Characterization of three aminopeptidases purified from maternal serum.

The biochemical characteristics of aminopeptidase A (EC 3.4.11.7), oxytocinase (EC 3.4.11.3) and alanyl aminopeptidase (EC 3.4.11.2) purified from serum of pregnant women were compared. Aminopeptidase A hydrolysed only acidic amino acid derivatives, whereas oxytocinase and alanyl aminopeptidase had partially overlapping broad substrate specificities. Oxytocinase showed the highest Vmax value with LeuNA but the lowest Km value with ArgNA (Km 0.059 +/- 0.08 mmol/l). Alanyl aminopeptidase hydrolysed AlaNA most rapidly, but showed the highest affinity for LysNA (Km 0.054 +/- 0.006 mmol/l). The enzymes were sensitive to EDTA. Co2+, Ni2+ and Zn2+ were able to reactivate all suppressed enzymes, but Mn2+ reactivated only aminopeptidase A after EDTA inhibition. The alkaline earth metals were activators of aminopeptidase A, while Co2+ activated only alanyl aminopeptidase. This enzyme was the most sensitive to L-amino acids. Acidic amino acids inhibited aminopeptidase A but had no effect on the two other enzymes. Oxytocinase was most sensitive to thermal treatment. Amastatin did not inhibit oxytocinase, whereas aminopeptidase A was more resistant than alanyl aminopeptidase to this effector.

Purification and partial characterization of aminopeptidase A from the serum of pregnant and non-pregnant women.

Aminopeptidase A (L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7) was purified from human maternal and control sera using CM-cellulose chromatography, DEAE-Sephacel chromatography, Sephacryl S-300 gel filtration and hydroxyapatite chromatography. The purification coefficients were 3069 and 5210 and the yields 6.3 and 6.1% for the maternal and control serum, respectively. The purified enzymes appeared free from other serum aminopeptidases in polyacrylamide gel electrophoresis. The biochemical and physical characteristics of the enzymes from maternal and control sera were similar. A molecular weight of 260 000, an optimum at pH 6.75-7.25 and a fairly good stability of the enzymes at 4 and -18 degrees C were recorded. The alkaline earth metals (Ca2+, Ba2+, Sr2+) were the activators of alpha-L-glutamyl-beta-naphthylamide hydrolysis, while alpha-L-aspartyl-beta-naphthylamide hydrolysis was markedly potentiated with Ca2+ but not with Ba2+ at all. The most rapid hydrolysis was shown with GluNA (Km with Ba2+ 0.156 +/- 0.014 mM and 0.136 +/- 0.009 mM in maternal and control serum, respectively), while only minimal hydrolysis of some neutral and basic amino-acid-beta-naphthylamides were observed. The contribution of the placenta to the elevated aminopeptidase A levels in the pregnancy plasma could not be solved on the basis of the present observations.

Age-dependent association of apolipoprotein E genotype with coronary and aortic atherosclerosis in middle-aged men: an autopsy study.

BACKGROUND: Apolipoprotein E (apoE) polymorphism is one of the genetic determinants of serum cholesterol values. The apoE epsilon4 allele has been associated with advanced coronary heart disease (CHD) diagnosed by angiography, but the role of the apoE genotype in atherosclerosis has not been confirmed at vessel-wall level, nor is any age-dependent effect of the apoE genotype on the development of CHD known. METHODS AND RESULTS: The right and left anterior descending coronary arteries (RCA and LAD) and the aorta from 700 male autopsy cases (Helsinki Sudden Death Study) in 1981-1982 and 1991-1992 (average age 53 years, range 33 to 70 years) were stained for fat, and all areas covered with fatty streaks, fibrotic plaques, and complicated lesions were measured. In the RCA and LAD, the apoE genotype was significantly associated with the area of total atherosclerotic lesions in men <53 years old but not with that in older men (P=0.0085 and P=0.041, respectively, for age-by-genotype interaction). Men <53 years old with the epsilon4/3 genotype showed 61% larger total atherosclerotic lesion area in the RCA (P=0.0027) and 26% larger area in the LAD (P=0.12) than did men with the epsilon3/3. The apoE epsilon4/3 was also associated with atherosclerotic lesions in the abdominal (P=0.014) and thoracic (P=0.12) aorta, but this effect, unlike that of the coronary arteries, was not age-related. CONCLUSIONS: In men, the apoE epsilon4 allele is a significant genetic risk factor for coronary atherosclerosis in early middle age. This suggests that at older age, other known risk factors of CHD play a more important role in the atherosclerotic process than apoE polymorphisms.

