Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin

The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements. Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone. This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant. The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region. The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C. The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25. This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant. Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein. We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76.     

A probability matrix for the identification of species of Vibrio and related genera

A matrix for the probabilistic identification of species of Vibrio and related genera has been constructed using the data from 1091 strains collected throughout the world and classified. Thirty-eight phenons are included in the matrix, 31 of these represent previously identified species or biovars and seven represent phenons which could not be identified and may represent new species. The identification matrix incorporates 81 characters although a subset of 30 tests can be used to distinguish the 38 phenons from each other. The additional 51 tests were included to assist the identification of some strains for which the initial 30 tests were inadequate. No significant cluster overlap was found at the 5% level and the identification score for the Hypothetical Median Organism of each cluster exceeded 0.9999 in all cases.     

Stereospecific induction of starfish oocyte maturation by (8R)-hydroxyeicosatetraenoic acid

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. This induction of meiotic divisions can be triggered also by four fatty acids: 5,8,11-20:3; 5,8,11,14-20:4 (arachidonic acid); 6,9,12,15-20:4; 5,8,11,14,17-20:5, all other fatty acids being completely inactive. This maturation triggered by eicosanoids occurs in the micromolar range and is facilitated by the presence of calcium. A variety of arachidonic acid derivatives (esters, epoxides, etc.) and metabolites (cyclooxygenase and lipoxygenase products) has been tested; the biological activity is restricted to 8-hydroxyeicosatetraenoic acid (8-HETE), other mono- and poly-HETEs being completely inactive. Maturation triggered by 8-HETE occurs around 10 nM and is insensitive to the presence of calcium. 8-HETE methyl ester and 8-hydroperoxyeicosatetraenoic acid are able to induce maturation at higher concentrations. Both (8S) and (8R) stereoisomers have been tested; the biological activity is strictly restricted to the (8R) isomer. 8-HETE triggers a complete maturation, i.e. maturation-promoting factor appearance, germinal vesicle breakdown, emission of the polar bodies, and formation of a female pronucleus. (8R)-HETE, but not (8S)-HETE, triggers the typical decrease in cyclic AMP concentration induced by 1-methyladenine and the burst of protein phosphorylation associated with maturation. Starfish oocytes oxidize exogenous arachidonic acid into 8-HETE and other HETEs. 8-HETE was identified, after high pressure liquid chromatography purification, by gas chromatography mass spectrometry. Furthermore, it was found that the starfish oocytes only produce the (8R)-HETE isomer. This highly stereospecific induction of oocyte maturation by (8R)-HETE suggests that this fatty acid, or a very closely related fatty acid, may play a role in the transduction of the 1-methyladenine message at the plasma membrane level.     

ATP-independent renaturation of complementary DNA strands by the mutant recA1 protein from Escherichia coli

In an effort to clarify the requirement for ATP in the recA protein-promoted renaturation of complementary DNA strands, we have analyzed the mutant recA1 protein which lacks single-stranded DNA-dependent ATPase activity at pH 7.5. Like the wild type, the recA1 protein binds to single-stranded DNA with a stoichiometry of one monomer per approximately four nucleotides. However, unlike the wild type, the mutant protein is dissociated from single-stranded DNA in the presence of ATP or ADP. The ATP analogue adenosine 5'-O-3' (thiotriphosphate) appears to stabilize the binding of recA1 protein to single-stranded DNA but does not elicit the stoichiometry of 1 monomer/8 nucleotides or the formation of highly condensed protein-DNA networks that are characteristic of the wild type recA protein in the presence of this analogue. The recA1 protein does not catalyze DNA renaturation in the presence of ATP, consistent with the dissociation of recA1 protein from single-stranded DNA under these conditions. However, it does promote a pattern of Mg2+-dependent renaturation identical to that found for wild type recA protein.     

Effects of psoralen on replicon size and mean rate of DNA synthesis in partially synchronized cells of Pisum sativum L.

We have examined by fibre autoradiography the spacing of replicons in pea root meristems during synchronized entry into S phase from arrest at the G1/S boundary. Pretreatment with the DNA cross-linking agent, psoralen, produces a marked shortening of replicon spacing, suggesting that premature arrest of the replication fork results in the recruitment of additional initiation points within a given replicon family. This is discussed in relation to models for the control of DNA replication.     

