In vitro drug screening system using membrane alteration in macrophages byMycobacterium leprae

The observation that liveMycobacterium leprae on entry into macrophages from lepromatous leprosy patients reduced the number of EA rosetting macrophages, was extended to macrophages from Swiss white mice also. Further, the fact that deadMycobacterium leprae do not bring about such a change in macrophages from mice, allowed us to develop this into a bacterial viability testing system. Thus drug treated macrophages in the presence ofMycobacterium leprae showed normal rosetting ability ifMycobacterium leprae are inactivated by the drug, but showed reduced level of rosetting when bacteria were not susceptible to the drug. It was shown that a drug like dapsone, does act onMycobacterium leprae based on its permeability, quantity available inside the macrophages and inhibition of its action by Para amino benzoic acid. The inactivation ofMycobacterium leprae by sulphone and rifampicin was also proved by the flourescence diacetate method, which showed poorly viable bacteria after exposure to drugs. Thus it has been possible to develop a rapid drug screening method for testing the activity of unknown compound againstMycobacterium leprae.     

Importance of determining viability ofMycobacterium leprae inside macrophages—anin vitro method using uracil

It has been demonstrated thatMycobacterium leprae, are caPable of taking uP uracil and incorPorating it into trichloroacetic acid-insoluble materials, both as free susPension of bacteria, as well as when they are inside the macrophages, a host cell for theirin vivo survival. Same amount of bacteria show better incorPoration inside macroPhages than as free bacterial susPension. Both tyPes of incorPoration are inhibited by rifamPicin an antileProsy drug and an RNA synthesis inhibitor. Thus uracil uPtake byMycobacterium lePrae inside macroPhages has been used for standardising a raPidin vitro viability assay for the leProsy causing bacteria.     

Mycobacterium leprae binds to a major human peripheral nerve glycoprotein myelin P zero (P0)

We have previously shown that a major phosphorylated 25-kDa glycoprotein of the human peripheral nerve binds to Mycobacterium leprae. In the present study, we confirm that the 25-kDa glycoprotein of the human peripheral nerve is myelin P zero (P₀) by immunoprecipitation and Western blot experiments using monoclonal antibodies to myelin P₀. Immunohistochemical studies on human nerve using these antibodies to myelin P₀ exhibited a strong immunoreactivity to the myelin and Schwann cells. Myelin P₀ is a peripheral nerve specific protein; therefore it could likely be one of the key target molecules for M. leprae binding/internalization or even contact-dependent demyelination. This finding of M. leprae binding to myelin P₀ adds to the present understanding on neural predilection of M. leprae.     

Correlation of bacterial viability with uptake of [14C] acetate into phenolic glycolipid-1 ofMycobacterium leprae within Schwannoma cells

The viability ofMycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of [¹⁴C] acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays,viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellularMycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellularMycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, asMycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host’s protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a uniqueMycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets.     

Functional insights by comparison of modeled structures of 18kDa small heat shock protein and its mutant in Mycobacterium leprae

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52^nd position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52^nd position is observed in β4 strand and Proline in 52^nd position is observed in loop. The number of residues contributing α helix at N-terminal region varies in both models. In 18S more number of residues is present in α helix when compared to 18P. The loop regions between β3 and β4 strands of both models vary in number of residues present in it. Number of residues contributing β4 strand in both models vary. β6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation.     

Adherence ofMycobacterium leprae to the nasal mucosa is influenced by surface integrity and viability

The intranasal route is one of the main routes of Mycobacterium leprae infection and there is paucity of information regarding the mode of spread of the pattern. The adherence of M.leprae to the nasal mucosa, its trapping within the sinuses of the head, and its fate after entry into the host was studied using mouse model. A comparison of the adherence profile of M. leprae and Mycobacterium tuberculosis showed that while larger numbers of M. tuberculosis were demonstrated within lungs, greater numbers of M.leprae were present within the sinuses of the head. Adherence of M.leprae to the nasal mucosa was dependent on surface integrity since opsonization and heat killing resulted in decreased numbers of M.leprae in the nasal sinuses and a greater amount entering the lungs. The adherence appeared to the independent of the viability of the bacilli, as similar numbers of formalin-fixed, rifampicin-treated and viable M.leprae entered the lungs in the initial stages. However the numbers of rifampicin-treated M. leprae in the nasal sinuses were 12-fold lower than the numbers of viable M.leprae. These results indicated that both viability and surface integrity were important in the entry of M.leprae and it’s consequent dissemination.     

