In vitro cytochemical studies of the golgi apparatus of pancreatic acinar cells exposed to drugs that inhibit the transport of membranes and proteins.

In a preceding article, we described alterations occurring in rat pancreas acinar cells at successive post-mortem (PM) intervals. In ultra-thin sections from samples obtained from 0.5, 1, 2, 4, 8 and 12 h, we observed in the Golgi apparatus the appearance of an anomalous membrane bound structure. Such structures are formed by tubules and vesicles that we have called tubular vesicular structure (TVS), and they are frequently located in the position corresponding to the 4th cisterna of the Golgian cisternal pile. Lobules of rat pancreas, incubated in vitro with metabolic inhibitors (such as antimycin A, sodium fluoride, sodium azide and potassium cyanide), were processed in order to be compared with the PM samples of the rat acinar cells. In sliced pieces of lobules, acid phosphatase (AcPase) and tiaminopirophosphatase (TPPase) activity were evaluated. Except for the potassium cyanide treatment, we frequently observed the TVS located at the position corresponding to the 4th cisternae (similar to those observed in the PM acinar cells). These TVS's are predominantly TPPase positive. Based on this result and the fact that the TVS's are surrounded by a membrane (as confirmed by the freeze-fracture replica results) with no structural elements inside, they seem not to correspond to autophagosomes. The TVS's, observed either at PM consecutive times or incubated with metabolic inhibitors, seem to be structures formed in response to ATP deprivation. In 0,5 h PM cells and in cells incubated for 30 and 60 min with metabolic inhibitors, the subcellular structures reacted for AcPase in the rigid lamellae, CV and lysosomes.

Analysis of DNA in minute volumes of blood from stains and crusts.

As the first step, the locus D1S80 was amplified by the polymerase chain reaction technique from genomic DNA extracted from artificial bloodstains and crusts with different amount of blood (32 microl, 16 microl, 8 microl, 4 microl, 2 microl, and 1 microl). In all samples of bloodstains and crusts, identification by DNA analysis was possible. As the second step, the locus HLA-DQA1 was amplified from genomic DNA extracted from diluted blood samples (640, 320, 160, 80, 40, 20, 10, and 5 leukocytes). DNA amplification was possible in diluted blood samples with at least 10 leukocytes. Considering the conditions in which the present study was carried out, it was possible to conclude that 1 microl of bloodstains or crusts was enough for identification. It was also concluded that five leukocytes are not enough material to render consistent DNA identification.

Parentage testing on blood crusts from firearms projectiles by DNA typing settles and insurance fraud case.

We describe a case of a fraudulent insurance claim. The family of an adult white male (DLF) notified the police of their son's disappearance. After a few weeks, a corpse that presented characteristics similar to those of the DLF was found in advanced stages of decay and was identified by the family as being DLF. The family then filed a claim for the life insurance that DLF had taken out just before he disappeared. Suspicions were raised about the identification of the corpse, because it had been done only visually, and because the insurance policy had been taken out just prior to DLF's disappearance. The insurance company requested a postmortem examination for identification. As the corpse had been cremated immediately after identification by the family, the biological material that was encrusted on the two projectiles removed from the body was used for analysis. The blood crusts provided enough genomic DNA for us to carry out PCR base typing of HLA-DQA1, D1S80, HUMCSF1PO, HUMTPOX, HUMTH01, D3S1744, D12S1090, D18S849, and amelogenin. Results from all loci typing from the corpse presumed to be that of DLF were then compared with that of his alleged biological parents, revealing genetic incompatibility.

Allele and genotype frequencies for D1S80 locus in a Brazilian population sample.

Gene and genotype frequencies in relation to the D1S80 locus were determined in a sample of 197 unrelated individuals (144 Caucasians and 53 Mulattoes), living in the city of São Paulo, Brazil. The Mulatto group was composed by mixed individuals who presented at least one negroid physical characteristic or declared themselves to be of mixed (Black-White) ancestry. Nineteen different alleles were detected in the Caucasian sample and 15 among Mulattoes. Alleles 18 and 24 were found to be the most common ones in the Caucasian population with frequencies of 0.173 and 0.357 respectively; the sample heterozygote frequency was estimated in 0.824. Alleles 18, 24, and 28 were found to be the most common alleles among Mulattoes with respective frequencies of 0.150, 0.349, and 0.113; the sample heterozygote frequency was 0.759. Fifty-five different genotypes were detected among Brazilian Caucasians whereas the respective figure among Mulattoes was 31. No significant deviations from Hardy-Weinberg equilibrium were found in both population samples.

Post-mortem forensic identity testing: application of PCR to the identification of fire victim.

CONTEXT: DNA analysis has been used with success in the identification of carbonized corpses and victims of large accidents. The analysis requires relatives of crash victims to donate blood for analysis. The relatives are generally willing contribute to the identification by giving a blood sample. OBJECTIVE: To describe the use of the polymerase chain reaction (PCR) for genetic characterization of one victim extensively burned by fire. DESIGN: Case report. CASE REPORT: DNA was extracted from blood of the cardiac chamber, and 15 different loci (D1S80, ApoB, D17S30, D3S1744, D18S849, D12S1090, FGA, D7S820, D1S533, D9S304, HUMCSF1PO, HUMTPOX, HUMTHO1, amelogenin and HLA-DQA1) were analyzed using the PCR technique. Results from all loci typing of the corpse were then compared to that of his alleged biological parents, revealing a genetic compatibility.

Potential definition of the time of death from autolytic myocardial cells: a morphometric study.

