Branched chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase activity in isolated rat pancreatic islets

Branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase in isolated rat pancreatic islets were shown to be regulated by a phosphorylation/dephosphorylation mechanism. Broad-specificity phosphoprotein phosphatase treatment stimulated and ATP addition inhibited their activities. The kinases responsible for inactivating these complexes were shown to be sensitive to inhibition by known inhibitors, alpha-chloroisocaproate and dichloroacetate. Total activity (nmol/min/islet / 37 degrees C) of branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase was 0.86 and 5.09, with a % active form (activity before phosphatase treatment divided by activity after phosphatase treatment X 100) of 36% and 94%, respectively. Incubation of intact isolated islets with alpha-chloroisocaproate affected neither insulin release nor flux through branched-chain alpha-ketoacid dehydrogenase.     

Phosphorylation sites and inactivation of branched-chain alpha-ketoacid dehydrogenase isolated from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle

Branched-chain alpha-ketoacid dehydrogenase complex was isolated from rat heart, bovine kidney, and rabbit liver, heart, kidney, brain, and skeletal muscle. Phosphorylation to approximately 1 mol Pi/mol alpha-subunit of the alpha-ketoacid decarboxylase component was linearly associated with 90-95% inactivation. The complex from some tissues (i.e., from rabbit kidney and heart, and rat heart) showed 30-40% more phosphate incorporation for an additional 5-10% inactivation. Reverse-phase HPLC analysis of tryptic digests of 32P-labeled complexes from all of the above tissues revealed two major (peaks 1 and 2) and one minor (peak 3) phosphopeptide which represent phosphorylation sites 1, 2, and a combination of 1 and 2, respectively. These phosphopeptides, numbered according to the order of elution from reverse-phase HPLC, had the same elution time regardless of the tissue or animal source of the complex. The amino acid sequence of site 1 from rabbit heart branched-chain alpha-ketoacid dehydrogenase was Ile-Gly-His-His-Ser(P)-Thr-Ser-Asp-Asp-Ser-Ser-Ala-Tyr-Arg. Regardless of the source of the complex, both sites were almost equally phosphorylated until total phosphorylation was approximately 1 mol Pi/mol of alpha-subunit and the rate of inactivation was correlated with the rate of total, site 1, or site 2 phosphorylation. Phosphorylation beyond this amount was associated with greater site 2 than site 1 phosphorylation. alpha-Chloroisocaproate, a potent inhibitor of branched-chain alpha-ketoacid dehydrogenase kinase activity, greatly reduced total phosphorylation and inactivation; however, phosphorylation of site 2 was almost abolished and inactivation was directly correlated with phosphorylation of site 1. Thus, the complex isolated from different tissues and mammals had an apparent conservation of amino acid sequence adjacent to the phosphorylation sites. Both sites were phosphorylated to a similar extent temporally although site 1 phosphorylation was directly responsible for inactivation.     

Carboranyl peptide-antibody conjugates for neutron-capture therapy: preparation, characterization, and in vivo evaluation.

Two model peptides rich in boron and prepared by Merrifield syntheses, dansyl.(nido-CB)2, (1) and dansyl.(nido-CB)10.Lys.Ac (2), where nido-CB represents the alpha-amino acid [nido-7-CH3-8-(CH2)3CH-(NH2)COOH-7,8-C2B9H10]-, were conjugated with the anti-CEA mAb T84.66 using peptide active ester reagents. The dansyl groups provided a means of fluorimetric analysis of mAb conjugates which was augmented by conventional amino acid analyses for nido-CB. The conjugate of 1 contained an average of 63 B atoms per mAb molecule. The mAb conjugate of 2 was chromatographically separated into a strongly fluorescent high molecular weight aggregated fraction (HMW) and a less intensely fluorescent monomeric fraction. Both fractions retained immunoreactivity. The HMW species contained an average of ca. 490 B atoms/mAb molecule, as determined by amino acid analysis. Biodistribution data were collected using nude mice bearing LS174T xenografts and 125I-labeled mAb conjugates. While the lightly B-loaded dipeptide conjugate gave biodistribution results which resembled those of native T84.66 mAb, the undecapeptide conjugate displayed greatly enhanced liver uptake and decreased tumor accretion. These results suggest that as the boron-containing burden on the supporting immunoprotein is greatly increased, as in the case of the T84.66-2 conjugate, loss of circulating conjugate to liver effectively competes with the desired tumor localization. Means which might be taken to circumvent this difficulty have been described elsewhere (ref 15).     

