2,4 Dinitrophenol as Medicine.

In the sanctity of pure drug discovery, objective reasoning can become clouded when pursuing ideas that appear unorthodox, but are spot on physiologically. To put this into historical perspective, it was an unorthodox idea in the 1950's to suggest that warfarin, a rat poison, could be repositioned into a breakthrough drug in humans to protect against strokes as a blood thinner. Yet it was approved in 1954 as Coumadin® and has been prescribed to billions of patients as a standard of care. Similarly, no one can forget the horrific effects of thalidomide, prescribed or available without a prescription, as both a sleeping pill and "morning sickness" anti-nausea medication targeting pregnant women in the 1950's. The "thalidomide babies" became the case-in-point for the need of strict guidelines by the U.S. Food & Drug Administration (FDA) or full multi-species teratogenicity testing before drug approval. More recently it was found that thalidomide is useful in graft versus host disease, leprosy and resistant tuberculosis treatment, and as an anti-angiogenesis agent as a breakthrough drug for multiple myeloma (except for pregnant female patients). Decades of diabetes drug discovery research has historically focused on every possible angle, except, the energy-out side of the equation, namely, raising mitochondrial energy expenditure with chemical uncouplers. The idea of "social responsibility" allowed energy-in agents to be explored and the portfolio is robust with medicines of insulin sensitizers, insulin analogues, secretagogues, SGLT2 inhibitors, etc., but not energy-out medicines. The primary reason? It appeared unorthodox, to return to exploring a drug platform used in the 1930s in over 100,000 obese patients used for weight loss. This is over 80-years ago and prior to Dr Peter Mitchell explaining the mechanism of how mitochondrial uncouplers, like 2,4-dinitrophenol (DNP) even worked by three decades later in 1961. Although there is a clear application for metabolic disease, it was not until recently that this platform was explored for its merit at very low, weight-neutral doses, for treating insidious human illnesses and completely unrelated to weight reduction. It is known that mitochondrial uncouplers specifically target the entire organelle's physiology non-genomically. It has been known for years that many neuromuscular and neurodegenerative diseases are associated with overt production of reactive oxygen species (ROSs), a rise in isoprostanes (biomarker of mitochondrial ROSs in urine or blood) and poor calcium (Ca2+) handing. It has also been known that mitochondrial uncouplers lower ROS production and Ca2+ overload. There is evidence that elevation of isoprostanes precedes disease onset, in Alzheimer's Disease (AD). It is also curious, why so many neurodegenerative diseases of known and unknown etiology start at mid-life or later, such as Multiple Sclerosis (MS), Huntington Disease (HD), AD, Parkinson Disease, and Amyotrophic Lateral Sclerosis (ALS). Is there a relationship to a buildup of mutations that are sequestered over time due to ROSs exceeding the rate of repair? If ROS production were managed, could disease onset due to aging be delayed or prevented? Is it possible that most, if not all neurodegenerative diseases are manifested through mitochondrial dysfunction? Although DNP, a historic mitochondrial uncoupler, was used in the 1930s at high doses for obesity in well over 100,000 humans, and so far, it has never been an FDA-approved drug. This review will focus on the application of using DNP, but now, repositioned as a potential disease-modifying drug for a legion of insidious diseases at much lower and paradoxically, weight neutral doses. DNP will be addressed as a treatment for "metabesity", an emerging term related to the global comorbidities associated with the over-nutritional phenotype; obesity, diabetes, nonalcoholic steatohepatitis (NASH), metabolic syndrome, cardiovascular disease, but including neurodegenerative disorders and accelerated aging. Some unexpected drug findings will be discussed, such as DNP's induction of neurotrophic growth factors involved in neuronal heath, learning and cognition. For the first time in 80's years, the FDA has granted (to Mitochon Pharmaceutical, Inc., Blue Bell, PA, USA) an open Investigational New Drug (IND) approval to begin rigorous clinical testing of DNP for safety and tolerability, including for the first ever, pharmacokinetic profiling in humans. Successful completion of Phase I clinical trial will open the door to explore the merits of DNP as a possible treatment of people with many truly unmet medical needs, including those suffering from HD, MS, PD, AD, ALS, Duchenne Muscular Dystrophy (DMD), and Traumatic Brain Injury (TBI).

