Identification of a gene, closely linked to dnaK, which is required for high-temperature growth of Escherichia coli.

We have constructed four deletion derivatives of the cloned dnaK gene. Plasmid pDD1, in which the last 10 amino acids of the DnaK protein have been replaced by three different amino acids derived from the pBR322 vector, was as effective as plasmid pKP31, from which it was derived, in restoring the ability of a dnaK null mutant, Escherichia coli BB1553, to plate lambda phage and to grow at high temperatures. The other three mutations, involving much larger deletions of the dnaK gene, did not restore the ability to plate lambda phage or the ability to grow at high temperatures. Plasmid pKUC2, which contains the whole dnaK gene and its promoters, was capable of restoring the ability of E. coli BB1553 to plate lambda phage but, surprisingly, it did not restore the ability to grow at high temperatures, even though it was shown that the DnaK protein was efficiently expressed in these cultures. By transposon mutagenesis and sub-cloning, we have shown the presence of a second gene in plasmid pKP31 which is required for high-temperature growth of E. coli BB1553. This gene, which we call htg A, is presumably also defective in the dnaK null mutant E. coli BB1553. We have also demonstrated that the inability of E. coli K756 to grow above 43.5 degrees C is complemented by sub-clones which contain the htg A gene, but not by plasmid pKUC2.     

Efficacy of intermittent or continuous administration of GnRH in inducing ovulation in early and late seasonal anoestrus in the Père David's deer hind (Elaphurus davidianus)

Père David's deer hinds were treated with GnRH, administered as intermittent i.v. injections (2.0 micrograms/injection at 2-h intervals) for 4 days, or as a continuous s.c. infusion (1.0 micrograms/h) for 14 days. These treatments were given early (February-March) and late (May-June) in the period of seasonal anoestrus. The administration of repeated injections of GnRH increased mean LH concentrations from pretreatment values of 0.54 +/- 0.09 to 2.10 +/- 0.25 ng/ml over the first 8 h of treatment in early anoestrus, and from 0.62 +/- 0.11 to 2.73 +/- 0.49 ng/ml in late anoestrus. The mean amplitude of GnRH-induced LH episodes was greater (P less than 0.01) in late (4.03 +/- 0.28 ng/ml) than in early (3.12 +/- 0.26 ng/ml) anoestrus, but within each replicate (early or late anoestrus), neither mean LH episode amplitude nor mean plasma LH concentrations differed significantly between the four periods of intensive blood sampling. On the basis of their progesterone profiles, 6/12 hinds had ovulated in response to treatment with injections of GnRH (1/6 in early anoestrus and 5/6 in late anoestrus), and oestrus and a preovulatory LH surge were recorded in all of these animals. Oestrus and a preovulatory LH surge were also recorded in one other animal treated in early anoestrus in which progesterone concentrations remained low. The mean times of onset of oestrus (91.0 +/- 1.00 and 62.4 +/- 0.98 h) and of the preovulatory LH surge (85.8 +/- 3.76 and 59.4 +/- 0.25 h) both occurred significantly earlier (P less than 0.001) in animals treated in late anoestrus. Continuous infusion of GnRH to seasonally anoestrous hinds resulted in an increase in mean plasma LH concentrations, but this response did not differ significantly between early (2.15 +/- 0.28 ng/ml) and late (2.48 +/- 0.26 ng/ml) anoestrus. Ovulation, based on progesterone profiles, occurred in 2/7 hinds in early anoestrus and in 4/6 hinds in late anoestrus. Oestrus was detected in all except one of these hinds. The mean time of onset of oestrus occurred earlier in animals treated in late anoestrus (66.2 +/- 0.32 h and 46.7 +/- 0.67 h, P less than 0.01). The administration of GnRH, given either intermittently or continuously, will induce ovulation in a proportion of seasonally anoestrous Père David's deer. However, more animals ovulate in response to this treatment in late than in early anoestrus (75% compared with 23%).     

5''-Nucleotidases in rat heart. Evidence for the occurrence of two soluble enzymes with different substrate specificities.

Chromatography of soluble proteins from rat heart on phosphocellulose columns separates two 5'-nucleotidases. The first to emerge from the column shows a preference for AMP over IMP as substrate, whereas the second shows a preference for IMP over AMP. The properties of the IMP-preferring enzyme, including the conditions under which it is eluted from phosphocellulose columns, show it to be the enzyme studied by Itoh, Oka & Ozasa [Biochem. J. (1986) 235, 847-851]. The kinetic properties of the AMP-preferring enzyme indicate that it is likely to be the enzyme responsible for the production of adenosine under conditions of hypoxia and increased work load, and with metabolic stresses such as a high load of acetate.     

