Identification of a novel HLA-B*35:42:02 allele in a Chinese bone marrow donor.

The nucleotide sequence of HLA-B*35:42:02 has a single nucleotide difference at position 141 C>T compared with that of HLA-B*35:42:01, which does not result in an amino acid change.

[Fibrosis-driving cells in patients with primary myelofibrosis and myelodysplastic syndromes with myelofibrosis].

Objective: To compare fibrosis-driving cells in patients with primary myelofibrosis (PMF) and patients with myelodysplastic syndromes (MDS) with myelofibrosis (MF) (MDS-MF) . Methods: Bone marrow biopsy sections of patients with newly diagnosed PMF and MDS (10 each randomly selected for MF-0/1, MF-2, and MF-3) were stained with specific immunofluorescence antibodies to label Gli1, LeptinR, alpha smooth muscle actin (α-SMA) , CD45, and ProcollagenⅠ. Images captured by confocal microscopy were analyzed by Fiji-ImageJ to calculate the cell counts of Gli1(+), LeptinR(+) cells, and fibrosis-driving cells including α-SMA(+), α-SMA(+)/Gli1(+), α-SMA(+)/LeptinR(+), and ProcollagenⅠ(+)/CD45(+) cells. Results: Patients with PMF and MDS with MF-2/3 had higher LeptinR(+), α-SMA(+), α-SMA(+)/Gli1(+), and Procollagen Ⅰ(+)/CD45(+) cell counts compared with those with MF-0/1 (all P values<0.05) . However, patients with PMF with MF-2/3 presented with higher Gli1(+) and α-SMA(+)/LeptinR(+) cell counts than those with MF-0/1 (P=0.001 and 0.006) , whereas these cells were similar between patients with MDS with MF-0/1 and MF-2/3 (P=0.169 and 0.067) . In patients with MF-0/1, all fibrosis-driving cells did not differ between PMF and MDS (all P>0.05) . However, in patients with MF-2/3, Procollagen Ⅰ(+)/CD45(+) cell counts were higher in patients with PMF compared with those with MDS (P=0.007) , while other fibrosis-driving cell counts were similar between these two groups (all P>0.05) . MF grade and fibrosis-driving cell counts were not correlated with overall survival in patients with either PMF or MDS. Conclusion: α-SMA(+) cells in patients with PMF originated from both Gli1(+) and LeptinR(+) cells, whereas α-SMA(+) cells in patients with MDS-MF only originated from Gli1(+) cells; patients with PMF had higher ProcollagenⅠ(+)/CD45(+) cell counts than those with MDS-MF.

[Pathological characteristics of megakaryocytes in myeloproliferative neoplasms and their correlation with driver gene mutations].

Objective: To investigate the pathological characteristics of megakaryocytes in myeloproliferative neoplasms(MPN)and their correlations with driver gene mutations. Methods: Trephine specimens administered for 160 patients with MPN from February 2012 to October 2017 were reevaluated according to the World Health Organization(WHO)'s(2016)diagnostic criteria. Results: This cohort of patients included 72(45.0%)men, with the median age of 59(range, 13-87)years, comprising 39 with polycythemia vera(PV), 33 with essential thrombocythemia(ET), 37 with prefibrotic/early-primary myelofibrosis(pre-PMF), 37 with overt PMF, 1 with post-ET MF, 2 with post-PV MF, and 11 with MPN-unclassifiable(MPN-U)after the re-diagnosis. With PV, ET, pre-PMF, and overt PMF changes, proportions of dense clusters, hypolobulated nuclei, and naked nuclei of megakaryocytes gradually increased, whereas erythropoiesis gradually decreased. Proportions of reticulin, collagen, and osteosclerosis grades of ≥1 also increased. Dense clusters, hypolobulated nuclei, and naked nuclei of megakaryocytes were negatively correlated with erythropoiesis and positively correlated with granulopoiesis and fibrosis. In patients with pre- and overt PMF, dense clusters and naked nuclei of megakaryocytes were positively correlated with fibrosis. Patients with JAK2V617F MPN had significantly increased erythropoiesis(P=0.022). Patients with CALR-mutated MPN were characterized by increased loose and dense clusters; paratrabecular distribution and naked nuclei of megakaryocytes(P=0.055, P=0.002, P=0.018, P=0.008); and increased reticulin, collagen, and osteosclerosis(P=0.003, P<0.001, P=0.001). In patients with pre- and overt PMF, patients with JAK2V617F had increased cellularity(P=0.037). CALR-mutated patients had increased dense clusters and giant sizes of megakaryocytes, collagen, and osteosclerosis(P=0.055, P=0.059, P=0.011, P=0.046). Conclusion: Megakaryocytes showed abnormal MPN morphology and distribution, which were related to fibrosis. CALR mutation was probably associated with abnormal morphology and distribution of megakaryocytes and fibrosis.

