Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.     

Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315–43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1. 80H.3. and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (lgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (lgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence. i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited BFU-GM and CFU-E. Antigens recognized by 80H.3. 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.     

The nuclei of origin of brain stem enkephalin and substance P projections to the rodent nucleus raphe magnus

The sites of origin of brain stem enkephalin and substance P projections to the rodent nucleus raphe magnus were studied utilizing the combined horseradish peroxidase retrograde transport-peroxidase-antiperoxidase immunohistochemical technique. Several brain stem areas were found to contain both enkephalin- and substance P-like immunoreactive double labeled neurons following injection of horseradish peroxidase into the raphe magnus. Nuclei providing both enkephalin and substance P inputs to the raphe magnus include the nucleus reticularis paragigantocellularis, the nucleus cuneiformis, the nucleus solitarius and the trigeminal subdivision of the lateral reticular nucleus. Enkephalin projections to the raphe magnus were also found to originate from the dorsal parabrachial nucleus, the nucleus reticularis gigantocellularis pars α and from an area which corresponds to the A5 group of Dahlström &; Fuxe. Additional neurons containing substance P-like immunoreactivity and horseradish peroxidase reaction product were identified in the superior central raphe nucleus and the nucleus pontis oralis. The midbrain periaqueductal gray contributes very few enkephalin and substance P fibers to the raphe magnus.The nucleus raphe magnus is a key structure in the intrinsic analgesia system and it has also been implicated in other diverse and non-nonciceptive functions. The present study identifies several brain stem sites which provide enkephalin and substance P input to this raphe nucleus. Several of these nuclei have been implicated in central analgesic mechanisms or in non-nociceptive autonomic functions. The present investigation raises the possibility that these brain stem regions may modulate neuronal activity in the raphe magnus via enkephalin or substance P projections and thus influence the involvement of the raphe magnus in both opiate related mechanisms of pain control and non-nociceptive functions.     

Localization of class i histocompatibility molecule assembly by subfractionation of the early secretory pathway

Class I molecules of the major histocompatibility complex bind peptides derived from cytosolic proteins and display them on the cell surface. This function alerts cytotoxic T cells to the presence of intracellular pathogens. Class I molecule assembly requires the association of the heavy chain with β₂-microglobulin, accompanied by peptide loading via specific transporters. This study localizes where these assembly steps take place, using monoclonal antibodies recognizing class I molecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded class I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molecules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RER) membranes. These data suggest that subunit assembly and peptide acquisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, which contained comparatively less peptide-loaded molecules than the high density RER fractions, at steady state. Following a 15 °C temperature block, class I molecules accumulated in these less dense membrane fractions, indicating that these fractions represent the intermediate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules recycling to the RER was detected, suggesting empty molecules recycle to acquire peptide.     

磷酸盐型低密度脂蛋白亲和吸附剂制备与体外吸附试验研究

研究目的是制备低密度脂蛋白亲和吸附剂,用于去除高脂血症患者血液中高含量的低密度脂蛋白。首先以悬浮分散法制备壳聚糖微球和纤维素微球载体,然后固定磷酸吡哆醛和磷酸盐配基,制备出三种吸附剂,并进行体外静态吸附性能测试。结果表明,壳聚糖-磷酸吡哆醛吸附剂对LDL最大吸附量为1.30mg/mL,磷酸盐型壳聚糖和纤维素吸附剂对LDL的最大吸附量分别为2.72mg/mL和3.12mg/mL,并且吸附量随配基含量的增加呈增大趋势,3种吸附剂对高密度脂蛋白均无明显吸附。对吸附剂的灭菌和储存稳定性的研究表明,磷酸盐型纤维素吸附剂具有较好的稳定性。本研究第一次报道磷酸盐型低密度脂蛋白吸附剂。     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