C13orf31 (FAMIN) is a central regulator of immunometabolic function.

Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.

Interaction of ATP with a small heat shock protein from Mycobacterium leprae: effect on its structure and function.

Adenosine-5'-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of "HSP18-ATP" interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that ß4-ß8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.

Growth and metabolic activity of conventional and non-conventional yeasts immobilized in foamed alginate.

The aim of this research was to study how the cell immobilization technique of forming foamed alginate gels influences the growth, vitality and metabolic activity of different yeasts. Two distinct strains were used, namely conventional yeast (exemplified by Saccharomyces cerevisiae) and a non-conventional strain (exemplified by Debaryomyces occidentalis). The encapsulation of the yeast cells was performed by the traditional process of droplet formation, but from a foamed alginate solution. The activities of two key enzymes, succinate dehydrogenase and pyruvate decarboxylase, together with the ATP content were measured in both the free and immobilized cells. This novel method of yeast cell entrapment had some notable effects. The number of living immobilized cells reached the level of 10(6)-10(7) per single bead, and was stable during the fermentation process. Reductions in both enzyme activity and ATP content were observed in all immobilized yeasts. However, S. cerevisiae showed higher levels of ATP and enzymatic activity than D. occidentalis. Fermentation trials with immobilized repitching cells showed that the tested yeasts adapted to the specific conditions. Nevertheless, the mechanical endurance of the carriers and the internal structure of the gel need to be improved to enable broad applications of alginate gels in industrial fermentation processes, especially with conventional yeasts. This is one of the few papers and patents that describe the technique of cell immobilization in foamed alginate and shows the fermentative capacities and activities of key enzymes in immobilized yeast cells.

Mechanisms of the antinociceptive action of (-) epicatechin obtained from the hydroalcoholic fraction of Combretum leprosum Mart & Eic in rodents.

BACKGROUND: The mechanisms of the antinociceptive activity of (-) epicatechin (EPI), a compound isolated from the hydroalcoholic fraction of Combreum leprosum Mart & Eicher. METHODS: were assessed in the model of chemical nociception induced by glutamate (20 µmol/paw). To evaluate the mechanisms involved, the animals , male Swiss mice (25-30 g), received EPI (50 mg/kg p.o.) after pretreatment with naloxone (2 mg/kg s.c. opioid antagonist), glibenclamide (2 mg/kg s.c. antagonist K + channels sensitive to ATP), ketanserin (0.3 mg/kg s.c. antagonist of receptor 5-HT(2A)), yoimbine (0.15 mg/kg s.c. α2 adrenergic receptor antagonist), pindolol (1 mg/kg s.c. 5-HT1(a)/1(b) receptor antagonist), atropine (0.1 mg/kg s.c. muscarinic antagonist) and caffeine (3 mg/kg s.c. adenosine receptor antagonist), ondansetron (0.5 mg/kg s.c. for 5-HT(3) receptor) and L-arginine (600 mg/kg i.p.). RESULTS: The antinociceptive effect of EPI was reversed by pretreatment with naloxone and glibenclamide, ketanserin, yoimbine, atropine and pindolol, which demonstrates the involvement of opioid receptors and potassium channels sensitive to ATP, the serotoninergic (receptor 5HT(1A) and 5HT(2A)), adrenergic (receptor alpha 2) and cholinergic (muscarinic receptor) systems in the activities that were observed. The effects of EPI, however, were not reversed by pretreatment with caffeine, L-arginine or ondansetron, which shows that there is no involvement of 5HT(3) receptors or the purinergic and nitrergic systems in the antinociceptive effect of EPI. In the Open Field and Rotarod test, EPI had no significant effect, which shows that there was no central nervous system depressant or muscle relaxant effect on the results. CONCLUSIONS: This study demonstrates that the antinociceptive activity of EPI in the glutamate model involves the participation of the opioid system, serotonin, adrenergic and cholinergic.

ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ɛ and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy.

Maintenance and growth requirements in the metabolism of Debaryomyces hansenii performing xylose-to-xylitol bioconversion in corncob hemicellulose hydrolyzate.