Various levels of copra meal supplementation with ß-Mannanase on growth performance, blood profile, nutrient digestibility, pork quality and economical analysis in growing-finishing pigs.

BACKGROUND: To reduce use of main feed ingredient like corn, soy bean meal (SBM) and wheat, alternative ingredients has been studied like copra meal (CM). Production amount of CM which has been high makes CM to be an alternative feed stuff. However, low digestibility on AA and low energy content by high fiber content can be an obstacle for using CM. This experiment was conducted to evaluate the effects of CM supplementation with ß-mannanase on growth performance, blood profile, nutrient digestibility, pork quality and economic analysis in growing-finishing pigs. METHODS: A total of 100 growing pigs ([Yorkshire × Landrace] × Duroc) averaging 31.22 ± 2.04 kg body weight were allotted to 5 different treatments by weight and sex in a randomized complete block (RCB) design in 5 replicate with 4 pigs per pen. Treatments were 1) Control (corn-SBM based diet + 0.1% of ß-mannanase (800 IU)), 2) CM10 (10% copra meal + 0.1% ß-mannanase (800 IU)), 3) CM15 (15% copra meal + 0.1% ß-mannanase (800 IU)), 4) CM20 (20% copra meal + 0.1% ß-mannanase (800 IU)) and 5) CM25 (25% copra meal + 0.1% ß-mannanase (800 IU)). Four phase feeding program was used: growing I (week 1-3), growing II (week 4-6), finishing I (week 7-9) and finishing II (week 10-12). RESULTS: In growth performance, there was no significant difference among treatments during whole experimental period. In growingI phase, G:F ratio tended to increase when CM was increased (P = 0.05), but ADG and ADFI tended to decrease in finishingII phase (linear, P = 0.08). Also, increasing CM reduced ADG (linear, P = 0.02) and feed efficiency (linear, P = 0.08) during the whole finishing period. In blood profiles, BUN was linearly increased as CM increased (linear, P = 0.02) at growingII period. In digestibility trial, there was no significant difference in dry matter, crude fat, crude ash and nitrogen digestibility. However, crude protein digestibility was decreased linearly (linear, P = 0.02). In economic analysis, feed cost per weight gain and total feed cost per pig were reduced in overall period when CM was provided by 25% (linear, P = 0.02). CONCLUSION: CM with 0.1% of ß-mannanase (800 IU) could be supplemented instead of corn and SBM up to 25% without detrimental effects on growth performance and pork quality of growing-finishing pigs.

Evaluation of dry feeding and liquid feeding to lactating sows under high temperature environment.

BACKGROUND: Liquid feeding system has been introduced to domestic swine farms, but negative cognition about liquid feeding system has been remained for feed waste decay related with poor management and microbial contamination. For these reasons, this study was conducted to evaluate the effects of feeding method in lactating sows. METHODS: A total of 30 mixed-parity (average 4.13) lactating sows (Yorkshire × Landrace) with an initial BW of 218.8 ± 19.5kg was used in a 3 week trial. Sows were allotted to 1 of 2 treatments in a completely randomized design by their body weight, backfat thickness, parity and alive litter weight. One of treatments was dry feeding and the other was liquid feeding (water to feed ratio, 1:1). Experimental diets contained 3265 kcal ME/kg, 12.6 % CP, 5.76 % EE, 1.09 % total lysine, 0.25 % total methionine, as fed basis. RESULTS: Dry feeding treatment had high body weight loss rather than liquid feeding treatment (P = 0.04). Dry feeding treatment had tendency to increase litter weight at 21d of lactation (P = 0.06) and litter weight gain (P = 0.04) during lactation period (0-3 week). Sows fed dry feeding method made milk containing high content of casein and total solid rather than sows fed liquid feeding method (P = 0.04). In addition, dry feeding treatment had tendency to higher content of milk fat, protein and solid not fat on 21d of lactation (P = 0.07). Sows fed dry feeding type also showed higher milk energy content in milk of 21d lactation (P = 0.05). Furthermore, liquid feeding treatment showed high occurrence in feed waste during lactation period (P < 0.01). CONCLUSION: Dry feeding method was more suitable feeding method to lactating sows under high temperature environment like lactating barn.

Effects of neuroblastoma tumor gangliosides on platelet adhesion to collagen.