Nitrogen concentration of cauliflower organs as determined by organ size,N supply,and radiation environment

Data from field experiments carried out in three consecutive years under contrasting N supply and radiation environment altered by artificial shading were used to identify (a) the relationship between N concentration and organ size under conditions of unrestricted N supply and (b) critical levels of soil nitrate (Nmin_crit), where nitrogen concentration of cauliflower organs begin to decline because of N limitations. The decline of N concentrations in cauliflower was analysed at different levels of morphological aggregation, i.e., the whole shoot level, the organ level (leaves, stem, and curd), and within different leaf groups within the canopy. Nmin_crit values (0–60 cm soil depth) for total nitrogen concentration of cauliflower organs leaves, stem and curd were estimated at 85, 93 and 28 kg N ha^–1, respectively. Within the canopy, Nmin_crit values for total N of leaves increased from the top to the bottom from 44 to 188 kg N ha^–1. Nmin_crit values for protein N in leaves from different layers of the canopy were much lower at around 30 kg N ha^–1, without a gradient within the canopy. It is discussed that these differences in Nmin_crit values are most likely a consequence of N redistribution associated with nitrogen deficiency. The decline of average shoot nitrogen concentrations, [Nm] (%N DM), with shoot dry matter, W _sh, (t ha^–1) under conditions of optimal N supply was [Nm]= 4.84 (±0.071) W _sh ^{–0.089(± 0.011)}, r ²=0.67 (±S.E.). The reduction of radiation intensity by artificial shading (60% of control) had no significant influence on total nitrogen concentrations of leaves and only a small influence on protein nitrogen concentrations in lower layers of the canopy. The leaf nitrate nitrogen fraction of nitrogen, f _nitr (–), within the canopy decreased linearly with increased average incident irradiance in different canopy layers (I _av, W PAR m^–2) (f _Nitr. = 0.2456(±0.0188) – 0.0023(±0.0004)I _av, r ² = 0.67.     

ATP synthases: bioinformatic based insights into how their electrochemically driven motor comprised of subunits a and c might serve as a drug target

ATP synthases, widely distributed in bacteria, eukaryotic mitochondria and chloroplasts, are highly conserved multi-subunit complexes. Although the conserved acidic residue in the transmembrane helix of the c subunit functions in H⁺ transport, the surrounding residues differ among species. Such divergence could lead to different regulatory modes since pH-dependent H⁺ transport has been demonstrated in E. coli with a c subunit carrying an additional acidic residue in the helix. There is further divergence in the number of c subunits that form the ring structure which is determined by the higher ordered structure. Recently, it was suggested that certain chemicals recognize the a and c subunits of pathogenic bacterial F₀. Since there may be structural divergence even in well-conserved ATP synthases, the c subunit-ring as well as the a subunit in F₀ could be targets for drugs for specific bacterial species.     

The coupling of the relative movement of thea andc subunits of the F0 to the conformational changes in the F1-ATPase

F₀F₁-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F₀F₁-ATPase. Because of the subunit stoichiometry and the involvement of both thea- andc-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed.     

H+ transport and coupling by the F0 sector of the ATP synthase: Insights into the molecular mechanism of function

The F₀ sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F₁ sector, couples proton transport to ATP synthesis. The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on theEscherichia coli F₀ sector is reviewed here. TheE. coli F₀ is composed of three types of subunits (a, b, andc) and current information on their folding and organization in F₀ is reviewed. The structure of purified subunitc in chloroform-methanol-H₂O resembles that in native F₀, and progress in determining the structure by NMR methods is reviewed. Genetic experiments suggest that the two helices of subunitc must interact as a functional unit around an essential carboxyl group as protons are transported. In addition, a unique class of suppressor mutations identify a transmembrane helix of subunita that is proposed to interact with the bihelical unit of subunitc during proton transport. The role of multiple units of subunitc in coupling proton translocation to ATP synthesis is considered. The special roles of Asp61 of subunitc and Arg210 of subunita in proton translocation are also discussed.     

Effect of peripheral benzodiazepine receptor (PBR/TSPO) ligands on opening of Ca<Superscript>2+</Superscript>-induced pore and phosphorylation of 3.5-kDa polypeptide in rat brain mitochondria

The effect of nanomolar concentrations of PBR/TSPO ligands—Ro 5-4864, PK11195, and PPIX—on Ca²⁺-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 μM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [γ-³²P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca²⁺ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of F_oF₁-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of F_oF₁-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca²⁺- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.     