In order to improve the biotechnological production of xylitol, the metabolism of Debaryomyces hansenii NRRL Y-7426 in corncob hemicellulose hydrolyzate has been investigated under different conditions, where either maintenance or growth requirements predominated. For this purpose, the experimental results of two sets of batch bioconversions carried out alternatively varying the starting xylose concentration in the hydrolyzate (65.6 < or = S(0) < or = 154.7 g L(-1)) or the initial biomass level (3.0 < or = X(0) < or = 54.6 g(DM) L(-1)) were used to fit a metabolic model consisting of carbon material and ATP balances based on five main activities, namely fermentative assimilation of pentoses, semi-aerobic pentose-to-pentitol bioconversion, biomass growth on pentoses, catabolic oxidation of pentoses, and acetic acid and NADH regeneration by the electron transport system. Such an approach allowed separately evaluating the main bioenergetic constants of this microbial system, that is, the specific rates of ATP and xylose consumption due to maintenance (m(ATP) = 21.0 mmol(ATP) C-mol(DM) (-1)h(-1); m(Xyl) = 6.5 C-mmol(Xyl) C-mol(DM) (-1)h(-1)) and the true yields of biomass on ATP (Y(ATP) (max) = 0.83 C-mol(DM) mol(ATP) (-1)) and on xylose (Y(Xyl) (max) = 0.93 C-mol(DM) C-mol(Xyl) (-1)). The results of this study highlighted that the system, at very high S(0) and X(0) values, dramatically increased its energy requirements for cell maintenance, owing to the occurrence of stressing conditions. In particular, for S(0) > 130 g L(-1), these activities required an ATP consumption of about 2.1 mol(ATP) L(-1), that is, a value about seven- to eightfold that observed at low substrate concentration. Such a condition led to an increase in the fraction of ATP addressed to cell maintenance from 47% to 81%. On the other hand, the very high percentage of ATP addressed to maintenance (> 96%) at very high cell concentration (X(0) > or = 25 g(DM) L(-1)) was likely due to the insufficient substrate to sustain the growth.

Long term follow-up results of 1 year MDT in MB leprosy patients treated with standard MDT + once a month Minocycline and Ofloxacin.

BACKGROUND: This study was initiated in consultation with the National Leprosy Eradication Programme (NLEP) in mid nineties to try new treatment regimens for leprosy which were more robust in terms of control of reactions, long term relapses, operationally easier to undertake and feasible in field conditions. It was also envisaged to see if the addition of newer bactericidal drugs would be beneficial. OBJECTIVES: (i) To test the feasibility, safety and response of the patients to the new regimen. (ii) To observe the incidence of reactions during and after stoppage of therapy, for a period of 8-10 years after release from treatment. MATERIALS AND METHODS: A total of one hundred skin smear positive MB patients (15 LL, 35 BL and 50 BB) patients were included in this study. All the patients received the standard MDT + once a month supervised 100 mg of Minocycline and 400 mg of Ofloxacin for 12 months during the treatment phase. Thereafter, the treatment was stopped in all the patients which were followed-up on placebo (B complex tablets). Of these, 70 patients completed the treatment schedule of one year therapy and the post treatment follow-up of 9 to 10 years. RESULTS: All the patients tolerated the drugs well. The clinical response of the patients to the treatment was very good of which 32.85% of cases had history of reactions before starting treatment. During treatment, the incidence of reactions increased marginally to 38.5%, but these were easily controlled with concurrent administration of steroids. After completion of treatment the incidence was much less i.e. 10% and 3% after 1 and 2 years of post treatment follow-up respectively. The overall relapse rate is 5.7% (4/70) with an incidence density of 0.05/100 patient years. Relapses were confirmed by clinical, bacteriological, molecular biological (rRNA probes and 36 kD targeting PCR) as well as ATP bioluminescence. The relapsed patients presented with the appearance of new lesions, slit-skin smears were again found to become positive after becoming negative. Three of the four cases who relapsed had the initial mean BI of 2 to 2.9+ whereas one had the initial mean BI of 1.5+. Also, 2 of the 4 relapsed patients had positive PCR signals at the time of stoppage of treatment. CONCLUSION: The addition of Minocycline and Ofloxacin to the standard FDT has been observed to be a well tolerated. Overall as of now, the incidence of reactions observed with the newer treatment regimen is found to be significantly lower than that of 2 years fixed duration MB-MDT. The efficacy of this regimen regarding bacteriological clearance and relapse rates could not be compared due to non-availability of the results of experience with standard 1 year MDT regimen. However, this regimen appears to be operationally feasible and safe for the users.