Gangliosides, sialic acid-containing glycosphingolipids, enhance tumor formation in experimental animals and are associated with tumor progression and metastasis in humans. The mechanism(s) for this activity is (are) unknown. One possibility is enhanced platelet activation, since the interaction of platelets with tumor cells contributes to tumor cell arrest in the vascular compartment. We have previously shown that neuroblastoma tumor gangliosides (NBTG) enhance platelet adenosine triphosphate (ATP) secretion, aggregation, and adhesion. We determined that these NBTG effects are specific for collagen and are mediated through an alpha2 beta1 integrin-dependent mechanism. This report describes the effects of NBTG on a physiologically relevant model of collagen-alpha2 betal interaction. Platelet adhesion to immobilized native collagen fibers similar to those found in the extracellular matrix of blood vessels was determined. Platelet adhesion is enhanced by NBTG in a concentration-dependent manner. Incubation with concentrations of 1 and 10 microM NBTG increased platelet adhesion by 9% and 52%, respectively, compared to less than 1% in controls not incubated with gangliosides (P = 0.001 and P < 0.0001, respectively). In addition to increasing the number of adherent platelets, NBTG promoted more rapid attachment. In NBTG-incubated platelets, platelet adhesion began after a 5-min lag phase and was maximal at 30 min compared to a 20-min lag phase and maximal adhesion at 60 min for control platelets. At 30 min this difference was significant (P = 0.017); however, by 120 min there was no difference between NBTG and controls (P = 0.259). NBTG also induces platelet adhesion at collagen concentrations (0.1 microg) that failed to support adhesion of control platelets. These effects of NBTG require Mg2+ or Mn2+ ions but are not supported by Zn2+ or Ca2+ ions. Furthermore, preincubation of platelets with a blocking antibody (6F1) to the integrin collagen receptor alpha2 beta1 abrogates all of the effects of NBTG. These results indicate that tumor gangliosides enhance platelet adhesion to extracellular matrix collagen and promote rapid stabilization of the collagen-alpha2 beta1 interaction, the initial steps in platelet activation.

Antagonism of morphine-induced antinociception by tetrandrine is dependent on serotonergic mechanisms.

'Tetrandrine (TET), a non-specific calcium antagonist, inhibited morphine-induced antinociception in the tail-flick test in mice. This study was undertaken to assess the mechanism of the antagonism of morphine-induced antinociception by TET. Morphine-induced antinociception was prevented by pretreatment with TET in the tail-flick but not in the tail-pinch tests carried out in mice and this antagonism was abolished by pretreatment of a serotonin precursor, 5-hydroxytryptophan (5-HTP), but not by the pretreatment of a noradrenaline precursor, L-dihydroxyphenylalanine (L-DOPA), in the tail-flick test. These results indicate that serotonergic mechanisms are involved in the antagonism of morphine-induced antinociception by TET. On the other hand, the development of morphine-induced analgesic tolerance was not prevented by TET. This result, in agreement with other reports, also indicates the possible dissociation of morphine analgesic effect from its tolerance-inducing effect. In addition, TET suppressed the 5-hydroxytryptamine (5-HT)-induced head twtch response. This result provides additional evidence that TET may modulate serotonergic function and the antagonism of morphine-induced antinociception by TET is dependent on serotonergic mechanisms.

Effects of tetrandrine and fangchinoline on human platelet aggregation and thromboxane B2 formation.

Tetrandrine (TET) and fangchinoline (FAN) are two major components of the radix of Stephania tetrandra. The effects of TET and FAN on human platelet aggregation and formation of thromboxane (TX) B2, a stable metabolite of TXA2, were examined in the aspect of platelet aggregation. TET and FAN inhibited platelet-activating factor (PAF)-induced human platelet aggregation. IC50 values for TET and FAN were 28.6+/-3.24 microM and 21.7+/-2.61 microM, respectively. In the PAF-receptor binding assay, neither TET nor FAN showed any inhibitory effects on the specific bindings of PAF to its receptor. TET and FAN also inhibited PAF-, thrombin- and arachidonic acid-induced thromboxane B2 formation in human washed platelet. These results indicate that TET and FAN inhibit the platelet aggregation by interfering with the intracellular messengers system, but not by inhibiting the binding of PAF to PAF-receptor on the platelet membrane directly, and the suppression of TXA2 formation by TET and FAN may be responsible for their inhibitory activities on the platelet aggregation and further on the thrombosis.

Effects of ginsenosides on bovine adrenal tyrosine hydroxylase.

The present study was undertaken to investigate the effects of ginsenosides on bovine adrenal tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. Ginsenoside-Rb1, Rc, Re and Rg, inhibited the TH activity by 51.5, 25.4, 31.3, 44.3 and 43.3%, respectively, at a concentration of 80 microg/ml. Ginsenoside-Rb1, Rc, Re and Rg1 exhibited noncompetitive inhibition of TH activity with a substrate L-tyrosine. From these results, it is presumed that the effects of ginsenosides on TH activity observed in vitro might be also produced in vivo, and thereby the inhibitory effects of ginsenosides on TH activity may be partially responsible for the antidopaminergic action of ginsenosides by reducing the availability of dopamine at the presynaptic dopamine receptor in vivo.

