Molecular studies on ancient M. tuberculosis and M. leprae: methods of pathogen and host DNA analysis.

Humans have evolved alongside infectious diseases for millennia. Despite the efforts to reduce their incidence, infectious diseases still pose a tremendous threat to the world population. Fast development of molecular techniques and increasing risk of new epidemics have resulted in several studies that look to the past in order to investigate the origin and evolution of infectious diseases. Tuberculosis and leprosy have become frequent targets of such studies, owing to the persistence of their molecular biomarkers in ancient material and the characteristic skeletal lesions each disease may cause. This review examines the molecular methods used to screen for the presence of M. tuberculosis and M. leprae ancient DNA (aDNA) and their differentiation in ancient human remains. Examples of recent studies, mainly from Europe, that employ the newest techniques of molecular analysis are also described. Moreover, we present a specific approach based on assessing the likely immunological profile of historic populations, in order to further elucidate the influence of M. tuberculosis and M. leprae on historical human populations.

Mitochondrial DNA genetic diversity and LCT-13910 and deltaF508 CFTR alleles typing in the medieval sample from Poland.

We attempted to confirm the resemblance of a local medieval population and to reconstruct their contribution to the formation of the modern Polish population at the DNA level. The HVR I mtDNA sequence and two nuclear alleles, LCT-13910C/T SNP and deltaF508 CFTR, were chosen as markers since the distribution of selected nuclear alleles varies among ethnic groups. A total of 47 specimens were selected from a medieval cemetery in Cedynia (located in the western Polish lowland). Regarding the HVR I profile, the analyzed population differed from the present-day population (P = 0.045, F(st) = 0.0103), in contrast to lactase persistence (LP) based on the LCT-13910T allele, thus indicating the lack of notable frequency changes of this allele during the last millennium (P = 0.141). The sequence of the HVR I mtDNA fragment allowed to identify six major haplogroups including H, U5, T, K, and HV0 within the medieval population of Cedynia which are common in today's central Europe. An analysis of haplogroup frequency and its comparison with modern European populations shows that the studied medieval population is more closely related to Finno-Ugric populations than to the present Polish population. Identification of less common haplogroups, i.e., Z and U2, both atypical of the modern Polish population and of Asian origin, provides evidence for some kind of connections between the studied and foreign populations. Furthermore, a comparison of the available aDNA sequences from medieval Europe suggests that populations differed from one another and a number of data from other locations are required to find out more about the features of the medieval gene pool profile.

Frequency of P2RX7 A1513C and TLR2 -196 to -174 ins/del in healthy Polish individuals.

Polymorphisms within genes coding innate immune response proteins are involved in genetic susceptibility to various conditions. We investigated the frequency of P2RX7 A1513C and TLR2 -196 to -174 ins/del polymorphisms in healthy Polish population. Frequency of minor alleles was relatively similar to the pattern presented by Caucasian populations while it differed significantly when compared to non-European populations, which could be a result of variable selection pressure put upon studied alleles or hindered gene flow between populations.

Changes in frequency of IDDM-associated HLA DQB, CTLA4 and INS alleles.

The incidence of type 1 diabetes is increasing worldwide. In Poland, the number of cases tripled during the last two decades. The aim of this study was to test the hypothesis that the increase may be at least partly explained by a shift in predisposing alleles' frequencies - resulting from treating the otherwise lethal disease, generally better health care as well as selective pressure imposed by pathogens affecting humankind throughout history. The source of DNA was skeletal remains of 232 individuals excavated in four burial sites, dating back to 11th-14th centuries. With all necessary precautions required in ancient DNA analysis, frequencies of HLA DQB(57), CTLA4+49A/G and INS -23A/T alleles were assessed and compared with available data, characterising contemporary Polish population. Frequency of HLA DQB(57-Asp) protective allele is much higher in present-day population of Poland (50.6%) than in the group of 155 medieval specimens successfully typed for this polymorphism (28.4%, P < 0.001). Out of 86 medieval individuals typed for CTLA4+49A/G, 29.1% were homozygous for the predisposing G allele, which is significantly more than contemporarily - 7.6% (P < 0.001). No statistically significant difference was found in alleles and genotypes frequencies of INS-23A/T polymorphic site. Contrary to the initial assumptions, genetic predisposition towards type 1 diabetes, conferred by HLA DQB(57), CTLA4+49A/G and INS -23A/T alleles is much lower contemporarily than it was approximately 700 years before present. This suggests involvement of other than genetic factors in the fast growing incidence of the disease.

