Disorder of gamma-motor neuronal activity in experimental thyrotoxicosis]

The activity of gamma-motor neurons was studied in the anesthetized rats following per os administration of thyroidin for 3 weeks. In experimental thyrotoxicosis against the backgrounds of an increased amount of mute filaments isolated from the stem of the n. gastrocnemius there was an increase in the frequency of discharge of the flexors and extensors of the gamma-motor neurons. A high frequency not peculiar to that of intact animals was recorded in the flexors of the gamma-motor neurons of the animals receiving thyroidin.     

COUMARIN THERAPY—Prothrombin Activity after Termination of Treatment

Twelve patients receiving coumarin type hypoprothrombinemic agents were studied before, during and after termination of therapy, the prothrombin proconvertin method having been used to assay the prothrombin activity complex.In no instance was post treatment “rebound” demonstrated.Prothrombin activity levels returned to pretreatment values only after ten days following termination of coumarin or Dicumarol administration.If a reactivation of thrombotic tendency occurs following discontinuance of anticoagulant therapy, it would not appear to be related to a “rebound” of prothrombin activity above that which is “normal” for the individual patient.Patients tend to return to the same level of prothrombin activity present before initiation of coumarin therapy.     

Restriction of stress-induced activation of lipid peroxidation by small doses of thyroid hormones]

The experiments on 57 female rats demonstrated that small doses of thyroid hormones (thyroidin) significantly (55-118%) restrict stress induced increase in the concentration of initial and terminal products of lipid peroxidation (LP) in the myocardium and in the blood plasma. After hormone injection stress decreases the activity of key antioxidant enzyme, superoxide dismutase of erythrocytes (SOD), to a lesser degree and increases the rate of malonyldialdehyde (MDA) production induced by Fe2+ in homogenates of the myocardium to the same degree as well in comparison with rats that had not been injected thyroidin. In normal rates thyroidin does not influence the concentration of products of LP, increases the activity of SOD and decreases increment of MDA induced by Fe2+ in homogenates of the myocardium. Thus, small doses of thyroid hormones restrict significantly stress induced activity of LP membranes, increasing the power of antioxidant systems both in the myocardium and in the organism.     

Purification and some characteristics of the human coagulation factor VII.

1. A purification procedure for factor VII (proconvertin) from human plasma is described. The procedure involves barium sulphate adsorption and elution. DEAE-Sephadex column chromatography, barium sulphate adsorption and elution, heparin-Sepharose column chromatography, preparative disc gel electrophoresis and finally adsorption with antiserum to prothrombin coupled to Sepharose and antiserum to albumin coupled to Sepharose. This procedure gave an approximately 8 . 10(5)-fold purification. 2. The factor VII obtained from the electrophoresis step was mainly a single-chain protein with an apparent molecular weight of 53000 +/- 2000. 3. After the final purification step, additional forms of factor VII, resulting from a fragmentation of the factor VII molecule were detected. 4. Amino acid composition data of the purified factor VII are given. 5. Antisera were raised in two different rabbits by injection of the purified factor VII. The antisera obtained gave a good titre against the factor VII activity and were not directed against any of the three other vitamin-K-dependent coagulation factors.     

Activation of prothrombin, modified by maleic anhydride, some properties of N-maleyl-thrombin with free alpha-amino groups]

Conditions for the acylation of bovine prothrombin by maleic anhydride are worked out. The reaction is shown to modify not more than 95% of amino groups. The changes in hydrodynamic and electrophoretic properties testify about structural changes of prothrombin as a result of the modification of free amino groups. The activation of maleyl-prothrombin to maleyl-thrombin took place in 25% sodium citrate only in the presence of thrombin and the Xa factor. The increase of modified amino groups in prothrombin resulted in the decrease of the activity of generated maleyi-thrombin. The main fraction of maleyl-thrombin with free alpha-amino groups had a sedimentation coefficient of 2.1 S and possessed a residual esterase activity.     

