Identification of a suitable promoter for the sigma factor of Mycobacterium tuberculosis.

Promoter binding specificity is one of the important characteristics of transcription by Mycobacterium tuberculosis (Mtb) sigma (σ) factors, which remains unexplored due to limited structural evidence. Our previous study on the structural features of Mtb-SigH, consisting of three alpha helices, and its interaction with core RNA polymerase has been extended herein to determine the little known DNA sequence recognition pattern involving its cognate promoters. Herein, high resolution X-ray crystallographic structures of the protein-DNA complexes were inspected to determine the tentative DNA-binding helix of the σ factor. The binding interface in the available crystal structures is found to be populated mainly with specific residues such as Arg, Asn, Lys, Gln, and Ser. We uncovered the helix 3 of Mtb-SigH containing most of these amino acids, which ranged from Arg 64 to Arg 75, forming the predicted active site. The complex of Mtb-SigH:DNA is modelled with 20 promoter sequences. The binding affinity is predicted by scoring these protein-DNA complexes through proximity and interaction parameters obtained by molecular dynamics simulations. The promoters are ranked considering hydrogen bonding, energy of interaction, buried surface area, and distance between centers of masses in interaction with the protein. The ranking is validated through in vitro transcription assays. The trends of these selected promoter interactions have shown variations parallel to the experimental evaluation, emphasizing the success of the active site determination along with screening of the promoter strength. The promoter interaction of Mtb-SigH can be highly beneficial for understanding the regulation of gene expression of a pathogen and also extends a solid platform to predict promoters for other bacterial σ factors.

Lupus cystitis: An unusual presentation of systemic lupus erythematosus.

Lupus cystitis is a rare complication of systemic lupus erythematosus (SLE) and occurs in association with gastrointestinal symptoms. This rare disorder has been reported mainly from Japan. We report a 20 year old female who diagnosed as having SLE associated with paralytic ileus and chronic interstitial cystitis. Treatment with intravenous methylprednisolone, cyclophosphamide pulse therapy followed by oral prednisolone and azathioprine led to amelioration of manifestations. Later she developed lupus nephritis which was treated with mycophenolate mofetil.

Unusual presentation of brain abscess with uncommon organism in an immunocompetent person.

Multiple brain abscesses due to Listeria monocytogenes was detected in a 55 year old immunocompetent person who had history of progressive weakness for last three months with difficulty in micturition and abnormal behavior for the same duration. The patient was diagnosed six months back as tubercular meningitis and was put on ATD (now in continuation phase). After being diagnosed with Listeria, the patient was put on antibiotic therapy and responded dramatically. The abscess, by virtue of being relatively large and superficially located, was drained surgically.

Changes in vagal tone with posture in asthma are related to changes in ventilatory pattern.

BACKGROUND: Posturally induced changes in minute ventilation in patients may be autonomically driven. This study aimed to test whether changes in autonomic tone with posture differed between normal and asthmatic subjects and whether this related to changes in indices of ventilation. METHODS: Ten patients with type 1 brittle asthma (BA), 10 with non-brittle, severe asthma (SA), 10 with mild asthma (MA) and 10 normal individuals were studied lying flat, at 60 degrees head up tilt and flat again each for 30min, assessing end-tidal CO(2) (ETCO(2)), respiratory rate and autonomic tone by heart rate variability. RESULTS: Parasympathetic tone (as high-frequency [HF] power) fell on tilt in all four groups, all showing increases in LF (low frequency)/HF ratio (sympatho-vagal balance) on tilt (BA, 2.03-4.9; SA, 3.3-8.2; MA, 1.5-4.9; normals, 2.3-6.6). ETCO(2) on tilt fell significantly in all 3 asthma groups (BA, DeltaETCO(2) -0.4, 95% CIs -0.18 to -0.75, P=0.005; SA, -0.7, 95% CIs -0.27 to -1.16, P=0.004; MA, -0.5, 95% CIs -0.14 to -0.78, P=0.01) but not in normals (-0.1, 95% CIs +0.23 to -0.49). The fall in ETCO(2) on tilt correlated with the fall in HF power in all three asthma groups but not in normal subjects. CONCLUSION: Changes in vagal tone posture are seen on tilt in both normal and asthmatic subjects which relate to changes in ETCO(2) only in asthmatic subjects. This provides support for the hypothesis that hyper-ventilatory response to postural change in asthma is autonomically influenced.

Translocation of sigma(70) with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA.

Using fluorescence resonance energy transfer, we show that, in the majority of transcription complexes, sigma(70) is not released from RNA polymerase upon transition from initiation to elongation, but, instead, remains associated with RNA polymerase and translocates with RNA polymerase. The results argue against the presumption that there are necessary subunit-composition differences, and corresponding necessary mechanistic differences, in initiation and elongation. The methods of this report should be generalizable to monitor movement of any molecule relative to any nucleic acid.

