Effect of iodide on estrogen-induced uterine peroxidase

Iodide administered in the drinking water for 5–7 days increased the activity of estradiol-induced uterine peroxidase in the immature rat. This effect was specific for iodide and could not be mimicked by chloride, bromide, thiocyanate, perchlorate or iodate. Sodium iodide also increased peroxidase activity in the parotid gland but had no effect on glucose-6-phosphate dehydrogenase in the uterus, thyroid or parotid even though estradiol produced a 2-fold increase in the activity of this enzyme in the uterus. ¹²⁵I was taken up more readily by the uterus than by muscle but this process was not influenced by prior treatment of the animals with estrogen. The in vitro effect of sulfhydryl reagents on uterine peroxidase was also investigated and proposals made for possible mechanisms of action of iodide on this enzyme in the intact animal.     

Role of methyl groups in dynamics and evolution of biomolecules

Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.     

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.     

Structural and functional variability among DQ β alleles of DR2 subtypes

Homozygous lymphoblastoid cell lines representing various Dw subtypes of DR2 were examined for polymorphism at the DQ locus by molecular and cellular techniques. The subtypes studied included Dw2, Dw12, and a group heterogenous by cellular typing that we shall refer to as non-Dw2/non-Dw12. Restriction fragment length polymorphism analysis of cell lines representing these subtypes revealed DQ -specific patterns consistent with cellular typing. Two-dimensional gel electrophoresis of DQ molecules from representative cell lines revealed a structural polymorphism of DQ among the three subtypes. The DQ chain migrated to a position that was unique to each subtype and was consistent among various representative cell lines of each subtype. Nucleotide sequence analysis of cDNA clones of DQ from Dw2, Dw12, and non-Dw2/non-Dw12 lines confirmed that the variability resided at the genetic level. Variability was found in the form of numerous scattered nucleotide substitutions throughout the first domain of these alleles. The DQ gene of the non-Dw2/non-Dw12 cell line AZH was further found to be almost identical with the DQ gene of a DR1 line (Bell et al. 1985b), implicating a common evolutionary origin of these alleles. The only difference between these two sequences was due to an apparent gene conversion event at amino acid 57. T-cell cloning experiments resulted in the derivation of Epstein-Barr virus-specific, DQw1-restricted clones that proliferated against only those cell lines that exhibited the DQ gene common to AZH and the DR1 cell line. Thus, the polymorphism among DQ alleles within DR2 results in subtype-specific restriction.     

Insulin effects on apolipoprotein B production by normal, diabetic and treated-diabetic rat liver and cultured rat hepatocytes.

1. The effect of insulin on apolipoprotein (apo B) secretion was studied in 24 h recirculating liver perfusions of isolated normal, diabetic and insulin-treated diabetic rats. In single perfusions from each group apo B accumulated in the media in a linear fashion. 2 In perfusions of normal rat livers, when the medium contained insulin plus cortisol, apo B production was significantly inhibited (by 35.8%), demonstrating a hormone effect on apo B secretion. 3. In perfusions of diabetic-rat livers, apo B production was decreased to 11.8% of normal when the medium contained no hormones, and was not significantly changed by the addition of insulin plus cortisol to the medium, suggesting that the hormone effect on apo B secretion is missing in long-term hypoinsulinaemic states. 4. Treatment of diabetic rats with daily insulin injection restored apo B production and restored the effect of insulin plus cortisol in the medium to inhibit apo B secretion during perfusion. 5. Parallel studies of apo B secretion with insulin alone, cortisol alone and insulin plus cortisol in the medium were performed in primary cultures of hepatocytes to compare results from liver perfusions. 6. Apo B secretion by hepatocytes from normal, diabetic and treated-diabetic rats was inhibited (by 36.8%, 57.1% and 57.9% respectively) when insulin alone was added to the medium. 7. Insulin plus cortisol inhibited apo B secretion by hepatocytes from normal and treated diabetic rats (by 30.2% and 47.2% respectively), but failed to inhibit apo B secretion by hepatocytes from diabetic rats.     

Staphylococcal nuclease: sequential assignments and solution structure

Sequential assignments are reported for backbone 15N and 1H of nearly all residues of staphylococcal nuclease (Nase) complexed with thymidine 3',5'-diphosphate and Ca2+. Because of the relatively large size of the Nase ternary complex, Mr 18K, the crucial element of our assignment strategy was the use of isotope-edited two-dimensional NMR spectra, particularly 15N-edited nuclear Overhauser enhancement spectroscopy (NOESY), 15N-edited J-correlated spectroscopy (COSY), and 1H/15N or 1H/13C heteronuclear multiple quantum shift correlation spectroscopy (HMQC). These experiments, together with the more conventional NOESY, COSY, and homonuclear Hartmann-Hahn spectra of natural abundance or deuteriated samples, yielded backbone assignments of 127 of the 136 residues in the structured part of the protein. Using the NOESY data, we identified three helical domains and several beta-sheets which were in close correspondence with secondary structure identified in the crystal structure. Moreover, many long-range NOESY connectivities were identified that were in agreement with distances derived from the crystal structure. The region of the sequence in the neighborhood of residue 50 appears to be more flexible and disordered in solution than in the crystal. Very slowly exchanging amide protons are those found to be hydrogen bonded in the crystal structure; however, even hydrogen-bonded amides located within similar types of regular secondary structures, e.g., alpha-helices, exchange with greatly different rates.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

Effect of pH on growth and ethanol production byZymomonas

Summary In a mineral salts medium containing yeast extract, NH₄Cl and glucose (50g/L), the pH range producing the fastest growth ofZ. mobilis was 5.5–6.5 with an apparent optimum at 6.5. At constant growth rate of 0.15hr^–1, the specific rates of glucose utilization (q_s) and ethanol production (q_p) were relatively unaffected by pH over the range 7.0–5.5 but increased sharply as the pH was further decreased below 5.5 to 4.0. Under these conditions the ethanol yield was unaffected by pH over the range 4.0–6.5 but decreased markedly at pH of 7.     

Effects of medium composition on extracellular proteinase stability and yield in batch cultures of a Thermus sp.

Summary Thermus sp. Rt41A produces an extracellular proteinase that is produced concomitant with growth and l-glutamate catabolism. Calcium-chelating medium components were shown to decrease the half-life of the proteinase in growing cultures. Medium modifications avoiding these components resulted in an increase in the half-life and in the peak level of proteinase. By adding the inorganic phosphate requirement for growth in anabolic amounts to pH-controlled batch cultures, stability of the proteinase in the medium was greatly enhanced and there was consequent improvement in the total proteinase yield. This approach also allowed a balanced increase in substrate and phosphate concentrations to increase the cell and proteinase yield in batch culture in an almost stoichiometric manner.Offprint requests to: H. W. Morgan