An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites

Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad‐spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild‐type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3–4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild‐type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.     

Acute colonic inflammation triggers detrusor instability via activation of TRPV1 receptors in a rat model of pelvic organ cross-sensitization

Chronic pelvic pain of unknown etiology is a common clinical condition and may develop as a result of cross-sensitization in the pelvis when pathological changes in one of the pelvic organs result in functional alterations in an adjacent structure. The aim of the current study was to compare transient receptor potential vanilloid 1 (TRPV1) activated pathways on detrusor contractility in vivo and in vitro using a rat model of pelvic organ cross-sensitization. Four groups of male Sprague-Dawley rats (N = 56) were included in the study. Animals received intracolonic saline (control), resiniferatoxin (RTX, TRPV1 agonist, 10(-7) M), 2,4,6-trinitrobenzene sulfonic acid (TNBS, colonic irritant), or double treatment (RTX followed by TNBS). Detrusor muscle contractility was assessed under in vitro and in vivo conditions. Intracolonic RTX increased the contractility of the isolated detrusor in response to electric field stimulation (EFS) by twofold (P ≤ 0.001) and enhanced the contractile response of the bladder smooth muscle to carbachol (CCh). Acute colonic inflammation reduced detrusor contractility upon application of CCh in vitro, decreased bladder capacity by 28.1% (P ≤ 0.001), and reduced micturition volume by 60% (P ≤ 0.001). These changes were accompanied by an increased number of nonmicturition contractions from 3.7 ± 0.7 to 15 ± 2.7 (N = 6 in both groups, P ≤ 0.001 vs. control). Desensitization of intracolonic TRPV1 receptors before the induction of acute colitis restored the response of isolated detrusor strips to CCh but not to EFS stimulation. Cystometric parameters were significantly improved in animals with double treatment and approximated the control values. Our data suggest that acute colonic inflammation triggers the occurrence of detrusor instability via activation of TRPV1-related pathways. Comparison of the results obtained under in vitro vs. in vivo conditions provides evidence that intact neural pathways are critical for the development of an overactive bladder resulting from pelvic organ cross talk.     

Normalization of deranged signal transduction in lymphocytes of COPD patients by the novel calcium channel blocker H-DHPM

Investigations on the role of intracellular Ca²⁺ ion concentration in the mechanism of development of COPD in smokers and non-smokers were carried out. The intracellular Ca²⁺ levels were found to be increased in human lymphocytes in patients with COPD as compared to non-smokers and smokers without COPD. The investigations reveal an association in altered intracellular Ca²⁺ regulation in lymphocytes and severity of COPD, by means of significant activation of Protein kinase C and inducible nitric oxide synthase (iNOS). The effect of a novel calcium channel blocker ethyl 4-(4′-heptanoyloxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylate (H-DHPM) as a potential candidate for the treatment of COPD was also investigated. H-DHPM treated cells showed a decrease in intracellular Ca²⁺ level as compared to the control cells. Molecular studies were carried out to evaluate the expression profile of NOS isoforms in human lymphocytes and it was shown that H-DHPM decreases the increased iNOS in COPD along with reestablishing the normal levels of endothelial nitric oxide synthase (eNOS). The results of H-DHPM were comparable with those of Amlodipine, a known calcium channel blocker. Calcium channel blocker H-DHPM proves to be a potential candidate for the treatment of COPD and further clinical studies are required to prove its role in the treatment of pulmonary hypertension (PH).     

Transformation through agroinfection on decapitated shoot apex of field-growing <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

A simple and easy transformation strategy was accomplished on field growing plants of Phyllanthus amarus, an anti-hepatitis B drug plant. Infection of Agrobacterium rhizogenes strains A₄M70GUS and ATCC 15834 on decapitated shoots of field growing P. amarus induced hairy roots and crown gall, respectively. Infection with A₄M70GUS yielded a mean of 23.2 roots from 40% plants in 40-day period. The crown gall induced on 30% plants after infection with ATCC 15834 grew to 5–10 mm in diameter. The roots and crown galls established in vitro on Murashige and Skoog (MS) basal medium grew well. The hairy roots yielded fivefold (6.91 g) biomass in half-strength MS liquid medium to that of the adventitious roots derived from internode explants in MS medium with 8.0 μM α-naphthaleneacetic acid (1.39 g). Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the hairy roots induced by A₄M70GUS. The crown galls induced by ATCC 15834 were confirmed by PCR analysis using rolB gene primers. The protocol enables an easy and early accomplishment of hairy roots.     

