Ultrastructural changes in cells of the inner enamel epithelium, stratum intermedium and reticulum stellatum in the enamel organ of the upper incisor of the Wistar rat at different phases of amelogenesis]

An electronmicroscopical study of the enamel organ of the upper incisors germs of Wistar rats was performed to analyse the ultrastructural features of the cells of the inner epithelium, the intermediate layer and the stellate reticulum, during preimary, young, transitional and mineralized enamel phases of amelogenesis. So, it was observed that the mitochondria in the ameloblasts are ovoid before the beginning of the enamel matrix formation and in the primary and young enamel phases. However, in the transitional and mineralized phases, these organelles are long and tortuous and some are characterized by a compact structure. In the cells of intermediate layer and stellate reticulum, the mitochondria are ovoid until the beginning of the mineralized phase. At the ending of this phase, these organelles are very long and present irregular form; many of them show also a compact structure. The "zonula adhaerens" could be observed only in the ameloblasts of the primary and young enamel phase. The cytoplasm of ameloblasts, during primary and young enamel phases is characterized by an abundance of free ribosomes and a branular endoplasmic reticulum; but during transitional and mineralized enamel phases, the cytoplasm of these cells shows little granular endoplasmic reticulum and free ribosomes, but ehe agranular endoplasmic reticulum is present. The granular endoplasmic reticulum and free ribosomes are abundant in the cells of the intermediate layer and stellate reticulum at the ending of the young enamel phase, in the transitional enamel phase and in the beginning of the minieralized phase. During different phases of amelogenesis, in the three above referred layers of the enamel organ, were also studied the features of the Golgi apparatus the presence and topographic distribution of the pigment granules, as well as the lysosomes, desmosomes and the tonophibriles.     

Evaluation of mutagenic activity in an extract of pepper tree stem bark (Schinus terebinthifolius Raddi)

An extract (decoction) from pepper tree stem bark (Schinus terebinthifolius Raddi) is widely used in Brazil as a topical antiinflammatory agent and to cicatrize wounds. The extract contains catechin, tannins, terpenes, flavonoids, and saponins; of these components, both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study, we evaluated an extract of S. terebinthifolius in a series of cell-free and bacterial assays in order to determine its genotoxic potential. The extract was negative in a cell-free plasmid DNA test, indicating that it did not directly break DNA. Positive results, however, were obtained in the SOS chromotest, in a forward mutagenesis assay employing CC104 and CC104mutMmutY strains of Escherichia coli, and in the Salmonella reversion assay, using strains TA97, TA98, TA100, and TA102. All the bacterial tests were performed without exogenous metabolic activation due to the topical use of this preparation. The results indicate that pepper tree stem bark extract produces DNA damage and mutation in bacteria, and that oxidative damage may be responsible for the genotoxicity.     

Differential effects of eight metal ions on lymphocyte differentiation antigens in vitro

In vitro studies were conducted to determine the effects of metal ions known to be released from metallic implants in vivo on the expression of lymphocyte surface antigens. Normal human peripheral blood lymphocytes were exposed to various concentrations of metal ions (Fe3+, Ni2+, Co2+, Mo6+, V5+, Cr6+, Cr3+, and Ti3+) for 30 min at 37 degrees C in a 5% CO2 atmosphere, and then analyzed for their ability to form rosettes with sheep red blood cells. Following this preliminary analysis, lymphocytes were exposed to the metal ions found to inhibit the E-rosette reaction (Fe3+, Ni2+, and Co2+) in order to determine which of the following surface antigens were affected: CD2, CD3, CD4, CD8, CD1, CD22, CD10, and HLA-DR. Our results showed that the in vitro treatment of lymphocytes with Fe3+ or Co2+ caused inhibition of CD2 only, whereas Ni2+ caused inhibition of both CD2 and CD3 antigens. These findings suggest that Fe3+, Co2+, and Ni2+ ions may interfere with T cell activation since both CD2 and CD3 are involved in that process.     