Enzymic hydrolysis of placental cell wall pectins and cell separation in watermelon (Citrullus lanatus) fruits exposed to ethylene

Watermelon [ Citrullus lanatus (Thunb.) Matsum and Nakai, cv. Charleston Gray] fruits were examined to determine the effect of ethylene on cell wall hydrolases. pectin degradation, and cell wall ultrastructure. Enzymic studies showed that activity of polygalacturonase (EC 3.2.1.15) increased in placental tissue following 1 day of ethylene treatment and was 10 times higher after 6 days of treatment. The increase in polygalacturonase activity was accompanied by the appearance in ethanol powders of low-molecular-weight pectic polymers and a decrease in total pectin. The enhanced enzyme activity and decrease in total pectins were observed only in fruits exposed to ethylene. Ultrastructural studies of ethylene-treated tissue revealed an early disintegration of the middle lamella. The onset of wall separation coincided with the first notable increase in polygalacturonase activity. Cell wall of untreated fruit showed no evidence of structural changes. The results indicate that initiation of enzymic activity and cell wall separation in response to ethylene are not characteristic phenomena of normal ripening and senescence in watermelon fruit.     

Changes of prothrombin activity by plasmin immobilized on L-lysine Sepharose 4B]

It is demonstrated that the method of immobilization of human plasmin by its adsorption on L-lysine-Sepharose 4B can be successfully applied for bovine plasmin isolation. Proteolysis of bovine prothrombin by bovine prothrombin by bovine plasmin preparations of high caseinolytic activity results in the increase of prothrombin level. Prothrombin proteolysis by bovine plasmin preparations of high caseinolytic activity results in the decrease of prothrombin level.     

Growth inhibitory actions of prothrombin on normal hepatocytes: influence of matrix

Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-gamma-carboxy-prothrombin (DCP), the function of which is unknown. We considered that the prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin and DNA synthesis and cytoskeletal changes were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH), concomitant with the peak of DNA synthesis in the hepatocytes at 24h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP.     

Effects of thyrotoxicosis and selective hepatic autonomic denervation on hepatic glucose metabolism in rats

Thyrotoxicosis is known to induce a broad range of changes in carbohydrate metabolism. Recent studies have identified the sympathetic and parasympathetic nervous system as major regulators of hepatic glucose metabolism. The present study aimed to investigate the pathogenesis of altered endogenous glucose production (EGP) in rats with mild thyrotoxicosis. Rats were treated with methimazole in drinking water and l-thyroxine (T(4)) from osmotic minipumps to either reinstate euthyroidism or induce thyrotoxicosis. Euthyroid and thyrotoxic rats underwent either a sham operation, a selective hepatic sympathetic denervation (Sx), or a parasympathetic denervation (Px). After 10 days of T(4) administration, all animals were submitted to a hyperinsulinemic euglycemic clamp combined with stable isotope dilution to measure EGP. Plasma triiodothyronine (T(3)) showed a fourfold increase in thyrotoxic compared with euthyroid animals. EGP was increased by 45% in thyrotoxic compared with euthyroid rats and correlated significantly with plasma T(3). In thyrotoxic rats, hepatic PEPCK mRNA expression was increased 3.5-fold. Relative suppression of EGP during hyperinsulinemia was 34% less in thyrotoxic than in euthyroid rats, indicating hepatic insulin resistance. During thyrotoxicosis, Sx attenuated the increase in EGP, whereas Px resulted in increased plasma insulin with unaltered EGP compared with intact animals, compatible with a further decrease in hepatic insulin sensitivity. We conclude that chronic, mild thyrotoxicosis in rats increases EGP, whereas it decreases hepatic insulin sensitivity. Sympathetic hepatic innervation contributes only to a limited extent to increased EGP during thyrotoxicosis, whereas parasympathetic hepatic innervation may function to restrain EGP in this condition.     

Water Extract of Ashwagandha Leaves Has Anticancer Activity: Identification of an Active Component and Its Mechanism of Action

Background

Cancer is a leading cause of death accounting for 15-20% of global mortality. Although advancements in diagnostic and therapeutic technologies have improved cancer survival statistics, 75% of the world population live in underdeveloped regions and have poor access to the advanced medical remedies. Natural therapies hence become an alternative choice of treatment. Ashwagandha, a tropical herb used in Indian Ayurvedic medicine, has a long history of its health promoting and therapeutic effects. In the present study, we have investigated an anticancer activity in the water extract of Ashwagandha leaves (ASH-WEX).