Morphometric methods and transmission electron microscopy were used to quantify modifications occurring in the mitochondria of dog myocardium during the first four hours of autolysis. Myocardial fragments were obtained from the outer free wall of the left ventricle, during anesthesia (control-zero) and at 15, 45, 120, and 240 min after cardiac arrest, maintaining the heart "in situ" at 22 degrees C. During the 240 min of autolysis, the main parameters evaluated showed: (a) a decrease in the number of mitochondria from 0.31 to 0.12 per micron 3 of cytoplasm. The decrease over the first 45 min reached 50% of the initial value; (b) an increase in mitochondrial volume, three times greater after the first 45 min (from 0.92 to 2.68 micron 3) and four times greater after 240 min (from 0.92 to 3.79 micron 3); (c) an increase in mitochondrial outer membrane surface area from 5.51 to 12.54 micron 2; (d) an increase in the surface area of individual mitochondria inner membrane and cristae from 27.60 to 56.96 micron 2. The progressive nature of the alterations and the difference in the numerically expressed values allow correlation with the time of somatic death. The authors emphasize the need for further studies in order to complement the present study.

Gene and genotype frequencies for HLA-DQA1 in Caucasians and Mulattoes in Brazil.

Gene and genotype frequencies of the HLA-DQA1 locus were determined in a sample of 197 unrelated individuals (144 Caucasians and 53 Mulattoes), living in the city of São Paulo, Brazil. The Mulatto group consisted of mixed individuals who presented at least one negroid physical characteristic or declared themselves to be of mixed ancestry. A total of six different alleles were identified with frequencies ranging from 0.087 to 0.316 in the Caucasian population and from 0.066 to 0.330 in the Mulatto population. We observed an increased frequency of allele 1.2 among Mulattoes in relation to Caucasians. The sample heterozygote frequency was 0.722 among Caucasians and 0.736 among Mullatoes. No significant deviations from Hardy-Weinberg equilibrium were found either in the Caucasian or in the Brazilian Mullato population samples.

Morphological and morphometric studies in pancreatic acinar cells of the rat at successive post-mortem intervals. The early appearance of a previously undescribed Golgi complex associated tubular vesicular structure.

Structural alterations in rat pancreatic acinar cells were studied in thin section at 0.5, 1, 4, 8, 12, 24 and 48 h post-mortem (PM). Morphometric analyses were performed both by light and electron microscopy, at 0.5 and 1 h PM. The parameters evaluated were: a) nuclear, cytoplasmic and cellular volumes; b) volume density and absolute volume of the rough endoplasmic reticulum (RER), mitochondria, zymogen granules (ZG), Golgi complex and its subcompartments [cisternae, condensing vacuole (CV) and 56-nm diameter vesicles], dense bodies (lysosome-like structures, electron-dense vacuoles and unidentifiable granules) and cytoplasmic matrix; c) surface density, surface/volume ratio and total surface area of the RER, mitochondria, ZG, Golgi cisternae, 56-nm diameter vesicles lying at the rough ER-Golgi interface, CV, and apical and basolateral membranes. Between 0.5 and 48 h, the mitochondria were dilated, junctional complexes were preserved and autophagic vacuoles were rare or absent. Flocculent densities were present in the mitochondria and chromatin condensation was observed at 4 h PM. In thin sections from samples obtained between 0.5 and 12 h, we consistently observed a membrane bounded structure formed by tubules and vesicles, designated as a tubular vesicular structure (TVS). These TVS's were observed at positions corresponding to the 4th Golgi cisterna. Fibrillar aggregates and a reduction in the number of 56-nm vesicles on the cis side of the Golgi were seen. Morphometry revealed a 60-70% reduction in the numerical density of the 56-nm vesicles between zero (control) and 0.5 h PM. These analyses also showed a 70% increase in the total volume and 57% increase in the total membrane surface of the Golgi cisternae in the PM period. The current results suggest that during the early PM (0.5 h) there is transport between Golgi compartments, and the 56-nm diameter vesicles fuse with the cisternae.

A three-dimensional reconstruction study of the rough ER-Golgi interface in serial thin sections of the pancreatic acinar cell of the rat.

Distinctive views of the tubulo-vesicular elements interposed between the endoplasmic reticulum (ER) and the Golgi apparatus were obtained in thin sections. The tubules that protrude from the transitional rough ER (tRER) are of dissimilar length. The numbers of tubules and of the nearby omega- and pear-shaped profiles decrease after fasting and are partially restored by refeeding. This formation is designated herein as the budding chamber of the tRER. Close to the budding chamber, clusters of 56 nm diameter vesicles are consistently observed. In some of the cells, convoluted tubules appear enmeshed with the presumptive transport vesicles of 56 nm diameter and with irregular, vesicular formations. Apparently structureless, electron-lucent ellipsoidal areas are found adjacent to these membranous elements. Serial and semi-serial sections show that the budding chamber, the sinuous tubules, the irregular vesicles, the structureless regions and the 56 nm vesicles fill tunnel-like spaces limited by the outermost Golgi cisterna (OGC) on one side and by the tRER on the other. Curved tubules appear to link the lumen of the OGC with that of smooth membranous occupants of these tunnel-like spaces. A presumptive luminal connection between these membranous occupants and the tubules of the budding chamber can also be seen. The predominant configuration of the OGC is that of a perforated, flat saccule. However, OGC regions exhibiting progressively lower densities of fenestrae, including smooth surfaced sectors eventually accumulating an intraluminal content are seen. Two such dilated, saccular portions of the OGC were analyzed through reconstruction of serial sections. Bundles of microtubules run closely apposed to the cis side of the OGC.