The Oligomeric Subunit c Rotor in the Fo Sector of ATP Synthase: Unresolved Questions in Our Understanding of Function

We have proposed a model for the oligomeric c-rotor of the F_o sector of ATP synthase and its interaction with subunit a during H⁺-transport driven rotation. The model is based upon the solution structure of monomeric subunit c, determined by NMR, and an extensive series of cross-linking distance constraints between c subunits and between subunits c and a. To explain the complete set of cross-linking data, we have suggested that the second transmembrane helix rotates during its interaction with subunit a in the course of the H⁺-translocation cycle. The H⁺-transport coupled rotation of this helix is proposed to drive the stepwise movement of the c-oligomeric rotor. The model is testable and provides a useful framework for addressing questions raised by other experiments.     

Observations of rotation within the FoF1-ATP synthase: deciding between rotation of the Foc subunit ring and artifact

F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.     

Nitric oxide scavenging by Mycobacterium leprae GlbO involves the formation of the ferric heme-bound peroxynitrite intermediate

Ferrous oxygenated (Fe(II)O2) hemoglobins (Hb's) and myoglobins (Mb's) have been shown to react very rapidly with NO, yielding NO3(-) and the ferric heme-protein derivative (Fe(III)), by means of the ferric heme-bound peroxynitrite intermediate (Fe(III)OONO), according to the minimum reaction scheme: Fe(II)O2 + NO (k(on))--> Fe(III)OONO (h)--> Fe(III) + NO3(-). For most Hb's and Mb's, the first step (indicated by k(on)) is rate limiting, the overall reaction following a bimolecular behavior. By contrast, the rate of isomerization and dissociation of Fe(III)OONO (indicated by h) is rate limiting in NO scavenging by Fe(II)O2 murine neuroglobin, thus the overall reaction follows a monomolecular behavior. Here, we report the characterization of the NO scavenging reaction by Fe(II)O2 truncated Hb GlbO from Mycobacterium leprae. Values of k(on) (=2.1x10(6) M(-1) s(-1)) and h (=3.4 s(-1)) for NO scavenging by Fe(II)O2 M. leprae GlbO have been determined at pH 7.3 and 20.0 degrees C, the rate of Fe(III)OONO decay (h) is rate limiting. The Fe(III)OONO intermediate has been characterized by optical absorption spectroscopy in the Soret region. These results have been analyzed in parallel with those of monomeric and tetrameric globins as well as of flavoHb and discussed with regard to the three-dimensional structure of mycobacterial truncated Hbs and their proposed role in protection from nitrosative stress.     

Tumorigenesis of rat mammary epithelial cells by N-nitroso-N-methylurea in anin vitro system: Characterization of the microtumors

Summary Chemical carcinogenesis is a lengthy process that involves the rather loosely defined stages of initiation, promotion, and progression. Several model systems of mammary carcinogenesis have been designed to elucidate the mechanisms of chemical carcinogenesis. Most of these systems have included animal models. While organ specific chemical carcinogenesis can be initiated in these systems, the subsequent stages of promotion and progression are difficult to study in detail. Investigations onin vitro carcinogenesis have shown transformation of mammalian cells in culture; the transformational event, however, is difficult to discern within the monolayer culture. We have recently reported the development of anin vitro carcinogenesis system that allows both the initiation as well as the progression of mammary cells in a collagen gel matrix culture system. The cells transformed by a chemical carcinogen develop into discernible microtumors with the three dimensions of a collagen gel culture. Isolation of these microtumors from the collagen gel an subsequent culture in monolayer has produced cells capable of colony formation in soft agar. The present study further characterizes these microtumors originatedin vitro by analysis of cell growth kinetics versus parallel control cells. In addition, flow cytometric and cytogenetic studies have been performed to investigate the chromosomal stability of these cells. It was also observed that the microtumors, producedin vitro from mammary epithelial cells of an inbred strain of rats, show the ability to form tumors upon transplantation into the fat pad of syngeneic hosts.     

Inhibition of proton-translocating ATPases of Streptococcus mutans and Lactobacillus casei by fluoride and aluminum