Sex diagnosis of subadult specimens from Medieval Polish archaeological sites: metric analysis of deciduous dentition.

The subject of this work is the characterisation of the metric features of deciduous dentition in a Medieval population of central Poland with the use of the jackknife technique leave one out (LOO)-supporting multivariate methods, which are important for deriving discrimination equations that would result in sex determination of children's skeletal remains. The sex of the individuals was assessed through analysis of sex-specific DNA sequences (AMELY/AMELX, SRY and alpha satellite sequences). Discriminant analysis concerned only teeth of those individuals whose sex was confirmed by the primary structure of three DNA sequences. The deciduous tooth diameters of males were found to be significantly larger than those of females in four respects: MD diameter of the maxillary second molar, MD and BL diameters of the mandibular first molar and BL diameter of the mandibular second molar. A two-group discriminant analysis considered all those measurements as independent variables. A multiple regression procedure produced a linear equation predicting the sex of children's skeletons with a significant probability amounting to approximately 78%. The accuracy of the sex assessment of an individual, using dental measurements, was established at 69% in deciduous male and 88% in deciduous female teeth.

Extremely high frequency of autoimmune-predisposing alleles in medieval specimens.

The precise etiology and reasons for the increase in incidence of autoimmune disorders still remain unclear, and although both genetic and environmental factors have been proven to shape individual predisposition, it is not known which of the factors, if not both, is responsible for the boom observed during the last decades. In order to establish whether a higher frequency of autoimmune-predisposing alleles may explain this increase we took advantage of ancient DNA methodology to establish the genetic predisposition, conferred by cytotoxic T lymphocyte associated antigen-4 (CTLA4) +49A/G and human leukocyte antigens (HLA) DQB1(57), in population inhabiting Poland in the Middle Ages. After successful typing of 42 individuals from a 12th approximately 14th's century archeological burial site, we found that frequencies of the predisposing alleles in the medieval population were higher than they are at present, suggesting thus that the recently observed incidence increase results most probably from factors of other than genetic nature.

Do determinants of platelet function co-segregate with genetic markers of type 1 diabetes mellitus?

This study examined the significance of selected parameters of primary haemostasis to discriminate between relatives of children with insulin-dependent diabetes mellitus (IDDM). Platelet function, including markers of spontaneous and agonist-induced platelet activation (CD62), platelet consumption (microparticles) and clumping (aggregates), as well as selected parameters of the fibrinolytic system (t-PA and PAI-1), were studied in IDDM children ( n = 45), their parents ( n = 65), siblings ( n = 17) and unrelated healthy controls ( n = 51). The fraction of activated platelets circulating in whole blood amounted to 4.3 +/- 2.1% in IDDM children, and significantly exceeded the level found in parents (1.3 +/- 0.7%, P < 0.002), siblings (1.2 +/- 1.0%, P < 0.002), and controls (1.2 +/- 0.6%, P < 0.002). Furthermore, an enhanced formation of platelet microparticles was observed in the IDDM group, both in resting platelets and also when platelets were stimulated with thrombin. Significantly decreased total PAI-1 occurred in IDDM children ( P < 0.02 versus parents); also slightly lowered active PAI-1 and t-PA antigen were noticed in IDDM subjects compared to other groups, however, the differences were not statistically significant. To assess dissimilarities between the groups of subjects we applied the forward stepwise model of discriminant function analysis, which included platelet flow cytometry parameters. The best separation and the highest discrepancy (expressed as the so called squared Mahalanobis distances, d ) was M revealed between controls and IDDM patients ( P < < 0.0001) and between controls and parents ( P < < 0.0001). The values of d found between IDDM children and their siblings (P < 0.001), as well as parents ( P < 0.01), were M of much lower significance. The finding that the control group, representing unrelated subjects, remains particularly well separated from the other groups, more or less clustered together, implies the possible involvement of genetic factor(s) which might potentially affect platelet activation and reactivity. In addition, the distinguished distribution of HLA DQAI(52) and HLA DQBI(57) genotypes in the groups further validates the suspicion that the altered platelet function and response in diabetes might be associated with some independent genetic factor(s), and is not likely to result from HLA DQAI(52) and HLA DQBI(57) impact.