Comparison of radiorespirometric Budemeyer assay with ATP assay and mouse foot pad test in detecting viable Mycobacterium leprae from clinical samples.

PURPOSE: This study compares the results of radiorespirometric Buddemeyer assay with adenosine triphosphate (ATP) assay and mouse foot pad (MFP) test to validate the sensitivity of Buddemeyer assay in detecting viable M. leprae in clinical samples. METHODS: Viability was assessed using all the three methods in 60 skin biopsy specimens, including 20 untreated lepromatous leprosy (BL-LL), 13 treated BL-LL, 12 untreated borderline tuberculoid to mid borderline (BT-BB) and 15 treated BT-BB cases. RESULTS: Of the 20 untreated BL-LL cases tested, positivity indicating the presence of viable M. leprae was detected in 85, 60 and 85% with Buddemeyer, ATP and MFP test, respectively. Among the 13 treated BL-LL cases, scores were 61, 54 and 0%; among the 12 untreated BT-BB cases, the scores were 58, 16 and 16% and among the 15 treated BT-BB cases, the scores were 46, 20, 0%, respectively. CONCLUSION: The detection sensitivity (positive scores) with three tests were closely comparable in the two untreated groups of cases. On the other hand, in the two treated groups, a good proportion of cases scored positive in the in vitro tests but none in the MFP test. Among the two in vitro methods, the Buddemeyer assay emerged as a better test, in terms of sensitivity and specificity.

A glucose kinase from Mycobacterium smegmatis.

Carbon metabolism and regulation is poorly understood in mycobacteria, a genus that includes some major pathogenic species like Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report the identification of a glucose kinase from Mycobacterium smegmatis. This enzyme serves in glucose metabolism and global carbon catabolite repression in the related actinomycete Streptomyces coelicolor. The gene, msmeg1356 (glkA), was found by means of in silico screening. It was shown that it occurs in the same genetic context in all so far sequenced mycobacterial species, where it is located in a putative tricistronic operon together with a glycosyl hydrolase and a putative malonyl-CoA transacylase. Heterologous expression of glkA in an Escherichia coli glucose kinase mutant led to the restoration of glucose growth, which provided in vivo evidence for glucose kinase function. GlkA(Msm) was subsequently overproduced in order to study its enzymatic features. We found that it can form a dimer and that it efficiently phosphorylates glucose at the expense of ATP. The affinity constant for glucose was with 9 mM about eight times higher and the velocity was about tenfold slower when compared to the parallel measured glucose kinase of S. coelicolor. Both enzymes showed similar substrate specificity, which consists in an ATP-dependent phosphorylation of glucose and no, or very inefficient, phosphorylation of the glucose analogues 2-deoxyglucose and methyl alpha-glucoside. Hence, our data provide a basis for studying the role of mycobacterial glucose kinase in vivo to unravel possible catalytic and regulatory functions.

Carbon material and bioenergetic balances of xylitol production from corncobs by Debaryomyces hansenii.

The effect of oxygenation on xylitol production by the yeast Debaryomyces hansenii has been investigated in this work using the liquors from corncob hydrolysis as the fermentation medium. The concentrations of consumed substrates (glucose, xylose, arabinose, acetate and oxygen) and formed products (xylitol, arabitol, ethanol, biomass and carbon dioxide) have been used, together with those previously obtained varying the hydrolysis technique, the level of adaptation of the microorganism, the sterilization procedure and the initial substrate and biomass concentrations, in carbon material balances to evaluate the percentages of xylose consumed by the yeast for the reduction to xylitol, alcohol fermentation, respiration and cell growth. The highest xylitol concentration (71 g/L) and volumetric productivity (1.5 g/L.h) were obtained semiaerobically using detoxified hydrolyzate produced by autohydrolysis-posthydrolysis, at starting levels of xylose (S(0)) and biomass (X(0)) of about 100 g/L and 12 g(DM)/L, respectively. No less than 80% xylose was addressed to xylitol production under these conditions. The experimental data collected in this work at variable oxygen levels allowed estimating a P/O ratio of 1.16 mol(ATP)/mol(O). The overall ATP requirements for biomass production and maintenance demonstrated to remarkably increase with X(0) and for S(0) >or= 130 g/L and to reach minimum values (1.9-2.1 mol(ATP)/C-mol(DM)) just under semiaerobic conditions favoring xylitol accumulation.

