Symmetry breaking in C. elegans: another gift from the sperm

Polarization of the C. elegans embryo depends on the sperm-contributed centrosome, which cues a retraction of the actomyosin cortex to the opposite end of the embryo by an unknown mechanism. New evidence reveals that the sperm donates a second polarizing cue that may locally relax the actomyosin cortex near the point of sperm entry.     

The interface between caldesmon domain 4b and subdomain 1 of actin studied by nuclear magnetic resonance spectroscopy

The ability of caldesmon to inhibit actomyosin ATPase activity involves the interaction of three nonsequential segments of caldesmon domain 4 (amino acids 600-756) with actin. Two of these contacts are located in the C-terminal half of this region of caldesmon which has been designated domain 4b (658-756). To investigate the spatial relationship between the two sites and to determine whether their corresponding contacts on actin are sequentially distinct, we have used NMR spectroscopy to compare the actin binding properties of the minimal inhibitory peptide LW30 comprising residues 693-722 with those of the recombinant domain 4b constructs 658C (658-756) and Cg1 (a mutant of 658C in which the sequence (691)Glu-Trp-Leu-Thr-Lys-Thr(696) is changed to Pro-Gly-His-Tyr-Asn-Asn). Cg1 retains dual-sited actin attachment but displays lowered actin affinity. In the presence of tropomyosin, domain 4b-actin contacts were stronger but not qualitatively different, indicating that tropomyosin affected the conformational equilibrium of caldesmon binding. Simultaneous dual-sited attachment of domain 4b to actin is enabled by the conformational properties of the site-spanning sequence common to 658C, Cg1, and LW30 as reflected in the corresponding NOE and other NMR spectral parameters. A backbone turn region ((713)Gly-Asp-Val-Ser(716)) preceded by an extended segment (Ser(702)-Pro-Ala-Pro-Lys-Pro) acts to constrain the relative disposition of the flanking actin contact sites of domain 4b. In tests with a library of actin peptides, only the C-terminus, 350-375, bound to 658C and LW30. The use of Cu(2+) as a paramagnetic spectral probe bound to the unique His-371 provided evidence of a well-defined geometry for the complex between LW30 and actin residues 350-375 with the N-terminal, site B of domain 4b close to the C-terminal residues of actin. The data are discussed in the context of the potentiation of inhibitory activity by tropomyosin.     

Antifungal diterpenes from Hypoestes serpens (Acanthaceae)

Two new diterpenes, fusicoserpenol A and dolabeserpenoic acid A, with antifungal activity, were isolated from leaves of Hypoestes serpens (Acanthaceae). Their structures were elucidated by means of spectrometric methods including 1D and 2D NMR experiments and MS analysis. X-ray crystallographic analysis confirmed the structure of fusicoserpenol A and established the relative configuration.     

Signal pathway profiling of prostate cancer using reverse phase protein arrays

Reverse phase protein arrays represent a new proteomics microarray technology with which to study the fluctuating state of the proteome in minute quantities of cells. The activation status of cell signaling pathways controls cellular fate and deregulation of these pathways underpins carcinogenesis. Changes in pathway activation that occur between early stage prostatic epithelial lesions, prostatic stroma and the extracellular matrix can be analyzed by obtaining pure populations of cell types by laser capture microdissection (LCM) and analyzing the relative states of several key phosphorylation points within the cellular circuitry. We have applied reverse phase protein array technology to analyze the status of key points in cell signaling involved in pro-survival, mitogenic, apoptotic and growth regulation pathways in the progression from normal prostate epithelium to invasive prostate cancer. Using multiplexed reverse phase protein arrays coupled with LCM, the states of signaling changes during disease progression from prostate cancer study sets were analyzed. Focused analysis of phospho-specific endpoints revealed changes in cellular signaling events through disease progression and between patients. We have used a new protein array technology to study specific molecular pathways believed to be important in cell survival and progression from normal epithelium to invasive carcinoma directly from human tissue specimens. With the advent of molecular targeted therapeutics, the identification, characterization and monitoring of the signaling events within actual human biopsies will be critical for patient-tailored therapy.     

The Cdc14 phosphatase and the FEAR network control meiotic spindle disassembly and chromosome segregation

During meiosis, DNA replication is followed by two consecutive rounds of chromosome segregation. Cells lacking the protein phosphatase CDC14 or its regulators, SPO12 and SLK19, undergo only a single meiotic division, with some chromosomes segregating reductionally and others equationally. We find that this abnormal chromosome behavior is due to an uncoupling of meiotic events. Anaphase I spindle disassembly is delayed in cdc14-1, slk19Delta, or spo12Delta mutants, but the chromosome segregation cycle continues, so that both meiotic chromosome segregation phases take place on the persisting meiosis I spindle. Our results show that Cdc14, Slk19, and Spo12 are not only required for meiosis I spindle disassembly but also play a pivotal role in establishing two consecutive chromosome segregation phases, a key feature of the meiotic cell cycle.     

GENETIC VARIABILITY AND POTENTIAL SOURCES OF GRATELOUPIA DORYPHORA (HALYMENIACEAE,RHODOPHYTA), AN INVASIVE SPECIES IN RHODE ISLAND WATERS (USA)1

Grateloupia doryphora (Montagne) M.Howe is an invasive foliose alga that was reported for the first time in Rhode Island, USA in 1997. The population has since increased in size and expanded in range. In this study, the genetic variation and potential sources of the Rhode Island G. doryphora population were examined using three types of molecular markers: randomly amplified polymorphic DNA (RAPD), nuclear internal transcribed spacer (ITS) sequences, and mitochondrial cox2–cox3 intergenic spacer (COX) sequences. No variation was detected in ITS or COX sequences among Rhode Island G. doryphora individuals. RAPDs, however, did reveal genetic variation, although banding patterns were similar, with RAPD genetic distances between individuals ranging from 0.00 to 0.17. The low level of genetic diversity observed within the Rhode Island population may be due to a small founder population or a founder population derived from a genetically uniform source. To identify possible sources of the Rhode Island invasion, individuals from nine geographically diverse populations of foliose Grateloupia were compared. Phylogenetic trees inferred from RAPD distances and ITS and COX sequences had similar topologies; thus there was phylogenetic congruence among these independent loci. The Rhode Island G. doryphora specimens were genetically similar to specimens from G. doryphora populations located in Portsmouth, England; Tholen Island, The Netherlands; and Brittany and Hérault, France. Interestingly, the G. doryphora population in each of these locations is itself due to an introduction event within the past 40 years.     

Von Hippel-Lindau disease: identification of deletion mutations by pulsed-field gel electrophoresis

Von Hippel-Lindau disease (VHL) is an inherited multisystem neoplastic disorder. We prepared a 2.5-megabase (Mb) restriction map of the region surrounding the VHL gene and identified and characterized overlapping deletions in three unrelated patients affected with VHL. The smallest nested deletion (100 kb) was located within a 510-kb NruI fragment detected by 19–63. The rearrangements detected will be useful in isolating and evaluating candidate cDNAs for the VHL gene. The detailed physical map will be useful in studying the organization and structure of genes in the VHL region.