Nonlinear mechanical responses of mouse cochlear hair bundles.

The stiffness of sensory hair bundles of both inner (IHC) and outer (OHC) hair cells was measured with calibrated silica fibres in mouse cochlear cultures to test the hypothesis that the mechanical properties of the hair bundle reflect processes underlying mechanotransduction. For OHCs, the displacement of the hair bundle relaxed with time constants of 6 ms for displacements which open transducer channels and 4 ms for displacements which close the channels. The corresponding values of the time constants for IHCs were 10 ms and 8 ms, respectively. A displacement-dependent change in the stiffness of the hair bundle was not observed when the bundle was displaced orthogonally to the direction of excitation. The stiffness of the hair bundle as a function of nanometre displacements from the resting position was remarkably nonlinear. The stiffness declined to a minimum from the resting stiffness by about 12% for OHCs and 20% for IHCs when the hair bundle was displaced by about 20 nm in the excitatory direction, and it increased by a similar amount when the bundle was displaced by 20 nm in the inhibitory direction. The displacement at which the stiffness reached a minimum was within the most sensitive region of the hair-cell transducer function (receptor potential as a function of hair-bundle displacement), and the displacement at which the stiffness reached a maximum was at the point of saturation of the transducer function in the inhibitory direction. The nonlinear displacement-dependent compliance change is reversibly abolished, and the time constant of relaxation of the bundle for excitatory displacements is reversibly reduced, when mechanotransduction is blocked by the addition of either neomycin sulphate or cobalt chloride to the solution bathing the hair cells. The displacement-dependent compliance change was not apparently reduced when the receptor potential was attenuated through the substitution of sodium in the bathing solution with a less permeant cation, tetraethylammonium. These findings suggest that the nonlinear mechanical properties of the hair bundle are associated with aspects of the hair-cell mechanotransducer process. The mechanical properties of the hair bundle are discussed in relation to the 'gating-spring' hypothesis of hair-cell transduction.     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

Biotransformation of 2-acetylthiophene by micromycetes

Summary The biotransformation of 2-acetylthiophene by 800 strains of micromycetes has been investigated. Among them, 460 strains have been selected on solid media and cultivated in a liquid synthetic medium. Of these, 106 strains were able to completely deplete 2-acetylthiophene with or without production of intermediary metabolites. 2-Thienylglyoxylic acid was not detected but 72 strains produced 2-thiophenecarboxylic acid. Among them, eight strains have been selected for further optimization of this bioconversion.     

Semisynthetic des-(B27-B30)-insulins with modified B26-tyrosine

Semisynthetic des-(B27-B30)-insulins containing modified B26-tyrosine residues were prepared to refine the understanding of the importance of position B26 with regard to biological and structural properties of the hormone. The following shortened insulin analogues were synthesized by trypsin-catalysed peptide-bond formation between the C-terminal amino acid ArgB22 of des-(B23-B30)-insulin and synthetic tetrapeptides as amino components: des-(B27-B30)-insulin, des-(B27-B30)-insulin-B26-methyl ester, -B26-carboxamide with varying C-terminal hydrophobicity of the B-chain, and [Tyr(NH2)B26]-, [Tyr(NO2)B26]-, [Tyr(I2)B26]-, [D-TyrB26]des-(B27-B30)-insulin-B26-carboxamide containing non-proteinogenic amino acids in position B26. Starting from insulin and an excess of synthetic Gly-Phe-Phe-Tyr-OMe as nucleophile, des-(B27-B30)-insulin-B26-methyl ester--the formal transpeptidation product at ArgB22--was formed in one step. Biological in vitro properties (binding to cultured human IM-9 lymphocytes, relative lipogenic potency in isolated rat adipocytes) of all semisynthetic analogues are reported, ranging from slightly decreased to two-fold receptor affinity and nearly three-fold biopotency relative to insulin. If the C-terminal tetrapeptide B27-B30 is removed, full relative insulin activity is still preserved, while the shortening results in the loss of ability to associate in solution. Only after carboxamidation or methyl esterification of TyrB26 the self-association typical of native insulin can be observed, and the CD-spectral effects in the near UV spectrum related to association and hexamerization of the native hormone are qualitatively reestablished. The results of this investigation underline the importance of position B26 to the modulation of hormonal properties and solution structure of the shortened insulins.     

