In vivo changes in plasma acute phase protein levels in the rat induced by slow release of IL-1, IL-6 and TNF

Administration of large doses of cytokines by injection is required to induce changes in acute phase protein levels. Comparisons were made in the rat of the effects of administering recombinant human cytokines by injection with continuous release from implanted osmotic minipumps. Continuous release of interleukin-1beta (0.2-2.1 ng h(-1)) induced dose-related changes in the plasma levels of albumin, seromucoid proteins, haptoglobin and caeruloplasmin; interleukin-1alpha had similar effects but required higher doses (2-21 ng h(-1)). Tumour necrosis factor alpha (50 ng h(-1)) only significantly increased seromucoid levels, whereas IL-6 (3-30 ng h(-1)) induced haptoglobin and caeruloplassynthesis. This method provides a better technique for studying the in rive effects of cytokines which may be relevant to the release mechanisms in inflammation.     

Cutting and closing without recombination in V(D)J joining.

Open and shut junctions are rare (V(D)J joining products in which site-specific recognition, cleavage and re-ligation of joining signals has been uncoupled from recombination. Here, we investigate the relationship of opening and shutting to recombination in two ways. First, we have tested a series of substrates containing one or two joining signals in an in vivo assay. Opening and shutting can be readily observed in substrates that have only one consensus joining signal. Thus, unlike recombination, the majority of open and shut events do not require interactions between two canonical joining signals. Next we examined two-signal substrates to investigate the effect of signal proximity on the frequency of dual open and shut events. These experiments indicate that at least some of the time opening and shutting can be a two-signal transaction. Together these results point to two mechanistically related, but distinct origins for open and shut joining events. In one case, cutting and closing may occur without interaction between two signals. In the other, we suggest that interaction of a canonical signal with 'cryptic' signal-like elements whose sequence is extensively diverged from canonical signals, may bias the V(D)J recombination machinery towards opening and shutting rather than recombination. Open and shut operations could in this way provide a means whereby mistakes in target recognition by the V(D)J recombination machinery produce a non-recombinant outcome, avoiding deleterious chromosomal rearrangements in lymphoid tissues.     

Genetic and molecular analysis of spontaneous respiratory deficient (res-) mutants of Escherichia coli K-12

Respiratory deficient (res-) mutants of E. coli are slow growing microcolonial, anaerobic, catalase and benzidine negative strains whose broad phenotypic alteration may result from pleiotropic mutations in genes of the hemin biosynthetic pathway. They are easily recovered from platings of sensitive cells on concentrations of gentamicin higher than the minimal inhibitory concentration. These mutants show a dramatic change in their biochemical diagnostic profile resulting primarily from deficiencies in the active transport mechanisms of the cell. Using well-marked F- and Hfr strains, 157 mutants were analyzed from 3 different parent strains; all but 2 resulted from mutations in 3 loci of the hemin biosynthetic pathway. Of these a marked skew to hemB- mutations was seen, with more than 80% mapping there. The possibility that this hot spot resulted from transpositional activity was tested by Southern hybridization of EcoRI digests of the chromosomal DNA, using as a probe, a 2.8-kb fragment containing the hemB gene. The WT and other hemB+ control strains contained a 14.6-kb fragment. Of 18 hemB strains tested, 14 showed deletion and insertion mutations which fell into four classes based on the variation in the size of the fragment or on the absence of hybridization. The latter resulted from complete deletion of the hemB gene. An increase in fragment size from 1.5-kb to 3.4-kb was observed in some of the strains.     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples.

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

D-serine dehydratase from Escherichia coli. DNA sequence and identification of catalytically inactive glycine to aspartic acid variants

We have identified two glycyl residues whose integrity is essential for the catalytic competence of a model pyridoxal 5'-phosphate requiring enzyme, D-serine dehydratase from Escherichia coli. This was accomplished by isolating and sequencing the structural gene from wild type E. coli and from two mutant strains that produce inactive D-serine dehydratase. DNA sequencing indicated the presence of a single glycine to aspartic acid replacement in each variant. The amino acid replacements lie in a glycine-rich region of D-serine dehydratase well removed from pyridoxal 5'-phosphate-binding lysine 118 in the primary structure of the enzyme. The striking effect of these two glycine to aspartic acid replacements on catalytic activity, the conservation of the glycine-rich region in several pyridoxal 5'-phosphate-dependent enzymes that catalyze alpha/beta-eliminations, and the placement of similar glycine-rich sequences in well-characterized active site structures suggest that the glycine-rich region interacts with the cofactor at the active site of the enzyme.     

Intermolecular histone H4 interactions in core nucleosomes

Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the pyrene-labeled nucleosome was characteristic of excited dimer (excimer) fluorescence, indicating that the single pyrene groups on the two H4 molecules are in close proximity in the reconstituted particle. Histone H4 was also labeled randomly at lysines with a group that contains two pyrene moieties separated by 12 A at most. Incorporation of this histone into nucleosome-like particles provides an excimer standard which does not depend on intermolecular interactions. The properties of the pyrene-containing nucleosome were examined as a function of ionic strength. It was found that the H4-H4 pyrene excimer fluorescence exhibited a cooperative disruption centered at 0.1 M NaCl which preceded increases in accessibility and environment polarity revealed by other fluors attached at the same site.     

Primary structure of the chromosomal protein HMb from the archaebacteria Methanosarcina barkeri

The amino acid sequence of the protein HMb, a protein of 93 residues (Mr 10757) which represents the major acid-soluble component of the Methanosarcina barkeri nucleoprotein complex, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid, arginine and methionine residues. The protein HMb is mainly characterized by a high amount of charged residues (15% of acidic residues and 26.8% of basic residues) which are distributed all along the polypeptide chain. The amino acid sequence of the protein HMb is not homologous to any eubacterial, archaebacterial or eukaryotic chromosomal proteins known up to now.     

An intermediate polymer in the assembly of clathrin baskets

Clathrin (8 S) is known to polymerize into two varieties of basket structures (150 S or 300 S) under the normal buffer conditions [100 mM 2-(N-morpholino)ethanesulfonic acid (Mes), pH 5.9-6.7] used for the isolation of coated vesicles. However, it is now observed that under very low salt conditions (2 mM Mes, pH 5.9), it forms a homogeneous species with a sedimentation coefficient of 27 S. Increasing the salt concentration to 50 mM Mes completely converts all the 27S species into 150S baskets. Sedimentation equilibrium data show that this 27S species has a molecular weight that is 6 times that of the clathrin protomer and is the result of highly cooperative reversible self-association of the 8S protomer. Light-scattering studies show that the stabilities of 27S species and baskets (150 S or 300 S) are comparable. Fluorescent labeling of sulfhydryl groups with N-(1-anilinonaphthalenyl)maleimide indicates that the conformation of clathrin in 27S species and baskets (150 S or 300 S) is similar. Trypsin digestion reveals that in the 27S species clathrin has a conformation differing from that in both the 8S species and baskets.     

Tubulin pseudogenes as markers for hominoid divergence

Processed pseudogenes arise via unimolecular events that result in the integration of nonfunctional (and therefore non-selected) regions of DNA into the germ line. The sequence of such pseudogenes can be used as a novel form of evolutionary clock: the older a particular pseudogene, the more mutations it has acquired relative to the selectively constrained functional gene from which it was originally derived. We have used specific beta-tubulin gene probes to assay for the presence of fully sequenced processed pseudogenes in genomic DNA from various hominoid species. The data suggest that orangutan is more closely related to human, chimpanzee and gorilla than is generally believed.