Effect of non-ß-lactams on stable variants of inhibitor-resistant TEM ß-lactamase in uropathogenic Escherichia coli: implication for alternative therapy.

AIMS: ß-lactamase inhibitor resistance (BLIR) among the uropathogenic Escherichia coli (UPEC) minimizes treatment options. This study aimed to identify inhibitor-resistant TEM (IRT) ß-lactamase that impart BLIR phenotype and explore non-ß-lactams as alternative therapeutics. METHODS AND RESULTS: Thirty BLIR UPEC isolates were detected by Kirby-Bauer disc diffusion technique using ß-lactam-ß-lactamase inhibitor combination. Conjugal transfer of BLIR was successful from 17 isolates. PCR and sequencing of the TEM ß-lactamases from the transconjugants indicated 14 TEM-84 (IRT) and three novel IRT variants (pUE184TEM, pUE203TEM, pUE210TEM). Three-dimensional models of the latter were predicted and validated. Molecular docking of selected non-ß-lactams (morin, catechin, naringenin triacetate) with the variants using AutoDock 4.2 showed comparable docking scores with significant hydrogen bond and hydrophobic interactions. Molecular dynamics simulation study confirmed stability of the non-ß-lactams inside the catalytic pocket of the enzymes. Moreover, all three non-ß-lactams were found to inhibit the purified TEM ß-lactamase variants in vitro. Microbroth dilution method indicated naringenin triacetate 64 µg ml-1 in combination with ceftazidime (CAZ) 30 µg ml-1 to be most effective against the BLIR transconjugants. CONCLUSIONS: BLIR phenotypes were primarily attributed to the production of IRT ß-lactamases. Administration of the non-ß-lactams with CAZ demonstrated an alternative therapeutic strategy against the IRT ß-lactamase producers. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates high risk of transmission of IRT ß-lactamases and suggests ß-lactam-non-ß-lactam combination therapy to combat BLIR.

Molecular characterization of the Rep protein of the blackgram isolate of Indian mungbean yellow mosaic virus.

The complete nucleotide sequence of the blackgram isolate of mungbean yellow mosaic virus, IMYMV-Bg, which infects legumes in India, was determined and compared at the amino acid level with those of other whitefly-transmitted geminiviruses. The genome organization of IMYMV-Bg was similar to that of the begomoviruses. A unique feature of the genome organization was the sequence divergence of the common region (CR) between DNA-A and DNA-B. In order to understand the mechanism of viral DNA replication, the replication initiator protein, Rep, of IMYMV-Bg was overexpressed in E. coli. The recombinant and refolded Rep bound to CR-sequences of IMYMV-Bg in a specific manner. In this study, evidence is presented for ATP-upregulated cleavage function and ATP-mediated conformational change of Rep. It is hypothesized that, although ATP is not required for cleavage, ATP-mediated conformational changes may result in better access of Rep to the DNA-cleavage site. Evidence is also presented for a site-specific topoisomerase function of Rep, which has not been demonstrated before. The Rep protein can be classified as a type-I topoisomerase because of its nicking activity and sensitivity towards camptothecin, a topoisomerase type-I inhibitor.

Pea chloroplast FtsZ can form multimers and correct the thermosensitive defect of an Escherichia coli ftsZ mutant.

This paper reports the isolation and characterization of a cDNA encoding the FtsZ protein of pea. The protein is synthesised as a precursor molecule of 423 amino acids with a molecular mass of 44 kDa. When translated in vitro, the protein is translocated efficiently into isolated, intact pea chloroplasts, demonstrating that the protein is localised in the chloroplast. Pea FtsZ synthesised in vitro formed multimers in a calcium-dependent manner. The pea cDNA complemented the thermosensitive defect of an E. coli ftsZ mutant in vivo and converted the filamentous phenotype of the E. coli mutant into the normal wild-type morphology at 42 degrees C. However, pea FtsZ mutants that were defective in multimerisation in vitro failed to correct the phenotype of the E. coli ftsZ mutant in vivo. The pea ftsZ transcripts were abundantly present in the young leaves, but barely detectable in roots and stems and undetectable in older leaves. Light stimulated transcription of the gene significantly in young and dark-grown leaves. This study strongly suggests that the division mechanisms used by chloroplasts and bacteria show considerable similarity.