Effect of ginseng total saponin on bovine adrenal tyrosine hydroxylase.

Ginseng total saponin (GTS) can modulate dopaminergic activity at both presynaptic and postsynaptic dopamine receptors (Kim et al., 1998). The present study investigated the effect of GTS on the bovine adrenal tyrosine hydroxylase (TH), which catalyze L/tyrosine to DOP. GTS inhibited the bovine adrenal TH by 42.4, 51.5 and 55.3% at concentrations of 40, 80 and 100 micrograms/ml, respectively. The IC50 value of GTS was 77.5 micrograms/ml. GTS exhibited noncompetitive inhibition with a substrate L-tyrosine. The Ki value was 155 micrograms/ml.

Fangchinoline inhibited the antinociceptive effect of morphine in mice.

Fangchinoline (FAN), a non-specific calcium antagonist, is a major alkaloidal component of the creeper Stephania tetrandra S. Moore (or fenfangji). It has been shown to possess antagonistic activity on morphine-induced antinociception in mice. This study was undertaken to assess the antagonistic mechanism. The results demonstrated that FAN (IP) attenuated morphine (SC)-induced antinociception in a dose-dependent manner with significant effect at doses of 30 and 60mg/kg body wt. (IP) in the tail-flick test but not the tail-pinch tests, carried out in mice. This antagonism was abolished by pretreatment with a serotonin precursor, 5-hydroxytryptophan (5-HTP, IP), but not by pretreatment with a noradrenaline precursor, L-dihydroxyphenylalanine (L-DOPA, IP) in the tail-flick test. On the other hand, the development of morphine-induced analgesic tolerance was not prevented by FAN. These results suggest that the serotonergic pathway may be involved in the antagonism of morphine-induced antinociception by FAN and, in agreement with other reports, also indicates the possible dissociation of the morphine analgesic effect from its tolerance-development mechanism.

Inhibitory effect of tetrandrine on dopamine biosynthesis and tyrosine hydroxylase in PC12 cells.

The effect of tetrandrine, a bis-benzylisoquinoline alkaloid, on dopamine biosynthesis in PC12 cells was investigated. Tetrandrine at a concentration of 3.0 microM decreased dopamine content by 59.4% (IC50 = 2.4 microM) and intracellular tyrosine hydroxylase (TH) activity was inhibited by the treatment of tetrandrine (49.8% inhibition at 3.0 microM) compared with control. We next examined the effects of tetrandrine on the kinetics of PC12 TH. The PC12 TH was obtained from PC12 cells with minor purification. Tetrandrine inhibited the PC12 TH activity by 40.6% at a concentration of 45 microM and exhibited noncompetitive inhibition on the enzyme using L-tyrosine as a substrate (Ki = 60.8 microM). These results suggest that the inhibition of TH activity by tetrandrine may partially contribute to the decrease in dopamine biosynthesis in PC12 cells.

Endothelium-dependent vasorelaxant and antiproliferative effects of apigenin.

This study was designed to determine whether the relaxant effect of apigenin was endothelium dependent and to examine the possible antiproliferative effect of apigenin. Apigenin relaxed the phenylephrine-precontracted endothelium-intact aortic rings with IC(50) value of 3.7+/-0.5 microM and removal of a functional endothelium significantly attenuated this relaxation (IC(50)=8.2+/-0.9 microM). However, apigenin did not affect the 0.1 microM phorbol 12,13-dibutyrate-induced contraction (IC(50)=34.6+/-1.2 microM) within the concentration range that relaxed the phenylephrine-contracted arteries, suggesting that apigenin did not influence protein kinase C-mediated contractile mechanisms in rat aorta. Pretreatment of apigenin significantly potentiated the relaxant effect of acetylcholine on phenylephrine-induced contraction. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) or methylene blue reduced the relaxant effect of apigenin. Apigenin (10 microM) increased the guanosine 3',5'-cyclic monophosphate (cGMP) content of endothelium-intact tissues. Pretreatment with L-NAME (100 microM) or removal of endothelium significantly suppressed the effect of apigenin on cGMP production. In addition, apigenin significantly inhibited [3H]thymidine incorporation into DNA of primary cultured rat aortic smooth muscle cell in a dose-dependent manner. These findings suggest that besides influx and release of Ca(2+), nitric oxide (NO) and cGMP may account for the apigenin-induced endothelium-dependent relaxation and hypotensive activity. Both vasorelaxant and antiproliferative activities may contribute to a benefit of apigenin in the vascular system.