Estimation of carrier density and turnover rate of the Na+/H+ exchanger in human platelets using 5-(N-methyl-N-[3H]isobutyl)- amiloride.

We used the radiolabelled inhibitor of Na+/H+ exchange 5-(N-methyl-N-[3H]isobutyl)amiloride ([3H]-MIA) for assessment of the amount of Na+/H+ exchanger in intact human blood platelets. The inhibition constant, KI, of unlabelled MIA toward the antiport was determined at 100 nM. Washed platelets were incubated for 5 s with different concentrations of [3H]-MIA in the presence or absence of an excess concentration of unlabelled amiloride (400 microM). The platelets were rapidly centrifuged and the radioactivity in the pellet was determined. Scatchard analysis revealed one single class of specific binding sites (KD = 63 nM) and a maximum binding capacity of 500 sites/cell. The turnover rate of the Na+/H(+)-exchanger in unstimulated platelets was estimated at 800/s at 25 degrees C.     

Transcellular metabolism of neutrophil-derived leukotriene A4 by human platelets. A potential cellular source of leukotriene C4

Transformation of leukotriene (LT) A4 into leukotriene C4 has been found to be carried out by human platelets in a rather efficient manner. LTC4 was characterized by a combination of high performance liquid chromatography, UV spectrophotometry, use of labeled precursor, guinea pig ileum bioassay, and enzyme immunoassay. LTA4 metabolism was found to be substrate-dependent, time-dependent, and proportional to platelet concentration even at sub- or supraphysiological levels (0.0019-1 X 10(9) platelets/ml). Neither plasma alone nor the supernatant of resting or activated platelets was found to catalyze the production of LTC4 in the presence or in the absence of reduced glutathione. These data suggest that platelets contain a glutathione S-transferase specific for LTC4 biosynthesis. The formation of LTC4 was greatly enhanced when LTA4 was incubated with platelets in the presence of albumin. Low concentrations of albumin (2-4 g/liter) stabilized LTA4 to an extent that conversion into LTC4 by the platelets could be detected after 1 h of incubation. The possible intercellular transfer of LTA4 between neutrophils and platelets was tested. The production of LTC4 by neutrophils was greatly enhanced in the presence of platelets. Furthermore, the supernatant of neutrophils stimulated with the calcium ionophore contained a short-lived acid-labile substance which was converted by the platelets into LTC4. When platelets were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]glutathione, the coincubation of both cell types challenged with the calcium ionophore resulted in the production of [35S] LTC4. These data indicate that platelets can produce large amounts of LTC4 from neutrophil-derived LTA4. They also suggest that such interactions may occur in vivo and that platelets could be an important contribution to the generation of the biologically active LTC4.     

Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. II. Pig platelets.

Since 1966 the domestic pig has served as the animal model in Malignant Hyperthermia (MH) research [1]. The use of genetically well-defined pigs rendered it possible to test the method for diagnosing MH-susceptibility of patients presented in the preceding paper. Thus, the effect of halothane on intracellular calcium movements was studied in Quin-2- and chlorotetracycline-loaded pig platelets. In 'Ca(2+)-free' suspensions the resting level of free cytosolic Ca2+ was about 60 nM. In contrast to the results with human platelets there were no significant differences between pig genotypes either in the absence or in the presence of external calcium. After addition of halothane, a mobilization of intracellular membrane-bound calcium can be observed. However, the calcium mobilization is not accompanied by a marked increase in fluorescence intensity of Quin-2-loaded platelets. Thus, in the absence of external calcium, halothane produces only a slight increase in free cytosolic Ca2+. Nevertheless, the calcium rises measured in platelets from affected animals were statistically significantly higher than those from normal subjects. However, in the presence of 1 mM external calcium, a rapid increase in free cytosolic calcium can be detected after halothane addition. This suggests that halothane causes a marked, dose-dependent increase in Ca2+ permeability of the plasma membrane. Compared to the control group, significantly enhanced calcium permeability was found, not only in homozygous positive pigs, but also in heterozygous animals.     

