Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis

Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.     

Guanylyl cyclases in unicellular organisms

Guanylyl cyclases in eukaryotic unicells were biochemically investigated in the ciliates Paramecium and Tetrahymena, in the malaria parasite Plasmodium and in the ameboid Dictyostelium. In ciliates guanylyl cyclase activity is calcium-regulated suggesting a structural kinship to similarly regulated membrane-bound guanylyl cyclases in vertebrates. Yet, cloning of ciliate guanylyl cyclases revealed a novel combination of known modular building blocks. Two cyclase homology domains are inversely arranged in a topology of mammalian adenylyl cyclases, containing two cassettes of six transmembrane spans. In addition the protozoan guanylyl cyclases contain an N-terminal P-type ATPase-like domain. Sequence comparisons indicate a compromised ATPase function. The adopted novel function remains enigmatic to date. The topology of the guanylyl cyclase domain in all protozoans investigated is identical. A recently identified Dictyostelium guanylyl cyclase lacks the N-terminal P-type ATPase domain. The close functional relation of Paramecium guanylyl cyclases to mammalian adenylyl cyclases has been established by heterologous expression, respective point mutations and a series of active mammalian adenylyl cyclase/Paramecium guanylyl cyclase chimeras. The unique structure of protozoan guanylyl cyclases suggests that unexpectedly they do not share a common guanylyl cyclase ancestor with their vertebrate congeners but probably originated from an ancestral mammalian-type adenylyl cyclase.     

The radiation of the Cape flora, southern Africa

The flora of the south-western tip of southern Africa, the Cape flora, with some 9000 species in an area of 90,000 km2 is much more speciose than can be expected from its area or latitude, and is comparable to that expected from the most diverse equatorial areas. The endemism of almost 70%, on the other hand, is comparable to that found on islands. This high endemism is accounted for by the ecological and geographical isolation of the Cape Floristic Region, but explanations for the high species richness are not so easily found. The high species richness is accentuated when its taxonomic distribution is investigated: almost half of the total species richness of the area is accounted for by 33 'Cape floral clades'. These are clades which may have initially diversified in the region, and of which at least half the species are still found in the Cape Floristic Region. Such a high contribution by a very small number of clades is typical of island floras, not of mainland floras. The start of the radiation of these clades has been dated by molecular clock techniques to between 18 million years ago (Mya) (Pelargonium) and 8 Mya (Phylica), but only six radiations have been dated to date. The fossil evidence for the dating of the radiation is shown to be largely speculative. The Cenozoic environmental history of southern Africa is reviewed in search of possible triggers for the radiations, climatic changes emerge as the most likely candidate. Due to a very poor fossil record, the climatic history has to be inferred from larger scale patterns, these suggest large-scale fluctuations between summer wet (Palaeocene, Early Miocene) and summer dry climates (Oligocene, Middle Miocene to present). The massive speciation in the Cape flora might be accounted for by the diverse limitations to gene flow (dissected landscapes, pollinator specialisation, long flowering times allowing much phenological specialisation), as well as a richly complex environment providing a diversity of selective forces (geographically variable climate, much altitude variation, different soil types, rocky terrain providing many micro-niches, and regular fires providing both intermediate disturbances, as well as different ways of surviving the fires). However, much of this is based on correlation, and there is a great need for (a) experimental testing of the proposed speciation mechanisms, (b) more molecular clock estimates of the age and pattern of the radiations, and (c) more fossil evidence bearing on the past climates.     

Mouse nomenclature and maintenance of genetically engineered mice

Modern genetic engineering technologies enable us to manipulate the mouse genome in increasingly complex ways to model human biology and disease. As a result, the number of mouse strains carrying transgenes or induced mutations has increased markedly. Thorough understanding of strain and gene nomenclature is essential to ensure that investigators know what kind of mouse they have, and what to expect in terms of phenotype. Genetically engineered mice alter gene function by over-expressing, eliminating, or modifying a gene product. The resulting phenotype is often unexpected and not completely understood, necessitating special care and potentially complex breeding and husbandry strategies. Animal care technicians responsible for routine maintenance of the colony, facility managers, veterinarians, and research personnel working with mice should be well informed about the nature of the mutation, distinguishing characteristics, and necessary precautions in handling the mice. Personnel working with mice also must be aware of the multitude of factors intrinsic to the mouse and present in the environment that can influence reproductive performance. Finally, diligent adherence to the maintenance of genetic quality in conjunction with cryopreservation of germplasm is the best insurance against loss of a colony.     

A guanylyl cyclase from Paramecium with 22 transmembrane spans. Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.     