Methodology/Principal Findings

Anticancer activity in the water extract of Ashwagandha leaves (ASH-WEX) was detected by in vitro and in vivo assays. Bioactivity-based size fractionation and NMR analysis were performed to identify the active anticancer component(s). Mechanism of anticancer activity in the extract and its purified component was investigated by biochemical assays. We report that the ASH-WEX is cytotoxic to cancer cells selectively, and causes tumor suppression in vivo. Its active anticancer component was identified as triethylene glycol (TEG). Molecular analysis revealed activation of tumor suppressor proteins p53 and pRB by ASH-WEX and TEG in cancer cells. In contrast to the hypophosphorylation of pRB, decrease in cyclin B1 and increase in cyclin D1 in ASH-WEX and TEG-treated cancer cells (undergoing growth arrest), normal cells showed increase in pRB phosphorylation and cyclin B1, and decrease in cyclin D1 (signifying their cell cycle progression). We also found that the MMP-3 and MMP-9 that regulate metastasis were down regulated in ASH-WEX and TEG-treated cancer cells; normal cells remained unaffected.

Conclusion

We provide the first molecular evidence that the ASH-WEX and TEG have selective cancer cell growth arrest activity and hence may offer natural and economic resources for anticancer medicine.     

Anticoagulant activity of M-LAO,L-amino acid oxidase purified from Agkistrodon halys blomhoffii,through selective inhibition of factor IX

One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.     

血栓弹力图指导食管癌患者临床输血的价值及其与常规凝血实验检测指标的相关性分析

目的:探讨血栓弹力图(TEG)指导食管癌患者临床输血的价值及其与常规凝血实验检测指标的相关性。方法:选取2017年1月-2019年3月在我院收治的食管癌手术治疗需输血的99例患者作为研究对象,将99例患者随机分为常规凝血功能检测组和TEG组,常规凝血功能检测组采用常规凝血实验检查结果指导输血,TEG组采用TEG检查结果指导输血,对比两组输血前后的常规凝血实验检测指标以及临床用血量,对比TEG组输血前后的TEG指标,分析TEG指标与常规凝血实验检测指标的相关性。结果:两组患者输血前凝血四项和血小板计数(PLT)差异无统计学意义(P>0.05),输血后两组活化凝血酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)、纤维蛋白原(FIB)均有不同程度的改善(P<0.05),TEG组PT、TT较常规凝血功能检测组低(P<0.05);输血后,TEG组患者凝血反应时间(R值)、血凝块形成时间(K值)较输血前降低,最大血凝块强度(MA值)、凝血综合指数(CI值)升高,凝血形成速率(Angle角)增大,差异有统计学意义(P<0.05);Pearson相关性分析结果显示,R值与APTT呈正相关(P<0.05),K值与PLT呈负相关,与FIB呈正相关(P<0.05),Angle角、MA值、CI值与FIB、PLT呈正相关(P<0.05);TEG组新鲜冰冻血浆、冷沉淀输注量少于常规凝血功能检测组,差异有统计学意义(P<0.05)。结论:TEG能更好地指导食管癌手术患者各种血液成分的合理输注,有效改善凝血异常情况,减少输血用量,TEG指标与常规凝血实验检测指标存在一定的相关性。     

Anticoagulant activity of M-LAO, l-amino acid oxidase purified from Agkistrodon halys blomhoffii, through selective inhibition of factor IX

One of haemorrhagic toxins present in snake venoms is l-amino acid oxidase (LAO), which catalyzes the oxidative deamination of l-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.     

Effect of thyroxine on the function of rabbit skeletal muscle sarcoplasmic reticulum

Thyrotoxicosis in rabbits was induced by prolonged intraperitoneal injection of L-thyroxin. The development of thyroxicosis was assoiated with a decreased Ca2+ accumulation rate by sarcoplasmic reticulum (SR) fragments and a lowered Ca2+ dependent ATPase activity. As compared to the analogous parameters in normal animals. Ca2+ accumulation rate and ATPase activity of thyrotoxicosis animals decreased by 60 and 25%, respectively. The changes in the specific parameters of SR were also observed during incubation of normal SR samples in the medium containing thyroxin (10-5 M). The changes seen in SR functioning in thyrotoxicosis animals are likely to be related to structural rearrangements of lipoprotein surroundings of Ca-ATPase.     