Estimation of the genetic contribution of presenilin-1 and -2 mutations in a population-based study of presenile Alzheimer disease

Two closely related genes, the presenilins ( PS ), located at chromosomes 14q24.3 and 1q42.1, have been identified for autosomal dominant Alzheimer disease (AD) with onset age below 65 years (presenile AD). We performed a systematic mutation analysis of all coding and 5'-non-coding exons of PS -1 and PS -2 in a population-based epidemiological series of 101 unrelated familial and sporadic presenile AD cases. The familial cases included 10 patients of autosomal dominant AD families sampled for linkage analysis studies. In all patients mutations in the amyloid precursor protein gene ( APP ) had previously been excluded. Four different PS -1 missense mutations were identified in six familial cases, two of which where autosomal dominant cases. Three mutations resulted in onset ages above 55 years, with one segregating in an autosomal dominant family with mean onset age 64 years (range 50-78 years). One PS -2 mutation was identified in a sporadic case with onset age 62 years. Our mutation data provided estimates for PS -1 and PS -2 mutation frequencies in presenile AD of 6 and 1% respectively. When family history was accounted for mutation frequencies for PS -1 were 9% in familial cases and 18% in autosomal dominant cases. Further, polymorphisms were detected in the promoter and the 5'-non-coding region of PS -1 and in intronic and exonic sequences of PS -2 that will be useful in genetic association studies.      

Compartmentation of glycolysis and glycogenolysis in the perfused rat heart

Developing methods that can detect compartmentation of metabolic pathways in intact tissues may be important for understanding energy demand and supply. In this study, we investigated compartmentation of glycolysis and glycogenolysis in the isolated perfused rat heart using (13)C NMR isotopomer analysis. Rat hearts previously depleted of myocardial glycogen were perfused with 5.5 mm [U-(13)C]glucose plus 50 mU/mL insulin until newly synthesized glycogen recovered to new steady-state levels ( approximately 60% of pre-depleted values). After a short wash-out period, the perfusate glucose was then switched to [1-(13)C]glucose, and glycolysis and glycogenolysis were stimulated by addition of glucagon (1 microg/ml). A (13)C NMR multiplet analysis of the methyl resonance of lactate provided an estimate of pyruvate derived from glucose vs glycogen while a multiplet analysis of the C4 resonance of glutamate provided an estimate of acetyl-CoA derived from glycolytic pyruvate vs glycogenolytic pyruvate. These two indices were not equivalent and their difference was further magnified in the presence of insulin during the stimulation phase. These combined observations are consistent with functional compartmentation of glycolytic and glycogenolytic enzymes that allows pyruvate generated by these two processes to be distinguished at the level of lactate and acetyl-CoA.     

Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen,but not by the LACK antigen,are protective against experimental Leishmania (Leishmania) amazonensis infection

Leishmania amazonensis is one of the major etiologic agents of a broad spectrum of clinical forms of leishmaniasis and has a wide geographical distribution in the Americas, which overlaps with the areas of transmission of many other Leishmania species. The LACK and A2 antigens are shared by various Leishmania species. A2 was previously shown to induce a potent Th1 immune response and protection against L. donovani infection in BALB/c mice. LACK is effective against L. major infection, but no significant protection against L. donovani infection was observed, in spite of the induction of a potent Th1 immune response. In an attempt to select candidate antigens for an American leishmaniasis vaccine, we investigated the protective effect of these recombinant antigens (rLACK and rA2) and recombinant interleukin-12 (rIL-12) against L. amazonensis infection in BALB/c mice. As expected, immunization with either rA2-rIL-12 or rLACK-rIL-12 induced a robust Th1 response prior to infection. However, only the BALB/c mice immunized with rA2-rIL-12 were protected against infection. Sustained gamma interferon (IFN-gamma) production, high levels of anti-A2 antibodies, and low levels of parasite-specific antibodies were detected in these mice after infection. In contrast, mice immunized with rLACK-rIL-12 displayed decreased levels of IFN-gamma and high levels of both anti-LACK and parasite-specific antibodies. Curiously, the association between rA2 and rLACK antigens in the same vaccine completely inhibited the rA2-specific IFN-gamma and humoral responses and, consequently, the protective effect of the rA2 antigen against L. amazonensis infection. We concluded that A2, but not LACK, fits the requirements for a safe vaccine against American leishmaniasis.     

