The use of acid phosphatase test papers for DNA profiling.

The acid phosphatase (AP) test is a routine assay used to screen casework items for the possible presence of semen. This colour test is carried out on filter paper which is retained after testing. Two-year-old AP test papers were found to contain sufficient DNA for short tandem repeat (STR) profiling. Prior to polymerase chain reaction (PCR) amplification, the DNA was preferentially separated into sperm depleted and sperm enriched cell fractions. The implication of these findings for past and present cases is discussed.

Use of chelating agents as terminators of alkaline phosphatase activity in enzyme-linked immunosorbent assay (ELISA) tests.

For a long time chelating agents have been known to be inhibitors of alkaline phosphatase activity. However, the use of chelating agents in stopping alkaline phosphatase activity in ELISA reactions is a novel application with several advantages over conventionally used acids or bases.

Use of human lice in forensic entomology.

Hematophagus arthropod bloodmeals may be useful in identifying individual hosts. To examine the application of human lice as a forensic tool, that is, as evidence of physical contact between individuals, body lice from a laboratory colony and head lice, collected from the head of infested children, were studied. The DNA profile of an individual was detectable in the pooled bloodmeals of two body lice, up to 20 h postfeeding. A mixed DNA profile of two hosts was identifiable in the pooled bloodmeals of three lice, for 3 h postfeeding. By pooling the bloodmeals of three adult and three nymphal head lice, a mixed DNA profile was obtained. These results indicate that, in criminal cases where there has been close contact between assailant and victim, louse bloodmeals should not be overlooked as potentially critical evidence.

Energy metabolism of adipose tissue--physiological aspects and target in obesity treatment.

Body fat content is controlled, at least in part, by energy charge of adipocytes. In vitro studies indicated that lipogenesis as well as lipolysis depend on cellular ATP levels. Respiratory uncoupling may, through the depression of ATP synthesis, control lipid metabolism of adipose cells. Expression of some uncoupling proteins (UCP2 and UCP5) as well as other protonophoric transporters can be detected in the adipose tissue. Expression of other UCPs (UCP1 and UCP3) can be induced by pharmacological treatments that reduce adiposity. A negative correlation between the accumulation of fat and the expression of UCP2 in adipocytes was also found. Ectopic expression of UCP1 in the white fat of aP2-Ucp1 transgenic mice mitigated obesity induced by genetic or dietary factors. In these mice, changes in lipid metabolism of adipocytes were associated with the depression of intracellular energy charge. Recent data show that AMP-activated protein kinase may be involved in the complex changes elicited by respiratory uncoupling in adipocytes. Changes in energy metabolism of adipose tissue may mediate effects of treatments directed against adiposity, dyslipidemia, and insulin resistance.

Haptoglobin phenotyping of bloodstains by horizontal electrophoresis on a compact polyacrylamide gradient gel.

A method is described for phenotyping haptoglobin by horizontal electrophoresis on a small polyacrylamide gradient gel. This method employs the same apparatus used in the separation of many red cell enzyme phenotypes and thereby eliminates the necessity for specialized vertical electrophoresis equipment.

The evidence notwithstanding--a case report on a rape.

This report concerns the rape of a woman by a stranger. The complainant stated that, during the course of the rape, penetration took place without ejaculation: these details were later confirmed by a suspect. This suspect, who was identified by the complainant, confessed and was convicted. Laboratory findings, however, indicated that the suspect could not have been the source of the semen found on the complainant's underpants. Further, a vaginal swab taken from the complainant contained intact sperm cells, an indication of a recent sexual contact. In spite of the presence of semen on the underwear which could not have come from the suspect, and the nonoccurrence of an ejaculation during the rape, the complainant maintained that she had not had a sexual contact for a month prior to the rape. This apparent contradiction and its implications are discussed.

DNA typing and blood transfusion.

As the result of a traffic accident, a man was seriously injured. Investigators found him outside the vehicle he had presumably driven. He was taken to the hospital in an unconscious state and there received a number of blood transfusions. Bloodstains found inside the car were collected and sent for comparison with a posttransfusion blood sample of the victim (suspect). As the car involved in the accident had been stolen, the police wished to ascertain whether there was a link between the suspect and the car. Furthermore, being unconscious, the suspect was unable to give a statement. The bloodstains from the car and the blood of the victim were tested by conventional blood group assays and DNA (RFLP and PCR). By conventional blood group assays, the effect of the blood transfusion were seen. On the other hand, the effect of the transfusion were not at all evident in the DNA assays. The implications of these results are discussed. Transfused blood, even in large quantities, did not alter the DNA profile of the recipient in this case.

A condom--the critical link in a rape.

