The effect of stabilizer composition on the thrombocytes and thrombocyte function in stored whole blood. II. Results of the thrombocyte spreading test]

The behaviour of thrombocyte spreading in ACD, AcD-A and AcD-AG stabilized blood was examined in 18 apparently healthy test persons for a storage period of 9 days. Due to an improved energy metabolism of thrombocytes the addition of adenin or guanosin respectively in ACD stabilized blood will cause the thrombocyte spreading to be widely preserved during storage.     

The effect of stabilizer composition on thrombocytes and thrombocyte function in stored whole blood. I. The thrombocyte count, thrombocyte aggregation and retraction during storage]

The behaviour of thrombocyte number and thrombocyte function aggregation and retraction in ACD, AcD-A and AcD-AG stabilized blood was examined in 18 apparently healthy test persons for a period of 9 days. On the one hand the addition of adenin or guanosin respectively increased the thrombocyte aggregation, on the other hand, however, a decrease of free, haemostatically efficient thrombocytes could be observed. Under the test conditions chosen retraction does not allow any statement to be made on the degree of the storage damage.     

Immunoenzymatic analysis and genetic detection of Cryptosporidium parvum in lambs from Italy

Cryptosporidiosis is a worldwide-diffused protozoan disease causing important economic losses to animal husbandry and livestock production. Additionally, several species/genotypes of Cryptosporidium have a relevant zoonotic potential and ruminants may be important sources of infection for human beings. Nonetheless, in Europe, little is known of the presence of Cryptosporidium in sheep nor on the species/genotypes involved. To obtain information on the occurrence of cryptosporidiosis in lambs and the potential zoonotic role of the Cryptosporidium isolates, one hundred and forty-nine faecal samples individually collected from lambs in central Italy have been examined for the presence of Cryptosporidium. All faecal specimens were processed with a commercial ELISA kit immunoassay and all ELISA-positive samples were further analyzed genetically. Twenty-six ELISA-positive samples scored positive at the PCR and the sequences obtained displayed 100% identity with the zoonotic Cryptosporidum parvum. This work suggests for the first time that lambs in Italy may shed C. parvum, thus representing a potential public health hazard.     

Immunoenzymatic techniques for the detection of Phoma exigua in infected potato tissues

Antisera to Phoma exigua var. foveata and var. exigua were prepared by injecting rabbits and mice with protein solutions from mycelium. Specific antibodies were isolated and immunoenzymatic techniques (double antibody sandwich ELISA and indirect ELISA) were used to test for the fungus in inoculated tubers and sprouts and in stems grown from these tubers. The fungus was detected in these different tissues, with var. foveata being more aggressive, demonstrating the applicability and sensitivity of the techniques. The antibodies produced to the two varieties of the fungus were not specific to their own varieties. They also reacted with Phoma tracheiphila but did not react with several other common potato pathogens. Preliminary results obtained with antibodies from mouse ascite liquid suggest the possibility of producing specific monoclonal antibodies.     

Immunoenzymatic detection of expressed gene fragments cloned in the lac Z gene of E. coli.

We describe a method which permits the detection of exon fragments. Such DNA was cloned and expressed in the promoter proximal part of the lac Z gene of Escherichia coli. The resulting antigen-beta-galactosidase chimeras are bound to their respective antibodies fixed to polyvinyl sheets. The beta-galactosidase part of the chimera permits detection of such clones by histochemical staining. As model DNA, we used the lac I gene cleaved with HaeIII, HhaI, or HpaII. Fragments were tailed with poly(dC) and inserted into the poly(dG)-tailed promoter proximal part of the lac Z gene. Recombinant clones, isolated on lactose-agar plates, were replica-plated and lysed with chloroform. Polyvinyl sheets coated with antibody against lac repressor were placed onto the top of the lysed colonies for immunoadsorption. The immune complexes were made visible after washing by incubation with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside in buffered agar. The beta-galactosidase activity of the chimera cleaves the colourless histochemical compound to a blue dye at those positions where clones produce the antigen. In the case of the lac I gene two types of clones were isolated, carrying the NH2-terminal part of the lac repressor up to codons 27 and 75.     

Binding of 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine (thrombocyte activating factor) by plasma components. Exchange of the thrombocyte activating factor between lipoproteins and thrombocytes

The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a platelet activating factor (PAF), to plasma components was studied. Gel filtration and lipoprotein fractionation revealed the presence in the plasma of PAF-binding fractions corresponding to plasma albumin as well as of low and high density lipoproteins. Incubation of PAF-containing lipoproteins with rabbit platelets resulted in a transfer of PAF to the platelets. PAF bound to plasma albumin is less exchangeable than PAF bound to lipoproteins. The PAF-transferring efficiency of high density lipoproteins (HDL) and of low density lipoproteins (LDL) correlates with the amounts of HDL- and LDL-receptors on the platelet surface. It may thus be assumed that PAF released by various cells interacts with lipoproteins which further transport the bound PAF to target cells carrying lipoprotein receptors.     

Prenatal diagnosis using fetal cells from the maternal circulation.