Substructural analysis of the insulin receptor by microsequence analyses of limited tryptic fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence or presence of dithiothreitol

Human placental insulin receptor contains 47 Cys per an alpha beta dimer. Most of the 94 Cys in an intact alpha 2 beta 2 receptor are expected to form interchain or intrachain disulfide bonds, since there appears to be only one free cysteine residue in each beta subunit. In order to gain more insight into the three-dimensional organization of the insulin receptor, we have used limited trypsin digestion, SDS-PAGE, and protein microsequencing. The present study revealed the following; major tryptic cleavages occurred at alpha 164, alpha 270, alpha 582, and beta 1115, generating Mr 175,000, 130,000, 100,000, 70,000, and 55,000 disulfide-linked complexes. Under reducing conditions, tryptic fragments of Mr values = 30,000, 70,000, 20,000, 55,000, and 20,000 were identified to be alpha(1-164), alpha(165-582), alpha(165-270), alpha(271-582), and alpha(583-C-terminal), respectively. The major beta subunit tryptic fragment of Mr = 55,000 was assumed to have beta(724-1115) or beta(N-terminal-392). The Mr 175,000 complex appeared to contain two alpha(1-164) and two alpha(165-582), whereas the Mr 70,000 complex contained alpha(583-C-terminal) and beta(724-1115). Tryptic cleavage at alpha 582 apparently produced one Mr 175,000 and two Mr 70,000 complexes, suggesting that the alpha(583-C-terminal) domain interacts with the extracellular domain of the beta subunit by disulfide bonds. Tryptic cleavage at alpha 270 resulting in a formation of one Mr 100,000 complex consisting of two alpha(1-270) and two Mr 130,000 complexes consisting of alpha(271-C-terminal) and beta(724-1115) suggests that Cys residues involved with disulfide bonds between the two alpha subunits are located in the alpha(1-270) domain. The identification of the Mr 55,000 complex consisting of small tryptic fragments between alpha(122-270) indicates that 40 Cys residues in the two alpha(122-270) domains are inter- and intramolecularly associated by disulfide bonds. The alpha(1-121) domain does not appear to be linked to any other domains by disulfide bonds. These results are consistent with the structural model that the N-terminal domains of alpha subunits (122-270) are disulfide-linked together while the C-terminal domain (583-C-terminal) of the alpha subunit is linked to the N-terminal domain of the beta subunit by disulfide bonds.     

Role of lymphography in carcinoma of the prostate.

The results of bilateral pedal lymphography in 83 patients with adenocarcinoma of the prostate gland are presented. The patients were divided into two groups: 45 new cases and 38 late or old cases presenting several years after the onset of the disease. Altogether 25 of the new patients and 29 of the late patients had lymphographic evidence of lymph node metastases. The lymphogram results in relation to local tumour size, histological grade, the presence of skeletal metastases, and acid phosphatase levels are discussed. Of the new patients with T1 and T2 tumors--that is, those still localized within the prostatic capsule--41% had positive lymphograms. The inaccuracy of acid phosphatase estimations in detecting early extraprostatic spread is shown and compared with the greater accuracy of lymphography. Lymphography should be used as an initial investigation in all cases where aggressive therapy is being considered, and the importance of regular follow-up radiographs is emphasized.     

鼠李糖乳杆菌LV108及其发酵乳免疫调节作用的比较

目的探讨鼠李糖乳杆菌LV108及其发酵乳对免疫抑制小鼠免疫功能的调节作用。方法将BALB/c小鼠随机分为5组,每组10只,即空白组(正常小鼠)、模型组(免疫抑制小鼠)、药物组(免疫抑制小鼠食物中添加左旋咪唑)、LV108菌悬液组(免疫抑制小鼠食物中添加LV108菌悬液)和LV108发酵乳组(免疫抑制小鼠食物中添加LV108发酵乳),除空白组外其余组构建免疫抑制小鼠模型。干预4周后,分别测定各组小鼠体质量和脏器指数,血清中白细胞介素2(IL2)、肿瘤坏死因子α(TNFα)和免疫球蛋白G(IgG)含量,血清溶血素含量、耳肿胀度和肝、脾巨噬细胞吞噬能力。结果相比模型组,LV108菌悬液组和LV108发酵乳组小鼠体质量增长速度、脏器指数、血清IL2与IgG水平、血清溶血值、耳肿胀度和巨噬细胞吞噬能力显著升高(均P<0.05);在脾脏指数、血清IL2与TNFα水平、血清溶血素含量和耳肿胀度免疫指标上,LV108菌悬液组与LV108发酵乳组之间比较差异具有统计学意义(均P<0.05)。结论LV108菌体及发酵乳对免疫抑制小鼠具备较全面的免疫调节作用,均可提高小鼠的自身免疫力;LV108发酵乳对小鼠的免疫调节作用强于LV108菌体。     

Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene AVP1 in peanut to improve drought and salt tolerance

The Arabidopsis gene AVP1 encodes an H⁺-pyrophosphatase that functions as a proton pump at the vacuolar membranes, generating a proton gradient across vacuolar membranes, which serves as the driving force for many secondary transporters on vacuolar membranes such as Na⁺/H⁺-antiporters. Overexpression of AVP1 could improve drought tolerance and salt tolerance in transgenic plants, suggesting a possible way in improving drought and salt tolerance in crops. The AVP1 was therefore introduced into peanut by Agrobacterium-mediated transformation. Analysis of AVP1-expressing peanut indicated that AVP1-overexpression in peanut could improve both drought and salt tolerance in greenhouse and growth chamber conditions, as AVP1-overexpressing peanuts produced more biomass and maintained higher photosynthetic rates under both drought and salt conditions. In the field, AVP1-overexpressing peanuts also outperformed wild-type plants by having higher photosynthetic rates and producing higher yields under low irrigation conditions.     

Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells

One of the hallmarks of HIV-1 disease is the association of heightened CD4⁺ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4⁺ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4⁺ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4⁺ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4⁺ Th-cells induced by exposed DCs.