Effects of low electric treatment on yeast microflora.

AIMS: To contribute to the understanding of phenomena related to different intensity electric current treatments on the growth and metabolism of selected micro-organisms using laboratory samples of pure and co-cultures (Saccharomyces cerevisiae strain 404 and Hanseniaspora guilliermondii strain 465). METHODS AND RESULTS: Low electric current (10, 30, 50 and 100 mA) was applied to prepared samples. Parameters, such as polarity, treatment duration (18-48 h) and type of inoculum yeast, were varied one at a time to highlight their cause-effect relationships. The effects on cell activity as well as microflora viability were assessed. Bioindicators capable of describing the phenomena caused by the electric current on the microflora were identified. CONCLUSIONS: Results demonstrated that a low voltage treatment using graphite electrodes had a greater effect on the viable S. cerevisiae strain 404 microflora. There was less bactericidal activity in the S. cerevisiae strain 404 than in the H. guilliermondii strain 465. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may be of significant importance in the development of new technological processes in the fields of agriculture and food, particularly new fermenting process controls.

[Reasons why Mycobacterium leprae cells do not multiply under the cell-free condition].

Our previous paper reported that the intracellular ATP content in cells of M. leprae consistently increased in the medium containing adenosine after 4-6 weeks of cultivation and decreased thereafter. The reason why ATP generation ceased 4-6 weeks after cultivation is not clear, but it was determined that the termination in ATP generation was not a result of deterioration in the culture medium during cultivation because a renewal trial of the old culture medium by freshly prepared culture medium had no effect further maintenance or progressive increase in ATP generation. From the results obtained in a renewal trial of the culture medium, I would like to speculate that the reason why M. leprae cells do not multiply in vitro might be due to the characteristic property of the cell wall of M. leprae, i.e., fragility.

Analysis of quantitative relationship between viability determination in leprosy by MFP, ATP bioluminescence and gene amplification assay.

Two hundred twenty-one untreated, borderline lepromatous/lepromatous (BL/LL) leprosy patients have been investigated for viability by the mouse foot pad method (MFP), adenosine triphosphate (ATP) and polymerase chain reaction (PCR). The biopsies were collected at the beginning of and 12/24 months after treatment. The patient group was treated with a) immunotherapy (BCG/Mw) + MDT; b) MDT + pyrazinamide; c) control MDT; d) MDT + minocycline 100 mg once a month supervised + ofloxacin 400 mg once a month supervised. Biopsies were divided in three parts for use in the mouse foot pad, molecular and ATP investigations. In untreated and treated patients (at 12 and 24 months), there was a general agreement among all three techniques, and PCR and ATP showed higher positivity as compared to MFP. Further, there was good correlation among the viable biomass estimated by bacillary ATP levels, PCR assay and growth in mouse foot pads. The positivity was observed by MFP as well as PCR assay (18-kDa and 36-kDa) from all of the specimens when the ATP content was more than 3.6 pg/million. When the ATP content was below 3.5 pg/million, the positive takes in MFP decreased but the PCR positivity correlated with ATP bioluminescence up to 0.04 pg/million. When the ATP content was even lower, the uptake in the MFP was possibly a matter of chance, while PCR positivity was observed in 96% of the cases. For specimens with undetectable ATP, positivity was seen in 1% of the cases, showing the inability of ATP bioluminescence method to detect low background due to host ATP. PCR signals in some cases could be due to the higher sensitivity of the method or persistence of DNA after bacterial death in some cases. On the whole, the PCR methods even though targeting DNA have shown good correlations with biomass which confirm their usefulness in monitoring therapeutic responses in leprosy.

Species variation in ATP-dependent protein degradation: protease profiles differ between mycobacteria and protease functions differ between Mycobacterium smegmatis and Escherichia coli.

