Molecular genetics of cell death in the nematode Caenorhabditis elegans

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insightinto the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells. © 1992 John Wiley & Sons, Inc.     

Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

The enzyme that cleaves apolipoprotein A-II upon in vitro incubation of human plasma high-density lipoprotein-3 with blood polymorphonuclear cells is an elastase

The proteolytic activity directed against apolipoprotein A-II (apo-A-II) which is released from human blood polymorphonuclear cells (PMN) when they are incubated with human plasma high-density lipoprotein-3 (HDL3) was studied to assess the properties and site specificity of the enzyme. When 125I-apo-A-II-labeled HDL3 was incubated with the PMN protease at 37 degrees C, a complete cleavage of apo-A-II was observed which paralleled the formation of bands of approximately 11,000 and 7,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 7,000-dalton component had the following N-terminal sequence: NH2-Thr-Asp-Tyr-Gly-Lys-Asp-Leu-Met-Glu-Lys. This corresponds to residues 19 through 28 of the intact apo-A-II monomer. Methoxysuccinyl (MeO-Suc)-Ala-Ala-Pro-Val-chloromethylketone-(CH2Cl) caused a 90% inhibition of apo-A-II hydrolysis at the highest concentration tested (6 X 10(-4)M). Besides apo-A-II, the PMN enzyme also hydrolyzed a synthetic substrate, MeO-Suc-Ala-Ala-Pro-Val-4-nitroanilide and its 4-methylcoumaryl-7-amide analogue. The protease appeared to have a mass of 28,000 daltons as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]diisopropylfluorophosphate-labeled PMN enzyme. That the PMN enzyme which cleaves apo-A-II is an elastase was derived from the following criteria: 1) cleavage at the Val-X bond in apo-A-II and in the two synthetic substrates studied; 2) prevention of the cleavage by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a known specific elastase inhibitor; and 3) a mass comparable to that reported for a pure PMN elastase. These studies establish that apolipoproteins can be suitable substrates for enzymes of the elastase family.     

A preliminary investigation of prediction of mean arterial pressure after self-regulatory treatments.

This study investigated the ability of pretreatment variables from three different domains (social-demographic, psychological, and psychophysiological) to predict posttreatment mean arterial pressure (MAP) for 59 unmedicated males with mild hypertension who were participating in a cross-cultural (USA-USSR) comparison of autogenic training and thermal biofeedback to a self-relaxation control. The overall multiple regression equation consisted of two variables and indicated that higher diastolic blood pressures during a cold pressor task were predictive of greater MAP reductions while higher scores on the Irritability subscale of the Buss-Durkee Hostility Scale were predictive of less MAP reductions. Suggestions for future research in this area are provided.