Apoptosis and oxidative stress in the aging brain.

DNA is a primary site of damage during oxidative stress in the brain. DNA fragmentation occurs within minutes of induction of oxidative stress. This DNA fragmentation probably results from the attack of free radicals on DNA and from the activation of endonucleases. Oxidative stress was induced by intracerebroventricular injection of t-butylhydroperoxide. This results in a very rapid flux of t-butylhydroperoxide, which is cleared from the brain within minutes. This flux of t-butylhydroperoxide results in the formation of hydroxyl radical in the brain and probably in the nuclei of brain cells. Necrosis results from extensive DNA fragmentation caused by massive oxidative stress. Cresyl violet stained brain sections demonstrated necrosis in many brain regions. In addition, previous electron microscopy studies showed degradation of cellular nuclei caused by tBuOOH toxicity. Low doses of t-butylhydroperoxide can induce apoptosis, which is a delayed form of cell death. Apoptosis was found in brains stained to visualize apoptotic DNA fragments. Experiments performed in mice aged 2, 8 or 24 months will be discussed. We have also found that apoptosis and DNA fragmentation can be prevented by pretreating mice with the vitamin micotinamide. Nicotinamide is a precursor for NAD. DNA repair requires high levels of NAD in the nucleus for the activity of poly(ADP-ribose) polymerase. Oxidative stress in the brain produces both necrosis and apoptosis, probably as the result of DNA fragmentation. Senescence is associated with an increase in the production of DNA fragments during brain oxidative stress, which probably leads to more necrosis and apoptosis than in younger mice.

Apoptosis and DNA fragmentation as induced by tertiary butylhydroperoxide in the brain.

In this study, the effect of intracerebroventricular administration of the free radical generator, tertiary butylhydroperoxide, on DNA, was quantitated. Previous studies had established DNA as a very important site of free radical attack. The purpose of the study was to detect whether DNA was one of the primary targets of the toxin as well as to detect any apoptosis that may have been induced by the toxin. The DNA fragmentation assay clearly showed DNA damage within 20 min of administration of 109.7 mg/kg t-BuOOH almost in all brain regions in both 2-month and 8-month-old C57BL/6 mice. In Situ Apoptosis Detection assay, where brain sections were stained with Apoptag, demonstrated that t-BuOOH induces apoptosis in many brain regions. Electron microscopy was done to show nuclear damage and DNA fragments appearing in the cytoplasm. Cresyl violet staining was done to show that while low dose (21.9 mg/kg) t-BuOOH induces apoptosis, it may also induce necrosis in other cells of the same brain region. Thus, from this study we can conclude that DNA may be one of the primary target sites of free radical attack in the brain, and results in both necrosis and apoptosis. This can have a profound effect on neurodegeneration.

Characterisation and mode of in vitro replication of pea chloroplast OriA sequences.

A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5'-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of replication. When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement-loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast-DNA-specific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo.

Purification and characterization of a eukaryotic type 1 topoisomerase from pea chloroplast.

A 69-kDa protein with topoisomerase I activity has been homogeneously purified from the chloroplasts of pea leaves. The topoisomerase properties are detected in crude lysate of pea chloroplasts using the technique of transferring 32P radioactivity from the 32P-labeled DNA to the protein. The purified enzyme relaxes both positive and negative supercoils in topological steps of unity without requiring magnesium ions. The enzyme is sensitive to topoisomerase I-specific inhibitors like camptothecin and berenil, and unaffected by reagents like novobiocin and doxorubicin at the topoisomerase II-inhibitory dosage. In the presence of the enzyme, supercoiled DNA is nicked, and the 3'-phosphoryl end of the nick becomes covalently linked with the enzyme. A tyrosine residue of the enzyme is responsible for the covalent linkage. Rabbit antiserum raised against the 16-mer peptide spanning the active residues of human topoisomerase I recognizes the 69-kDa protein within the crude lysate of pea chloroplasts as does the antiserum to the purified 69-kDa protein. From the enzymatic characteristics, the protein has been classified as a eukaryotic type I topoisomerase.