Distribution of phlebotomine sand fly genotypes (Lutzomyia shannoni, Diptera: Psychodidae) across a highly heterogeneous landscape.

Genetic variability of eight Colombian field populations and two laboratory colonies of a tropical forest sand fly, Lutzomyia shannoni Dyar, was assessed by comparing allozyme frequencies at 20 enzyme loci. Substantial genetic variability was noted in all strains, with mean heterozygosities of 13-21% and alleles per locus of 2.0-2.8. Four loci were monomorphic. Six populations in north and central Colombia showed close genetic similarity (Nei's distances, 0.01-0.09), despite mountainous environment, discontinuous forest habitat, and elevation differences from 125 to 1,220 m. Two samples representing the Orinoco (near Villavicencio) and Amazon (near Leticia) river basins were similar (Nei's distance, 0.08) but diverged substantially from the central six samples (Nei's distances, 0.26-0.40). Although the range of L. shannoni extends from the southeastern United States to northern Argentina, three genetically distinct, geographically discrete, groups were discerned by the current analysis: Orinoco-Amazon river basins, north-central Colombia, and eastern United States.

A novel RNA polymerase binding site upstream of the galactose promoter in Escherichia coli exhibits promoter-like activity.

RNA polymerase is known to bind and utilize the overlapping promoters P1 and P2 in Escherichia coli galactose operon. We have identified an additional specific site upstream of P2, where RNA polymerase binds in a heparin-resistant manner. Binding of polymerase to this site, termed P3, occurs simultaneous to its binding at P1/P2. We have located this P3 site by DNase I footprinting. A 63 base pair region centered around position - 100 with respect to galP1 is protected by polymerase. Interestingly, a Pribnow box TATAAT is present within this protected region (-103 to -108). We have shown that transcription occurs from P3 in vitro. Primer extension analysis provides direct evidence that P3 is transcribed in vivo. The start site of transcription has been mapped at -96 position relative to galP1. beta-galactosidase assays with different gal promoter constructs reveal that while P3 alone functions as a weak in vivo promoter, it has a synergistic effect on transcription from the gal operon, since deletion of P3 or specifically mutating its -10 region result in a substantial reduction in the gal promoter activity.

Micropropagation of Paulownia fortuneii through in vitro axillary shoot proliferation.

Primary cultures were established with nodal segments from juvenile shoots of two- year-old Paulownia fortuneii trees from a clonal plantation in Andhra Pradesh. A medium containing half-strength MS salts + RAP (1 mg/L) + sucrose (2%) produced optimum bud break in nodal explants. The same basal medium with reduced hormone level (0.5 mg/L) supported maximum multiplication of secondary cultures of P. fortuneii (1:6 in 6 weeks). Specific treatments were tested to enhance this rate of multiplication. In one approach, five to six week old in vitro grown shoots were ratooned (cutting the main shoot at the bottom leaving one node). The stumps (ratooned basal node) produced 2 to 3 axillary shoots, which grew into 4 to 5 nodes by 3 weeks; thus, providing additional shoots from the same explant. This provided 30% additional shoots in 4 cycles. Secondly, reducing the light intensity to 1200 lux resulted in higher shoot elongation, i.e, formation of 8 nodes in 5 weeks with healthier shoots than the normal intensity of 3000 lux under which only 6 nodes were produced in 6 weeks. In vitro-grown shoots could be successfully rooted ex vitro in vermiculite + cocopeat mixture (1:1 v/v) under 90% humidity, transferred to soil in polybags for hardening in the green house for 2 weeks and shifted to shade net for further hardening. After one month, the plants could be successfully transplanted to field with 95% survival. Micropropagated plants showed an excellent growth in the field attaining a height of 1.5 m and a collar diameter of 2.8 cm in 3 months.

Identification of cutaneous Leishmaniasis vectors, Phlebotomus papatasi and P. duboscqi using random amplified polymorphic DNA.

Phlebotomus papatasi and P. duboscqi are two closely related, morphologically similar sandfly species that are established vectors of zoonotic cutaneous leishmaniasis in the Old World. We have developed a Random Amplified Polymorphic DNA (RAPD) method to find species-specific DNA profiles of these two species. It was found that using a single 10-mer primer a 'species specific' amplification band of about 0.49 kb was produced in all specimens of P. duboscqi while it was absent in P. papatasi. The 0.49 kb diagnostic band was consistently present in both males and females of P. duboscqi. The suitability of this primer for Phlebotomus species identification will help to find the true vector-parasite relationship in the epidemiology of leishmaniasis, particularly in the African countries where both species are prevalent.

Morphology of larval antennae and mouthparts of four Indian sand flies (Diptera: Psychodidae) by scanning electron microscopy.

