Evaluation of genetic diversity of <Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Acanthaceaea) using RAPD,ISSR and RAMP markers

Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.     

A proteomic approach to cisplatin resistance in the cervix squamous cell carcinoma cell line A431

Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin-resistant subline, A431/Pt, were used as a model system. The experimental set-up involved not just a two-way comparison of the control vs. the drug-resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four-way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat-shock proteins HSP60, HSP90, HSC71, heat-shock cognate 71 kDa protein), Ca2+-binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione-S-transferase, GST), anti-apoptotic proteins (such as 14-3-3 switched on in cisplatin-exposed cells) and ion channels (such as VDAC-1, voltage-dependent anion-selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up-regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up-regulated the anti-apoptotic 14-3-3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug-induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.     

Cor Triatriatum Sinister diagnosed in adult life with three dimensional transesophageal echocardiography

Background

Cor triatriatum is a very rare congenital abnormality, usually symptomatic during childhood, diagnosis in adult age is less common.

Case Presentation

We report the case of a 40 years old woman referred to our hospital for atrial flutter ablation, transthoracic cardiac bidimensional echocardiography showed an abnormal membrane bisecting the left atrium, the diagnosis of cor triatriatum was fully made via three dimensional transesophageal echocardiography. More interstingly three other cardiac anomalies were associated: ostium secundum atrial septal defect, dilated coronary sinus due probably to persistent left superior vena cava and normally functioning bicuspid aortic valve.

Conclusions

Cor triatriatum sinister in adult life is important to recognize because it may be easily surgically correctable when hemodynamically significant. Three Dimensional transesophageal echocardiography is a minimally invasive and highly sensitive diagnostic modality.     

Leishmania major: low infection dose causes short-lived hyperalgesia and cytokines upregulation in mice

Neural involvement was traditionally associated with leprosy. However, more recent studies have shown the presence of a persistent hyperalgesia in cutaneous leishmaniasis caused by the infection of BALB/c mice with a high dose of Leishmania major. In this study, we report the presence of hyperalgesia within the first two weeks of infection caused by a low dose of the parasite. Using BALB/c mice, we demonstrate the presence of hyperalgesia during the first 10 days of infection as assessed by thermal pain tests. After 10 days these decreased pain thresholds start to recover resulting in similar levels to those in uninfected controls during the third week of infection. This hyperalgesia is accompanied by a sustained upregulation of interleukin-1beta (IL-1beta) and an early upregulation of interleukin-6 (IL-6) which is restored to normal levels after five days of infection. In conclusion, this study shows that, during early infection, the low dose of L. major causes hyperalgesia accompanied by an upregulation of IL-1beta and IL-6 and that these effects are reversed within the first two weeks of infection.     

Determination of susceptibility/resistance to antifungal drugs of Trichophyton mentagrophytes isolates by a macrodilution method

Onychomycosis is a common adult human mycosis, and dermatophytes of the Trichophyton genera are the most frequently isolated microorganism. Globally, from 3% to 10% of the human population is attacked by ony cho mycosis, and many cases involve toenails. The aim of this work was to determine the minimal inhibitory concentrations (MICs) of antifungal drugs (fluconazole, ketoconazole, itraconazole, terbinafine, and griseofulvin) often used for the treatment of ungueal dermatophytosis caused by Trichophyton mentagrophytes. The MICs were determined by the broth medium macrodilution method. The results showed that activities of terbinafine and itraconazole were significantly higher (MIC <0.007-0.015 microg.mL -1 and MIC = 0.062-1.000 microg.mL -1, respectively). All isolates had reduced susceptibility to fluconazole (MIC = 16 to >64 microg.mL -1). The MICs of ketoconazole and griseofulvin varied among strains, ranging from 0.125 to 2.000 microg.mL -1 for ketoconazole and from 0.25 to 2.00 microg.mL -1 for griseofulvin. These MICs were higher than those of other studies cited, possibly because of differences in culture medium used in the other studies.     

Identification of an agonist-induced conformational change occurring adjacent to the ligand-binding pocket of the M(3) muscarinic acetylcholine receptor

To study the conformational changes that convert G protein-coupled receptors (GPCRs) from their resting to their active state, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system. Specifically, we employed a recently developed in situ disulfide cross-linking strategy that allows the formation of disulfide bonds in Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. At present, little is known about the conformational changes that GPCR ligands induce in the immediate vicinity of the ligand-binding pocket. To address this issue, we generated 11 Cys-substituted mutant M(3) muscarinic receptors and characterized these receptors in transfected COS-7 cells. All analyzed mutant receptors contained an endogenous Cys residue (Cys-532(7.42)) located within the exofacial segment of transmembrane domain (TM) VII, close to the agonist-binding site. In addition, all mutant receptors harbored a second Cys residue that was introduced into the exofacial segment of TM III, within the sequence Leu-142(3.27)-Asn-152(3.37). Disulfide cross-linking studies showed that muscarinic agonists, but not antagonists, promoted the formation of a disulfide bond between S151(3.36)C and Cys-532. A three-dimensional model of the inactive state of the M(3) muscarinic receptor indicated that Cys-532 and Ser-151 face each other in the center of the TM receptor core. Our cross-linking data therefore support the concept that agonist activation pulls the exofacial segments of TMs VII and III closer to each other. This structural change may represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface. To the best of our knowledge, this is the first direct demonstration of a conformational change occurring in the immediate vicinity of the binding site of a GPCR activated by a diffusible ligand.     

Transmission Properties of Human PrP 102L Prions Challenge the Relevance of Mouse Models of GSS

Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene (PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Sträussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease.