Cryptococcus neoformans var. grubii - Pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile

The pathogenicity of Cryptococcus neoformans is heterogeneous and is associated with the expression of virulence factors. This study aimed to correlate the pathogenicity of C. neoformans var. grubii in BALB/c mice with in vitro virulence factors, fluconazole minimal inhibitory concentrations (MICs) and molecular profiles, before and after animal passage. Ten environmental isolates and one ATCC strain of C. neoformans var. grubii mating type α were evaluated. Most isolates (91%) killed 50% or more of the infected animals by day 24 postinfection and were recovered from the lungs and brains of surviving animals on days 7 and 14 postinfection. The burden of yeast in the lungs was more variable than that in the brain. The differences in the expression of virulence factors (growth at 37ºC, presence and size of the capsule and production of melanin, urease, proteinase and phospholipase) by most isolates pre and postpassage in animals were not statistically significant. The fluconazole MICs in postpassaged lines differed by a one-dilution from the MIC of the corresponding prepassaged line for six isolates. Using molecular typing [polymerase chain reaction-fingerprinting with (GACA)4 and M13], eight isolates were identified as VNI and three as VNII. We concluded that different isolates with the same molecular and phenotypic profiles, including isolates that are markedly hypervirulent, span a wide range of virulence and there were no changes in virulence factors in the postpassaged lines when compared with the corresponding nonpassaged lines.

Molecular aspects of hepatic carcinogenesis

Exogenous agents correlated with hepatocellular carcinoma (HCC) have been identified and well characterized. These agents, including the different viruses that cause chronic hepatitis and cirrhosis, can lead to regenerative nodules and dysplastic nodules/adenomatous hyperplasia. These conditions associated with several molecular alterations of hepatocyte ultimately culminate in hepatocellular carcinoma. Recently, there has been a great progress in the identification of somatic and germinative mutations that may be correlated with the development of HCC, justifying a review on the subject. Hence, the factors involved in the process of hepatic carcinogenesis, such as infection by the hepatitis B and C viruses, with a special focus in the molecular alterations described in recent years are discussed herein, pointing out areas potentially relevant for clinical development.

Molecular approaches to malaria and Babesisosis diagnosis

The development of additional methods for detecting and identifuing Babesia and Plasmodium infections may be useful in disease monitoring, management and control efforts. To preliminarily evaluate sunthetic peptide-based serodiagnosis, a hydrophilic sequence (DDESEFDKEK)was selected from published BabR gene of B. bovis. Immunization of rabbits and cattle with the hemocyanin-conjugated peptide elicited antibody responses that specifically detected both P. falciparum and B. bovis antigens by immunofluorescence and Western blots. Using a dot-ELISA with this peptide, antisera from immunized and naturally-infected cattle, and immunized rodents, were specifically detected. Reactivity was weak and correlated with peptide immunization or infection. DNA-based detection using repetitive DNA was species-specific in dot-blot formats for B. bovis DNA, and in both dot-blot and in situ formats for P. falciparum; a streamlined enzymelinked synthetic DNA assay for P. falciparum detected 30 parasites/mm(cúbicos) from patient blood using either colorimetric (2-15 h color development) or chemiluminescent detection (0.5-6-min. exposures). Serodiagnostic and DNA hybridization methods may be complementary in the respective detection of both chronic and acute infections. However, recent improvements in the polymerase chain reaction (PCR) make feasible a more sensitive and uniform approach to the diagnosis of these and other infectious disease complexes, with appropriate primers and processing methods. An analysis of ribosomal DNA genes of Plasmodium and Toxoplasma identified Apicomplexa-conserved sequence regions. Specific and distinctive PCR profiles were obtained for primers spanning the internal transcribed spacer locus for each of several Plasmodium and Babesia species.

Transmission of tuberculosis in Havana, Cuba: a molecular epidemiological study by IS6110 restriction fragment length polymorphism typing

The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55%) had unique IS6110 restriction fragment length polymorphism (RFLP) patterns, while isolates from 23 others (45%) had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.

Molecular characterization of soybean cultivars by microsatellite markers with universal tail sequence

The objective of this work was to standardize a semiautomated method for genotyping soybean, based on universal tail sequence primers (UTSP), and to compare it with the conventional genotyping method that uses electrophoresis in polyacrylamide gels. Thirty soybean cultivars were genotypically characterized by both methods, using 13 microsatellite loci. For the UTSP method, the number of alleles (NA) was 50 (2-7 per marker) and the polymorphic information content (PIC) ranged from 0.40 to 0.74. For the conventional method, the NA was 38 (2-5 per marker) and the PIC varied from 0.39 to 0.67. The genetic dissimilarity matrices obtained by the two methods were highly correlated with each other (0.8026), and the formed groups were coherent with the phenotypic data used for varietal registration. The 13 markers allowed the distinction of all analyzed cultivars. The low cost of the UTSP method, associated with its high accuracy, makes it ideal for the characterization of soybean cultivars and for the determination of genetic purity.

Genetic diversity of irrigated barley based on molecular and quantitative data and on malting quality

The objective of this work was to quantify the genetic diversity of elite genotypes of irrigated barley in the Brazilian savanna. Thirty elite barley genotypes from Embrapa Cerrados' collection were evaluated using 160 RAPD markers, 12 agronomic traits related to yield components, and 10 malting quality parameters. The genetic dissimilarity matrices based on molecular markers, quantitative traits, and malting quality characters were calculated and a cluster analysis was performed using the unweighted pair-group method with arithmetic mean (UPGMA) as grouping criterion. High genetic diversity among accessions were observed. The estimated genetic dissimilarities were weakly correlated, showing the complementarity of the different character groups. Selection indices and graphical dispersion analysis allowed the selection of promising genotypes and the indication of suitable crosses for maximizing the heterotic effects in breeding programs for irrigated barley in the Brazilian savanna.

Entendendo estrutura molecular com a molécula de hidrogênio ionizada

In this paper a general view about the modern molecular structure theory is developed discussing the ionized hydrogen molecule. We introduce some necessary approximation methods for the electronic and nuclear spectra study adopting a systematic approach. In addition though, we have performed calculations in order to illustrate these methods.

Determinação da estrutura molecular do ciclooctano por métodos ab initio e difração de elétrons na fase gasosa

The determination of the molecular structure of molecules is of fundamental importance in chemistry. X-rays and electron diffraction methods constitute in important tools for the elucidation of the molecular structure of systems in the solid state and gas phase, respectively. The use of quantum mechanical molecular orbital ab initio methods offer an alternative for conformational analysis studies. Comparison between theoretical results and those obtained experimentally in the gas phase can make a significant contribution for an unambiguous determination of the geometrical parameters. In this article the determination of the molecular structure of the cyclooctane molecule by electron diffraction in the gas phase and ab initio calculations will be addressed, providing an example of a comparative analysis of theoretical and experimental predictions.

Spectroscopic analysis of C80 doping with group III and group V elements using semiempirical PM3 molecular modelling technique

Semiempirical calculations at the level of PM3 of theory were carried out to study the structural and electronic properties of C80 and some of its doped derivatives with the elements of group III and V at the level of PM3 of theory. We have selected these elements to be substituted in the fullerene-C80 cage in order to show the effect of such structural change on the electronic properties of the molecules studied. The theoretical IR spectra, some of physical and chemical properties of the molecules studied are obtained and discussed.

The maspin expression in canine mammary tumors: an immunohistochemical and molecular study

The serpin maspin, a tumor suppressor in breast cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumor metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other types of cancer. Little is known about expression, regulation and function of maspin in canine mammary tumors. It was demonstrated in this study, a loss of maspin expression in malignant canine mammary cells compared with a pool of normal canine mammary tissue, analyzed by quantitative real-time PCR; weak maspin expression in malignant canine mammary tumors were observed by immunohistochemistry. It was also demonstrated that a correlation with nuclear maspin expression and a good prognosis. It is suggested that maspin could be used as a prognostic marker in canine mammary neoplasia.