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients.

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy on order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure on conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules ( the increased h+1/h0 parameter of maleimide spin label, MSL; 0.277 +/- 0.021 in diabetics vs 0.338 +/- 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (tau c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 +/- 0.42 in diabetics, n = 21 vs 4.79 +/- 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 +/- 1.08, n = 28 vs 7.31 +/- 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h+1/h0 parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.

Cystic fibrosis--V. Does cystic fibrosis alter the values of dynamic parameters of erythrocyte membrane ghosts?

1. The dynamic properties of erythrocyte membranes in CF children have been investigated by means of fluorescence and ESR techniques. 2. It has been revealed that the apparent distance separating the membrane protein tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased in CF children which results in a significant increase of the maximum energy transfer efficiency. 3. The slight increase in the ratio hw/hs of maleimide bound to membrane protein-SH groups of erythrocytes in cystic fibrosis may ensue the lowered membrane protein immobilization in the plane of lipid bilayer, especially at the intrinsic, more slowly reacting thiol groups.

The association between erythrocyte internal viscosity, protein non-enzymatic glycosylation and erythrocyte membrane dynamic properties in juvenile diabetes mellitus.

The association of intracellular viscosity of red blood cells and the dynamic properties of erythrocyte membranes in children suffering from diabetes has been investigated by means of ESR spectroscopy. It has been revealed that the slight decrease in the ratio hw/hs of maleimide bound to membrane protein-SH groups of erythrocytes in diabetes may ensue from the enhanced membrane protein immobilization in the plane of lipid bilayer. These alterations were accompanied by a corresponding increase in the relative rotational correlation time (tau c) of iodoacetamide spin label, thus suggesting that the conformational changes in membrane proteins may occur at both the intrinsic and more exposed thiol groups. The membranes of diabetic red blood cells were more glycosylated than those of relevant controls, and the extent of glycosylation was found to correlate significantly with h + 1/h0 and tau c (r = -0.652, P < 0.01 and r = 0.609, P < 0.01). Further, the conformational alterations in erythrocyte membranes from diabetic subjects were accompanied by a significant increase in the mobility parameter (h + 1/h0) of haemoglobin molecules in diabetic erythrocytes. The latter changes correlated well with the enhanced intracellular viscosity of diabetic red blood cells and the level of glycosylated haemoglobin. We conclude that the alterations in membrane lipid-protein interactions together with the increased glycosylation-derived internal viscosity may consequently imply altered viscoelastic properties of erythrocyte membranes and, underlying the impaired deformability of red blood cells in the diabetic state, contribute to the development of late diabetic sequelae.

Cystic fibrosis. IV. Structural analysis of a chloroform soluble compound isolated from gastric juice.

Cystic fibrosis (CF) is the most common autosomal disorder yet its cause is unknown. During an investigation of chloroform soluble fraction compounds of samples of gastric secretions from children with CF, a new substance was isolated. The new compound is concerned in alteration of membrane phospholipid composition, and changes in the activity of the enzyme transferring long-chain acyl moieties. Its structure is investigated, evaluated and discussed in relation to CF.

Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets.

The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.

Cystic fibrosis: II. Altered microviscosity of erythrocyte membrane.

Erythrocyte membrane fluidity alterations in cystic fibrosis are described. The relative flexibility of the membrane was studied using lipid spin label, i.e. methyl-5-doxylpalmitate (M5DP), and pyrene as a fluorescence probe. It was found that there was a decrease of membrane fluidity in the hydrophobic midzone of the membrane, probed by pyrene, as well as at the hydrophilic surface region, probed by M5DP.

Paraquat as an agent affecting antioxidant enzymes of common carp erythrocytes.

The effects of paraquat on the superoxide dismutase, catalase, glutathionine peroxidase activities and lipid peroxidation at different times of paraquat exposure of Cyprinus carpio morph L. erythrocytes were studied. Typical characteristics were observed in the changes of the enzyme activities of the erythrocytes after exposure to paraquat. The haemoglobin concentration of common carp haemolysates was decreased by exposure to paraquat.

Comparative studies on superoxide dismutase and catalase activities in livers of fish and other Antarctic vertebrates.