The mouthparts and antennae of the fourth-instar larvae of four sand fly species were studied using scanning electron microscopy. The morphology of the clypeus, labrum, mandible, maxilla, mentum, and antennae were compared for Phlebotomus argentipes Annandale & Brunetti, P. papatasin (Scopoli), Sergentomyia babu babu (Annandale), and S. bailyi (Sinton). Most of structures exhibited species-specific features, particularly the characteristics of the antennae. P. papatasin larvae had heart-shaped antennae, a long mandible, stout maxilla, and a heavy mentum with large teeth. In contrast, P. argentipes larvae had dumbbell-shaped antennae and a singular club-shaped labrum. The antennae of S. b. babu were ovoid, whereas those of S. bailyi were elliptical. The labrum of S. b. babu was lanceolate, whereas that of S. bailyi was rounded and exhibited a small, thick projection with several folds. The teeth of the mentum of both Sergentomyia species were shorter than those of the Phlebotomus species. Species-specific differences in the morphology of larval mouthparts and antennae indicate that it may not always be necessary to rely on adult morphology to identify sympatric phlebotomines.

Vector potential of Phlebotomus duboscqi and P. papatasi: a comparison of feeding behaviour, reproductive capacity and experimental infection with Leishmania major.

Phlebotomus duboscqi and P. papatasi are the incriminated vectors of Leishmania major, the causative agent of zoonotic cutaneous leishmaniasis (ZCL) in various parts of the world. Factors contributing to the vector potential of these two species were investigated in the laboratory. It was found that 90.1% of the P. duboscqi females tested completed feeding in 1 h and they could complete up to eight gonotrophic cycles in their maximum adult life-span in the laboratory of almost 2 months. In contrast, only 70.3% of the female P. papatasi took bloodmeals within 1 h and they could only complete a maximum of four gonotrophic cycles in the maximum adult life-span of just 1 month. Biting habit (proportion feeding/day of the gonotrophic cycle), survival (proportion surviving first oviposition), gonotrophic discordance (proportion refeeding on blood before first oviposition) and experimental infection (proportion of flies offered a bloodmeal containing L. major which became infected) were all higher for P. duboscqi (0.18, 0.91, 0.50 and 0.65, respectively) than for P. papatasi (0.14, 0.71, 0.12 and 0.56, respectively). It therefore seems likely that P. duboscqi is a much more effective vector of L. major than P. papatasi in several endemic foci of ZCL.

Population ecology of Phlebotomus argentipes (Diptera: Psychodidae) in West Bengal, India.

The population abundance of Phlebotomus argentipes Annandale & Brunetti was studied between January 1986 and December 1987 at 2 sites in West Bengal, India, in relation to 8 ecological parameters (air temperature, rainfall, windspeed, relative humidity, soil moisture, soil temperature, soil pH, and soil organic carbon). Sand flies were present throughout the year with minimum abundance in winter months and maximum during monsoon and postmonsoon months. Correlation analysis examined pairwise relationships among the 8 ecological parameters and P. argentipes abundance. Multiple linear regression of sand fly abundance on the 8 parameters showed that average soil temperature and soil moisture, both recorded 1 mo earlier, were associated positively with sand fly abundance. These findings have important implications for Indian kala-azar disease control and prevention. Effective vector management programs are needed most when weather conditions favor increased sand fly abundance, given that greater sand fly abundance increases the likelihood of host-vector contact and the transmission of Leishmania.

Functional roles of the two cyclic AMP-dependent forms of cyclic AMP receptor protein from Escherichia coli.

The cyclic AMP receptor protein activates transcription in Escherichia coli, only when complexed with cyclic AMP. The cyclic AMP receptor protein-cyclic AMP complex formed at low concentrations of cyclic AMP has a different conformation from either cyclic AMP receptor protein alone or its complex with cyclic AMP formed at high cyclic AMP concentrations. Various biophysical data suggest that the latter complex resembles free cyclic AMP receptor protein. We have examined the conformational and biological properties of cyclic AMP receptor protein as a function of cyclic AMP concentrations, using the gal operon of E. coli. A biphasic behavior is observed. It is shown that only the complex formed at lower concentrations of cyclic AMP is the transcriptionally active form. This difference between the complexes at different levels of cyclic AMP arises from a decreased ability of the cyclic AMP receptor protein-cyclic AMP complex at high cyclic AMP concentrations to bind to DNA at specific sites.

Interspecific hybridization and genetic variability of Phlebotomus sandflies.