All current methods of fetal karyotyping are invasive and carry a definite, albeit small, procedure-related risk. Because of this and testing costs, only women older than 35 years who have a greater risk for fetal aneuploidy are currently offered prenatal testing. But this detects only 20% to 25% of fetuses with Down syndrome. It would be a tremendous advance to find a noninvasive technique for prenatal diagnosis that carries no procedure-related risk and could be offered to all pregnant women. We describe a possible technique for noninvasive prenatal diagnosis that aims to identify fetal cells in the peripheral maternal circulation and successfully garner them for prenatal testing. Early attempts at fetal karyotyping were hampered by inaccurate diagnostic methods and cumbersome cell-counting techniques. Today, improved capabilities of identifying and enriching for fetal cells, coupled with sensitive methods of analysis such as the polymerase chain reaction, bring renewed enthusiasm to this task. Many technical issues, as well as serious questions regarding the test''s utility, still exist, however, and must be explored and answered before the capture of fetal cells in the maternal circulation translates into reality for noninvasive prenatal diagnosis.     

A carbohydrate-containing antigen from Trypanosoma cruzi and its detection in the circulation of infected mice.

A polysaccharide-containing fraction has been prepared from the water-soluble material of a phenol-water extract of epimastigote forms of Trypanosoma cruzi. Sera from rabbits immunized with this material have been used to detect trypanosome-derived polysaccharide in plasma of mice acutely infected with this parasite. The polysaccharide nature of the circulating antigen is suggested by its heat stability, resistance to proteinase and nucleases, and destruction by sodium metaperiodate, as well as by its periodic acid-Schiff staining and precipitation with concanavalin A. The antigen appears to be present in different strains of the parasite and in different stages in the life cycle of the parasite.     

Comparison of a nested polymerase chain reaction--restriction fragment length polymorphism method, the PATH antigen detection method, and microscopy for the detection and identification of malaria parasites.

A nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, the PATH antigen detection method, and light microscopy were compared for their capacity to detect and identify Plasmodium species. One hundred and thirty-six blood specimens obtained from patients suspected of having malaria were examined by each of the three methods. Forty-four specimens were positive for malaria using microscopy as the "gold standard". The sensitivity for nested PCR was 100%, and the specificity was 98%. For the detection of Plasmodium falciparum, the antigen detection method had a sensitivity of 100% and a specificity of 97%. Species identification obtained using PCR-RFLP was identical or superior to light microscopy in 42 cases (96%). Although the nested PCR-RFLP method was more sensitive and specific, the rapid turnaround time and high sensitivity of the antigen detection method makes it a useful adjunct to standard microscopy.     

DNA restriction fragment length polymorphisms in differential diagnosis of genetic disease: application in neuromuscular diseases

Summary Three mutations causing alpha-1-antitrypsin defiiency have been identified by gene amplification and direct DNA sequencing. In the Pi (proteinase-inhibitor) null_cardiff gene, the codon for aspartate at position 256 has mutated to encode valine. In Pi M_malton and Pi I, the respective mutations are the deletion of the codon for a phenylalanine residue at position 51 or 52, and a single base substitution resulting in arginine being replaced by cysteine at position 39. Examination of the protein tertiary structure suggests that all of these mutations are likely to result in folding abnormalities that may explain the deficiency states.     

Carbonic anhydrase II deficiency: diagnosis and carrier detection using differential enzyme inhibition and inactivation.

Carbonic anhydrase (CA) I and II are soluble isozymes that represent the major nonhemoglobin proteins in the erythrocyte. We recently identified a deficiency of CA II as the enzymatic basis for the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Virtual absence of the CA II peak on high-performance liquid chromatography, of CA II esterase activity, and of immunoprecipitable CA II were demonstrated on extracts of red cell lysates from all patients studied. Reduced levels of CA II were found in obligate heterozygotes. Here, we present evidence that CA II in red cell lysates can be quantitated by measuring CO2 hydratase activity in the presence of inhibitors that selectively inhibit the activity of CA I to a much greater extent than that of CA II. This was done with iodide (anion binding) and bromopyruvic acid (alkylation), and the respective assays evaluated as diagnostic tools for CA II deficiency in human red cells. These techniques greatly simplify the quantitation of CA II in hemolysates and should make genetic diagnosis and counseling for the newly described inborn error of metabolism due to CA II deficiency generally available. They also allow quantitation of CA I in red cell lysates.     

Structure of the glycoprotein Ib.IX complex from platelet membranes

The glycoprotein Ib.IX complex is a major component of the platelet membrane. It mediates the adhesion of platelets to exposed subendothelium and provides an attachment site for the membrane skeleton on the plasma membrane. The present study was designed to characterize the structure of the glycoprotein Ib.IX complex. Electron microscopy of purified glycoprotein Ib.IX complex in detergent showed that each complex existed as a flexible rod with a globular domain on either end. The overall length of the complex was approximately 59.5 nm. The smaller globular domain had a diameter of approximately 8.9 nm; the larger, a diameter of approximately 15.9 nm. In the absence of detergent, the glycoprotein Ib.IX complexes tended to self-associate through the larger globular domain, suggesting that this domain contained the hydrophobic region that inserts into the membrane. Proteases known to cleave glycoprotein Ib alpha close to its membrane-insertion site released the larger globular domain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that this domain was composed of glycoprotein Ib beta, glycoprotein IX, and a Mr = 25,000 fragment of glycoprotein Ib alpha. Proteolysis at the external end of glycoprotein Ib alpha reduced the size of the smaller globular domain. This study shows that the glycoprotein Ib.IX complex has an elongated shape, with a globular domain on the end that inserts into the membrane and a smaller globular domain on the end of glycoprotein Ib alpha that is oriented external to the plasma membrane.