Amino-Terminal Charge Affects the Periplasmic Accumulation of Recombinant Heregulin/EGF Hybrids Exported Using theEscherichia coliAlkaline Phosphatase Signal Sequence

AnEscherichia coliexpression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA–hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated byin vitroreplacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein inE. coliperiplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed inE. coliusing the PhoA signal sequence for protein export.     

Age-related variability in occlusal wear planes

Investigation of two populations of 136 individuals shows several patterns of occlusal wear plane change which are positively correlated with age. For individuals up to the age of 18, there is a characteristic pattern in which the occlusal wear planes of the mandibular teeth are lingually sloped and those of the maxillary dentition buccally sloped, with the exception of the maxillary premolars, which are also lingually sloped. The long axes of the mandibular teeth give them a lingual orientation relative to the maxillary teeth, and the long axes of the maxillary molars, by contrast, are buccally oriented. In the 18-30 age range for all sexes, the mandibular M1 becomes buccally sloped on its occlusal surface while the occlusal wear plane on the maxillary M1 becomes lingually sloped. Later age changes indicate a trend for the mandibular premolars to become buccally sloped, while the wear planes of the maxillary premolars remain lingually sloped. There is a corresponding tendency for the maxillary and mandibular second molars to undergo changes in the initial orientation of the occlusal wear planes.     

Studies on anaerobic bacteria

Summary A new chromogenic anaerobe, Clostridium roseum nov. spec., has been found. It is characterized by: red-orange pigment, turning purplish on oxidation; gelatin liquefaction and other evidence of proteolysis; nitrate reduction; fermentation of various carbohydrates including pectin; close resemblance to Cl. acetobutylicum in corn mash fermentation, with the same neutral products, acetone, ethyl alcohol and butyl alcohol, in nearly the same ratios; agglutinative specificity and separation from Cl. acetobutylicum and Cl. felsineum, as well as several less nearly physiologically related butyric anaerobes.     

Cell-wall lysing enzymes and products of cell-wall digestion elicit ethylene in citrus

Ethylene production was induced in Valencia oranges [ Citrus sinensis (L.) Osbcck] by injection of the fungal enzyme mixture Pectolyase ( Aspergillus japonicus ) which contains pectolytic enzymes into the peel. The mixture also stimulated production of 1-aminocyclopropane-1-carboxylic acid (ACC). Cycloheximide partially inhibited the Pectolyase-induced ethylene response. Pectin fragments, resulting from partial acid hydrolysis or Pectolyase digestion, caused an increase in ethylene production when injected into the peel of intact orange fruits. Pectic fragments produced by fungal enzymes are known to be elicitors of phytoalexins and in this study are shown to elicit ethylene in citurs.     

Comparison of different methods of determining cell viability after exposure to cytotoxic compounds

Cell-survival (of DON and L1210 cells) after treatment with cytotoxic compounds was assessed by measuring cloning efficiency, exclusion of trypan blue and erythrosin B, [⁵¹Cr] release, and attachment of DON cells to glass. Cell survival as measured by cloning efficiency did not correlate with survival measured by any of the other methods. We found that the stainability of cells after drug exposure depended on the cell line used. For example, after 3 h exposure to tubercidin although 100% of both DON and L1210 cells were killed (on basis of cloning efficiency), only 11% of DON cells and 68% of L1210 cells were dead as indicated by staining with erythrosin B. The stainability of cells also depended on the particular drug used. For example, after 24 h exposure of L1210 cells to adriamycin and tubercidin (both killed >99% of cells on basis of cloning efficiency) 21% of cells exposed to adriamycin and 99% of cells exposed to tubercidin were stained. The results obtained with several other cytotoxic compounds are discussed.