Side reactions in the synthesis of phosphotyrosine-containing peptides

Summary A series of phosphopeptides Tyr(PO₃H₂)-Val-Pro-Xxx-Leu (Xxx=Met, Met(O), Nle, Dab or Cys), derived from the native platelet-derived growth factor- receptor (PDGF-) sequence, has been prepared to study their interaction with the src-homology 2 (SH2) domains of the p85 subunit of PI3 kinase. The phosphopeptides were synthesized using Fmoc methodology incorporating N-Boc dibenzyl-protected phosphotyrosine (Boc-Tyr[PO₃(Bzl)₂]) as the N-terminal amino acid, since the benzyl groups can be removed during resin cleavage with TFA. Only peptides containing methionine were found to exist partially as S-benzyl sulfonium salts after TFA cleavage from the resin. The desired peptide could be obtained from the S-benzyl sulfonium salt by hydrogenolysis.     

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has proven to be a useful tool for examining noncovalent complexes between proteins and a variety of ligands. It has also been used to distinguish between denatured and refolded forms of proteins. Surfactants are frequently employed to enhance solubilization or to modify the tertiary or quaternary structure of proteins, but are usually considered incompatible with mass spectrometry. A broad range of ionic, nonionic, and zwitterionic surfactants was examined to characterize their effects on ESI-MS and on protein structure under ESI-MS conditions. Solution conditions studied include 4% acetic acid/50% acetonitrile/46% H2O and 100% aqueous. Of the surfactants examined, the nonionic saccharides, such as n-dodecyl-beta-D-glucopyranoside, at 0.1% to 0.01% (w/v) concentrations, performed best, with limited interference from chemical background and adduct formation. Under the experimental conditions used, ESI-MS performance in the presence of surfactants was found to be unrelated to critical micelle concentration. It is demonstrated that surfactants can affect both the tertiary and quaternary structures of proteins under conditions used for ESI-MS. However, several of the surfactants caused significant shifts in the charge-state distributions, which appeared to be independent of conformational effects. These observations suggest that surfactants, used in conjunction with ESI-MS, can be useful for protein structure studies, if care is used in the interpretation of the results.     

On the relationship between food, reproduction and survival of two carabid beetles: Calathus melanocephalus and Pterostichus versicolor

Abstract.

• 1 The relative influences of temperature and availability of food on reproduction, survival and growth of all developmental stages of two carabid beetle species are discussed with special reference to the suggested relationship between availability of food, size of egg production and survival of adults from one breeding season to the next.
• 2 Temperature as well as food supply influence the length of larval growth and adult body size. Beetles grown at low temperatures and low amounts of food are smaller than those grown at higher temperature and with more food.
• 3 The number of eggs laid per female was correlated with the amount of food gathered. There was no inverse relationship (trade-off) between reproductive output and survival in the field until the next breeding season.
• 4 In 1980 no significant relationship was found between winter mortality and the amounts of food gathered by beetles in the period after reproduction and before winter diapause. However, in 1981 in C. melanocephalus a lower number of starved beetles survived the winter than the fed ones and‘field’beetles.
• 5 Only in the first part of the feeding activity period in autumn can enough food be gathered by C.melunocephalus for successful hibernation. In the second part of this period there is not enough food to build up the fat reserves needed to survive the winter.
• 6 Difference in population fluctuations of both species are discussed in relation to their life histories.

     

Fine structure of aphid stylet routes in plant tissues in correlation with EPG signals

Abstract. Plant penetration by Aphis fabae (Scopoli) was recorded by the electrical penetration graph (EPG) technique and followed by stylectomy during wave-forms that were suspected of indicating sieve element punctures. The severed stylets in the plant tissue were subsequently processed for transmission electron microscopy (TEM) and sectioned either transverse or longitudinal to the stylets. Two completely serially sectioned probes from the epidermis to the phloem were reconstructed.
In one probe the stylet pathway went to a sieve element and showed many empty branches of salivary sheath material. Breaks in cell walls filled with sheath material demonstrated that the majority of cells bordering the track had been punctured, which supports earlier evidence from EPGs. All types of cells showed punctures and the highest number was found inside the vascular bundle. Very few cells died, which would appear to be important for virus transmission, and in others cellular reactions remained limited to some callose formation. The route of the stylets was intercellular and passed through the secondary wall material. The role of pectinase in intercellular penetration, and previous evidence for intracellular tracks are discussed. Most sieve elements had been punctured but only one was eventually accepted. Thus, reaching a sieve element in a host plant does not automatically imply its acceptance though the reason remains unclear. Gelation of phloem proteins was shown in the stylet canal.
In a second probe, plant cytological and morphological correlations with the EPG were emphasized. Probes by other aphid-plant combinations showed great similarity.     

Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle.

The yeast Cdc7 function is required for the G1/S transition and is dependent on passage through START, a point controlled by the Cdc28/cdc2/p34 protein kinase. CDC7 encodes a protein kinase activity, and we now show that this kinase activity varies in the cell cycle but that protein levels appear to remain constant. We present several lines of evidence that periodic activation of CDC7 kinase is at least in part through phosphorylation. First, the kinase activity of the Cdc7 protein is destroyed by dephosphorylation of the protein in vitro with phosphatase. Second, Cdc7 protein is hypophosphorylated and inactive as a kinase in extracts of cells arrested at START but becomes active and maximally phosphorylated subsequent to passage through START. The phosphorylation pattern of Cdc7 protein is complex. Phosphopeptide mapping reveals four phosphopeptides in Cdc7 prepared from asynchronous yeast cells. Both autophosphorylation and phosphorylation in trans appear to contribute to this pattern. Autophosphorylation is shown to occur by using a thermolabile Cdc7 protein. A protein in yeast extracts can phosphorylate and activate Cdc7 protein made in Escherichia coli, and phosphorylation is thermolabile in cdc28 mutant extracts. Cdc7 protein carrying a serine to alanine change in the consensus recognition site for Cdc28 kinase shows an altered phosphopeptide map, suggesting that this site is important in determining the overall Cdc7 phosphorylation pattern.     

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.     

The cytologic diagnosis of Mycobacterium kansasi tuberculosis by fluorescence microscopy of Papanicolaou-stained specimens

The cytologic diagnosis of Mycobacterium kansasi tuberculosis by fluorescence microscopy of Papanicolaou-stained specimens
The sensitivities of (i) Papanicolaou fluorescence, (ii) auramine rhodamine fluorescence, and (iii) Ziehl-Neelsen staining were compared for their ability to detect the atypical mycobacterium Myco. kansasi in cytological samples. Ninety-two cases were investigated, and the sensitivities of the three methods of detection were found to be 36.9%, 12.0%, and 20.7%, respectively. The control groups consisted of 30 specimens from cases of bronchial carcinoma and 30 of pneumonia. All cases were proved by microbiology. No false-positive results were recorded using Papanicolaou fluorescence. An important but coincidental finding arising from this study was that infection by the atypical mycobacterium Myco. kansasi causes cytological patterns corresponding to those normally associated with acute pneumonia and not to tuberculosis.     

Protection of neonatal mice against herpes simplex virus infection: probable in vivo antibody-dependent cellular cytotoxicity

Infant mice are extremely susceptible to fatal Herpes simplex virus (HSV) infection. They are unable to produce antibody to HSV, and their leukocytes cannot mediate antibody-dependent cellular cytotoxicity (ADCC) to HSV-infected cells. In order to avoid H-2-dependent effector mechanisms and instead analyze possible in vivo ADCC, a murine model employing adoptive transfer of antibody and human leukocytes was developed. Administration of either human immune globulin or leukocytes i.p. from HSV immune or nonimmune humans could not protect infant C57BL/6 mice from fatal HSV infection. In contrast, a combination of a subneutralizing dilution of globulin and leukocytes from nonimmune or immune human donors, given one day before inoculation, was highly protective against lethal HSV infection. The cells involved included lymphocytes or monocyte-macrophages. At least 5 X 10(6) viable leukocytes (or 1 X 10(6) monocyte-macrophages) and immune serum globulin concentrations as low as 10(-8) were protective. Infected cell monolayer adsorption and DEAE column fractionation demonstrated that the protection by globulin was due to specific antiviral IgG antibody. Protection was n ot seen in animals receiving virus before immune transfer. Protection did not involve synergistic viral neutralization by antibody and cells, as shown by in vitro experiments. Animals receiving globulin and cells, unlike normal infant mice, had circulating antiviral antibody and peritoneal leukocytes able to mediate ADCC to HSV-infected cells. This is the first in vivo evidence for the role of human ADCC. This model also allows for the in vivo evaluation of the ability of cells from immunocompromised humans to curb viral infection.