Characterization of three aminopeptidases purified from human placenta.

Three aminopeptidases purified from the human placenta were characterized and compared with each other. Aminopeptidase II1 preferred L-arginine- and L-lysine-beta-naphthylamides or p-nitroanilides as substrate, with low or negligible hydrolysis of other amino acid derivatives. It was inhibited by L-arginine, L-lysine and L-methionine. This enzyme activity was highly sensitive to heat treatment, N-ethylmaleimide, p-chloromercuribenzoate, puromycin, bestatin, epsilon-amino-n-caproic acid (EACA) and EDTA. After EDTA, this enzyme could be reactivated by Co2+. It is concluded that aminopeptidase II1 is identical with arginine aminopeptidase (EC 3.4.11.6) from other mammalian tissues. Aminopeptidase II2 preferred L-alanine-beta-naphthylamide and p-nitroanilide as substrates. It was also able to hydrolyse L-leucine, L-arginine, L-methionine and L-lysine derivatives but only very weakly L-cystine and Bz-L-cysteine substrates. This enzyme was inhibited by L-arginine, L-alanine, L-lysine and most strongly by L-leucine and L-methionine. It was resistant to bestatin and heat treatment but sensitive to EACA. EDTA caused a marked suppression, which could be prevented by Co2+ and Zn2+. These characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-) found in other tissues. The third enzyme was the only one clearly particle bound and was therefore called PB-aminopeptidase. It preferred L-leucine derivatives as substrate but also readily hydrolysed other amino acid-beta-naphthylamides and p-nitroanilides including L-cystine and Bz-L-cysteine substrates. Among the amino acids L-cysteine, L-leucine and L-methionine were inhibitory. Bestatin and thiol reagents were without effect and EACA was only moderately inhibitory. EDTA caused a strong suppression, which could be prevented by Co2+ and Zn2+. These properties are equal to those previously described for the placental cystine aminopeptidase (oxytocinase) (EC 3.4.11.3). All three enzymes had an optimum close to neutral pH but apparent differences in their Km and Vmax values with various substrates. These findings suggest that the three purified aminopeptidases are distinct enzymes. Two of these (aminopeptidases II1 and II2) have not previously been isolated and characterized in the human placenta.

Purification and partial characterization of human placental particle-bound aminopeptidase.

Particle-bound aminopeptidase was purified from the human term placenta by separation of the particles from the soluble fraction by centrifugation and solubilization of the particle fraction with 0.5 per cent (v/v) Triton X-100 followed by CM-cellulose chromatography, Sepharose 6B gel filtration, DE-cellulose and Con A-Sepharose 4B affinity chromatography. Polyacrylamide gel electrophoresis indicated enzymatic and protein homogeneity of the purified enzyme. The enzyme seemed to be a glycoprotein with a molecular weight of 320 000. The purified enzyme did not endure freezing but was fairly stable at 4 degrees C. At 60 degrees C more than half and at 65 degrees C all enzyme activity disappeared in 15 min. The pH dependence of the purified enzyme varied between 6.75 and 7.5, and 6.0 and 6.5, depending on the substrate used. The enzyme hydrolysed most rapidly LeuNA (Km 0.095 +/- 0.008 mmol) followed by beta-naphthylamide derivatives of alanine (Km 0.222 +/- 0.02 mmol), arginine (Km 0.041 +/- 0.002 mmol), lysine (Km 0.084 +/- 0.005 mmol), methionine (Km 0.085 +/- 0.004 mmol) and cystine (Km 0.048 +/- 0.004 mmol). Thus LeuNA was the most specific among the substrates for the enzyme. The purification process revealed, however, that CysNA was the most selective substrate for the particulate enzyme, which readily hydrolysed also Leu-p-na (Km 0.865 +/- 0.023 mmol) and Bz-Cys-p-na (Km 0.248 +/- 0.034 mmol).

Biochemical studies on dipeptidyl peptidases I to IV of the human placenta.