Calorimetric and Fourier transform infrared spectroscopic studies on the interaction of glycophorin with phosphatidylserine/dipalmitoylphosphatidylcholine-d62 mixtures

Glycophorin has been isolated in pure form from human erythrocyte membranes and reconstituted into lipid vesicles composed of binary mixtures of bovine brain phosphatidylserine (PS) and acyl-chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The effect of protein on lipid melting behavior and order has been monitored with differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). The phase diagram for PS/DPPC-d62 is consistent with that previously reported for PS/DPPC (Stewart et al. (1979) Biochim. Biophys. Acta 556, 1-16) and indicates that acyl chain perdeuteration does not greatly alter the lipid mixing characteristics. The use of deuterated lipid allows the examination of lipid order by FT-IR of each lipid component in the binary mixtures as well as in the ternary (lipid/lipid/protein) systems. Addition of glycophorin to a 30:70 PS/DPPC-d62 binary lipid mixture results in a preferential glycophorin/PS interaction leading to bulk lipid enriched in DPPC-d62. This is revealed in two ways: first, through cooperative calorimetric transitions increased in temperature from the binary lipid system and second, through FT-IR melting curves of the DPPC-d62 component which shows transitions increased in both onset and completion temperatures in the presence of protein. In addition, non-cooperative melting events are observed at temperatures below the onset of phase separation. The FT-IR data are used to assign these non-cooperative events to the melting of the PS component. For the 50:50 lipid mixture with protein, two transitions are observed in the DSC experiments. The IR results indicate that both lipid components are involved with the lower temperature event.     

Comparison of physicochemical properties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosis and asthmatic patients

The major nonreduced mucus glycoproteins (mucins) from sputa of cystic fibrosis (CF) and asthmatic patients have been purified to electrophoretic homogeneity and subjected to physical and chemical characterization. The sputum specimens were solubilized in buffer containing 0.22 M KSCN and fractionated on Bio-Gel A-5m, followed by digestion with DNase, rechromatography on the same column, and chromatography on hydroxylapatite. Sodium dodecyl sulfate gel electrophoresis of purified mucins gave a single band. Carbohydrate analyses of the purified mucins showed no significant differences in the sugar components from the two mucins. However, the CF mucin contained substantially higher (11%) sulfate content than that observed for the asthmatic mucin (5.9%). Amino acid analyses indicated that the CF mucin had higher levels of serine plus threonine (35%) as compared to the asthmatic mucin (29%). In contrast, CF mucin contained a lower content of aspartate, glutamate, and glycine than that observed for the asthmatic mucin. Molecular weights of 3.8 X 10(6) and 3.5 X 10(6) were obtained for CF and asthmatic mucins, respectively, from light-scattering studies of mucins in the presence of 6 M guanidine hydrochloride. Reduction of the disulfide bonds of the two mucins did not alter their molecular weights. Liquid chromatographic studies on Sepharose CL2B showed that CF mucin forms aggregates sufficiently large to be excluded from the gel. As compared to the CF mucin, the asthmatic mucin formed fewer of these large aggregates under identical experimental conditions. Reduction and alkylation of the mucins resulted in their inability to form aggregates. The higher state of aggregation of CF mucin may influence the viscoelastic properties of the CF lung's mucus secretions.     

The analysis of recombinant interleukin-2 by thermospray liquid chromatography-mass spectrometry

The complete sequence of recombinant human interleukin-2 expressed in Escherichia coli has been confirmed by thermospray liquid chromatography-mass spectrometry (TS-LC-MS) of a tryptic digest derived from 100 micrograms (7 nmol) of reduced carboxymethylated interleukin-2. The preparation was shown by this method to contain predominantly unprocessed N-terminal initiator Met, with some authentic N-terminal Ala; the rest of the protein was as predicted from the DNA sequence, though some deamidated material was noted. TS-LC-MS proved to be a rapid and efficient method for surveying the protein tryptic peptide products allowing all the data to be collected in one chromatographic run; all tryptic fragments were identified by their molecular ions including those for the larger peptides (Mr 1500-3500) which, due to the presence of doubly and triply charged molecular ions, were brought within the mass range of the instrument (1800 Da). It is proposed that TS-LC-MS is a good general method for analyzing recombinant protein digests with respect to sequence confirmation, processing, and post-translational modification, and since each chromatographic peak is identified allows for subsequent monitoring of the protein by LC using uv detection. The method suffers from the disadvantages that all the sample is consumed during the experiment and that no fragment (sequence) ions are generally observed.     

Fluorometric determination of histamine with OPT: optimum reaction conditions and tests of identity

Histamine reacts with OPT at an alkaline pH giving fluorescent conjugation products. Optimum fluorophore formation was observed at pH 12.5 after 40 min reaction at 0°C under continuous gassing with nitrogen. After acidification with sulfuric acid to pH 2–5 the fluorescence was stable for hours. Reagent blanks were reduced by the lowering of the reaction temperature and by decreasing the amount of OPT added. These modifications permitted the determination of 2 ng histamine/ml and gave far better reproducibility than the original procedure of Shore, Burkhalter, and Cohn. The fluorometric assay for histamine is nonspecific; the major interfering OPT-reactive tissue component is believed to be spermidine. Specificity was secured by adding formaldehyde before acidification, thus abolishing the fluorescence of histamine but not that of spermidine, or by adding CdCl₂ or SrCl₂ together with OPT, thus preventing the formation of the spermidine fluorophore but not that of the histamine fluorophore.