An intact condom, reputedly used during a rape, was submitted for forensic examination. Conventional biochemistry results indicated that blood found on one side of the condom may have originated from the victim. Semen from the other side of the condom was not characterizable by conventional biochemical methods. Pubic hairs recovered from the condom matched those of the victim and not those of the suspect. Testing the blood and semen from the condom by DNA analysis gave the profile of the victim from the blood and the profile of the suspect from the semen.

DNA profiling of trace evidence--mitigating evidence in a dog biting case.

A young girl was the victim of a severe dog attack. An animal, suspected of having caused the attack, was later impounded for investigation. Microclots of blood, recovered from the dog's fur, were analyzed by STR DNA. Results showed that this blood was not related to the biting. Other forensic evidence--hairs, fibers, and odontology--failed to connect a particular animal to the attack. The implications of these findings for the dog and its owners are discussed as well as other forensic methods for resolving such cases.

The effects of blood transfusions on PCR DNA typing at the CSF1P0, TP0X, TH01, D1S80, HLA-DQA1, LDLR, GYPA, HBGG, D7S8 and GC loci.

Pre-transfusion and post-transfusion blood samples from eight individuals were typed at 10 PCR amplified loci. In no case did the PCR DNA profile of the post-transfusion blood sample differ from that of the pre-transfusion profile.

Wall hangings, hairs and the Holocaust.

Two wall hangings, from the time of the Second World War, were reportedly made from human hair. Laboratory examination of the hangings confirmed this fact. Fibers, holding the wall hangings together, and the weave of the hangings were also examined. Results and implications of the findings are discussed.

Chorionic villus sampling prior to pregnancy termination, a tool for forensic paternity testing.

Chorionic villus sampling (CVS), prior to pregnancy termination (pre-termination CVS), is suggested as a tool for forensic paternity testing. Unlike the abortion material, which consists of ruptured tissues of fetal and maternal origin, extra-embryonic membranes obtained through CVS can provide an uncontaminated source of fetal tissue for genotyping. We discuss the possibility of confined placental mosaicism (CPM) and its implications on the polymerase chain reaction (PCR) based analyses of short tandem repeats (STRs) and the D1S80 loci.

Polyunsaturated fatty acids of marine origin upregulate mitochondrial biogenesis and induce beta-oxidation in white fat.

AIMS/HYPOTHESIS: Intake of n-3 polyunsaturated fatty acids reduces adipose tissue mass, preferentially in the abdomen. The more pronounced effect of marine-derived eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on adiposity, compared with their precursor alpha-linolenic acid, may be mediated by changes in gene expression and metabolism in white fat. METHODS: The effects of EPA/DHA concentrate (6% EPA, 51% DHA) admixed to form two types of high-fat diet were studied in C57BL/6J mice. Oligonucleotide microarrays, cDNA PCR subtraction and quantitative real-time RT-PCR were used to characterise gene expression. Mitochondrial proteins were quantified using immunoblots. Fatty acid oxidation and synthesis were measured in adipose tissue fragments. RESULTS: Expression screens revealed upregulation of genes for mitochondrial proteins, predominantly in epididymal fat when EPA/DHA concentrate was admixed to a semisynthetic high-fat diet rich in alpha-linolenic acid. This was associated with a three-fold stimulation of the expression of genes encoding regulatory factors for mitochondrial biogenesis and oxidative metabolism (peroxisome proliferator-activated receptor gamma coactivator 1 alpha [Ppargc1a, also known as Pgc1alpha] and nuclear respiratory factor-1 [Nrf1] respectively). Expression of genes for carnitine palmitoyltransferase 1A and fatty acid oxidation was increased in epididymal but not subcutaneous fat. In the former depot, lipogenesis was depressed. Similar changes in adipose gene expression were detected after replacement of as little as 15% of lipids in the composite high-fat diet with EPA/DHA concentrate, while the development of obesity was reduced. The expression of Ppargc1a and Nrf1 was also stimulated by n-3 polyunsaturated fatty acids in 3T3-L1 cells. CONCLUSIONS/INTERPRETATION: The anti-adipogenic effect of EPA/DHA may involve a metabolic switch in adipocytes that includes enhancement of beta-oxidation and upregulation of mitochondrial biogenesis.

Multiple use of one-piece microtitration plates in ELISA (enzyme-linked immunosorbent assay) tests for the detection of cytomegalovirus and rubella virus antibodies.

Ninety-six-well, one-piece microtitration plates coated with rubella virus or cytomegalovirus (CMV) antigen can be used for multiple ELISA (enzyme-linked immunosorbent assay) testings. Only the number of test wells required per test need be used and the remaining unused test wells can be retained for subsequent assay. Consequently, as the one-piece microtitration plate is not a single-use, "all or none" element of the ELISA system, it is therefore as suitable for multiple ELISA testings as for one-time use. An alternate system of result interpretation for ELISA is introduced. Results are presented comparing the conventional optical density (OD) readings to values of the ratio: OD sample/OD low-positive sample.