Homogenates of human term placentae were utilized to demonstrate the presence of four dipeptidyl peptidases (DPP I to IV) after sequential fractionations with gel filtration and anion-exchange chromatography. DPP I was assayed with SerTyrNA as substrate and showed the characteristics of a thiol-dependent serine enzyme with optimum at pH 4.5 and a molecular weight of 210,000. DPP II, analysed with LysAlaNA as substrate, had an optimum at pH 5.5 and a molecular weight of 130,000 with no dependence on thiol groups or divalent ions. DPP III was studied with ArgArgNA as substrate. It was optimally hydrolysed at pH 8.8 and had a molecular weight of 84,000. The enzyme was highly suppressed by chelating agents but could be reactivated by Co2+ and some other divalent ions. DPP IV, with GlyProNA as substrate, displayed an optimum at pH 8.2. The fractionations displayed multiple forms of the enzyme, possibly indicating the presence of proto- and polymeric DPP IV activities. The protomer had a molecular weight of 260,000 and showed no dependence on thiol groups but was sensitive to Zn2+.

Lumbar disc degeneration and atherosclerosis of the abdominal aorta.

STUDY DESIGN: This study analyzed the relationship of disc degeneration to atherosclerotic changes in the abdominal aorta and stenosis of the ostia of the lumbar and middle sacral arteries, 86 plain radiographs of lumbosacral spines, and the corresponding abdominal aortas were evaluated in connection with routine autopsy. RESULTS: Disc degeneration, advanced atherosclerotic manifestations, and stenosis of the ostia of the lumbar and middle sacral arteries all increased with age (P < 0.001). After partial rank correlation analysis, keeping the effect of age constant, there was still a statistically significant association between the grade of disc degeneration and stenosis of the ostia of the arteries supplying the disc. The association was stronger at upper lumbar levels (0.001 < P < 0.01) than at lower ones (0.01 < P < 0.05). Furthermore, with complicated lesions in the abdominal aorta, disc degeneration at all lumbar levels increased. CONCLUSIONS: Atherosclerosis in the abdominal aorta and especially stenosis of the ostia of segmental arteries may play a part in lumbar disc degeneration.

Asbestos exposure and pulmonary fiber concentrations of 300 Finnish urban men.

OBJECTIVES: The aim of the study was to determine the pulmonary concentrations of mineral fibers in the Finnish male urban population and to evaluate the analysis of pulmonary fiber burden by scanning electron microscopy (SEM) as an indicator of past fiber exposure. METHODS: The pulmonary concentration of mineral fibers was determined by SEM and compared with occupational history for a series of 300 autopsies of urban men aged 33 to 69 years. RESULTS: The concentration of fibers (f) longer than 1 micron ranged from < 0.3 to 163.10(6) per gram of dry tissue (f.g-1). Asbestos fiber concentrations exceeding 1.10(6) f.g-1 were observed in 33% of the cases with probable occupational exposure to asbestos and 1% of the cases with unlikely occupational exposure. Even asbestos fiber concentrations of 0.3 to 1.10(6) f.g-1, especially of crocidolite-amosite fibers, were rare among the men with unlikely occupational exposure. Fiber concentrations exceeding or equaling 1.10(6) f.g-1 were 10 times more frequent among the men more than 60 years of age as compared with those less than 40 years of age. Inorganic fibers other than asbestos had a weaker correlation with occupational history and age. Smoking habits had no significant effect on the pulmonary fiber counts. CONCLUSIONS: Asbestos fiber concentrations exceeding 1.10(6) f.g-1 are highly indicative of past occupational exposure to asbestos. The distribution of fiber concentrations in the different age groups of this study indicated decreasing asbestos exposure in Finland since the 1970s.

Independent forensic autopsies in an armed conflict: investigation of the victims from Racak, Kosovo.