Increased apoptosis and increased clonogenic survival of 12V-H-ras transformed rat fibroblasts in response to cisplatin

Mutationally activated Ras is involved in tumor progression and likely also in drug resistance. Using survival, viability and apoptosis assays, we have here compared the cisplatin sensitivities of FR3T3 rat fibroblasts and a 12V-H-ras transformed subline (Ras2:3). Around 24 h after cisplatin treatment Ras2:3 cells showed higher apoptosis levels and lower viability than FR3T3. This increased sensitivity correlated with weaker cisplatin-induced activation of Jun N-terminal kinase (JNK). In contrast to apoptosis assays, colony formation assays showed that Ras2:3 were more resistant to cisplatin than were FR3T3. This was partly due to the increased cisplatin sensitivity of FR3T3 seeded at low densities, as required in colony formation assays. In addition, Ras2:3 cisplatin survivors had a higher relative proliferative capacity. Cell cycle analyses showed that FR3T3 cells initially responded with a dose-dependent G2 arrest, while Ras2:3 accumulated in S-phase. Experiments with an anti-apoptotic mutant of MEKK1 suggested that the apoptotic response of Ras2:3 cells is not specific to the S-phase fraction. In summary, the cisplatin response of ras-transformed fibroblasts is distinct from that of parental cells, in that they show increased apoptosis, a different cell cycle response and increased post-treatment proliferative capacity. The results illustrate the need to carefully consider methods and protocols for in vitro studies on chemotherapy sensitivity.     

RGS4 binds to membranes through an amphipathic alpha -helix

RGS4, a mammalian GTPase-activating protein for G protein alpha subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584-5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic alpha-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an alpha-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an alpha-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal alpha-helix.     

A universal molecular method for identifying underground plant parts to species

As part of a large project to determine rooting depth and resource uptake on the Edwards Plateau of central Texas, we developed a DNA-based technique that allows the below-ground parts of all plants to be identified to the level of genus and usually to species. Identification is achieved by comparing DNA sequences of the internal transcribed spacer (ITS) region of the 18S-26S nuclear ribosomal DNA repeat, derived from below-ground plant material, with a reference ITS region database for plants at a site. The method works throughout plants because the plant ITS region can be PCR amplified using a set of universal primers. Congeneric species can usually be identified because the ITS region evolves relatively rapidly. In our study, all roots were easily identified to the level of genus; most congeneric species were identified solely by ITS sequence differences but some required a combination of ITS sequence data and above-ground surveys of species at a site. In addition to showing the feasibility and efficacy of our technique, we compare it with another DNA-based technique used to identify below-ground plant parts. Finally, we also describe a DNA extraction and purification technique that reliably provides high-quality DNA of sufficient quantity from roots so that PCR can be readily accomplished. Our technique should allow the below-ground parts of plants in any system to be identified and thereby open new possibilities for the study of below-ground plant communities.     

Development of an anti-vitellin ELISA for the assessment of reproduction in the ridgeback shrimp, Sicyonia ingentis

To investigate the reproductive regulation of the ridgeback shrimp, Sicyonia ingentis, vitellin (Vn) synthesis was studied. Using gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Vn was found to have a molecular mass of 322 kDa and to be composed of three subunits of 182, 91 and 85 kDa. Purified Vn was used to prepare anti-Vn antiserum, which was used to develop an enzyme-linked immunosorbent assay (ELISA) with a dynamic range of 0.3-300 ng. The ELISA was used to measure hemolymph levels of yolk proteins. The mean hemolymph concentrations in fresh caught animals ranged from 256 (+/-36.6 S.E.M.) to 1073 (+/-87.6 S.E.M.) mg/ml in stage 2 and 4 animals, respectively. The ELISA was also used to determine the effects of steroid hormone injections in adult non-reproductive female shrimp. One milligram injections of progesterone, 17alpha-hydroxyprogesterone or 17beta-estradiol were administered for three consecutive days to individual females. There were no changes in hemolymph vitellogenin levels during the successive 7-day period following the first injection of any steroid.     

Milk ceruloplasmin and its expression by mammary gland and liver in pigs

Concentrations of ceruloplasmin and copper in milk and blood plasma, the nature of milk ceruloplasmin, and the effects of lactation and gestation on these parameters, as well as the expression of ceruloplasmin mRNA by the mammary gland, were examined in pigs. As seen previously in humans, ceruloplasmin and copper concentrations in sow milk were much higher a few days after birth than 1 month later, averaging 26.5 and 6.6 mg ceruloplasmin/L (by immunoassay) and 1.67 and 0.34 mg total Cu/L, on days 3 and 33 postpartum, respectively. Values for ceruloplasmin oxidase activity (measured with p-phenylene diamine) were 7.8 and 1.3 nmol/min/L, respectively. Daily milk ceruloplasmin production went from 61 to 22 mg/day and daily copper output from 38 to 12 mg/day. In contrast, there was little or no variation in serum ceruloplasmin concentration during lactation or gestation, although total plasma copper was high at the end of gestation. Milk ceruloplasmin was of the same apparent size as serum ceruloplasmin, as determined by SDS-PAGE and immunoblotting, and ceruloplasmin mRNAs of liver and mammary gland were indistinguishable by Northern analysis and RT-PCR of the various exons. Expression of total RNA and ceruloplasmin mRNA, as detected in biopsies of mammary gland, increased markedly upon onset of lactation and then declined during the next month in conjunction with a drop in milk ceruloplasmin production. The results indicate that milk ceruloplasmin, while being the same protein as in plasma, is not derived from the plasma but is produced by the mammary gland.