Interaction of nucleic acids with the DNA-dependent RNA polymerases of Drosophila

The interaction between the three Drosophila DNA-dependent RNA polymerases (EC 2.7.7.6) and the DNA template or the RNA product was investigated by photochemical cross-linking and binding studies, using RNA polymerase subunits immobilized on nitro-cellulose filters. It can be shown that the two largest subunits are responsible for the binding of the enzymes to both template and newly-synthesized RNA.     

Size fractionation of thermal aggregates of immunoglobulin G

Purified pooled human immunoglobulin G (IgG) in solution, when extensively heated at high temperatures or for long periods, irreversibly aggregates and insoluble precipitates result. However, when IgG solutions are heated in the temperature range 55-65 degrees C for more limited time periods, soluble turbid polydispersed aggregate mixtures are obtained. Gel filtration of such aggregate mixtures on calibrated Bio-Rad A-150m columns demonstrates a continuous size distribution from dimers to aggregates as large as 4 X 10(7) Da (200-mers) with no particular size predominant. Chromatographically reproducible cuts of narrow size heterogeneity can be obtained by short-time fraction collection. Elution-time reproducibility is excellent both for mixture and for individual cuts. Stability studies indicate that reproducible and stable aggregates may be made from purified IgG and that fractionated aggregates should be stored quick-frozen until needed. Sized IgG aggregates have proved useful in reactivity studies with rheumatoid factor, animal anti-IgG antibodies, and complement.     

Structure and ultrastructure of the frog motor endplate

Summary The frog motor endplate in its simplest form consists of an elongated, slender nerve ending embedded in a gutter-like depression of the sarcolemma. This nerve terminal contains the usual synaptic organelles. It is covered by a thin coating of Schwann cell cytoplasm which embraces the terminal with thin finger-like processes from both sides, thereby sub-dividing it into 300–1000 regularly spaced compartments. The individual synaptic compartments correspond to the strings of varicosities or grape-like configurations of motor nerve terminals in endplates of other species and in the cerebral neuropil of vertebrates.Each compartment contains one or more bar-like densities of the presynaptic membrane, active zones, which are associated with the attachment sites between synaptic vesicles and plasmalemma. Active zones have a regular transverse arrangement and occur at specific loci opposite the junctional folds. The attachment sites for synaptic vesicles are at the edges of the bars which are bilaterally delineated by a double row of 10 nm particles attached to the A-face. The structural appearance of vesicle attachment sites in freeze-etch replicas corresponds to that of micropinocytosis. The active zones are often fragmented and the frequency of their association with vesicle attachment sites is highly variable.The junctional folds are characterized by specific sites in which intramembranous particle aggregations occur at relatively high packing density (7500/m²). These sites are located opposite the active zones at the juxtaneural lips, a location where one would expect ACh-sensitive receptors on the postsynaptic membrane.This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 38, Projekt N), The Swiss National Foundation for Scientific Research (Grants Nr. 3 82372 and 3 77472) and the Dr. Eric Slack-Gyr Foundation Zürich.     

Analysis of three-dimensional cell imaging obtained with optical microscopy techniques based on defocusing

The properties of an optical microscope are analyzed and analytically evaluated with a simple and effective model in order to understand the true meaning, limitations, and real capabilities of a defocusing technique. Major emphasis is given to the applications related to microscopic objects of biological interest using fluorescence and absorption light microscopy. A procedure for three-dimensional viewing is analyzed and discussed.     

应用酶联免疫吸附试验检测不同类型乙型肝炎中激活补体类HBsAg循环免疫复合物

本文以抗人C_(?)的羊IgG为包被抗体,以HRP-HBs抗体为指示抗体,建立了可检测激活补体类HBsAg循环免疫复合物(HBsAg/C3-CIC)的C_3捕捉法酶联免疫吸附试验。检测了236例六种类型临床诊断为乙型肝炎的病人血清标本,其阳性率分别为:无症状携带者(ASC)12.9％(4/31),急性肝炎(AH)36.7％(22/60),慢性迁延性肝炎(CPH)33.3％(7/21),慢性活动性肝炎(CAH)59.6％(34/57),重型肝炎(SH)77.8％(14/18),肝炎后肝硬化(PLC)67.3％(33/49),阳性率与肝损严重程度明显相关(P<0.01)。认为HBs-Ag/C3-CIC可能在乙型肝炎病毒引起的慢性活动性肝炎、重型肝炎和肝炎后肝硬化的发病过程中起重要作用,并可作为乙型肝炎的诊断、临床分型和预后判断的指标之一。     

Transforming growth factor-beta does not alter interleukin-1 expression in cultured human macrophages

Transforming growth factor-beta (TGF beta) is a growth modulator that stimulates the growth of fibroblastic cells but inhibits the growth of cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins, and of proteases and the type 1 plasminogen activator inhibitor (PAI-1) by cultured cells. TGF beta appears also to have various immunoregulatory effects, suppressing both T- and B-cell activities. It has been proposed that it might increase the expression of interleukin-1 (IL-1) mRNA in cultured human monocytes, thus potentiating immune functions. To analyze the role of TGF beta in IL-1 production we have now quantitated the effect of this factor on the production of biologically active IL-1 as well as IL-1 beta mRNA expression. The effect of TGF beta on IL-1 production optimally activated with bacterial lipopolysaccharide (LPS) was also studied. It was found that IL-1 activity and mRNA levels were rapidly elevated by LPS but not by TGF beta. Culture fluids from monocytes treated with TGF beta alone or with TGF beta plus LPS inhibited the proliferation of the test thymocytes. After gel filtration, the media from TGF beta-treated cultures showed no activity in the molecular weight area of IL-1 (approx. 15 kD), while the supernatants from TGF beta plus LPS-induced cells contained IL-1 activity in these fractions, the magnitude of which was, however, at the same level as in the culture fluids derived from cells stimulated with LPS alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.     

Synthesis of polypeptides by microwave heating II. Function of polypeptides synthesized during repeated hydration-dehydration cycles

Summary Polypeptides, synthesized from a mixture of amino acid amides by microwave heating during repeated hydration-dehydration cycles, showed hydrolase- and oxidoreductase-like catalytic activities. Poly(GAVDH), polypeptides synthesized from an equimolar mixture (each 0.1 M) of glycinamide,l-alaninamide,l-valinamide,l-aspartic acid -amide, andl-histidinamide, catalyzed the hydrolysis of PNPAc. The hydrolytic rate of PNPAc with poly(GAVDH) was the quadruple of that ofl-histidine alone. Though the k_cat values of different resulting polypeptides were 10³–10⁶ times less than those of native hydrolases, the K_m value of the polypeptides further containing phenylalanine residues was nearly equal to that of the esterase. This result indicates the presence of hydrophobic interaction between a substrate and the polypeptides. Resulting polypeptides also showed catalytic activity for the reduction of ferricyanide ion [Fe(CN)^3–] with NADH. The polypeptides seemed to have a strong affinity for adenine nucleotides, because the reaction was inhibited by adenine derivatives such as NAD⁺ and AppA. A hypothesis for the emergence of primitive protein enzymes is discussed.