In-Vitro Activity of Doxycycline and β-Lactam Combinations Against Different Strains of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis>

Although doxycycline is active against Burkholderia pseudomallei and has been used in the eradication stage of melioidosis therapy, it is not regularly used during the initial intensive phase. In order to assess its potential use in intensive phase therapy, we investigated in vitro pharmacodynamic activity of doxycycline and β-lactams alone and in combination against four Malaysian strains of B. pseudomallei. Using a checkerboard assay, the combinations of doxycycline and imipenem, doxycycline and ceftazidime, and doxycycline and amoxicillin–clavulanate tested against four strains showed indifferent effects with summation fractional inhibitory concentration values ranging from 0.62 to 2.12. Time-kill experiments also indicated that the combinations of doxycycline/β-lactam antibiotics against four tested strains did not fulfil synergy criteria, in which all combinations showed indifferent effects with ? 1.36 to 1.26-log CFU/mL compared to the most active monotherapy regimen in each combination. No re-growth of bacteria was detected after the early killing in doxycycline/β-lactam combination regimens compared to β-lactam monotherapy regimens, in which 9 out of 10 were associated with re-growth of bacteria. As no synergistic activity was observed, this in vitro study showed that doxycycline offers no additional benefit to be used in combination with β-lactams in the intensive phase of therapy.     

Allicin-induced suppression of Mycobacterium tuberculosis 85B mRNA in human monocytes

Despite of encountering a robust immune response, Mycobacterium tuberculosis (MTB) successfully survives and persists in the human host. We investigated the early regulation of MTB 85B gene by allicin in MTB-infected human monocytes. During the first 24h of infection, levels of both MTB 85B intracellular mRNA and secreted protein were significantly down-regulated by allicin in a dose-dependent manner, which was mediated by inhibition of glutathione and NF-kappaB pathway. Allicin-induced MTB 85B suppression correlated with suppression of TNF-alpha released from infected monocytes. The allicin-induced up-regulation of glutathione and IFN-gamma with simultaneous decrease in TNF-alpha supports the anti-inflammatory property of allicin by elicitation of protective immune response. Thus, allicin may prove to be valuable in the containment of MTB and therefore be useful as an adjunct in treatment of tuberculosis.     

Physiological mechanisms of enhancing salt tolerance of oilseed rape plants with brassinosteroids

The ability of brassinosteroids, such as 24-epibrassinolide (EBL) to increase the resistance of oilseed rape plants (Brassica napus L.) to salt stress (175 mM NaCl) was investigated along with the possible mechanisms of their protective action. Seedlings were grown for three weeks on the Hoagland-Snyder medium under controlled conditions. The experimental plants were treated with either (1) 175 mM NaCl, or (2) 10^?10 M EBL, or (3) 175 mM NaCl plus 10^?10 M EBL by adding the corresponding components to the growth medium. The exposure was 7 and 14 days. As compared to the control, salinization inhibited plant height by 33–35%, reduced leaf area by 2.0–2.5 times, reduced 2.5- and 2-fold plant fresh and dry weight, respectively, reduced water content of plant tissues by 26–31% and, twofold, the content of chlorophylls a and b. Plants responded to NaCl by developing oxidative stress conditions, lowering the osmotic potential of the cell contents down to ?2 MPa, accumulating proline (by 43–52 times) and low-molecular-weight phenolics (by 1.9–2.7 times). Oilseed rape plants were shown to respond to salinization with an increase of endogenous content of steroid hormones: 24-epibrassinosteroids (24-epibrassinolide and 24-epicastasterone), 24S-methyl-brassinosteroids (brassinolide and castasterone), and 28-homobrassinosteroids (28-homobrassinolide and 28-homocastasterone); such evidence indirectly confirms the involvement of brassinosteroids in the development of salt tolerance. Adding EBL to the nutrient medium under optimal growth conditions did not significantly affect the indices under study. Under salt stress, EBL showed a pronounced protective effect: stem growth was fully restored, plant assimilation area increased by as much as 67–76% as compared to the control index, fresh and dry weight largely recovered (up to 85–92% of the control values), and the inhibitory effect of NaCl on photosynthetic pigments was diminished. Exogenous EBL impeded the development of NaCl-dependent lipid peroxidation and increased the osmotic potential of the leaf cell contents. The protective effect of EBL under salt stress was probably associated with EBL antioxidant effect, rather than the hormone-induced accumulation of proline and of low-molecula-weight phenolics, as well as with the ability to regulate water status by maintaining intracellular ion homeostasis.     