One of the major effects of fluoride on oral bacteria is a reduction in acid tolerance, and presumably also in cariogenicity. The reduction appears to involve transport of protons across the cell membrane by the weak acid HF to dissipate the pH gradient, and also direct inhibition of the F₁F₀, proton-translocating ATPases of the organisms, especially for Streptococcus mutans. This direct inhibition by fluoride was found to be dependent on aluminum. The dependence on aluminum was indicated by the protection against fluoride inhibition afforded by the Al-chelator deferoxamine and by loss of protection after addition of umolar levels of Al³⁺, which were not inhibitory for the enzyme in the absence of fluoride. The F₁ form of the enzyme dissociated from the cell membrane previously had been found to be resistant to fluoride in comparison with the F₁F₀ membrane-associated form. However, this difference appeared to depend on less aluminum in the F₁ preparation in that the sensitivity of the F₁ enzyme to fluoride could be increased by addition of umolar levels of Al³⁺. The effects of Al on fluoride inhibition were apparent when enzyme activity was assayed in terms of phosphate release from ATP or with an ATP-regenerating system containing phosphoenolpyruvate, pyruvate kinase, NADH and lactic dehydrogenase. Also, Be²⁺ but not other metal cations, e.g. Co²⁺, Fe²⁺, Fe³⁺, Mn², Sn²⁺, and Zn²⁺, served to sensitize the enzyme to fluoride inhibition. The differences in sensitivities of enzymes isolated from various oral bacteria found previously appeared also to be related to differences in levels of Al. Even the fluoride-resistant enzyme of isolated membranes of Lactobacillus casei ATCC 4646 could be rendered fluoride-sensitive through addition of Al³⁺. Thus, the F₁F₀ ATPases of oral bacteria were similar to E₁E₂ ATPases of eukaryotes in being inhibited by Al-F complexes, and the inhibition presumably involved formation of ADP-Al-F _inf3 ^sup- complexes during catalysis at the active sites of the enzymes.     

In-vitro evaluation of the antimicrobial activity of Bauhinia variegata,locally known as koiralo

Bauhinia variegata, commonly known as Koiralo is considered as medicinal plant in Nepal and India. The alcoholic extract of this plant was found to have antimicrobial activity against Bacillus subtilis (ATCC 6635) Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus (ATCC 29213) and Vibrio cholerae. The largest zone of inhibition (18 mm) was found to be exhibited against B. subtilis. For this organism the minimum bactericidal concentration (MBC) of the crude extract was 0.39 mg/ml. The extract was found to be more effective against gram-positive than gram-negative bacteria. The antimicrobial activity of the extract was found to be decreased during purification.     

Concentration levels of major and trace elements in rice from sri lanka as determined by the k 0 standardization method

Instrumental neutron activation (INAA) with k ₀ standardization has been used to determine the concentration levels of a variety of major and trace elements (Al, As, Br, Cd, Cl, Co, Cr, Cs, Cu, Fe, Hg, K, La, Mg, Mn, Mo, Na, Rb, Sc, Se, and Zn) in rice grains (raw and parboiled) and in rice flour collected from local markets in Sri Lanka. In addition, the energy-dispersive X-ray fluorescence (EDXRF) analysis has been used to determine the three elements Ca, P, and S in powdered samples. To evaluate and assure the accuracy and precision of the k ₀ standardization method, the IAEA standard reference material V-8 rye flour was analyzed. The results obtained in the present investigation were compared with the results reported from other countries. All of the elements detected in the rice matrices from Sri Lanka were very low in concentration or within normal limits for food plants. Approximate daily dietary intakes of the individual elements supplied through rice were calculated and compared with the available literature values of daily allowances.     

Rutaceous alkaloids as models for the design of novel antitumor drugs

The chemical diversity of alkaloids in the Rutaceae is correlated with biosynthetic pathways involving various aromatic amino acid precursors, tyrosine, tryptophan, histidine, and anthranilic acid. The interest of rutaceous polyheteroaromatic alkaloids as models for the development of anticancer agents relies on their frequent ability to interact with DNA or with systems involved in the control of its topology, repair, and replication. Fagaronine and nitidine, from Zanthoxylum, demonstrate antileukemic activity, associated with topoisomerases inhibition. Evodiamine from Euodia rutaecarpa, displays antimetastatic properties. The pyranoacridone acronycine, from Sarcomelicope, exhibits antitumor activity against a broad spectrum of solid tumors. Development of synthetic analogues based on this latter natural product template followed the isolation of the unstable acronycine epoxide, which led to a hypothesis of bioactivation of acronycine by transformation of the 1,2-double bond into the corresponding oxirane. 1,2-Diacyloxy-1,2-dihydroacronycine derivatives exhibited antitumor properties, with a broadened spectrum of activity and an increased potency. The demonstration that acronycine interacted with DNA led to develop benzo[a], [b], and [c]acronycine analogs. Benzo[a] and [b] derivatives displayed significant antitumor activities. 1,2-Dihydroxy-1,2-dihydrobenzo[b]acronycine esters and diesters were active in human orthotopic models of cancers xenografted in nude mice. The activity of these compounds was correlated with their ability to give covalent adducts with DNA, involving reaction between the N-2 amino group of guanines and the ester group at the benzylic position of the drug. Cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine, currently developed under the code S23906-1, successfully underwent phase I and is currently under phase II clinical trials.