 in vivo has not been clarified yet. Here, we report that calpain mediates pro-IL-33 processing in vivo. Pro-IL-33 was expressed by stimulating human epithelial cells with phorbol 12-myristate 13-acetate. Calcium ionophore induced pro-IL-33 cleavage and mature IL-33 production. This cleavage was inhibited by treatment with a calcium chelator and calpain inhibitors. Moreover, short interfering RNA-mediated knockdown of calpains suppressed pro-IL-33 cleavage. These results indicate that calpains play a critical role in pro-IL-33 processing in vivo.     

Changes in macrophage status in vivo during infection with and immunity to Leishmania enriettii

The changing status of peritoneal macrophages in guinea pigs infected with Leishmania enriettii has been examined. It was possible to demonstrate that, at certain times during a primary infection and following attempted reinfection of immune animals, the response of peritoneal macrophages to lymphokine contact in vitro was altered. At these times the harvested cells appeared to behave in vitro as if they had been at least partially activated in vivo before removal. They were unresponsive to lymphokine in the migration inhibition assay, and contact with lymphokine in culture caused a rapid increase in the level of glucose oxidation in these cells. It is suggested that changes in the response of macrophages to lymphokine in vitro may be one way of monitoring activation in vivo.     

An efficient synthetic route for preparation of antimycobacterial wollamides and evaluation of their in vitro and in vivo efficacy

A convenient solid phase peptide synthetic (SPPS) route is reported for the preparation of antimycobacterial wollamides. The method is based on on-resin head-to-tail cyclization and is fast, efficient and amenable to automation. The in vitro antimycobacterial activities of the newly synthesized wollamides were evaluated against M. tuberculosis H37Rv (Mtb H37Rv). To assess their drug-likeness, in vitro pharmacokinetic (ADME) profiling was also performed. For wollamides with potent extracellular potency, intracellular activities and in vivo efficacy were determined. The results disclose the potent antimycobacterial (MIC_{Mtb H37Rv}?=?1.1?µM) and suitable drug-like properties of wollamide A (4b). Out of the synthesized wollamides, four compounds (4b–e) exhibited potent intracellular activities against Mtb H37Rv infected human macrophages (IC₅₀?=?0.2–1.3?µM). Results of in vivo blood exposure and efficacy assays for 4d and 4e are discussed.