Gene map for the Cyanophora paradoxa cyanelle genome.

The genes for the following proteins were localized by hybridization analysis on the cyanelle genome of Cyanophora paradoxa: the alpha and beta subunits of phycocyanin (cpcA and cpcB); the alpha and beta subunits of allophycocyanin (apcA and apcB); the large and small subunits of ribulose-1,5-bisphosphate carboxylase (rbcL and rbcS); the two putative chlorophyll alpha-binding apoproteins of the photosystem I-P700 complex (psaA and psaB); four apoproteins believed to be components of the photosystem II core complex (psbA, psbB, psbC, and psbD); the two apoprotein subunits of cytochrome b-559 which is also found in the core complex of photosystem II (psbE and psbF); three subunits of the ATP synthase complex (atpA and atpBE); and the cytochrome f apoprotein (petA). Eighty-five percent of the genome was cloned as BamHI, BglII, or PstI fragments. These cloned fragments were used to construct a physical map of the cyanelle genome and to localize more precisely some of the genes listed above. The genes for phycocyanin and allophycocyanin were not clustered and were separated by about 25 kilobases. Although the rbcL gene was adjacent to the atpBE genes and the psbC and psbD genes were adjacent, the arrangement of other genes encoding various polypeptide subunits of protein complexes involved in photosynthetic functions was dissimilar to that observed for known chloroplast genomes. These results are consistent with the independent development of this cyanelle from a cyanobacterial endosymbiont.     

Improving the accuracy of burn-surface estimation

A user-friendly computer-assisted method of calculating total body surface area burned (TBSAB) has been developed. This method is more accurate, faster, and subject to less error than conventional methods. For comparison, the ability of 30 physicians to estimate TBSAB was tested. Parameters studied included the effect of prior burn care experience, the influence of burn size, the ability to accurately sketch the size of burns on standard burn charts, and the ability to estimate percent TBSAB from the sketches. Despite the ability for physicians of all levels of training to accurately sketch TBSAB, significant burn size over-estimation (p less than 0.01) and large interrater variability of potential consequence was noted. Direct benefits of a computerized system are many. These include the need for minimal user experience and the ability for wound-trend analysis, permanent record storage, calculation of fluid and caloric requirements, hemodynamic parameters, and the ability to compare meaningfully the different treatment protocols.     

Defense of winter-dormant Alaska paper birch against snowshoe hares

Summary Mature growth-phase internodes of Alaska paper birch (Betula resinifera) are preferred by the snowshoe hare (Lepus americanus) over juvenile growth-phase internodes due to the low food value of the latter. While the mature over juvenile preferencec cannot be explained by the levels of inorganic nutrients or gross chemical fractions (resins or phenols), it can be explained by the striking differences in secondary metabolites of the two growth phases. The principle compound which renders the juvenile phase internodes unpalatable is papyriferic acid, a triterpene which is a demonstrated feeding deterrent to snowshoe hares and which is present in juvenile internodes at concentrations 25 times greater than those in mature internodes.     

Isolation and characterization of the proteinase inhibitor-inducing factor from tomato leaves. Identity and activity of poly- and oligogalacturonide fragments

Mild acid hydrolysis of a small (Mr = 6 kDa) pectic polysaccharide isolated from tomato leaves, an inducer of the synthesis and accumulation of two proteinase inhibitors in excised tomato plants, yielded a alpha-D-polygalacturonic acid polymer with degree of polymerization = 20 that retained proteinase inhibitor-inducing activity. Enzymic and acid hydrolysis of this polygalacturonan yielded a series of alpha-1,4-D-galacturonic acid oligomers with degrees of polymerization from 2 to 6 which were purified to homogeneity and assayed for proteinase inhibitor-inducing activity in young excised tomato plants. All of the oligomers exhibited activity. The hexagalacturonide possessed the highest activity and the trimer the lowest. The evidence supports a possible role for plant cell wall fragments as systemic messengers that regulate the expression of proteinase inhibitor genes in plant leaves in response to pest attacks.