Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane.

A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder.     

Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians

Xanthomonas campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting.mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A⁺ RNA in rabbit reticulocyte lysate in the presence of ³⁵S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these had disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.     

Novel DNA structures resulting from dTam3 excision in tobacco

A Tam3 two-element system has been designed by combining an immobilized Tam3 element with a non-autonomous dTam3 element inserted into the HPT gene. The phenotypic assay employed, restored hygromycin resistance, indicated thattrans-activation of the non-autonomous dTam3 element occurred. Molecular analyses of the excision sites revealed that the ends of the dTam3 element remain in the empty donor sites. The predominant consequence of this type of excision appears to be that excised fragments fail to re-integrate into the tobacco genome. Only one case of dTam3 re-integration could be detected. The ends of this element had been degraded upon integration into the tobacco genome. Either the altered structure of the Tam3 derivatives or tobacco host factors are influencing thetrans-activation of a dTam3 element, resulting in aberrant excision.     

The influence of level of infection of rats with Nippostrongylus brasiliensis on the haematology and phospholipase activity and mast cell numbers in the small intestine and colon

The haematology and phospholipase activity and mast cell numbers of the small intestine and colon of rats was studied 10 days after infection with various numbers of larvae of N. brasiliensis. A significant reduction in the RBC occurred after infections with 200 and 5000 larvae but not with 1000 larvae. Hb was significantly reduced after infection with 200 larvae and increases in the MCV and MCH indicated the development of a macrocytic anaemia. Reticulocyte count was increased at all levels of infection except after 200 larvae. WBC was increased at all levels of infection except in the 5000 larvae group. Lymphocytes were significantly increased in all groups except those infected with 5000 larvae. Neutrophils increased only at the lower levels of infection. The most marked changes occurred in eosinophil numbers, significant increases occurring with increasing levels of infection. However, after infection with 5000 larvae the numbers were significantly lower than after infection with 200 or 1000 larvae. Phospholipase activity, which is believed to be related to tissue eosinophil levels, was significantly increased at all infection levels in the proximal small intestine. Significant increases in the distal ileum and colon occurred mainly after infection with 1000 and 5000 larvae. Mast cell numbers did not change significantly at any infection level. It is suggested that the pathology observed, here in the form of anaemia, is multifactorial in origin and is largely a function of the immune response, the development and expression of which is dependent on the level of infection, with suppression of immune damage occurring at the high levels of infection when pathogenesis may involve a direct effect of the worms.     

Quantitative estimation of the pathways followed in the conversion to glycogen of glucose administered to the fasted rat

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.     

Chloroplast targeting of spectinomycin adenyltransferase provides a cell-autonomous marker for monitoring transposon excision in tomato and tobacco

Antibiotic resistance genes can act as either cell autonomous or non-cell autonomous genetic markers with which to monitor the excision of plant transposons. To convert spectinomycin resistance from a noncell autonomous resistance to cell autonomous resistance, a transit peptide for chloroplast localization from a petunia ribulose bisphosphate carboxylase (rbcS) gene was fused in-frame to the aadA gene, which confers spectinomycin and streptomycin resistance. Constructs were generated in which the expression of this chimeric gene was prevented by the presence, in the 5 untranslated leader, of the maize transposons Activator (Ac) or Dissociation (Ds). When progeny of tobacco or tomato plants transformed with these constructs were germinated on spectinomycin-containing medium, germinally revertant and somatically variegated individuals could be distinguished.     

Use of the maize transposonsActivator andDissociation to show that phosphinothricin and spectinomycin resistance genes act non-cell-autonomously in tobacco and tomato seedlings

Cell-autonomous genes have been used to monitor the excision of both endogenous transposons in maize andAntirrhinum, and transposons introduced into transgenic plants. In tobacco andArabidopsis, the streptomycin phosphotransferase (SPT) gene reveals somatic excision of the maize transposonActivator (Ac) as green sectors on a white background in cotyledons of seedlings germinated in the presence of streptomycin. Cotyledons of tomato seedlings germinated on streptomycin-containing medium do not bleach, suggesting that a different assay for transposon excision in tomato is desirable. We have tested the use of the spectinomycin resistance (SPEC) gene (aadA) and a Basta resistance (BAR) gene (phosphinothricin acetyltransferase, or PAT) for monitoring somatic excision ofAc in tobacco and tomato. Both genetic and molecular studies demonstrate that genotypically variegated individuals that carry clones of cells from whichAc orDs have excised from either SPEC or BAR genes, can be phenotypically completely resistant to the corresponding antibiotic. This demonstrates that these genes act non-cell-autonomously, in contrast to the SPT gene in tobacco. Possible reasons for this difference are discussed.     

Depression of mitogen response in spleen cells from reticuloendotheliosis virus-infected chickens and their suppressive effect on normal lymphocyte response

Spleen cells and peripheral blood lymphocytes from chickens infected with reticuloendotheliosis virus (REV) were depressed in their responsiveness to phytohemagglutinin (PHA). When spleen cells from uninfected chickens were co-cultured with spleen cells from chickens infected with REV at 2 weeks of age, the PHA response by the normal cells was completely suppressed. Although spleen cells from chickens infected with REV at 6 or 9 weeks of age were also suppressed in their ability to respond to PHA, they did not suppress the mitogenic response of normal cells in mixed cultures.