Mitochondrial uncoupling and lipid metabolism in adipocytes.

Metabolism of white adipose tissue is involved in the control of body fat content. In vitro experiments indicated a dependence of lipogenesis on mitochondrial ATP production, as well as a reciprocal link between hormonal effects on metabolism and energetics of adipocytes. Therefore, mitochondrial uncoupling in adipocytes that results in stimulation of energy dissipation and depression of ATP synthesis may contribute to control of lipid metabolism and adiposity. This is supported by the expression of protonophoric proteins in adipocytes, e.g. uncoupling proteins (UCPs) 2 and 5, and some anion transporters, and induction of UCP1 and UCP3 in white fat by pharmacological treatments that reduce adiposity. Negative correlation between expression of UCPs in adipocytes and accumulation of white fat was also found. Expression of UCP1 from the adipose-specific promoter in aP2-Ucp1 transgenic mice mitigated obesity induced by genetic or dietary factors. The obesity resistance, accompanied by mitochondrial uncoupling in adipocytes and increased energy expenditure, resulted from ectopic expression of UCP1 in white but not in brown fat. Probably due to depression of ATP/ADP ratio in white fat of transgenic mice, both fatty acid synthesis and lipolytic action of noradrenaline in adipocytes were relatively low. These results support the role of protonophoric proteins in adipocytes in the control of adiposity. The main function of these proteins in white fat may be modulation of lipogenesis and intracellular hormone signalling. Augmentation of energy expenditure may be of relatively small importance, in accordance with the low oxidative capacity of white adipocytes.

Fast decline of hematopoiesis and uncoupling protein 2 content in human liver after birth: location of the protein in Kupffer cells.

Hepatic hematopoiesis is prominent during fetal life and ceases around birth. In rodent liver, the decline of the hepatic hematopoiesis starts abruptly at birth being accompanied by a decrease of mitochondrial uncoupling protein 2 (UCP2) expression in monocytes/macrophages, whereas hepatocytes may express UCP2 only under pathologic situations. The goals of this study were to characterize hepatic hematopoiesis in humans around birth, and to identify cells expressing UCP2. Hematopoiesis was evaluated histologically in the liver of 22 newborns (mostly very premature neonates), who died between 45 min and 140 d after birth, and one fetus. UCP2 expression was characterized by Northern blots, immunoblotting, immunohistochemistry, and by in situ hybridization. The number of hematopoietic cells started to decrease rapidly at birth, irrespectively of the gestational age (23-40 wk) of neonates. A similar decline was observed for UCP2 expression, which was relatively high in fetal liver. UCP2 was detected only in myeloid cells (mainly in Kupffer cells), but not in hepatocytes, although sepsis or other pathologies occurred in the critically ill newborns. Kupffer cells represent the major site of mitochondrial UCP2 expression in the human newborn. UCP2 may be essential for the differentiation and function of macrophages and serve as a marker for these cells in human liver during the perinatal period.

Role of energy charge and AMP-activated protein kinase in adipocytes in the control of body fat stores.

As indicated by in vitro studies, both lipogenesis and lipolysis in adipocytes depend on the cellular ATP levels. Ectopic expression of mitochondrial uncoupling protein 1 (UCP1) in the white adipose tissue of the aP2-Ucp1 transgenic mice reduced obesity induced by genetic or dietary manipulations. Furthermore, respiratory uncoupling lowered the cellular energy charge in adipocytes, while the synthesis of fatty acids (FA) was inhibited and their oxidation increased. Importantly, the complex metabolic changes triggered by ectopic UCP1 were associated with the activation of AMP-activated protein kinase (AMPK), a metabolic master switch, in adipocytes. Effects of several typical treatments that reduce adiposity, such as administration of leptin, beta-adrenoceptor agonists, bezafibrate, dietary n-3 polyunsaturated FA or fasting, can be compared with a phenotype of the aP2-Ucp1 mice. These situations generally lead to the upregulation of mitochondrial UCPs and suppression of the cellular energy charge and FA synthesis in adipocytes. On the other hand, FA oxidation is increased. Moreover, it has been shown that AMPK in adipocytes can be activated by adipocyte-derived hormones leptin and adiponectin, and also by insulin-sensitizes thiazolidinediones. Thus, it is evident that metabolism of adipose tissue itself is important for the control of body fat content and that the cellular energy charge and AMPK are involved in the control of lipid metabolism in adipocytes. The reciprocal link between synthesis and oxidation of FA in adipocytes represents a prospective target for the new treatment strategies aimed at reducing obesity.