Effects of very rapid versus vapor phase freezing on human sperm parameters

The aim of the present study was to compare the effects of two freezing methods, vapor phase and very rapid freezing, with and without cryoprotectant on semen parameters in men with normal semen criteria. Cryopreservation was done on semen samples from 31 men by two methods of vapor phase freezing and very rapid freezing, with and without Test Yolk buffered glycerol (TYBG) as cryoprotectant. The motility, viability, acrosome and DNA integrity were evaluated on fresh and post-thaw samples. Post-thaw sperm progressive motility was significantly higher in the presence of TYBG in the vapor phase cryopreservation (%6.30 ± 3.74) compared with the very rapid freezing method (%2.2 ± 1.97 and %4.00 ± 2.42 in the presence and absence of TYBG, respectively). There was no significant difference in viability, acrosome status and DNA integrity between two methods in presence or absence of TYBG. The very rapid freezing method in the absence of TYBG showed better sperm motility but viability, acrosome and DNA integrity were similar to the presence of TYBG. The results show that cryopreservation of human spermatozoa together with seminal plasma by using vapor phase method is better than very rapid freezing method to preserve sperm progressive motility; however very rapid freezing method is quick and simple and do not require special cryoprotectant. It can be used for cryopreservation of small number of spermatozoa in IVF centers.     

Absence of satellite III DNA in the centromere and the proximal long-arm region of human chromosome 14: analysis of a 14p- variant.

Cytogenetic methods and molecular probes derived from the centromere and short arm of chromosome 14 were used to investigate the structural properties of a chromosome 14 variant. Results of GTL, CBG, Ag-NOR, and non-banded Giemsa staining of the chromosomes suggested the complete absence of the short arm and possibly a large part of the centromere. Negative in situ hybridisation with an alpha satellite III probe confirmed the absence of the arm; the detection of normal amounts of alpha satellite DNA, however, indicated retention of the centromeric domain. The natural occurrence of a human acrocentric variant lacking a short arm was thus established. Within the detection limits of the methods used, the results demonstrate that satellite III DNA is not essential for normal centromeric activity and allow us to exclude the presence of this satellite DNA within the centromere and proximal long-arm region of human chromosome 14.     

The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.     

In vitro study of platelet concentrate preparations from whole blood using N-(2-mercaptopropionyl)-glycine

The sulfhydryl group containing drug N-(2-mercaptopropionyl)-glycine (MPG) which inhibits platelet aggregation in a reversible manner permits to prepare platelet concentrates in non-siliconized glass containers at pH 7.4. Resuspension of platelets is possible immediately after centrifugation. In vitro platelets tests were carried out after washing out the MPG in MPG-free plasma. Thereafter, no inhibitory effects on platelet functions were found. Platelets concentrated in presence of MPG were significantly better with respect to yield, maintenance of discoid shape, aggregability, and hypotonic shock response compared with control platelets concentrated in absence of MPG.     

Calcium handling by platelets from normal and malignant hyperthermia-susceptible pigs

Platelets from normal and malignant hyperthermia (MH)-susceptible pigs were evaluated for differences in 45calcium uptake in the absence or presence of caffeine (2-16 mM), halothane (0.05-0.5%), or halothane and caffeine together. There were no statistically significant differences in basal or halothane-inhibited calcium uptake by platelets from either source. There was a small statistically significant difference in calcium uptake between platelets from normal and MH-susceptible pigs in the presence of 16 mM caffeine and 0.5% halothane. Calcium uptake by platelets from one pedigree of MH-susceptible pigs were stimulated in a concentration-dependent manner by caffeine. These data suggest that exposure of platelets to caffeine may have potential for identifying MH-susceptibility.     

Effect of albumin and polyanion on the structure of DNA complexes with polycation containing hydrophilic nonionic block.