Acute systemic changes in blood cells, proteins, coagulation, fibrinolysis, and platelet aggregation after frostbite injury in the rabbit

Acute systemic blood changes were measured in New Zealand white rabbits after severe and mild frostbite injury to the foot. There were observed after 72 hr, in the severely frostbitten rabbits, a decrease in erythrocytes, hematocrit, lymphocytes, and albumin, and an increase in total leukocytes, neutrophils, platelets, fibrinogen, and antithrombin III. Mildly frostbitten rabbits showed similar changes except for no changes in the platelets, albumin, and antithrombin III. In severely frostbitten rabbits, after 72 hr, the changes in the plasma coagulation tests were a prolonged partial thromboplastin time, an accelerated prothrombin time, and increased activities of Factors VII, IX, X, and XI. In mildly frostbitten rabbits there were a prolonged partial thromboplastin time and an increased activity of Factor VII. No changes in fibrinolysis were seen in either group of rabbits. Platelet aggregation, studied only in the severely frostbitten rabbits, showed a change only by an increase in the slope of the collagen-induced platelet aggregation. The blood changes observed in the rabbit model are different than those reported in human frostbite cases. No disseminated intravascular coagulation was apparent in the rabbit model after frostbite injury.     

Metal and phospholipid binding properties of partially carboxylated human prothrombin variants

To study the specific role of gamma-carboxyglutamic acid (Gla) residues in prothrombin, we have isolated a series of partially carboxylated prothrombin variants from a patient with a hereditary defect in vitamin K-dependent carboxylation (Goldsmith, G. H., Pence, R. E., Ratnoff, O. D., Adelstein, D. A., and Furie, B. (1982) J. Clin. Invest. 69, 1253-1260). The three variant prothrombins, purified by DEAE-Sephacel, immunoaffinity chromatography, and preparative gel electrophoresis, were indistinguishable from prothrombin in molecular weight, amino acid composition, and NH2-terminal amino acid sequence, with the exception of Gla residues. Variant prothrombin 1, with 8 Gla residues, had 66% of the coagulant activity of prothrombin, one high affinity metal-binding site (Kd = 15 nM), and three lower affinity sites (Kd = 2.7 microM); prothrombin contained two high affinity (36 nM) and four lower affinity sites (Kd = 1 microM). Ca(II) induced a 23% decrease in the intrinsic fluorescence of variant prothrombin 1 fragment 1, compared to a 35% decrease in that of prothrombin fragment 1. The phospholipid binding activity of variant prothrombin 1 was 44% that of prothrombin. Variant prothrombin 2 and variant prothrombin 3, with 4 and 6 Gla residues, respectively, had about 5% of prothrombin coagulant activity and a single high affinity and two lower affinity metal-binding sites and exhibited no phospholipid binding activity. Variant prothrombin 3 fragment 1 and variant prothrombin 2 fragment 1 demonstrated 18 and 13% of Ca(II)-induced fluorescence quenching, respectively. Abnormal prothrombin, with 1 Gla residue, had 8% of prothrombin coagulant activity, a single lower affinity (1 microM) metal-binding site, and 13% Ca(II)-induced fluorescence quenching of the fragment 1 species and did not bind to phospholipid. These results indicate that Gla residues define the metal binding properties of prothrombin. Most, if not all, of the Gla residues are required for complete prothrombin function, and the prothrombin coagulant activity correlates to the phospholipid binding activity of the prothrombin species.     

Endotoxin-induced ATP depletion in thyrotoxic rats

Effect of endotoxin from E. coli on the ATP content in heart muscle, the liver and the kidney of thyrotoxic rats was studied. When endotoxin (200-400 micrograms) was intravenously injected to rats taking drinking water containing 2-7.5 micrograms T3 per ml, body temperature rose and the heart rate increased. At the same time, a marked decrease in the ATP content in heart muscle and the kidney was observed together with an increase in Na+-K+-ATPase activity. Such changes were not observed or seen only to a small extent in euthyroid rats after endotoxin administration. Endotoxin-induced ATP depletion in T3-treated rats was prevented by administration of 5 mg hydrocortisone just prior to endotoxin injection. These findings indicate that endotoxin easily causes ATP depletion in some tissue or organs in thyrotoxicosis, even if the dose of endotoxin is not enough to produce such an effect in the euthyroid. These observations are of interest in relation to thyroid storm associated with bacterial infection.     

