The neutral sterols of Megalotomus quinquespinosus (say) (hemiptera: Alydidae) and identification of makisterone a as the major free ecdysteroid

Last-stage nymphs of the broad-headed bug, Megalotomus quinquespinosus contain the C₂₈ ecdysteroid makisterone A as their major ecdysteroid. No ecdysone or 20-hydroxyecdysone was detected in whole body extracts analyzed by high performance liquid chromatography and radioimmune assay. Analyses of the neutral sterols of this phytophagous hemipteran revealed that the sterol composition of the nymphs was highly reflective of their dietary sterols. The most abundant nymphal sterols were sitosterol (46.6%), Δ⁷-stigmastenol (13.8%) and spinasterol (13.4%). Cholesterol accounted for only 0.2% of the total sterols and indicates that this species is incapable of converting 24-alkyl sterols to cholesterol.     

The ecdysteroid titres during the last-larval instar of Ephestia kuehniella Z. (Lepidoptera: Pyralidae)

The ecdysteroid titre and the body weight during the last-larval instar of Ephestia kuehniella were determined. Slightly elevated ecdysteroid titres occur during the first 12 h following the last larval-larval ecdysis (38 ng/g) and again some 120 h later, lasting about 48 h (33 ng/g). A high ecdysteroid peak (750 ng/g) with a maximum in prepupae of the eye-class A4 precedes the larval-pupal ecdysis. The basal levels between these increased ecdysteroid titres are between 13 ng/g and 15 ng/g. Compared with the body weight, the first sligtly increased ecdysteroid titre 12 h after ecdysis is associated with the beginning of food intake, the second increase at 144 h after ecdysis with reduced gain in body weight. The prepupal ecdysteroid peak occurs whilst the body weight remains constant. Correlations between the varying ecdysteroid titre and morphological and physiological events accompanying the progress in larval-pupal development are discussed.     

Immunoglobulin recognition of synthetic and natural left-handed Z DNA conformations and sequences

The relative immunogenicities of the poly[d(G-C)] and poly[d(A-C) · d(G-T)] families of helices have been determined. The specificities of the resultant immunoglobulins have been characterized for recognition of different synthetic and natural left-handed sequences and conformations. Certain modifications of poly[d(G-C)] in the sugar-phosphate bacbone and cytosine C-5 potentiate the right(R)-to-left(L) (B → Z) transition under physiological conditions. The resulting polynucleotides, poly[d(G^S-C)], poly[d(G-io⁵C)], poly[d(G-br⁵C)] and poly[d(G-m⁵C)], are also highly immunogenic. In contrast, DNAs incapable of assuming the left-handed conformation under physiological salt concentrations are weakly or non-immunogenic. These include unmodified poly[d(G-C)] as well as members of the poly[d(A-C) · d(G-T)] family of sequences bearing pyrimidine C-5 substitutions (methyl, bromo, iodo). These polynucleotides undergo the R → L isomerization under more stringent ionic and thermal conditions.The specificities of purified polyclonal and monoclonal anti-Z DNA immunoglobulins (IgG) were measured by binding to radiolabeled polynucleotides, by electrophoretic analysis of IgG bound to covalent closed circular DNAs, and by immunofluorescent staining of polytene chromosomes. The salt-induced left-handed forms of poly[d(G-C)] and its derivatives (including the cytidine C-5 methyl, bromo, iodo, and N-5 aza substituted polynucleotides) and of the modified poly[d(A-C) · d(G-T)] polymers are bound to varying degrees by different antibodies. The patterns of substrate recognition demonstrate the existence of several antigenic domains in left-handed DNAs, including the helix convex surface and the sugar-phosphate backbone. Substitutions in these regions can produce enhancing (required substitutions), neutral, or inhibitory effects on subsequent IgG binding. Additionally, certain modifications of either the convex surface of Z DNA at the C-5 position of cytidine (i.e. a methyl group) or of the backbone (i.e. phosphorothioate substitution) can lead to polymorphic lefthanded conformations that are compatible with antibody binding when present individually but not in combination. The recognition patterns exhibited with DNA substrates from the two DNA families indicate that some, but not all, IgGs show specificity for different nucleotide sequences.The anti-Z DNA IgGs were used to probe for specific left-handed Z DNA determinants on plasmid (e.g. pBR322) or viral (e.g. simian virus 40 (SV40)) DNAs and on the acid-fixed polytene chromosomes of dipteran larvae. At their extracted superhelical density, the negatively supercoiled form I, but not the relaxed, nicked, or linear forms of all tested plasmid and viral DNAs specifically bind sequence-independent anti-Z IgGs. Dimers, trimers and higher oligomers of form I DNA cross-linked by bivalent anti-Z IgGs are formed with numerous (e.g. φX174, SV40, pBR322) genomes. Their occurrence depends upon IgG concentration and specificity, the conditions of ionic strength and temperatures and the DNA genome. The IgG cross-linked DNA multimers are converted to monomers by dithiothreitol reduction. Sequence-independent monovalent anti-Z Fab fragments bind form I DNA but do not generate oligomeric species. Multimers of order >2 indicate the existence of at least two anti-Z Ig binding sites per molecule, as in the case of SV40. IgGs differ in their ability to form stable complexes with some sites on natural DNAs, presumably due to their sequence and conformation binding specificities. A differential binding of these antibodies is also observed in certain bands of polytene chromosomes, such as the telomeric regions that are involved in chromosome associations.     