Correlation between high molecular weight gluten subunits composition and bread-making quality in Brazilian wheat

Bread-making quality is one of the most important targets in the genetic improvement of wheat. Although extensive analyses of quality traits such as farinography, sodium dodecyl sulfate (SDS) sedimentation, alveography, and baking are made in breeding programs, these analyses require high amounts of seeds which are obtained only in late generations. In this experiment the statistical correlations between the high molecular weight subunit of glutenin and bread-making quality measured by alveograph, farinograph and SDS sedimentation were evaluated. Seventeen wheat genotypes were grown under the same conditions, each producing about 1 kg of seeds for the evaluations. The high molecular weight (HMW) glutenin subunits were analyzed by SDS-PAGE. Statistical correlations were highly significant between HMW glutenin subunits and alveograph and SDS sedimentation. These results indicate the possibility of manipulating major genes for wheat seed quality by coupling traditional breeding with non-destructive single seed analysis. Only half seed is necessary to perform the SDS-PAGE analysis. Therefore, the other half seed can be planted to generate the progeny. Seed yield and SDS sedimentation were statistically correlated, indicating the possibility of simultaneous selection for both traits

Experience with molecular and cytogenetic diagnosis of fragile X syndrome in Brazilian families

We report on the cytogenetic and DNA analysis of 55 families with the fragile X (FMR-1 locus) mutation (318 individuals and 15 chorionic villi samples). A total of 129 males were investigated, 54 mentally normal and 75 presenting mental retardation. Among the 54 normal males, 11 had the premutation, and none expressed the fragile site. The full mutation was detected in 73 retarded males, and 14 (18%) presented a premutation along with the full mutation (mosaics). All of them manifested the fragile site. The frequencies of fragile site expression correlated positively with the sizes of the expansion of the CGG repeats (D). Among 153 normal females, 85 were found to be heterozygous for the premutation and 15 had the full mutation. In the premutated females the fragile site was not observed or it occurred at frequencies that did not differ from those observed in 53 noncarriers. Cytogenetic analysis was thus ineffective for the diagnosis of premutated males or females. Among the 51 heterozygotes for the full mutation, 36 (70%) had some degree of mental impairment. As in males, a positive correlation was detected between the frequencies of fragile site manifestation and the size of the expansion. However, the cytogenetic test was less effective for the detection of fully mutated females, than in the case of males, since 14% false negative results were found among females. Segregation analysis confirmed that the risk of mental retardation in the offspring of heterozygotes increases with the length of D. The average observed frequency of mental retardation in the offspring of all heterozygotes was 30%. There was no indication of meiotic drive occurring in female carriers, since the number of individuals who inherited the mutation did not differ from the number of those inheriting the normal allele. No new mutations were detected in the 55 genealogies studied here.

Autoimmunity and molecular mimicry in tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy

Viruses share antigenic sites with normal host cell components, a phenomenon known as molecular mimicry. It has long been suggested that viral infections might trigger an autoimmune response by several mechanisms including molecular mimicry. More than 600 antiviral monoclonal antibodies generated against 11 different viruses have been reported to react with 3.5% of cells specific for uninfected mouse organs. The main pathological feature of tropical spastic paraparesis/human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (TSP/HAM) is a chronic inflammation of the spinal cord characterized by perivascular cuffing of mononuclear cells accompanied by parenchymal lymphocytic infiltration. We detected the presence of autoantibodies against a 98- to 100-kDa protein of in vitro cultured human astrocytes and a 33- to 35-kDa protein from normal human brain in the serum of HTLV-I-seropositive individuals. The two cell proteins exhibited molecular mimicry with HTLV-I gag and tax proteins in TSP/HAM patients, respectively. Furthermore, the location of 33- to 35-kDa protein cross-reaction correlated with the anatomical spinal cord areas (in the rat model) in which axonal damage has been reported in several cases of TSP/HAM patients. Our experimental evidence strongly suggests that the demyelinating process occurring in TSP/HAM may be mediated by molecular mimicry between domains of some viral proteins and normal cellular targets of the spinal cord sections involved in the neurodegeneration.

