Detection of ethambutol-resistant Mycobacterium tuberculosis strains by multiplex allele-specific PCR assay targeting embB306 mutations

We describe a multiplex allele-specific (MAS)-PCR assay to detect simultaneously mutations in the first and third bases of the embB gene codon 306ATG. These mutations are known to confer ethambutol (EMB) resistance in the majority of clinical Mycobacterium tuberculosis isolates worldwide. The mutated bases are revealed depending on the presence or absence of the respective indicative fragments amplified from the embB306 wild-type allele. Initially optimized on purified DNA samples, the assay was tested on crude cell lysates and auramine-stained sputum slide DNA preparations with the same reproducibility and interpretability of the generated profiles in agarose gel electrophoresis. Since EMB resistance is generally linked to multiple-drug resistance (MDR), the MAS-PCR assay for EMB resistance detection can be used in clinical laboratory practice in areas with a high prevalence and a high transmission rate of MDR-EMB-resistant tuberculosis.     

Apoptosis of myocytes and proliferation markers as prognostic factors in end-stage dilated cardiomyopathy.

INTRODUCTION: The aim of the study was to evaluate the role of apoptosis, proliferation markers, volume density of interstitium, and myofibril volume fraction for the prognosis in patients with end-stage dilated cardiomyopathy (DCM). METHODS: Endomyocardial biopsy was performed during open-heart surgery in 56 patients with end-stage DCM. Patients were divided into two groups, one group with shorter survival (24+/-9 months, mean+/-S.D.) and another group with survival of more than 7 years after operation. The TUNEL method was used for the detection of apoptosis, and immunohistochemical methods were used for the evaluation of inhibitor of apoptosis (bcl-2) and proliferation markers (PCNA and Ki-67). RESULTS: The increased percentage of apoptotic myocytes, decreased expression of bcl-2, and decreased expression of PCNA and Ki-67 antigen was found in the group with early mortality compared to that with longer survival. Myofibril volume fraction was lower and volume density of interstitium was higher in the group with early mortality compared to that with longer survival. CONCLUSION: Apoptosis, bcl-2 expression, and proliferation activity of myocytes, myofibril volume fraction, and volume density of interstitial tissue might be useful in predicting the prognosis (progressive vs. nonprogressive form) of patients with heart failure due to DCM.     

Update on Pneumocystis carinii f. sp. hominis Typing Based on Nucleotide Sequence Variations in Internal Transcribed Spacer Regions of rRNA Genes

Pneumocystis carinii f. sp. hominis isolates from 207 clinical specimens from nine countries were typed based on nucleotide sequence variations in the internal transcribed spacer regions I and II (ITS1 and ITS2, respectively) of rRNA genes. The number of ITS1 nucleotides has been revised from the previously reported 157 bp to 161 bp. Likewise, the number of ITS2 nucleotides has been changed from 177 to 192 bp. The number of ITS1 sequence types has increased from 2 to 15, and that of ITS2 has increased from 3 to 14. The 15 ITS1 sequence types are designated types A through O, and the 14 ITS2 types are named types a through n. A total of 59 types of P. carinii f. sp. hominis were found in this study.     

Loss of cell polarity causes severe brain dysplasia in Lgl1 knockout mice

Disruption of cell polarity is seen in many cancers; however, it is generally considered a late event in tumor progression. Lethal giant larvae (Lgl) has been implicated in maintenance of cell polarity in Drosophila and cultured mammalian cells. We now show that loss of Lgl1 in mice results in formation of neuroepithelial rosette-like structures, similar to the neuroblastic rosettes in human primitive neuroectodermal tumors. The newborn Lgl1(-/-) pups develop severe hydrocephalus and die neonatally. A large proportion of Lgl1(-/-) neural progenitor cells fail to exit the cell cycle and differentiate, and, instead, continue to proliferate and die by apoptosis. Dividing Lgl1(-/-) cells are unable to asymmetrically localize the Notch inhibitor Numb, and the resulting failure of asymmetric cell divisions may be responsible for the hyperproliferation and the lack of differentiation. These results reveal a critical role for mammalian Lgl1 in regulating of proliferation, differentiation, and tissue organization and demonstrate a potential causative role of disruption of cell polarity in neoplastic transformation of neuroepithelial cells.     

Relating cistrons and functions in bacteriophage PM2

An approach to correlate functions with cistrons in bacteriophage PM2 is presented. Two of the temperature-sensitive mutants obtained differed from the wild type in heat sensitivity and four differed in host range. Comparison of the electrophoretic patterns of DNA from cells infected at the restrictive temperature with those obtained in wildtype infection revealed that viral DNA bands were absent with three additional mutants. Complementation analysis assigned the four host range mutants to cistron I and the three mutants defective in DNA synthesis to cistron IV. Recombination frequencies were used to locate cistrons III and IV on the partial genetic map of PM2.     

Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations

A study set comprised 44 Mycobacterium tuberculosis strains of the Beijing family selected for their representativeness among those previously characterized by IS6110-RFLP and spoligotyping (Northwest Russia, 1997 to 2003). In the present study, these strains were subjected to mycobacterial interspersed repetitive units (MIRU) typing to assess a discriminatory power of the 12-MIRU-loci scheme (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). The 44 Russian Beijing strains were subdivided into 12 MIRU types with identical profiles: 10 unique strains and two major types shared by 10 and 24 strains. Thus, basically, two distinct sublineages appear to shape the evolution of the Beijing strains in Russia. Most of the MIRU loci were found to be (almost) monomorphic in the Russian Beijing strains; the Hunter-Gaston discriminatory index (HGDI) for all 12 loci taken together was 0.65, whereas MIRU26 (the most variable in our study) showed a moderate level of discrimination (0.49). The results were compared against all available published MIRU profiles of Beijing strains from Russia (3 strains) and other geographic areas (51 strains in total), including South Africa (38 strains), East Asia (7 strains), and the United States (4 strains). A UPGMA (unweighted pair-group method with arithmetic averages)-based tree was constructed. Interestingly, no MIRU types were shared by Russian and South African strains (the two largest samples in this analysis), whereas both major Russian types included also isolates from other locations (United States and/or East Asia). This implies the evolution of the Beijing genotype to be generally strictly clonal, although a possibility of a convergent evolution of the MIRU loci cannot be excluded. We propose a dissemination of the prevailing local Beijing clones to have started earlier in South Africa rather than in Russia since more monomorphic loci were identified in Russian samples than in South African samples (mean HGDI scores, 0.08 versus 0.17). To conclude, we suggest to use a limited number of MIRUs for preliminary subdivision of Beijing strains in Russian (loci 26 + 31), South African (10 + 26 + 39), and global settings (10 + 26 + 39).     

Mother-child interaction in autistic and nonautistic children: characteristics of maternal approach behaviors and child social responses

The nature of mother-child interaction in autism and the maternal approach characteristics that elicit social response in children with autism were examined in two studies. Mother-child play sessions of 24 preschool children with autism and 24 typically developing preschoolers were compared in Study 1, and play sessions of 9 mothers with their autistic child and with their nonautistic child were compared in Study 2. Mother-child interactions were coded using the Approach Withdrawal Interaction Coding System to quantify maternal approach behaviors and child responses. Results of Study 1 indicate that, although the quantity of approaches did not differ between mothers with their autistic children and mothers with their nonautistic children, there were qualitative differences. Mothers used more physical contact, more high-intensity behaviors, and fewer social verbal approaches with autistic children. Results of Study 2 replicated these findings with mothers showing a similar pattern of approach toward their autistic children but not their nonautistic children. Although autistic children displayed lower contingency to maternal approaches in general, they showed greater responsiveness to approaches involving increased physical proximity and/or containing nonverbal object use. Mothers socially engaged both autistic and nonautistic children. The implications for parent training and intervention are discussed.     

Adrenomedullin and vascular endothelial growth factor production by follicular fluid macrophages and granulosa cells

BACKGROUND: Human follicular fluid contains several substances, such as cytokines and growth factors, which may affect follicular growth and maturation. The present study examines the relative contribution of macrophages and granulosa cells in the production of vascular endothelial growth factor (VEGF) and adrenomedullin in the human ovulatory follicle. METHODS: Both follicular fluid samples and blood samples were obtained at the time of oocyte retrieval following ovarian stimulation from 20 women undergoing IVF treatment because of male infertility. Human follicular fluid macrophages and luteinized granulosa cells were obtained from pooled follicular fluid of individual patients. Accumulation of VEGF and adrenomedullin in the culture medium of the isolated macrophages and human granulosa cells was determined at variable time intervals ranging from 0 to 48 h. Plasma and follicular fluid concentrations of VEGF and adrenomedullin were also measured. RESULTS: The follicular fluid concentrations of VEGF and adrenomedullin were significantly higher than those found in plasma. After 48 h, accumulation of VEGF in the culture medium of follicular fluid macrophages was significantly higher than that released in the culture medium of luteinized granulosa cells. In contrast, the production rate of adrenomedullin by follicular fluid macrophages was similar to that found in granulosa cells. VEGF secreted by follicular fluid macrophages increased progressively within 48 h of cell culture. A similar response pattern was observed with the culture medium of luteinized granulosa cells, but with lower production rates. CONCLUSIONS: This study suggests for the first time that both luteinized granulosa cells and macrophages actively secrete VEGF and adrenomedullin into follicular fluid in the human ovary.     

Interoceptive sensitivity and emotion processing: an EEG study.

Theories of emotion consider the self-perception of visceral activity to play an important role in emotion. This study examined the relationship between interoceptive sensitivity and both the subjective emotional experience and the processing of emotional pictures. According to their results in a heartbeat detection task subjects were classified as good (N = 17) or poor (N = 20) heartbeat perceivers. Event-related potentials were recorded while subjects viewed pleasant, neutral and unpleasant pictures and SAM ratings were examined. Good heartbeat perceivers showed significantly greater P300 and slow wave amplitudes for emotional pictures at antero-inferior, medial and posterior electrode sites and experienced a greater arousal for emotional pictures compared to poor heartbeat perceivers. The heartbeat perception score correlated significantly positive both with emotional P300 and slow wave amplitudes as well as with the arousal ratings for emotional pictures. The results indicate that there is a significant and strong association between interoceptive sensitivity and the intensity of emotional experience as well as the central processing of emotional stimuli.