In January 1999, a team of Finnish forensic experts under the mandate of the European Union (EU forensic expert team, EU-FET) performed forensic investigations in a sovereign state, in Kosovo, the Federal Republic of Yugoslavia (FRY). The team served as a neutral participant in the forensic investigation of victims of an incident at Racak, which was receiving considerable international attention. The Finnish team performed forensic autopsies, monitored forensic autopsies performed by local experts and verified findings of earlier executed autopsies. The victims had sustained varying numbers of gunshot wounds, which were established to be the cause of death. The manner of death remained undetermined by the EU-FET, because the scene investigation and the chain of custody for the bodies from the site of the incident to the autopsy were impossible to verify by the team. The events at Racak were the first of those leading to charges by the International Criminal Tribunal for the former Yugoslavia (ICTY) against the highest authorities in power in the FRY for crimes against humanity and violations of the laws or customs of war.

Genotyping of five short tandem repeat loci via triplex and duplex PCR.

We have developed a triplex PCR method for D3S1359, HumTH01 and HumTPO tetranucleotide loci and a duplex PCR method for HumFES/FPS and HumvWA31A tetranucleotide loci using high resolution polyacrylamide gel electrophoresis and silver staining. The methods were evaluated for paternity testing and individual identification and allele frequencies at these loci are reported for 189-3387 unrelated individuals in the Finnish population. The D3S1359 locus, especially, was found to be a highly informative locus. Seventeen alleles were found in the D3S1359 locus with a highest observed allele frequency of 0.199, a high exclusion power (PE) in paternity testing (0.78) and a high observed heterozygosity (0.89). The combined PE for these five loci was 0.99.

Forensic osteological investigations in Kosovo.

A team of Finnish forensic experts performed investigations of alleged mass graves in Kosovo under the mandate of the European Union (EU). Human skeletal remains from two locations were examined. The remains contained three almost complete skeletons, and individual bones and bone fragments, part of which were burned. Injuries, pathological changes, and findings for identification purposes were examined and documented using standard methods of forensic pathology and osteology. Gunshot injuries were found in some cases, but reliable determination of the cause and manner of death was not possible. A discrepancy arose between the number of victims reported in information received from the presiding district court, and results of the investigations. The estimation of the minimum number of victims was mostly acquired by DNA analysis.

Radiology in forensic expert team operations.

Radiological methods are widely used in forensic pathology. Their most common applications are in complementing human identification, particularly in investigations of mass disasters and decomposed bodies, and in searching for foreign material inside corpses. A team of Finnish forensic experts investigated human skeletal remains in Bosnia and Herzegovina (1996) and in Kosovo, the Federal Republic of Yugoslavia (1998). It also investigated more recently deceased victims in Kosovo (1999). In these investigations, the benefit of X-ray was in the detection of foreign material inside victims and their remains. For identification purposes, X-rays were mainly used to provide the best evidence possible of any pathological changes, physical characteristics, and injuries present.

Practical and legal aspects of forensic autopsy expert team operations.

Finnish forensic experts have investigated remains of the victims of alleged mass violence in the former Yugoslavia in 1996 under the mandate of the United Nations, and in 1998 and 1999 under the mandate of the European Union. The investigative documents later were surrendered to the International Criminal Tribunal for the former Yugoslavia (ICTY). The indictments issued by the ICTY include charges even against the highest authorities of the Federal Republic of Yugoslavia. This study describes the experience gained in organising forensic expert team operations in a foreign state by the Finnish team. The establishment and operation of a forensic expert team in a foreign state involve, among other things, legal issues, often related to differing legal systems. From an independent forensic expert team, great objectivity and self-constraint are expected. Moreover, a clear and sufficiently detailed agreement on the mandate of the team and on the possibility for unhindered and safe access of the experts to the alleged mass graves, as well as the assembling and briefing of the team members and provision of the necessary equipment should be completed before the beginning of the mission. Furthermore, the application of adequate and internationally recognised methods for processing and documenting the examination is essential for team's credibility.

Peripheral zonal hepatic necrosis caused by accidental ingestion of methyl ethyl ketone peroxide.

Fatal massive peripheral zonal hepatic necrosis developed in a 47-year-old man who accidentally ingested a solution of methyl ethyl ketone peroxide (MEKP) in dimethyl phthalate. Such solutions contain about 10% active oxygen. The clinical course was characterized by temporary cardiac arrest, abdominal burns, severe metabolic acidosis, rapid hepatic failure, rhabdomyolysis and respiratory insufficiency. A fatal outcome resulted 4 d afterwards from hepatic coma associated with blood coagulation disorders. Microscopical examination revealed massive periportal hepatic necrosis accompanied by atypical pseudoductular proliferation. The proliferating cells were probably of bile duct origin and exhibited atypia and mitoses. The pathogenetic mechanism may involve lipid peroxidation caused by free oxygen radicals derived from MEKP.

