Seasonal changes in LH secretion in normal ewes and ewes which grazed oestrogenic clover

Plasma luteinizing hormone (LH) concentrations were measured in normal (control) Corriedale X Merino (comeback) ewes and in clover-infertile comeback ewes which had grazed oestrogenic Yarloop clover (Trifolium subterraneum L. cv. Yarloop) for more than 4 years. Plasma LH concentrations were measured in samples taken at 20-min intervals for 6 h during the dioestrous stage of the oestrous cycle in the breeding season (BS) and during the anoestrous season (AS). In the control ewes during BS, transitory elevation in plasma LH concentration (pulses) occurred, reflecting secretory episodes, with a frequency of one per 5.2 h. This frequency fell to one per 16.5 h during the anoestrous season. In clover-infertile ewes, LH pulses occurred with a frequency of one per 4.5 h during BS and one per 4.9 h during AS (difference not significant). In the controls, plasma LH levels were higher (P less than 0.05) during BS (mean +/- s.d. = 1.2 +/- 0.4 ng/ml, n = 9) than in AS (0.7 +/- 0.3 ng/ml, n = 5). In the clover-infertile ewes, plasma LH levels in BS (1.3 +/- 0.6 ng/ml, n = 12) were similar to those of controls. During AS, plasma LH levels in the clover-infertile ewes (1.0 +/- 0.6 ng/ml, n = 10) remained similar to their BS levels, being significantly (P less than 0.05) higher than LH levels in the controls at this time. These studies indicate that the higher plasma concentrations of LH which have been reported in clover-infertile ewes arise from more frequent LH pulses. Furthermore, in contrast to normal ewes, average plasma LH, reflecting pulse frequency, is not reduced in AS. This supports the view that ingestion of phytooestrogens affects neural centres involved in regulating LH secretion.     

马立克氏病毒单克隆抗体的研究

获得了4株分泌马立克氏病毒(MDV)特异性单克隆抗体(McAb)的杂交瘤细胞:4BS10对MDV所有毒株呈阳性反应;4CN8 对MDV血清1,3型毒株发生反应;2BN90和4CN24只对MDV血清1型毒株有阳性反应。3个McAb属IgG1,1个为IgG2b,均不中和MDV,免疫扩散试验也无沉淀线。对禽白血病毒(ALV)无交叉反应。 以2BN90和辣根过氧化物酶、异硫氰酸荧光素的结合物进行直接酶联免疫吸附试验和直接荧光抗体试验,均获得成功。抗体滴度前者为1/51,200,后者为1/640。对ALV无交叉反应。     

Inhibition of protein carboxyl methylation by S-adenosyl-L-homocysteine in intact erythrocytes. Physiological consequences

S-Adenosyl-L-homocysteine was used to inhibit the methylation of carboxylic acid residues of membrane proteins in intact human erythrocytes. Incubation of erythrocytes for 24 h with 5 mM each of adenosine and L-homocysteine resulted in the intracellular accumulation of S-adenosyl-L-homocysteine and substantially inhibited membrane protein carboxyl methylation. From the degree of inhibition and from the observed turnover of methylated proteins, we estimate that the number of protein methyl esters in cells incubated with adenosine and L-homocysteine for 20 h is less than 20% that of cells incubated without these inhibitors. No significant differences in the physical deformability properties of the membrane of these hypomethylated cells were detected. However, there was a small but significant (p less than 0.001) increase in the amount of membrane protein D-aspartyl residues in these cells compared to control cells. These observations are consistent with the hypothesis that methylation of membrane proteins at D-aspartyl residues may result in the selective removal or repair of these uncommon residues.     

A limited region within hen egg-white lysozyme serves as the focus for a diversity of T cell clones

The C57BL/6 (H-2b) mouse is a nonresponder to hen egg-white lysozyme (HEL) injected i.p., owing to a T suppressor cell-inducing determinant at the amino-terminal region. After immunization with a 93-amino acid fragment (a.a. 13-105) of HEL lacking this determinant, all clones from two independently derived C57BL/6 T cell lines were found to be specific for epitopes within a subregion of peptide 74-96. Three specificity patterns for the clones could be defined on the basis of cross-reactivities with only two other species variant lysozymes. Reactivities of all three specificity groups was consistent with the serine to threonine substitution at position 91, although reactivity of one of the groups could be affected by substitutions at position 84. The results confirm at the clonal level that even for distantly related antigens, only limited regions are recognized by T cells. They are consistent with the notion that specific sites on the antigen capable of interaction with Ia molecules lead to dominance of certain regions for T cell reactivity. Moreover, the diversity in specificity among clones suggests that the limiting feature of T cell responsiveness is not a lack of available T cells in the repertoire directed against a single antigenic site.     

Amino acid sequence of a phosphocholine-binding antibody from an immune defective CBA/N mouse employing the T15 VH region associated with unusual DH, JH, and V kappa segments

Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.     

