Quality validation of platelets obtained from the Haemonetics and Trima Accel automated blood-collection systems.

BACKGROUND: Platelet transfusion is required to treat haemo-oncology or trauma patients. Platelet apheresis (PA) performed with apheresis equipment has increased rapidly in recent years. Leucocyte-reduced platelet apheresis (LRPA) can reduce the risk of platelet refractoriness and febrile nonhemolytic transfusion reactions (FNHTRs) for transfusion. Accordingly, this study aimed to investigate and compare the platelet metabolic and functional responses between PA performed with Haemonetics and LRPA performed with Trima Accel cell separator. METHODS: The qualities of platelets collected through PA and LRPA were evaluated in terms of visual appearance, morphology, platelet-aggregation changes, metabolic activities, and bacterium-screening test during 5-day storage. Statistical analyses included two-sample t-test and generalised estimating equation(GEE) method. RESULTS: During 5-day storage in LRPA, residual leucocytes were all <1.0×106, and the parameters of platelet function were as follows: platelet aggregated to agonists such as adenosine 5'-diphosphate (ADP) and collagen, and the extent of shape change and pO2 showed no statistically significant difference between PA and LRPA. The hypotonic shock reaction (HSR) on days 0, 1, and 3 were significantly higher in LRPA than in PA (71.78±6.92 vs. 64.10±7.42; P=0.002; 71.53±8.98 vs. 62.96±9.84; P=0.007; 68.05±7.28 vs. 57.76±6.80; P<0.0001, respectively). Values of mean platelet volume (MPV) were statistically larger in PA than in LRPA on days 0, 1, and 3. On day 5, the swirling score was higher in LRPA than in PA. The mean lactate levels had no statistically significant difference between PA and LRPA. Moreover, no growth was observed through bacterium-screening test conducted on 40 samples. CONCLUSION: Comparison of LRPA and PA products collected from the Trima Accel and Haemonetics automated blood-collection systems, respectively, revealed that both products possessed good platelet qualities even though additional processes are needed to reduce leucocytes. Furthermore, investigating the outcomes of other apheresis instruments with focus on the safety of donors, products, and recipients is necessary.

Germline expression of the hunchback orthologues in the asexual viviparous aphids: a conserved feature within the Aphididae.

In animals, differentiation of germline from soma usually takes place during embryogenesis. Genes and their products that are preferentially expressed in the embryonic germ cells are regarded as candidates for maintaining germline fate or promoting germline identity. In Drosophila, for example, the protein encoded by the germline gene vasa is specifically restricted to the germ cells, while products of the gap gene hunchback (hb), a somatic gene, are preferentially expressed in the neuroblasts. In this study, we report the expression of both messenger RNA and protein encoded by Aphb, an hb orthologue in the asexual viviparous pea aphid Acyrthosiphon pisum, in germ cells as well as in neuroblasts. We infer that expression of Aphb messenger RNA in the germ cells during the formation of germaria is required for the anterior localization of Aphb in the protruding oocytes. Germarial expression and anterior localization of ApKrüppel was also identified but, unlike Aphb, its expression was not detected in the migrating germ cells. Very similar patterns of hb expression were also identified in the green peach aphid Myzus persicae, suggesting that germline expression of hb is conserved within the Aphididae. To date, this pattern of hb germline expression has not been reported in other insects.

Noncanonical expression of caudal during early embryogenesis in the pea aphid Acyrthosiphon pisum: maternal cad-driven posterior development is not conserved.

Previously we identified anterior localization of hunchback (Aphb) mRNA in oocytes and early embryos of the parthenogenetic and viviparous pea aphid Acyrthosiphon pisum, suggesting that the breaking of anterior asymmetry in the oocytes leads to the formation of the anterior axis in embryos. In order to study posterior development in the asexual pea aphid, we cloned and analysed the developmental expression of caudal (Apcad), a posterior gene highly conserved in many animal phyla. We found that transcripts of Apcad were not detected in germaria, oocytes and embryos prior to the formation of the blastoderm in the asexual (viviparous) pea aphid. This unusual expression pattern differs from that of the existing insect models, including long- and short-germ insects, where maternal cad mRNA is passed to the early embryos and forms a posterior-anterior gradient. The first detectable Apcad expression occurred in the newly formed primordial germ cells and their adjacent blastodermal cells during late blastulation. From gastrulation onward, and as in other insects, Apcad mRNA is restricted to the posteriormost region of the germ band. Similarly, in the sexual (oviparous) oocytes we were able to identify anterior localization of Aphb mRNA but posterior localization of Apcad was not detected. This suggests that cad-driven posterior development is not conserved during early embryogenesis in asexual and sexual pea aphids.