The activities of superoxide dismutase and catalase in livers of fish and other Antarctic vertebrates were examined. Significant differences between superoxide dismutase activities in livers of white-blooded and red-blooded fish species were observed. Superoxide dismutase seems to be involved more than catalase in protection processes against cell damage caused by oxygen free radicals.

Lipids associated with rat small-intestinal mucus glycoprotein.

The lipid content and composition of rat small-intestinal mucus, and the purified mucus glycoprotein before and after Pronase digestion were investigated. The mucus, obtained by the instillation of intestine with 2M NaCl, was fractionated on Bio-Gel A-50 in the presence of 6M urea and the mucus glycoprotein free of noncovalently bound protein was isolated. A portion of the purified glycoprotein was subjected to Pronase digestion to yield glycopeptides. The native mucus, and the purified glycoprotein and glycopeptides were extracted with chloroform-methanol, and the lipids contained in the extracts were analyzed. The lipids accounted for 17.6 of the dry weight of mucus, 26.4 of the mucus glycoprotein, and 25.3% of the glycopeptides. In comparison to mucus, the lipids associated with mucus glycoprotein contained 1.9 times more phospholipids and 2.1 times more glycolipids, showed a 26% increase in neutral lipids, and were virtually free of glycosphingolipids. Treatment of the purified glycoprotein with Pronase led to a moderate (22.3%) loss in neutral lipids, 4.3-fold decrease in phospholipids, and 52.3% increase in glyceroglucolipids. The results indicate that while the interaction of mucus glycoprotein with phospholipids involves its Pronase-susceptible region, the interaction with glyceroglucolipids occurs in the glycosylated region of the glycoprotein that is resistant to proteolysis.

Covalently linked fatty acids in gastric mucus glycoprotein of cystic fibrosis patients.

Undegraded mucus glycoprotein has been isolated in highly purified form from gastric secretion of cystic fibrosis patients. The purification procedure involved gel filtrations on Bio-Gel P-100 and Bio-Gel A-50 and lipid extractions with five mixtures of the organic solvents. The final preparation represented pure glycoprotein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cesium chloride density gradient centrifugation, and lipid analysis. Treatment of the pure and delipidated glycoprotein with methanolic KOH or hydroxylamine resulted in liberation of ester-bound fatty acids. Of the total released fatty acids, 95% were represented by hexadecanoate (36.5%), octadecanoate (48.7%), and octadecenoate (8.6%). The quantitative analysis established that, on the average, 12.2 nmol of fatty acids/mg of glycoprotein were released. The studies on cystic fibrotic glycoprotein susceptibility to proteolytic digestion indicated that fraction of glycoprotein which was resistant to pronase digestion contained on the average 33.1 nmol of fatty acids/mg of glycoprotein. After removal of the fatty acid residues from pronase-resistant glycoprotein, by treatment with hydroxylamine, the glycoprotein became susceptible to proteolytic digestion. Thus, in cystic fibrosis, the covalently bound fatty acids interfere with proteolytic degradation of mucus glycoprotein. Perhaps this is the major defect of cystic fibrosis glycoproteins and the cause of the obstruction of secretory glands and the accumulation of poorly soluble secretions.

Isolation of fatty acids covalently bound to the gastric mucus glycoprotein of normal and cystic fibrosis patients.

Covalently bound fatty acids were found in strictly purified and delipidated gastric mucus glycoprotein of normal and cystic fibrosis individuals. The susceptibility of this linkage to methanolic KOH and hydroxylamine treatment indicated the ester bond between fatty acids and glycoprotein. On the average, 2.9 nmol fatty acid/mg glycoprotein were found in normal samples, and 12.2 nmol/mg glycoprotein in samples derived from cystic fibrosis. In normal gastric mucus glycoprotein the covalently linked fatty acids consisted of hexadecanoate (47.0%), octadecanoate (22.0%), tetracosanoate (5.9%), octadecenoate (14.5%) and tetracosenoate (6.0%). In cystic fibrosis mucus glycoprotein the covalently bound fatty acids were comprised mainly of hexadecanoate (36.5%), octadecanoate (48.7%) and octadecenoate (8.6%). These data indicate that cystic fibrosis gastric mucus glycoprotein is highly acylated and perhaps this is the major defect of glycoproteins in this disease.