^32磷简易敷贴药膜治疗小儿皮肤血管瘤2450例疗效分析

目的　评价3 2 磷简易敷贴药膜治疗小儿皮肤血管瘤的效果 .方法　 2 4 5 0例不同年龄的血管瘤小儿接受3 2 磷简易敷贴药膜治疗 ,治疗次数为 1～ 9次 ,平均 3.5次 ;每次间隔 2个月 ,按不同年龄给不同的照射量 ,以治疗时间换算 ,每次治疗剂量为 0 .92 5MBq/cm2 × ( 12～ 4 2 )h .结果　血管瘤和血管痣治愈率 71.7% ,部分治愈 2 2 .5 % ,总有效率 94 .2 % ,无效者 5 .8% .其中血管瘤效果明显优于血管痣 (p<0 .0 1) ,并且年龄越小效果越明显 (p<0 .0 1) .结论　3 2 磷简易敷贴药膜治疗小儿皮肤血管瘤是一种治疗简便、疗效确切的方法 ,尽早治疗效果更明显     

AMPK as a metabolic switch in rat muscle,liver and adipose tissue after exercise

An increasing body of evidence has revealed that activation of adenosine monophosphate (AMP)‐activated protein kinase (AMPK)‐activated protein kinase increases fatty acid oxidation by lowering the concentration of malonyl coenzyme A (CoA), an inhibitor of carnitine palmitoyl transferase 1. Studies carried out primarily in skeletal muscle suggest that AMPK modulates the concentration of malonyl CoA by concurrently phosphorylating and inhibiting acetyl CoA carboxylase (ACC), the rate limiting enzyme in malonyl CoA synthesis, and phosphorylating and activating malonyl CoA decarboxylase (MCD), an enzyme involved in its degradation. We have recently observed that AMPK and MCD activities are increased and ACC activity diminished in skeletal muscle, liver and, surprisingly, in adipose tissue 30 min following exercise (treadmill run) in normal rats. In liver and adipose tissue these changes were associated with a decrease in the activity of glycerol‐3‐phosphate acyltransferase (GPAT), which catalyses the first committed reaction in glycerolipid synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. Similar changes in ACC, MCD and GPAT were observed following the administration of 5‐aminoimidazole 4‐carboxamide‐riboside (AICAR), further indicating that the exercise‐induced alterations in these enzymes were AMPK‐mediated. Conclusions: (1) AMPK plays a major role in regulating lipid metabolism in multiple tissues following exercise. (2) The net effect of its activation is to increase fatty acid oxidation and diminish glycerolipid synthesis. (3) The relevance of these findings to the regulation of muscle glycogen repletion in the post‐exercise state and to the demonstrated ability of AMPK activation to decrease adiposity and increase insulin sensitivity in rodents remains to be determined.     

医用磷酸钙骨水泥的制备及性能的实验研究

探索了磷酸四钙（Ca4（PO4）2O，TTCP）的制备，并合成了磷酸钙骨水泥（CPC），对CPC固化时间、引起浸泡液pH值的变化、抗压强度、产物物相组成及微观结构进行了研究。结果表明：在真空条件下、1500℃下煅烧6h可制得TTCP，并含有少量CaO。CPC初凝时间为4min、终凝时间为15min，浸泡1d和7d后的抗压强度分别为20MPa和35MPa，浸泡液的pH值在6．4～8．9之间变化，这些性能均符合临床用CPC的性能要求。CPC水化产物为片状或针状羟基磷灰石（Ca5（PO4）3OH，HA），相互交错呈连续分布的网状结构，这种结构有利于材料强度的提高。实验研制的CPC材料可用于骨缺损的修复治疗。     

Role of oxidative stress,angiogenesis and chemo-attractant cytokines in the pathogenesis of ischaemic protection induced by remote ischaemic conditioning: Study of a human model of ischaemia-reperfusion induced vascular injury

Aims

We explored the effect of remote ischaemic conditioning (RIC) on endothelial function and on circulating mediators.