The effect of paclitaxel on gene expression and activity of arylamine N-acetyltransferase and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cells.

N-Acetylation is recognized as the first step in arylamine metabolism. The enzyme responsible for N-acetylation is called arylamine N-acetyltransferase (NAT),which uses acetyl coenzyme A as the acetyl group donor. Paclitaxel has been shown to exhibit antineoplastic and anticancer activity. In this study, paclitaxel was selected to determine the inhibition of arylamine N-acetyltransferase activity, gene expression (NAT mRNA) and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cell line. Paclitaxel (0.01-l microM) did decrease the level of NAT mRNA in a dose-dependent manner. The results demonstrated that paclitaxel inhibited NAT activity and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cells in a dose-dependent manner. Using standard steady-state kinetic analysis, it was demonstrated that paclitaxel was a possible uncompetitive inhibitor to NAT activity in cytosols based on the decrease in apparent values of K(m) and V(max). This report is the first demonstration that paclitaxel affected human leukemia HL-60 cells NAT activity and DNA-2-aminofluorene adduct formation.

Determination of tetracycline and streptomycin in mixed fungicide products by capillary zone electrophoresis.

A method of capillary zone electrophoresis (CZE) was used to determine tetracycline and streptomycin content in commercial agriculture products. The results indicated that this method was capable of analyzing the mixed fungicide in formulated products with instrument detection limit (IDL) of 0.50 microg/mL and a method detection limit (MDL) of 0.52 microg/mL for tetracycline, and IDL of 1.00 microg/mL and MDL of 1.22 microg/mL for streptomycin. Precision expressed by relative standard deviation (RSD) ranged from 1.44 to 4.37% of tetracycline and 1.00 to 4.20% of streptomycin. Recoveries were in the region of 98.2-102.5% for tetracycline and 95.3--103.0% for streptomycin. The low detection limit, the low RSD values, and the high percentage of recovery confirmed that the CZE technique is a sensitive and selective method. And the CZE method can analyze both tetracycline and streptomycin at the same time without complicated extraction and further derivative reaction.

Determination of destruxins, cyclic peptide toxins, produced by different strains of Metarhizium anisopliae and their mutants induced by ethyl methane sulfonate and ultraviolet using HPLC method.

Metarhizium anisopliae produces a family of cyclic peptide toxins, destruxins (DTXs), which exhibit various insecticidal activity. Four major DTXs have been separated by HPLC and identified by the liquid chromatography electrospray mass spectrometry (LC-ESI-MS) methods. Strain F061 of M. anisopliae produced large amounts of (DTXs), especially DTX-A (12.84+/-0.04 microg/ml), DTX-B (66.89+/-2.57 microg/ml) and DMDB (1.41+/-0.13 microg/ml). High levels of DTX-E (4.19+/-0.13 microg/ml) were produced by strain F007 of M. anisopliae. The results of our studies also showed that either ethyl methane sulfonate (EMS) or ultraviolet (UV) can significantly increase the production of DTXs. Mutant 61E-9 produced high levels of DTX-A (30.05+/-1.97 microg/ml), DTX-B (110.37+/-10.02 microg/ml) and DMDB (8.30+/-0.45 microg/ml). High levels of DTX-E (20.59+/-2.65 microg/ml) were produced by mutant 7E-3. Both mutant strains are suitable for industrial fermentation processes and possess a wide range of potential applications in the area of metabolic toxin production.

Tamoxifen inhibits arylamine N-acetyltransferase activity and DNA-2-aminofluorene adduct in human leukemia HL-60 cells.

Genetic regulation of acetyl coenzyme A-dependent N-acetyltransferase (NAT)and O-acetyltransferase (OAT) activities may play an important role in the metabolic activation of arylamine chemicals and carcinogens. N-acetylation is thought to be the first step in arylamine metabolism. The enzyme responsible for N-acetylation is called NAT. In this study, synthetic non-steroidal antiestrogen tamoxifen was selected for determining the inhibition of arylamine NAT activity, gene expression (NAT mRNA) and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cell line. The results demonstrated that tamoxifen did not affect the level of NAT mRNA in HL-60 cells. But the results also showed that NAT activity and 2-Aminofluorene-DNA adduct formation in HL-60 cells were inhibited and decreased by tamoxifen in a dose-dependent manner when the doses of tamoxifen up to 100 micro M. We also examined the standard steady-state kinetic analysis, and the data showed that tamoxifen may be an uncompetitive inhibitor to NAT activity in cytosols based on the decrease apparent values of Km and Vmax. This report is the first finding that tamoxifen inhibited human leukemia HL-60 cells NAT activity and DNA-2-aminofluorene on adduct formation.