Self-assembling systems based on ionic complexes of DNA with block copolymer of N-(2-hydroxypropyl)methacrylamide with 2-(trimethylammonio)ethyl methacrylate were studied as systems suitable for gene delivery. In this study, the influence of albumin and polyanion on parameters of the DNA polyelectrolyte complexes in aqueous solutions was investigated. Static and dynamic light-scattering methods were used as a main tool for characterizing these interactions. It was found that albumin is not able to release free DNA, but it can rather bind to the complexes forming ternary DNA-polycation-albumin complexes with increased hydrodynamic radii of about 10 nm. Polyanion tested, sodium poly(styrenesulfonate), was able to release free DNA in the presence of a low-molecular-weight electrolyte. In the absence of a low-molecular-weight electrolyte, only formation of ternary complexes and no DNA release was observed. The in vivo biodistribution analysis of DNA complexes showed no effect of the presence of hydrophilic nonionic poly(HPMA) on the circulatory time or organ distribution. The interaction of DNA complexes with albumin and other plasma proteins was suggested to be a major reason for the short circulatory times.     

Leaf and stem epicuticular wax structures in Lonicera species (Caprifoliaceae)

Epicuticular waxes are found on the surface of the plant epidermis as crystal structures which show morphological diversity and may be useful in plant systematics. The aim of this study was to recognise the diversity of the epicuticular wax cover on leaves and stems of Lonicera species (Caprifoliaceae) and to estimate the taxonomic value of wax traits. Adaxial and abaxial sides of the leaves and the stem surface in 35 taxa were investigated through a scanning electron microscope (SEM). The assessment was prepared on the basis of the presence or absence of basic types of wax structures and their dimensions, using categorical response analysis, multiple correspondence analysis and cluster analysis. In the analysed species, we found differences in the wax layer structure on the surfaces of the leaves and stems. Apart from the smooth layer, tubules and platelets can be found. Tubules are considerably more frequent than platelets. In many cases, the same plant part produces more than just one wax type. Our analysis indicates that wax structures are not very informative for the taxonomy of Lonicera, because we observed only a weak correspondence between the presence or absence of some types of wax tubules and the infrageneric classification of the genus. However, in particular cases these new data can be used for species identification.     

DNA RNA and protein content of tissue during growth and embryogenesis in wild-carrot suspension cultures

Summary A highly selected population of cells (clumps from 63 to 125 μm in diameter), obtained by screening 14-day-old stock suspension cultures of wild carrot (Daucus carota L.), was used to initiate cultures in this study. Time-course changes in DNA, RNA and protein were followed when these cultures were grown in the presence or absence of 2.25 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The data show that growth of these cultures, particularly in the early part of the growth curve, is different from that in most other studies reported on suspension cultures initiated without screening. The gross compositional analysis shows that this difference stems from the very high RNA:DNA and protein:DNA ratios of the cellular material used as the inoculum in this study. The presence of 2,4-D in the medium promoted total RNA and protein levels. Correlations were sought between the appearance of embryos in the absence of exogenous 2,4-D and gross compositional differences developing in cultures grown in the presence and absence of 2,4-D. The handling of cultures during inoculation appeared to have led to a substantial loss of DNA. This had, however, little effect on dry weight or protein content of the tissue. This research was supported by the W. Alton Jones Foundation.     

Aggregation and/or oxygenated products of arachidonic acid are not required for collagen-induced deacylation of phosphatidylcholine in human platelets.

In the present study the effects of collagen on platelet aggregation and arachidonic acid (AA) mobilization, specifically from phosphatidylcholine (PC), were investigated in the presence and absence of BW755C, a selective inhibitor of cyclo-oxygenase and lipoxygenases. The inhibition of cyclo-oxygenase and lipoxygenase(s) by BW755C (75 microM) resulted in severe impairment in collagen-induced platelet aggregation. In the presence of BW755C, the aggregation response amounted to 14, 26, 37 and 49% of the corresponding controls (without BW755C) at 10, 25, 50 and 100 micrograms of collagen respectively. On the contrary, the amount of AA released from PC, which ranged from 3.5 to 8.6 nmol/10(9) platelets, in response to the action of collagen (10-100 micrograms) remained unaffected by the presence of BW755C. Substantial amounts of non-esterified AA were detected in the free fatty acid fractions obtained from collagen-stimulated platelets in the presence as well as in the absence of BW755C. However, the presence of BW755C caused a greater accumulation of free AA (mass) and this ranged from 4 to 16 nmol, depending upon the amount of collagen. In addition, small increases in free stearic and oleic acids were observed in collagen-stimulated platelets as compared with unstimulated platelets. The amount of AA lost from PC represented 67, 80, 49 and 52% of the free AA obtained at 10, 25, 50 and 100 micrograms of collagen respectively. Our results adhesion of platelets to collagen fibres may be responsible for much of the AA release from PC Furthermore, these results demonstrate that aggregation and/or cyclo-oxygenase/lipoxygenase metabolites are not obligatory for the release of AA from PC in collagen-stimulated human platelets.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