Ligula intestinalis (L.) (Cestoda: Pseudophyllidea): plerocercoid-induced changes in the spleen and pronephros of roach, Rutilus rutilus (L.), and gudgeon, Gobio gobio (L.)

The effects of the plerocercoid of Ligula intestinalis (L.) (Cestoda: Pseudophyllidea) on the major lymphoid organs, the spleen and pronephros, of roach, Rutilus rutilus (L.), and gudgeon, Gobio gobio (L.), are described. The spleen of ligulosed roach showed a significant decrease in weight. Differential cell counts suggested this was due to a reduction in erythrocytes, despite significant increases in macrophages and vacuolated granulocytes. The spleen of gudgeon, which consisted almost entirely of erythrocytes, showed a slight reduction in weight in ligulosed fish. In both roach and gudgeon this decrease was independent of parasite burden. Differential cell counts of the pronephros from ligulosed roach revealed a significant decrease in neutrophils and increase in vacuolated granulocytes. In the pronephros of gudgeon, however, cell counts were unaffected by ligulosis. Ultrastructural observations included an apparent disintegration of vacuolated granulocytes and increased pinocytic activity in specialized endothelial cells in the spleens of ligulosed roach. Also, melano-macrophage centres and melano-macrophages increased in the spleen of ligulosed roach and gudgeon, respectively. The marked changes in spleen weight and differential cell counts in ligulosed roach and lack of such changes in ligulosed gudgeon correlate with the differential response to the parasite by these two fish species.     

Chronic cardiac-specific thyrotoxicosis increases myocardial beta-adrenergic responsiveness

Whereas many cardiac symptoms of thyrotoxicosis resemble those of the hyperadrenergic state, circulating catecholamines are reduced or normal in this condition. To test the hypothesis that the thyrotoxic heart is hypersensitive to catechol-amines, we studied beta-adrenergic signaling in a transgenic (TG) mouse in which the human type 2 iodothyronine deiodinase (D2) gene is expressed in myocardium. Because D2 converts T4 to T3, the active form of thyroid hormone, the D2 TG mouse exhibits mild, chronic thyrotoxicosis that is limited to the myocardium. In the current study, we determined that cAMP accumulation in response to either norepinephrine or forskolin treatment was increased in isolated ventricular myocardiocytes and membrane-enriched fractions prepared from these D2 TG hearts as compared with wild type. This increase in adenylyl cyclase (AC) Vmax could not be explained by changes in AC isoform expression or changes in the long or short forms of stimulatory G-protein Gsalpha, which were approximately 10% decreased in D2 TG membranes. However, Western analysis and ADP-ribosylation studies suggest that the increase in AC Vmax is mediated by a decrease in the expression of inhibitory G proteins (Gialpha-3 and/or Goalpha). These data suggest that cardiac thyrotoxicosis leads to increased beta-adrenergic responsiveness of cardiomyocytes via alterations in the regulatory G-protein elements of the AC membrane complex.     

Role of thromboelastography in clinical emergency trauma patients

This study aimed to investigate dynamic changes in thromboelastography (TEG) and evaluate its value for emergency trauma patients. TEG values (R, K, Angle, MA and CI) were detected l, 3, and 7 days post trauma. Followup prognosis data were recorded among all patients and correlations between TEG changes and prognosis were assessed. The results revealed that the K and R volumes of the poor prognosis group were obviously higher than those of the good prognosis group at each time point; while the Angle, MA, and CI volumes of the poor prognosis group were relatively lower than those of the good prognosis group, and showed a little rising tendency. This study suggests that variables measured by TEG were closely associated with prognosis evaluation, and highlighted the dangers of coagulation disorder at early stage. TEG is of benefit in evaluating the function of coagulation in clinical emergency trauma patients.