Molecular cloning of the mouse 46-kDa mannose 6-phosphate receptor (MPR 46).

A cDNA clone for the mouse 46-kDa mannose 6-phosphate receptor (MPR 46) was isolated from an embryonic mouse cDNA library. Its single open reading frame codes for a protein of 278 residues. It shows an over-all amino-acid identity of 93% with the human receptor. Nine non-conservative amino-acid exchanges are found in the luminal domain, one non-conservative exchange of hydrophobic amino acids is in the transmembrane domain, while the cytoplasmic receptor tails are identical. All five potential N-glycosylation sites are conserved as well as amino acids that are important for ligand binding (Arg 137 and His 131) and disulfide pairing (Cys 32 and 78, Cys 132 and Cys 167, Cys 145 and Cys 179). The absolute identity in the cytoplasmic MPR 46 tail suggests the importance of this amino-acid sequence for the intracellular routing of the MPR 46.     

N-terminal amino-acid sequence of pig kidney dipeptidyl peptidase IV solubilized by autolysis.

The N-terminal amino-acid sequence of the intrinsic membrane protein dipeptidyl peptidase IV (DP IV) was determined. The protein was isolated from pig kidney and solubilized by autolysis at pH 3.8. The first 34 amino acids were sequenced and indicated approximately 78% identity to the N-terminal sequence of rat liver DP IV.     

Influence of tissue preparation techniques on p53 expression in bronchial and bladder carcinomas, assessed by immunofluorescence staining and flow cytometry.

In a series of bronchial and bladder carcinomas, p53 protein expression was examined. Samples from formalin-fixed, paraffin-embedded tissue (routine-treated) were compared with parallel samples of fresh tissue and tissue fixed in paraformaldehyde and ethanol. The expression of p53 was measured by immunofluorescence staining and dual parameter flow cytometry, with simultaneous monitoring of DNA content. For each tumor, p53 fluorescence with different fixatives was expressed relative to fresh tissue. The p53 fluorescence signals were on average brighter from routine-treated tissue than from fresh tissue. The tissue fixed in paraformaldehyde showed no difference from fresh tissue. In the ethanol-fixed tissue, however, fluorescence signals were reduced by nearly 70%, and the fraction of detectable p53 positive cells in tumor tissue was reduced by more than 50%. This loss of fluorescence was probably due to a leakage of the antigen from nucleus to cytoplasm. Pepsin treatment did not influence p53 fluorescence. Within the same tumor, the S-phase fraction in p53 positive cells was significantly higher than in p53 negative cells (13.1 +/- 2.0% vs. 6.5 +/- 0.8%). This pattern was not influenced by formalin fixation or pepsin treatment. Our study demonstrates that in measuring a nuclear antigen, tissue handling may influence the results, and care should be taken to optimize the preparation procedure. Using the antibody PAb 1801, p53 expression measured in archival material is not reduced as compared to fresh tissue.     

Comparison of two cytochromes P-450 from Candida maltosa: primary structures, substrate specificities and effects of their expression in Saccharomyces cerevisiae on the proliferation of the endoplasmic reticulum

cDNAs were cloned, sequenced and expressed which encode two different cytochrome P-450 forms of the alkane-assimilating yeast Candida maltosa, designated as P-450Cm1 and P-450Cm2. The amino acid sequences deduced were about 55% identical. Expression in Saccharomyces cerevisiae resulted in the formation of intact microsomal P-450 systems catalyzing the hydroxylation of n-hexadecane and lauric acid with significantly different substrate preferences. A massive proliferation of the endoplasmic reticulum was observed in the S. cerevisiae cells which produced P-450. Depending on the P-450 form expressed, distinctly organized stacks of paired membranes appeared and occupied considerable areas of the cytoplasm. As shown by immunoelectron microscopy for P-450Cm1, the protein expressed was highly concentrated within these newly formed membrane structures.