p53 immunostaining is correlated with reduced survival and is not correlated with gene mutations in resected pulmonary large cell carcinomas

Malignancy of pulmonary large cell carcinomas (LCC) increases from classic LCC through LCC with neuroendocrine morphology (LCCNM) to large cell neuroendocrine carcinomas (LCNEC). However, the histological classification has sometimes proved to be difficult. Because the malignancy of LCC is highly dependent on proteins with functions in the cell cycle, DNA repair, and apoptosis, p53 has been targeted as a potentially useful biological marker. p53 mutations in lung cancers have been shown to result in expression and protein expression also occurs in the absence of mutations. To validate the importance of both p53 protein expression (by immunostaining) and p53 gene mutations in lung LCC (by PCR-single strand conformational polymorphism analysis of exons 5, 6, 7, and 8) and to study their relationships with clinical factors and sub-classification we investigated the correlation of p53 abnormalities in 15 patients with LCC (5 classic LCC, 5 LCNEC, and 5 LCCNM) who had undergone resection with curative intent. Of these patients, 5/15 expressed p53 and none had mutant p53 sequences. There was a negative survival correlation with positive p53 immunostaining (P = 0.05). After adjustment for stage, age, gender, chemotherapy, radiotherapy, and histological subtypes by multivariate analysis, p53 expression had an independent impact on survival. The present study indicates that p53 assessment may provide an objective marker for the prognosis of LCC irrespective of morphological variants and suggests that p53 expression is important for outcome prediction in patients with the early stages of LCC. The results reported here should be considered to be initial results because tumors from only 15 patients were studied: 5 each from LCC, LCNEC and LCCNM. This was due to the rarity of these specific diseases.

Molecular adjuvant interleukin-33 enhances the antifertility effect of Lagurus lagurus zona pellucida 3 DNA vaccine administered by the mucosal route

It has been shown that cytokines can act as molecular adjuvant to enhance the immune response induced by DNA vaccines, but it is unknown whether interleukin 33 (IL-33) can enhance the immunocontraceptive effect induced by DNA vaccines. In the present study, we explored the effects of murine IL-33 on infertility induced by Lagurus lagurus zona pellucida 3 (Lzp3) contraceptive DNA vaccine administered by the mucosal route. Plasmid pcD-Lzp3 and plasmid pcD-mIL-33 were encapsulated with chitosan to generate the nanoparticle chi-(pcD-Lzp3+pcD-mIL-33) as the DNA vaccine. Sixty female ICR mice, divided into 5 groups (n=12/group), were intranasally immunized on days 0, 14, 28, and 42. After intranasal immunization, the anti-LZP3-specific IgG in serum and IgA in vaginal secretions and feces were determined by ELISA. The results showed that chi-(pcD-Lzp3+pcD-mIL-33) co-immunization induced the highest levels of serum IgG, secreted mucosal IgA, and T cell proliferation. Importantly, mice co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) had the lowest birth rate and mean litter size, which correlated with high levels of antibodies. Ovaries from infertile female mice co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) showed abnormal development of ovarian follicles, indicated by atretic follicles and loss of oocytes. Our results demonstrated that intranasal delivery of the molecular adjuvant mIL-33 with chi-pcD-Lzp3significantly increased infertility by enhancing both systemic and mucosal immune responses. Therefore, chi-(pcD-Lzp3+pcD-mIL-33) co-immunization could be a strategy for controlling the population of wild animal pests.