Molecular characterization of invasive and noninvasive Campylobacter jejuni and Campylobacter coli isolates

Campylobacter jejuni is one of the most common causes of bacterial diarrhea worldwide and is the primary bacterial cause of food-borne illness. Adherence to and invasion of epithelial cells are the most important pathogenic mechanisms of Campylobacter diarrhea. Molecular characterization of invasive and noninvasive Campylobacter isolates from children with diarrhea and symptom-free children was performed by random amplified polymorphic DNA techniques (RAPD). A distinct RAPD profile with a DNA band of 1.6 kb was observed significantly more frequently among invasive (63%) than among noninvasive (16%) Campylobacter isolates (P = 0.000005). The 1.6-kb band was named the invasion-associated marker (IAM). Using specifically designed primers, a fragment of 518 bp of the iam locus was amplified in 85% of invasive and 20% of noninvasive strains (P = 0.0000000). Molecular typing with a PCR-restriction fragment length polymorphism assay which amplified the entire iam locus showed a HindIII restriction fragment polymorphism pattern associated mainly with invasive strains. Although cluster analysis of the RAPD fingerprinting showed genetic diversity among strains, two main clusters were identified. Cluster I comprised significantly more pathogenic and invasive isolates, while cluster II grouped the majority of nonpathogenic, noninvasive isolates. These data indicate that most of the invasive Campylobacter strains could be differentiated from noninvasive isolates by RAPD analysis and PCR using specific primers that amplify a fragment of the iam locus.     

High resolution microarray comparative genomic hybridisation analysis using spotted oligonucleotides

BACKGROUND: Currently, comparative genomic hybridisation array (array CGH) is the method of choice for studying genome wide DNA copy number changes. To date, either amplified representations of bacterial artificial chromosomes (BACs)/phage artificial chromosomes (PACs) or cDNAs have been spotted as probes. The production of BAC/PAC and cDNA arrays is time consuming and expensive. AIM: To evaluate the use of spotted 60 mer oligonucleotides (oligos) for array CGH. METHODS: The hybridisation of tumour cell lines with known chromosomal aberrations on to either BAC or oligoarrrays that are mapped to the human genome. RESULTS: Oligo CGH was able to detect amplifications with high accuracy and greater spatial resolution than other currently used array CGH platforms. In addition, single copy number changes could be detected with a resolution comparable to conventional CGH. CONCLUSIONS: Oligos are easy to handle and flexible, because they can be designed for any part of the genome without the need for laborious amplification procedures. The full genome array, containing around 30000 oligos of all genes in the human genome, will represent a big step forward in the analysis of chromosomal copy number changes. Finally, oligoarray CGH can easily be used for any organism with a fully sequenced genome.     

Prime number variant of concealed trigeminy

A 53-year-old man with myocardial infarction was found to have frequent premature ventricular beats. The predominant pattern was classical concealed trigeminy; i.e., the number of conducted sinus beats, S, between extrasystoles satisfied the equation S = 3n + 2, where "n" is zero or any positive integer. Two other transient patterns also occurred. The first one was characterized by exceptional values of S, which satisfied the equation S = 3n + 3. In the second transient pattern, all values of S fitted the classical equation, but there were singularly absent values; i.e., the "n" in the equation was exclusively an odd number, giving rise to only prime numbers of interectopic conducted sinus beats. It is proposed in this last form that there are two sites of fixed block proximal to a variable distal block in a re-entry loop responsible for the ventricular extrasystoles.