Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin.

We have previously shown that replacing the P1-site residue (Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lys results in the acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively. However, the inhibitory activities thus induced are not strong. In the present study, we introduced additional amino acid replacements around the reactive site to try to make the P1-site mutants more effective inhibitors of chymotrypsin or trypsin. The amino acid replacement Asp-->Tyr at the P2' site of OMCHI3(P1Met) resulted in conversion to a 35000-fold more effective inhibitor of chymotrypsin with an inhibitor constant (K(i)) of 1. 17x10(-11) M. The K(i) value of OMCHI3(P1Met, P2'Ala) indicated that the effect on the interaction with chymotrypsin of removing a negative charge from the P2' site was greater than that of introducing an aromatic ring. Similarly, enhanced inhibition of trypsin was observed when the Asp-->Tyr replacement was introduced into the P2' site of OMCHI3(P1Lys). Two additional replacements, Asp-->Ala at the P4 site and Arg-->Ala at the P3' site, made the mutant a more effective inhibitor of trypsin with a K(i) value of 1. 44x10(-9) M. By contrast, Arg-->Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) resulted in a greatly reduced inhibition of chymotrypsin, and Asp-->Ala replacement at the P4 site produced only a small change when compared with a natural variant of OMCHI3. These results clearly indicate that not only the P1-site residue but also the characteristics, particularly the electrostatic properties, of the amino acid residues around the reactive site of the protease inhibitor determine the strength of its interactions with proteases. Furthermore, amino acids with different characteristics are required around the reactive site for strong inhibition of chymotrypsin and trypsin.     

Testing the accuracy of methods for reconstructing ancestral states of continuous characters.

Many methods are available for estimating ancestral values of continuous characteristics, but little is known about how well these methods perform. Here we compare six methods: linear parsimony, squared-change parsimony, one-parameter maximum likelihood (Brownian motion), two-parameter maximum likelihood (Ornstein-Uhlenbeck process), and independent comparisons with and without branch-length information. We apply these methods to data from 20 morphospecies of Pleistocene planktic Foraminifera in order to estimate ancestral size and shape variables, and compare these estimates with measurements on fossils close to the phylogenetic position of 13 ancestors. No method produced accurate estimates for any variable: estimates were consistently less good as predictors of the observed values than were the averages of the observed values. The two-parameter maximum-likelihood model consistently produces the most accurate size estimates overall. Estimation of ancestral sizes is confounded by an evolutionary trend towards increasing size. Shape showed no trend but was still estimated very poorly: we consider possible reasons. We discuss the implications of our results for the use of estimates of ancestral characteristics.     

Sequence analysis of a 101-kilobase plasmid required for agar degradation by a Microscilla isolate.

An agar-degrading marine bacterium identified as a Microscilla species was isolated from coastal California marine sediment. This organism harbored a single 101-kb circular DNA plasmid designated pSD15. The complete nucleotide sequence of pSD15 was obtained, and sequence analysis indicated a number of genes putatively encoding a variety of enzymes involved in polysaccharide utilization. The most striking feature was the occurrence of five putative agarase genes. Loss of the plasmid, which occurred at a surprisingly high frequency, was associated with loss of agarase activity, supporting the sequence analysis results.     

Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.     

Identification of clusterin sequences mediating renal tubular cell interactions.

Expression of the glycoprotein clusterin is markedly increased following tissue injury. One function of clusterin is to promote cell interactions which are perturbed in these pathologic settings. Clusterin causes cell aggregation and adhesion in vitro yet the molecular mechanism for this effect is not known. In order to identify the active site(s) of clusterin, 34 peptides, each 15 amino acid residues in length, were synthesized from hydrophilic regions of human clusterin. When studied individually, none of the peptides caused aggregation of LLC-PK1 cells, a porcine renal epithelial cell line. However, two out of the 34 peptides inhibited clusterin-induced cell aggregation in a dose-dependent manner. Scrambled versions of these two 'active' peptides did not inhibit cell aggregation. Seven peptides promoted cell adhesion. In conclusion, these findings provide evidence for novel amino acid sequences mediating clusterin-induced renal cell interactions.     

Physiological responses of exercised-fatigued individuals exposed to wet-cold conditions.

Thirteen healthy and fit men [age = 27 +/- 8 (SD) yr, height = 177 +/- 5 cm, mass = 75 +/- 7 kg, body fat = 14 +/- 5%, maximal O2 consumption = 51 +/- 4 ml. kg-1. min-1] participated in an experiment designed to test their thermoregulatory response to a challenging cold exposure after 5 h of demanding mixed exercise during which only water was consumed. Subjects expended 7,314 +/- 741 kJ on cycling, rowing, and treadmill-walking machines, performed 8,403 +/- 1,401 kg. m of mechanical work during resistance exercises, and completed 120 inclined sit-ups. Subjects then assumed a seated position in a 10 degrees C air environment while wearing shorts, T-shirt, rain hat, and neoprene gloves and boots. After 30 min the subjects were showered continuously with cold water ( approximately 920 ml/min at 10 degrees C) on their backs accompanied by a 6 km/h wind for up to 4 h. Blood samples were taken from the nondominant arm every 30 min during the exposure and assayed for energy metabolites, hormones, indexes of hydration, and neurotransmitters. Counterbalanced control trials without prior exercise were also conducted. Blood insulin was higher during the control trial, whereas values of glycerol, nonesterified fatty acids, beta-hydroxybutyrate, lactate, cortisol, free triiodothyronine, and thyroxine were lower. Three subjects lasted the maximum duration of 4.5 h for control and fatigue trials, with final rectal temperatures of 36.43 +/- 0.21 and 36.08 +/- 0.49 degrees C, respectively. Overall, the duration of 172 +/- 68 (SD) min for the fatigue trial was not significantly different from that of the control trial (197 +/- 72 min) and, therefore, was not affected by the preexposure exercise. Although duration was positively correlated to body fatness and shivering intensity, the latter was not correlated to any physical characteristic or the fitness level of the individual.     

The role of the cysteine-rich region of the β2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) heterodimer formation and ligand binding

The cysteine-rich region (CRR) of the β2 integrin subunit was replaced by that of β1 to give the chimera β2NV1. β2NV1 can combine with αL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the β2 interaction with αL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing αLβ2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic β2 CRR.