We report here that the existence of the potentially broad substrate specificity protease Lon (also called La), is evolutionarily discontinuous within the order Actinomycetales. Lon homologues were identified in the fast-growing species Mycobacterium smegmatis, and the slow-growing species Micobacterium avium and Mycobacterium intracellulare. However, Lon homologues were not detected in the slow-growing species Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium leprae; or in the non-mycobacterial Actinomycetale Corynebacterium glutamica. To characterize the function of the Lon protease within the Actinomycetales, a viable M. smegmatis Deltalon strain was constructed, demonstrating that lon is not essential under certain conditions. Surprisingly, lon was also dispensable in M. smegmatis cells already lacking intact 20S proteasome alpha- and beta-subunit genes (called prcA and prcB, respectively). Creation of the later double deletion strain (prcBA::kan Deltalon) necessitated use of a novel gene deletion strategy that does not require an antibiotic resistance marker. The M. smegmatis prcBA::kan Deltalon double mutants displayed wild type (wt) growth rates and wt stress tolerances. In addition, the M. smegmatis prcBA::kan Deltalon double mutants degraded at wt rates the broad spectrum of truncated proteins induced by treating cells with puromycin. This later result was in sharp contrast to those in Escherichia coli, where either lon or hslUV single mutants are strongly impaired in their degradation of puromycyl peptides (hslV is a prcB homologue). Overall these data suggested that mycobacterial species contain additional ATP-dependent proteases that have broad substrate specificity. Consistent with this suggestion, M. smegmatis and M. tuberculosis each contain at least one homologue of ClpP, the proteolytic subunit common to the ClpAP and ClpXP proteases.

Expression and functional characterisation of the clpC gene of Mycobacterium leprae: ClpC protein elicits human antibody response.

This paper reports the expression of a previously described gene [Nath and Laal, Nucleic Acids Res. 18 (1990) 4935], currently identified as the clpC gene of Mycobacterium leprae, using an in vitro rabbit reticulocyte lysate-coupled transcription/translation system. The produced protein moved as a 95-kDa band on SDS-PAGE. An additional band of 79 kDa was seen which may have resulted from a GTG codon downstream to the initiating ATG in the clpC sequence. A threefold increase in synthesis of the 95-kDa protein was achieved by altering the translation codon context sequence of the ATG start codon. The ClpC (caseinolytic protease C) amino acid sequence, which contained two nucleotide-binding sites, exhibited in vitro ATP binding. Of functional significance was its immunoreactivity in human subjects with mycobacterial infection. Leprosy and tuberculosis patients with active disease had antibodies which recognised ClpC in dot ELISA.

An improved assay for UDPglucose pyrophosphorylase and other enzymes that have nucleotide products.

UDPglucose pyrophosphorylase catalyses the interconversion UDPglucose plus pyrophosphate and glucose 1-phosphate plus UTP. Several assay methods for this enzyme have been described but the only one that can be used to investigate the specificity with respect to various UDPsugars is based on coupling to UTP formation. This assay employs phosphoglycerate kinase to catalyse the formation 1,3- bisphosphoglycerate which is then used to oxidise NADH in the presence of glyceraldehyde 3-phosphate dehydrogenase. We have found that the activity of phosphoglycerate kinase towards UTP is low which limits the usefulness of the assay to very low rates, in agreement with the published recommendation of Hansen et al. Here it is shown that the dynamic range of the assay is increased by more than five fold on addition of nucleoside diphosphate kinase and ADP, which convert UTP to the preferred phosphoglycerate kinase substrate, ATP. It is also shown that the improved assay is suitable for enzymes with other nucleotide triphosphate products.

Modeling the current-voltage characteristics of Chara membranes: I. The effect of ATP removal and zero turgor.