Kinetics and modelling of cell growth and substrate uptake inCentella asiatica cell culture

In this study, we have conducted kinetics and modelling studies ofCentella asiatica cell growth and substrate uptake, in an attempt to evaluate cell growth for a better understanding and control of the process. In our bioreactor cultivation experiment, we observed a growth rate of 0.18/day, a value only 20% higher than was seen in the shake flask cultivation trial. However, the observed maximum cell dry weight in the shake flask, 10.5 g/L, was 14% higher than was achieved in the bioreactor. Ninety seven percentage confidence was achieved via the fitting of three unstructured growth models; the Monod, Logistic, and Gompertz equations, to the cell growth data. The Monod equation adequately described cell growth in both cultures. The specific growth rate, however, was not effectively predicted with the Logistic and Gompertz equations, which resulted in deviations of up to 73 and 393%, respectively. These deviations in the Logistic and Gompertz models may be attributable to the fact that these models were developed for substrate-independent growth and fungi growth, respectively.     

Effects of losartan on the blood-brain barrier permeability in long-term nitric oxide blockade-induced hypertensive rats

Hypertension is closely associated with vascular endothelial dysfunction. The aim of this study was to investigate the effects of Angiotensin II (ANG II) receptor antagonist losartan on the blood-brain barrier (BBB) permeability in L-NAME-induced hypertension and/or in ANG II-induced acute hypertension in normotensive and hypertensive rats. Systolic blood pressure was measured by tail cuff method before, during and following L-NAME treatment (1 g/L). Losartan (3 mg/kg) was given to the animal for five days. Acute hypertension was induced by ANG II (60 microg/kg). Arterial blood pressure was directly measured on the day of the experiment. BBB disruption was quantified according to the extravasation of the albumin-bound Evans blue dye. Losartan significantly reduced the mean arterial blood pressure from 169 +/- 3.9 mmHg to 82 +/- 2.9 mmHg in L-NAME and from 171 +/- 2.9 mmHg to 84 +/- 2.9 in L-NAME plus losartan plus ANG II groups (p < 0.05). The content of Evans blue dye in the cerebral cortex significantly increased in L-NAME (p < 0.01). Moreover, the content of Evans blue dye markedly increased in the cerebellum (p < 0.001) and slightly increased in diencephalon region (p < 0.05) in L-NAME plus ANG II. Losartan reduced the increased BBB permeability to Evans blue dye in L-NAME (p < 0.01) and L-NAME plus ANG II (p < 0.001). These results indicate that L-NAME and L-NAME plus ANG II both lead to an increase in microvascular Evans blue dye efflux to brain, and losartan treatment attenuates this protein-bound dye transport into brain tissue presumably due to its protective effect on endothelial cells of brain vessels.     

EPR spectral investigation of radiation-induced radicals of gallic acid

In the present work, spectroscopic features of the radiation-induced radicals of gallic acid compounds were investigated using electron paramagnetic resonance (EPR) spectroscopy. While un-irradiated samples presented no EPR signal, irradiated samples exhibited an EPR spectrum consisting of an intense resonance line at the center and weak lines on both sides. Detailed microwave saturation investigations were carried out to determine the origin of the experimental EPR lines. It is concluded that the two side lines of the triplet satellite originate from forbidden “spin-flip” transitions. The spectroscopic and structural features of the radiation-induced radicals were determined using EPR spectrum fittings. The experimental EPR spectra of the two gallic acid compounds were consistent with the calculated EPR spectroscopic features of the proposed radicals. It is concluded that the most probable radicals are the cyclohexadienyl-type, \({\dot{\text{O}}}\left( {\text{OH}} \right)_{ 2} {\text{C}}_{ 6} {\text{H}}_{ 2} {\text{COOH}}\) radicals for both compounds.     

Evolutionary relationships among strains of Mycobacterium tuberculosis with few copies of IS6110

Molecular typing of Mycobacterium tuberculosis by using IS6110 shows low discrimination when there are fewer than five copies of the insertion sequence. Using a collection of such isolates from a study of the epidemiology of tuberculosis in London, we have shown a substantial degree of congruence between IS6110 patterns and both spoligotype and PGRS type. This indicates that the IS6110 types mainly represent distinct families of strains rather than arising through the convergent insertion of IS6110 into favored positions. This is supported by identification of the genomic sites of the insertion of IS6110 in these strains. The combined data enable identification of the putative evolutionary relationships of these strains, comprising three lineages broadly associated with patients born in South Asia (India and Pakistan), Africa, and Europe, respectively. These lineages appear to be quite distinct from M. tuberculosis isolates with multiple copies of IS6110.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.