Determination of the antibiotic fungicide validamycin A in formulated products by micellar electrokinetic chromatography.

A micellar electrokinetic capillary chromatographic method (MEKC) was used to determine validamycin A content in commercial products. The results indicated that this method was capable of analyzing the validamycin A content in formulated products with an instrument detection limit of 0.94 microg/mL and a method detection limit of 1. 70 microg/mL. Relative standard deviation (RSD) values of MEKC determination of validamycin A in formulated products ranged from 0. 61 to 2.09%. Recoveries of validamycin A in formulated products were in the region of 99.5-105.1%. All commercial products collected from markets contained validamycin A. The high percentage of recovery, the low detection limit, and the low RSD values confirmed that the MEKC technique is a senstivie and selective method.

Purification and characterization of tilapia (Oreochromis mossambicus) deoxyribonuclease I--primary structure and cDNA sequence.

DNase I of tilapia (Oreochromis mossambicus) was purified to homogeneity. Tilapia DNase I is most active at pH 8.5 with Mg2+ as activator. The Ca2+/Mg2+ pair has a synergistic effect on activation. The enzyme is readily inactivated by heating above 55 degrees C, but is not inactivated by trypsin or 2-mercaptoethanol under alkaline conditions, with or without CaCl2. Its isoelectric point is 6.0. The 258-amino-acid sequence of tilapia DNase I was derived from overlapping sequences of tryptic, chymotryptic and CNBr peptides. The purified enzyme has two variants differing by a single Lys-->Arg mutation at position 125. The polypeptide chain has one disulfide bridge and one carbohydrate side chain. By mass spectrometry, the purified enzyme shows many molecular mass forms differing by Lys/Arg substitution and sugar-chain length. The major form has a molecular mass of 30,914 Da. A 1061-bp nucleotide sequence for the cDNA of tilapia DNase I, obtained by gene cloning and DNA sequencing, contains an ORF coding for a putative 26-residue transmembrane peptide and the mature DNase I polypeptide.

Protein engineering of venom toxins by synthetic approach and NMR dynamic simulation: status of basic amino acid residues in waglerin I.

The tertiary structure of waglerin I has been determined by NMR and dynamic simulated annealing [Chuang et al., Biochim. Biophys. Acta 1292, 145-155 (1996)]. It is believed that the peptide basicity of waglerin may play an important role for its activity due to its high content of basic amino acids. In order to investigate the active site of the toxin, seven analogues of waglerin, [Ala3]-waglerin, [Ala7]-waglerin, [Ala10]-waglerin, [Ala14]-waglerin, [Ala18]-waglerin, [Ala20]-waglerin and [Ala22]-waglerin have been synthesized chemically by single replacement of basic amino acid residues one by one with Ala. By correlation of structures for each analogue with LD50 toxicity bioassays, it is found that the [Ala10]-waglerin exhibits no toxicity and the active site of the native toxin seems to reside in the proximity of the disulfide loop, which is spatially close to His10. Furthermore, the closer is the disulfide loop to the basic amino acid in waglerin, the more influential is the basic amino acid on the toxicity of waglerin. Based on the tertiary structure of waglerin, the structures of all synthetic analogues were derived based on computer-simulated modelling. By the pair-wise structural comparison, the disulfide loop in [Ala10]-waglerin analogue is found to be twisted as compared to the native form, in agreement with the lack of toxicity for this synthetic analogue.

Arthroscopy for acute knee haemarthrosis in road traffic accident victims.

The objective of this study is to show the characteristics of acute traumatic haemarthrosis of the knee due to road traffic accidents. A prospective study was undertaken of 47 knees in 46 road traffic accident victims (14 to 74 years old), who presented with an acute haemarthrosis of the knee over 12 months. Anterior cruciate ligament (ACL) tears occurred in 51.1 per cent (24 of 47). Lateral meniscus injuries occurred in 40.4 per cent (19 of 47). There were seven injuries of the posterior segment of the lateral meniscus associated with the 18 PCL tears. Road traffic accident victims had a higher incidence of PCL and lateral meniscus injuries than did a series of athletes with injuries. High-energy and multi-directional injury and the anatomy of the posterior horn of the lateral meniscus might account for a major part of the discrepancy.