We have obtained and modeled the electrical characteristics of the plasma membrane of Chara internodal cells: intact, without turgor and perfused with and without ATP. The cells were voltage and space-clamped to obtain the I/V (current-voltage) and G/V (conductance-voltage) profiles of the cell membrane. The intact cells yielded similar I/V characteristics with resting p.d.s. of -221 +/- 12 mV (cytoplasmic clamp, 5 cells) and -217 +/- 12 mV (vacuolar clamp, 5 cells). The cut unperfused cells were depolarized at -169 +/- 12 mV (7 cells) compared to the vacuole-clamped intact cells. The cells perfused with ATP fell into three groups: hyperpolarized group with resting p.d. -175 +/- 12 mV (4 cells) and I/V profile similar to the intact and cut unperfused cells; depolarized group with resting p.d. of -107 +/- 12 mV (6 cells) and I/V profiles close to linear; and excited cells with profiles showing a negative conductance region and resting p.d. at -59 +/- 12 mV (5 cells). The cells perfused with medium containing no ATP showed upwardly concave I/V characteristics and resting p.d. at -81 +/- 12 mV (6 cells). The I/V curves were modeled employing the "Two-state" model for the H+ pump (Hansen et al., 1981). The inward and outward rectifiers were fitted to exponential functions and combined with a linear background current. The excitation state in perfused cells was modeled by including an inward current, iexcit, with p.d.-dependence described by a combination of hyperbolic tangent functions. An inward current, ino-ATP, with a smaller amplitude, but very similar p.d.-dependence was also included in the simulation of the I/V curves from cells without ATP. This approach avoided I/V curve subtraction. The modeling of the total I/V and G/V characteristics provided more information about the parameters of the "Two-state" pump model, as well as more quantitative understanding of the interaction of the major transport systems in the plasmalemma in generation of the resting potential under a range of circumstances. ATP had little effect on nonpump currents except the excitation current; depolarization profoundly affected the pump characteristics.

[The genetic susceptibility to leprosy in humans]. / Susceptibilité génétique à la lèpre chez l'homme.

The capacity of certain individuals to resist certain diseases, including leprosy, has for a long time been considered as being influenced by genetic factors. The clinical and pathological spectrum of leprosy, epidemiological heterogeneity, both geographic and ethnic, in the prevalence of polar forms, may be explained by genetic differences in host resistance. While the specific genes in question have not been identified, recent studies suggest a genetic basis for differences in the capacity of macrophages in the host to reduce bacterial multiplication. Experimental models analyzing the reactions of antimycobacterial defence have underscored at existing differences in resistance or vulnerability to infection (M. bovis, BCG, M. lepraemurium, M. tuberculosis) were guided by a dominant gene which exists in two allelic forms, bcgr and bcg5. The bcgr allele confers resistance and is more dominant than the bcgs allele which represents greater vulnerability to infection. The murine candidate gene for the bcg gene has been named NRAMP (Natural Resistance-associated Macrophage Protein). Even though the exact function of NRAMP is not currently known, it has been demonstrated that this gene is expressed mainly in macrophages, and that it brings about increased bacteriostatic capacity in these cells. NRAMP is structurally homologous to the family of membranous proteins having a transport function linking ATP. NRAMP is similar to the membranous bacterial system transporting nitrites. The NRAMP protein is also involved as a signal of transduction during the activation of macrophages. It is therefore possible to conceive of genetic polymorphism at this locus intervening in specific and non-specific immune responses to infection. Apart from such potential polymorphism during the initial phase of infection, immunogenetic studies suggest that the polymorphism of class II HLA molecules could intervene in the evolution of secondary immune response to M. leprae. Knowing that HLA molecules are expressed in a co-dominant form, and attributing extraordinary allelic polymorphism to this locus, there may be a rather wide range of immune responses to the M. leprae antigens in subjects with discordant HLA and in populations which have varied genetic profiles. In general it has been acknowledged that HLA-DR isotypes are associated with protective response, while HLA-DQ isotypes are said to be associated with multibacillary lepromatous forms. The chief role of the HLA systems controlling cell-mediated immunity leads to the probability that differences in HLA haplotypes could contribute to the wide spectrum of immune responses observed in leprosy. Genetic determinants of resistance to leprosy cannot be described in a straightforward manner using a classic approach because the complex mechanisms of resistance, yet to be clarified and for which at least two loci are believed to be contributory, may be re-assessed like a multifactorial, multigenetic complex in which environmental events linked to the transmission of M. leprae, its duration, intensity and host factors, varying as a function of time, intervene. A close study of each element and better understanding of the physiological and pathological mechanisms of infection and disease are necessary in order to state the influence of genetic factors on each of them with greater precision.