Modulatory roles of RANTES in IL-4 production by human blood CD4(+)T cells

An increase in serum IgE levels has been reported in several thrombotic cardiovascular diseases. Since such diseases are associated with the activation of platelets, we hypothesized that platelets are implicated in a mechanism leading to heightened IgE synthesis. To this end, we performed an in vitro investigation of the effects on IL-4 production caused by several bioactive substances potentially released from platelets. Human blood CD4(+)T cells from blood donors were stimulated with anti-CD3 antibody and costimulatory signals delivered via CD58 and CD80 in the presence or absence of IL-4. One of the following test substances was also included in the culture: platelet factor-4, beta-thromboglobulin, platelet-derived growth factor, serotonin, platelet activating factor, or RANTES. The cells were restimulated in the absence of IL-4 and test substances. Among the six substances, RANTES alone exhibited significant effects on IL-4 production. RANTES enhanced IL-4 production in the presence of IL-4, whereas it suppressed IL-4 production in the absence of IL-4. The enhancing effect of RANTES was positively correlated with plasma IgE levels in the donors. We concluded that RANTES may induce IgE synthesis by increasing IL-4 production in individuals predisposed to high IgE responses. Our observations indicate a link between platelets and immune phenomena associated with increased IgE responses.     

Detection of plasmid DNA from all Chlamydia trachomatis serovars with a two-step polymerase chain reaction.

A polymerase chain reaction was used to amplify a 137-base-pair sequence of DNA from a Chlamydia trachomatis plasmid. Various parameters of the polymerase chain reaction were explored, and it was found that two short steps per reaction cycle were sufficient to achieve 10(12)-fold amplification in less than 1 h. By use of this procedure, 10(-18) g of a sequence of plasmid DNA, representing the amount of that sequence found in one C. trachomatis bacterium, was amplified to the point where it was clearly visible on an ethidium bromide-stained polyacrylamide gel under UV light. DNA from intact cells from each of the 15 serovars of C. trachomatis could also be amplified for visualization. With this procedure, the presence or absence of C. trachomatis DNA in a sample could be established in less than 1.5 h. The speed and extreme sensitivity of this detection procedure may make it a useful method for the detection of C. trachomatis, and similar techniques should be possible for any type of bacteria.     

Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee

Summary The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 11 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.     

Genetic Diversity of ‘Ubá’ Mango Tree Using ISSR Markers

In this study, the genetic diversity of ‘Ubá’ mango trees cultivated at the Zona da Mata of Minas Gerais State, Brazil, was assessed, to identify whether there is variability in the plants grown in the region, justifying the mass selection as a breeding method. We used 102 accessions. Leaves were collected for extraction of genomic DNA, which was amplified with nine ISSR primers. The data obtained by the analysis of electrophoretic patterns were arranged in a binary matrix, considering 0 for the absence and 1 for the presence of bands. Based on these data, we performed the analysis of genetic dissimilarity and carried out the cluster analysis by the methods of Tocher and graphical dispersion. The most similar accessions are 144 and 150, both coming from Ubá, while the most divergent ones are 29 and 97, from Visconde do Rio Branco. The grouping by the Tocher method separated the accessions into six groups, 94.1% of which were allocated in the first group and showed that there is no separation of accessions depending on the sampling sites. The 3D scatter plot reinforces this conclusion. There is genetic variability among the accessions of ‘Ubá’ mango tree evaluated. Therefore, it is possible to make mass selection in open-pollinated populations.