Mechanism of blood sugar lowering by a swerchirin-containing hexane fraction (SWI) of Swertia chirayita.

Mechanism of blood sugar lowering by the crude/impure swerchirin (SWI) isolated from the hexane fraction of Swertia chirayita was investigated. Single oral administration of SWI (50 mg/kg, body wt) to fed CF rats induced about 60% (max.) fall in blood glucose by 7 hr post-treatment. This was associated with marked depletion of aldehyde-fuchsin stained beta-granules and immunostained insulin in the pancreatic islets. In vitro, glucose uptake and glycogen synthesis by muscle (diaphragm) was significantly enhanced by the serum of SWI-treated rat. At 100, 10 and 1 microM final concentration, SWI greatly enhanced glucose (16.7 mM)-stimulated insulin release from isolated islets. It is therefore concluded that SWI lowers blood glucose level by stimulating insulin release from islets of Langerhans.

Hypoglycemic effect of swerchirin from the hexane fraction of Swertia chirayita.

A xanthone was isolated from the hexane fraction of the Swertia chirayita plant and identified as 1,8-dihydroxy-3,5-dimethoxyxanthone (swerchirin). It has a very significant blood sugar lowering effect in fasted, fed, glucose loaded, and tolbutamide pretreated albino rat models. The ED50 for 40% blood sugar lowering in CF male albino rats (body weight 140-165 g) is 23.1 mg/kg/oral. The possibility of its application in clinical therapy for diabetes mellitus needs exploration.

Hypoglycemic activity of Swertia chirayita (Roxb ex Flem) Karst.

Hexane fraction of S. chirayita (250 mg/kg body wt.) induced significant fall in blood sugar and significant increase in plasma IRI simultaneously after single oral administration without influencing liver glycogen concentration in albino rats. On the other hand, daily administration for 28 days resulted in significant lowering of blood sugar and increase in plasma IRI along with a significant rise in liver glycogen. Intestinal absorption of glucose was not inhibited by hexane fraction. It is suggested that hexane fraction of S. chirayita possibly acts through its insulin releasing effect.

Blood sugar lowering potentiality of selected Cucurbitaceae plants of Indian origin.

Using five experimental models, the blood sugar lowering efficacy of eight plants of Cucurbitaceae family has been assessed. The ethanolic extract of Cucumis sativus Linn, Cucumis melo utilissimum Roxb, Cucumis melo Linn, Benincasa hispida Thunb Cogn and Tricosanthes anguina Nees, when administered in 250 mg/kg dose, orally to rats failed to lower blood sugar or to depress the peak value, after glucose load. However, ethanolic extract of Momordica charantia Linn plant and Coccinia indica Whit and Arn root significantly lowered blood sugar in fasted model and depressed the peak value in glucose loaded model. Ethanolic extract of Tricosanthes dioica Roxb plant caused a significant lowering of blood sugar in fasted rats and depressed the peak value in glucose loaded single and longterm fed groups of rats. The ethanolic extract of the aerial part of T. dioica also induced significant depression in the peak values in the glucose loaded models.

Effect of different fractions of Swertia chirayita on the blood sugar level of albino rats.

A 95% ethanol extract and four fractions of Swertia chirayita were tested for blood sugar lowering activity in rats when given orally at 250 mg/kg. The hexane fraction caused maximum lowering although the ethanol extract was clearly active. Of the four dose levels of the hexane fraction tested, 250 mg/kg was found to produce the optimum effect and 300 mg/kg did not evoke a better response. The optimum dose produced significant lowering of blood sugar in fed, glucose-loaded and tolbutamide-pretreated animal models, but not in fasted rats.

Hypolipidemic effect of garlic and thyroid function.

Male Sprague-Dawley rats were maintained on cholesterol and garlic oil for 12 weeks. Cholesterol induced hyperlipidemia was controlled by garlic feeding. Garlic treatment did not alter the concentrations of circulating thyroid hormones and thyroidal uptake of radioiodine. The results indicate that the hypolipidemic effect of garlic is probably not mediated through the thyroid.