Radiographic demonstration of esophageal and tracheal fistulas at autopsy using a contrasting medium that vulcanizes at room temperature.

Esophageal and tracheal fistulas, which occur in 0.05% of medicolegal autopsies, were demonstrated in three cases by a postmortem radiographic technique using silicone rubber/lead oxide as a contrasting medium that vulcanizes at room temperature. In one 83-year-old male, a tracheoesophageal fistula was detected, which had developed after surgical repair of an esophageal rupture caused by a flexible fiberoptic endoscope. In a second case, carcinoma of the esophagus in a 78-year-old male had eroded the trachea and arcus of the aorta creating a fatal tracheoesophagoaortic fistula. In a third case, 55-year-old female developed a tracheobrachicephalic artery fistula as a result of an infiltrating cystic adenocarcinoma of the trachea, resulting in a fatal hemorrhage into the trachea. The results of this study indicate that diagnostic radiologic methods using a vulcanized contrasting medium are useful in supplementing normal dissection in autopsy cases with suspected fistulas of the esophagus or trachea.

Forensic investigations in Kosovo: experiences of the European Union Forensic Expert Team.

The international community has intervened ethnic hostilities in Kosovo, the Federal Republic of Yugoslavia. A team of Finnish forensic experts worked in December 1998 and January 1999 in Kosovo under the mandate of the European Union. The team investigated human remains from three locations of alleged ethnic violence. Concerning the events in Klecka, Volujak and Racak, the International Criminal Tribunal for the former Yugoslavia received documents of great gravity. The standard methods of forensic pathology were complemented with radiological, forensic odontological, physical anthropological and molecular genetic investigations.

Characterization of two thiol-dependent aminopeptidases partially purified from human placenta.

1. Two thiol-dependent aminopeptidases (I and III) were partially purified from the soluble fraction of human placenta. 2. Aminopeptidase I preferred L-alanine-beta-naphthylamide (AlaNA) as substrate at pH 7.0-7.5. It was sensitive to heat and some divalent metal ions (Co2+, Ni2+, Zn2+) but resistant to EDTA, and some amino acids. 3. Aminopeptidase III hydrolysed equally well AlaNA and ArgNA at pH 6.5-7.0. It was markedly suppressed by EDTA and reactivated by Co2+. It was inhibited by heat, some amino acids, benzamadine and puromycin.

Purification of three aminopeptidases from human maternal serum.

Three aminopeptidases (I--III) were purified from maternal serum using sequential chromatographic fractionations. Aminopeptidase I was specific for N-terminal alpha-L-dicarboxylic acid residues and activated by alkaline earth metals (Ba2+, Ca2+, Sr2+). It is concluded that aminopeptidase I is aminopeptidase A (L-alpha-aspartyl-(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7). Aminopeptidase II hydrolysed all tested substrates including L-cystine and Bz-L-cysteine derivatives but preferred L-leucine derivatives. The properties of aminopeptidase II are equal to those described for the cystine aminopeptidase (oxytocinase) (EC 3.4.11.3.). Aminopeptidase III preferred L-alanine derivatives as substrates. It was activated by Co2+, but strongly inhibited by amastatin, puromycin and L-methionine. The characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-).

Lectin binding of subunits of placental and serum oxytocinase after electrophoresis and transblotting to nitrocellulose.

1. Oxytocinase enzymes were purified from maternal serum and human placenta, run by SDS-PAGE and transferred onto nitrocellulose. Both enzymes were homogeneous in protein staining with Mr of 145,000. 2. Both serum and placental oxytocinases bound concanavalin A (Con A), limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA). The WGA-binding of the placental enzyme was more strongly inhibited by 0.2 M N-acetylglucosamine than that of the serum enzyme which may indicate a higher sialic acid content in the serum enzyme. 3. Neuraminidase treatment did not affect the binding of Con A but decreased the binding of WGA to serum and placental enzymes. Serum enzyme showed a pl 4.7 on isoelectric focusing.