Neuromedin B is a major bombesin-like peptide in rat brain: regional distribution of neuromedin B and neuromedin C in rat brain, pituitary and spinal cord

Neuromedin B and neuromedin C are the novel mammalian bombesin-like peptides isolated from porcine spinal cord. We have developed highly specific and sensitive radioimmunoassays for neuromedin B and neuromedin C, and determined their regional distribution in rat central nervous system. Prior to measurements of the tissue contents, immunoreactive neuromedin B and C were characterized by gel-filtration and high performance liquid chromatography. Neuromedin B and C immunoreactivities have similar regional distribution in rat brain, but the content of immunoreactive neuromedin B is 2-6 times higher than that of immunoreactive neuromedin C in every region. These results indicate that neuromedin B is a major endogenous bombesin-like peptide in rat brain and has specific functions of physiological importance.     

Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus

Neutralizing antibodies were raised in mice against notexin, the most toxic phospholipase A₂ (PLA₂) from Notechis scutatus scutatus venom, without the necessity of detoxifying the toxin prior to immunization. Using a sensitive radioimmunoassay we demonstrated that anti-notexin antibodies recognized (i) the parent antigen, (ii) closely related isoforms of notexin and (iii) venoms from Notechis genus snakes. In contrast, they failed to recognize other purified PLA₂ or PLA₂-containing venoms from other origins. Substitutions or chemical modifications occurring in the C-terminal part of the polypeptide chain of notexin altered the binding affinity for antibodies, implying that this region constitutes an antigenic domain of notexin.     

A direct-selection vector derived from pColE3-CA38 and adapted for foreign gene expression

The construction of a plasmid vector, pVT25, which allows an efficient and direct selection for transformed cells carrying recombinant plasmids is described. In this vector, the replicon and Ap^R gene from plasmid pBR327 are fused to the colE3 gene of pColE3-CA38, whereby positive selection is based on the inactivation of the lethal colicin E3 by the insertion of a foreign DNA fragment. However, pVT25 can be maintained within the Escherichia coli cells when complemented with another plasmid, pVT26, which expresses the colicin E3 immunity (imm) and the Tc^R phenotypes. Furthermore, pVT25 was used to regulate the expression of the synthetic human proinsulin gene fused to the colE3 gene at the single ClaI site. The production of the characteristic C-peptide of proinsulin, monitored by radioimmunoassay, was shown to be under the control of the inducible promoter of the colE3 gene.     

Limitations of direct estradiol and testosterone immunoassay kits

Estradiol (E2) and testosterone (T) are biologically active hormones that serve as important diagnostic markers in serum of premenopausal and postmenopausal women and in men. These hormones are measured frequently by immunoassay in clinical laboratories and the test results are used in the diagnosis and treatment of patients. For measuring the hormones by immunoassay, most laboratories utilize commercially available reagents that are packaged in the form of a kit and are used either in an automated instrument or manually. However, both the diagnostic kit manufacturer and testing laboratory seldom thoroughly validate the assay methods generated with these kits. This deficiency may lead to unreliable test results that could affect clinical evaluation and treatment of patients. The purpose of the present study was to assess the reliability of immunoassays that quantify serum E2 and T levels with commercial diagnostic kits. The data generally show wide differences in the apparent levels of each hormone in a given sample obtained with kits from different manufacturers. This was especially true when measuring postmenopausal E2 and T levels. However, a purification step, which included organic solvent extraction, prior to radioimmunoassay (RIA) of E2 gave values that compared well with those obtained by conventional RIA (with preceding extraction/chromatographic steps). Our results point out the importance of more thoroughly validating assays performed with commercial immunoassay kits, especially with respect to sensitivity and specificity, prior to their use for measuring hormone levels in patient samples.     

促甲状腺激素抗血清的研制

用较少的抗原量免疫绵羊,采用活体采血法,定期加强免疫,定期补铁,定期采血,获得抗人促甲状腺激素(TSH)抗血清.采用放免分析方法鉴定抗血清,结果为:抗血清滴度为28-205万,与促卵泡激素、促绒毛膜生长激素、促黄体生成素交叉都小于0.02‰,亲和常数K大于10¹⁰L/mol.     

Preferential utilization of endogenous arachidonate by cyclo-oxygenase in incubations of human platelets

Thromboxane B2 (TXB2) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formed from the endogenous and exogenous arachidonate during human platelet incubation, was evaluated by selected ion monitoring (SIM). TXB2 formed from endogenous substrate accounted for about one third of the total, whereas the great part of 12-HETE derived from exogenous arachidonate. These data indicate that under the tested conditions the pool of arachidonate that acts as substrate for cyclo-oxygenase is different from the pool that acts as substrate for lipoxygenase and that the arachidonate released from phospholipids is preferentially utilized by cyclo-oxygenase.