The first successful hybridization is reported between Phlebotomus papatasi and P. duboscqi, two important Old World sandfly vectors of leishmaniasis and other diseases. Laboratory strains of P. papatasi and P. duboscqi were separable by six diagnostic enzyme loci: Est-3, Idh-1, Mdh-2, Mpi, Tre-1 and Tre-3. Hybrids between the two species were verified by the recovery of heterozygous isozyme patterns for the diagnostic loci. No F2 or backcross progeny were obtained. P. papatasi was separated from P. bergeroti by three diagnostic enzyme loci: Est-3, Mpi and Pgd. The isozyme patterns of P. bergeroti contain elements of both P. duboscqi and P. papatasi, although seven diagnostic loci (Est-3, Idh-1, Me, Mpi, Pgd, Tre-1 and Tre-3) separated P. bergeroti from P. duboscqi. Genetic variability profiles of the three species were established for 20 enzyme loci. Three geographically distant strains of P. papatasi from Calcutta, Maharashtra and Israel had isozyme genetic distances of < 0.05. The recently established Calcutta strain showed an unexpectedly low genetic variability with only one (Idh-2) of 20 loci being polymorphic (average heterozygosity of 1.9%) in contrast to 5-8 polymorphic loci (10-12% heterozygosity) in the Maharashtra and Israel strains. Mass and single pair crosses between the three P. papatasi strains were fertile with normal progeny numbers. Thus we found no signs of speciation in P. papatasi.

Genetic polymorphism of morphological and biochemical characters in a Natal, Brazil, population of Lutzomyia longipalpis (Diptera: Psychodidae).

The phlebotomine sand fly, Lutzomyia longipalpis, is the vector of visceral leishmaniasis in the New World. Variability in its tergal spot morphology has led to conflicting interpretations of the species status of the various forms. An L. longipalpis field population from eastern Brazil was found with three co-occurring morphological variations--1-spot, 2-spot, and an intermediate form. Genetic profiles were established for each form. Fifteen isoenzyme loci provided the data matrix for comparison of genetic variation among the forms. Spot patterns and isoenzyme frequencies fit Hardy-Weinberg expectations, and no significant differences in isoenzyme frequencies were associated with morphological phenotype. The spot phenotype appears to be a polymorphic character not related to genetic isolation or differentiation at the species level.

The effect of anti-sandfly saliva antibodies on Phlebotomus argentipes and Leishmania donovani.

A study was undertaken to find the effect of repeated bites of the sandfly, Phlebotomus argentipes, on its host as well as on the vector itself. The study also aimed to find the effect of the immune serum on the parasite, Leishmania donovani, naturally transmitted by the vector. The hamster which was exposed to sandfly feeding showed good antibody titre against the sandfly salivary-gland secretion, which indicates that the salivary-gland secretion is immunogenic in nature. The result also revealed that the feeding attraction of the females, which has been expressed as the percentage of engorgement, gradually decreased as the mortality rate increased during the subsequent bites. Similar mortality rate was observed when the flies were fed with the immune sera through an artificial membrane feeding method. When the sandflies were fed both with the immune sera and the blood-parasite (L. donovani) suspension, in addition to the major loss of the number of vectors, there was an inhibition of development in the gut and a concomitant reduction in the migration of the parasite in the surviving females. These results indicate that the anti-sandfly saliva immune sera probably bind with the respective antigen-presenting sites of the sandfly salivary gland and, thus, cause the sandfly death. The possible explanation of the inhibition of the forward movement of the parasites is that the attraction of the parasites to the oesophagus, mediated by the sandfly saliva, is inhibited by the anti-saliva antibodies. The importance of anti-sandfly saliva antibodies as a tool of vector control and also to block the transmission of leishmaniasis has been indicated.

Genetic variability in biochemical characters of Brazilian field populations of the Leishmania vector, Lutzomyia longipalpis (Diptera: Psychodidae).

The phlebotomine sand fly Lutzomyia longipalpis is the insect vector of visceral leishmaniasis, a protozoan disease of increasing incidence and distribution in Central and South America. Electrophoretic allele frequencies of 15 enzyme loci were compared among the L. longipalpis populations selected across its distribution range in Brazil. The mean heterozygosity of two colonized geographic strains (one each from Colombia and Brazil) were 6% and 13% respectively, with 1.6-1.9 alleles detected per locus. In contrast, among the seven widely separated field populations, the mean heterozygosity ranged from 11% to 16% with 2.1-2.9 alleles per locus. No locus was recovered that was diagnostic for any of the field populations. Allelic frequency differences among five field strains from the Amazon basin and eastern coastal Brazil were very low, with Nei's genetic distances of less than 0.01 separating them. The two inland and southerly samples from Minas Gerais (Lapinha) and Bahia (Jacobina) states were more distinctive with genetic distances of 0.024-0.038 and 0.038-0.059, respectively, when compared with the five other samples. These differences were the consequence of several high frequency alleles (glycerol-3-phosphate dehydrogenase [Gpd1.69] and phosphoglucomutase [Pgm1.69]) relatively uncommon in other strains. The low genetic distances, absence of diagnostic loci, and the distribution of genes in geographic space indicate L. longipalpis of Brazil to be a single, but genetically heterogeneous, polymorphic species.