Identification of the dye gene product, mutational loss of which alters envelope protein composition and also affects sex factor F expression in Escherichia coli K-12

Summary The product of the dye gene of Escherichia coli, mapping at 99–100 min, is required for expression of the sex factor F, and also appears to be involved in the regulation of envelope proteins. Mutation of dye thus results in loss of expression of the F-factor (Fex^–, i.e. male sterility, and dye sensitivity (Dye^s). We have isolated a plasmid, pRB38, in which a 6 kb SalI fragment carrying the dye ⁺ gene was cloned into the plasmid pACYC184. This 6 kb SalI fragment also carries two nearby markers, chlG, involved in the synthesis of the molybdenum cofactor, and phoM, required for constitutive expression of alkaline phosphatase.Some of the polypeptides synthesised by pRB38 were identified using the maxi-cell procedure. The product of the dye gene was found to be a polypeptide of M_r=29,000. Thus derivatives of pRB38 in which the transposon was inserted into dye, resulting in a Dye^S Fex^– phenotype when these plasmids were in a dye strain, failed to produce this polypeptide and in some cases produced a truncated product. Such insertions also resulted in a Chl^r and Pho^– phenotype when the plasmid was in a (dye-chlG-phoM) phoR strain, although complementation tests suggested that the phoM ⁺ and chlG ⁺ genes were still intact. Insertions of into the promoter distal end of dye did not result in a Dye^S Fex^– phenotype, although a truncated Dye protein was synthesised, and a Chl^r Pho^– phenotype was produced.It has been suggested (Gaffney et al. 1983) that the dye (=sfrA) gene product is necessary for F-factor expression because it is required for translocation of the F-factor TraJ protein to the outer membrane. Our results suggest that the Dye protein is also required for expression of the molybdenum cofactor and of alkaline phosphatase, and could perhaps be involved in the translocation of these proteins to the membrane.     

Vaccination against schistosomiasis in mice with killed schistosomula without adjuvant

Attempts to develop a killed vaccine against schistosomiasis have generally resulted in failure. There are two recent reports, but unfortunately, harsh adjuvants were used in conjunction with the antigenic materials. In our laboratory, a killed vaccine was developed by freezing (-196 degrees C) and thawing the schistosomula of S. mansoni. The use of such a preparation without adjuvant was effective in vaccinating mice. A worm reduction of 36.4-41.1% was achieved by one vaccinating injection, a 60.2% worm reduction by 3 injections, and a 63.7-66.0% reduction by 5 injections. The sequence of the development and the expression of the immune reactions were similar to those previously found in hosts immunized with highly X-irradiated schistosome organisms. Delayed hypersensitivity was demonstrated in histological sections of the skin in the challenged mice after one vaccination, showing that an adjuvant was not necessary to initiate the induction of cellular immunity.     

Carboxyl methylation of human erythrocyte band 3 in intact cells. Relation to anion transport activity.

The anion transport protein of the human erythrocyte membrane, band 3, is reversibly methylated by an endogenous protein carboxyl methyltransferase. The physiological consequence of this modification was studied by measuring the rate of phosphate transport by intact erythrocytes incubated under conditions where protein methylation reactions are inhibited. No change in phosphate transport was detected when cells were treated with either methionine-free media or cycloleucine, whereas cells incubated with adenosine and homocysteine thiolactone displayed a marginally slower rate of transport, which was not reversed by subsequent remethylation of the membrane proteins. These results suggest that erythrocyte protein carboxyl methylation does not directly regulate this activity of band 3.     

Identification of the single gene for calmodulin in Dictyostelium discoideum.

We report the isolation and sequence determination of a cDNA containing most of the coding sequence for Dictyostelium discoideum calmodulin. The cloned cDNA was used as a probe to examine the complexity of D. discoideum genomic DNA. These studies indicated that D. discoideum cells possess a single calmodulin gene.     

The disappearance of isocitrate lyase from the green alga Chlorella fusca studied by immunoprecipitation

When acetate-adapted cultures of Chlorella fusca were transferred to nitrogen-free medium containing glucose, isocitrate lyase activity was lost over a period of about 25 h. Using a combination of in vivo isotope labelling and immunoprecipitation with anti-isocitrate lyase IgG it was shown that: 1. The onset of loss of enzyme activity preceeded the complete cessation of enzyme synthesis. 2. Disappearance of isocitrate lyase activity was accompanied by loss of enzyme protein, without accumulation of antigenic protein distinguishable from the normal subunit polypeptide of the enzyme, as judged by SDS gel electrophoresis of immunoprecipitated samples from supernatant cell-free extracts. 3. SDS gel electrophoresis of immunoprecipitated isocitrate lyase revealed the presence of antigenic protein bands of M_r about twice that of the normal subunit polypeptide, but the appearance of these apparent dimer forms did not obviously correlate with enzyme degradation. 4. Isoelectric focusing of immunoprecipitated isocitrate lyase showed that the enzyme became progressively more oxidised during the period of its degradation in vivo. 5. By titrating crude broken cell suspensions with anti-isocitrate lyase antibody, preliminary evidence was obtained for transfer of the enzyme from the soluble fraction to an insoluble form as part of the process of disappearance.