Thymic stromal lymphopoietin is a key mediator of breast cancer progression

Inflammation is a double-edged sword that can promote or suppress cancer progression. In this study, we report that thymic stromal lymphopoietin (TSLP), an IL-7-like type 1 inflammatory cytokine that is often associated with the induction of Th2-type allergic responses in the lungs, is also expressed in human and murine cancers. Our studies with murine cancer cells indicate that TSLP plays an essential role in cancer escape, as its inactivation in cancer cells alone was sufficient to almost completely abrogate cancer progression and lung metastasis. The cancer-promoting activity of TSLP primarily required signaling through the TSLP receptor on CD4(+) T cells, promoting Th2-skewed immune responses and production of immunosuppressive factors such as IL-10 and IL-13. Expression of TSLP therefore may be a useful prognostic marker, and its targeting could have therapeutic potential.     

Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant

Background

Despite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS).

Results

Archival strains and two “present day” type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the “military” isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of “military” isolates on lactate-containing media, and showed that the “military” strains exhibited a hypersporulating phenotype.

Conclusions

Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation.     

Successful Ablation of Antero-septal Accessory Pathway in the Non-Coronary Cusp in a Child

A 15-year-old boy with Wolff-Parkinson-White syndrome underwent an electrophysiology study for symptoms of palpitations and persistence of pre-excitation during peak exercise. He was detected to have right antero-septal accessory pathway with relatively long effective refractory period and no inducible tachycardia. He had only transient normalization with cryoablation. Eight months later, he presented again with two episodes of seizures with preceding palpitations. Neurology evaluation was unremarkable with a normal electroencephalogram. In view of his symptoms in association with evidence of pre-excitation, he underwent a second electrophysiology study with ablation. Cryoablation in the anterior septum again achieved only transient normalization. Mapping in the non-coronary cusp identified an earliest accessory pathway potential. RF ablation was performed in the non-coronary cusp with immediate normalization of his electrocardiogram. At 6 month follow-up, he continues to have no pre-excitation on his EKG. Ablation of the anteroseptal accessory pathway in the non-coronary cusp can be safely performed in patients' refractory to conventional ablation sites and techniques.     

Metabolic syndrome and acute hyperglycemia are associated with endoplasmic reticulum stress in human mononuclear cells

Endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR) have been implicated in a number of complications associated with diabetes mellitus including micro‐ and macrovascular dysfunction. In this study we examine ER stress levels in blood cells isolated from human subjects with metabolic syndrome and in healthy controls. Total RNA and protein were isolated from leukocytes and the levels of specific ER stress markers were quantified by real‐time‐PCR and immunoblot analysis. Our results indicate that, compared to healthy controls, individuals with metabolic syndrome have elevated mRNA levels of genes indicative of ER stress; including spliced XBP‐1 (sXBP‐1), Grp78, and CHOP. Induced ER stress levels correlate with blood glucose but not plasma lipid concentration. Furthermore, in healthy individuals, a standard 75 g oral glucose challenge produced a significant elevation in spliced XBP‐1 (1.3 fold), Grp78 (2.0 fold), and calreticulin (3.5 fold) mRNA 60 min post challenge and a significant increase in Grp78 (2.0 fold), calreticulin (2.7 fold) protein levels 2 h postchallenge, relative to fasting levels. The UPR was also activated ex vivo, in human leukocytes cultured in the presence of 15 mmol/l glucose, supporting a specific role for glucose. The oral glucose challenge was associated with a significant increase in the expression of inflammatory cytokines, including interleukin (IL)‐1α/β, IL‐6, and IL‐8, that may result from ER stress. These findings suggest that there is an association between both acute and chronic dysglycemia and ER stress in humans.