Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication.

The apoptotic program incorporates a paracrine component of importance in fostering tissue repair at sites of apoptotic cell deletion. As this paracrine pathway likely bears special importance in maladaptive intercellular communication leading to vascular remodeling, we aimed at further defining the mediators produced by apoptotic endothelial cells (EC), using comparative and functional proteomics. Apoptotic EC were found to release nanovesicles displaying ultrastructural characteristics, protein markers and functional activity that differed from apoptotic blebs. Tumor susceptibility gene 101 and translationally controlled tumor protein (TCTP) were identified in nanovesicle fractions purified from medium conditioned by apoptotic EC and absent from purified apoptotic blebs. Immunogold labeling identified TCTP on the surface of nanovesicles purified from medium conditioned by apoptotic EC and within multivesicular blebs in apoptotic EC. These nanovesicles induced an extracellular signal-regulated kinases 1/2 (ERK 1/2)-dependent antiapoptotic phenotype in vascular smooth muscle cells (VSMC), whereas apoptotic blebs did not display antiapoptotic activity on VSMC. Caspase-3 biochemical inhibition and caspase-3 RNA interference in EC submitted to a proapoptotic stimulus inhibited the release of nanovesicles. Also, TCTP siRNAs in EC attenuated the antiapoptotic activity of purified nanovesicles on VSMC. Collectively, these results identify TCTP-bearing nanovesicles as a novel component of the paracrine apoptotic program of potential importance in vascular repair.

Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis.

Apoptosis of endothelial cells (ECs) is an early pathogenic event in various fibrotic diseases. In this study, we evaluated whether paracrine mediators produced by apoptotic ECs play direct roles in fibrogenesis. C3H mice injected subcutaneously with serum-free medium conditioned by apoptotic ECs (SSC) showed increased skin thickness and heightened protein levels of alpha-smooth-muscle actin (alphaSMA), vimentin and collagen I as compared with mice injected with medium conditioned by non-apoptotic ECs. Fibroblasts exposed to SSC in vitro showed cardinal features of myofibroblast differentiation with increased stress fiber formation and expression of alphaSMA. Caspase-3 silencing in ECs prevented the release of mediators favoring myofibroblast differentiation. To identify the fibrogenic factor(s) released by ECs, the protein contents of media conditioned by either apoptotic or non-apoptotic ECs were compared using SDS-PAGE-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) and two-dimensional LC-MS/MS. Connective tissue growth factor (CTGF) was the only fibrogenic protein found increased in SSC. Pan-caspase inhibition with ZVAD-FMK or caspase-3 silencing in ECs confirmed that CTGF was released downstream of caspase-3 activation. The fibrogenic signaling signatures of SSC and CTGF on fibroblasts in vitro were similarly Pyk2-, Src-family kinases- and PI3K dependent, but TGF-beta-independent. CTGF-immunodepleted SSC failed to induce myofibroblast differentiation in vitro and skin fibrosis in vivo. These results identify caspase-3 activation in ECs as a novel inducer of CTGF release and fibrogenesis.

RNase H overproduction corrects a defect at the level of transcription elongation during rRNA synthesis in the absence of DNA topoisomerase I in Escherichia coli.

It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation.

The AIDS pandemic: changes in the believability and use of information sources.

Over 20,000 Americans under 25 years old are infected with HIV every year. In 1994, AIDS was the number one killer of adults between the ages of 25 and 44 years old. Education concerning HIV is critical to the prevention of AIDS. Both the public and private sectors, including government agencies, cause-related organizations, medical professionals, and promotion specialists must determine the best information sources to educate young people. However, with many television shows that appeal to young adults condoning casual sex, it is increasingly difficult to generate motivation to act responsibly. This paper examines the effectiveness of information sources, sponsored by both the government and private organizations, about HIV. Given the impact of media publicity regarding the AIDS virus and the "hard-hitting" messages and public service announcements about AIDS, implications about ethical issues, public policy issues, and media strategies to educate the public about AIDS are discussed. While publicity and specific types of television programming were found to be the most believable and most often used information sources for health issues, there are many strategic implications concerning promotion strategies, targeting, and positioning for both the public and private sectors.

Roles of DNA topoisomerases in the regulation of R-loop formation in vitro.

We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation. Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level. In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process. By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase. When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation. This result suggests that more stable R-loops are being produced in this orientation. Our results also suggest that DNA gyrase can participate in the process of R-loop elongation. The strong transcription-induced relaxing activity of E. coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping. In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.

The physical properties of blood--forensic considerations.

An informed understanding and appreciation of blood related crime scenes is only achieved through practical experimentation of simulated blood spatter. Because of the increasing frequency of the HIV, Hepatitis B and C viruses, the use of human blood may be hazardous for those involved in such experimentation. Porcine (pig) blood has been cited by some as a reasonable alternative, and this paper presents a comparison of the physical parameters (surface tension, viscosity and density) of ageing pig and fresh human blood, under a variety of conditions, as well as measurements of droplet trajectories and stain patterns. The results support the use of porcine blood (up to two weeks old) in representing the behaviour of freshly spilt human blood.

Oscillating blood droplets--implications for crime scene reconstruction.

Traditionally, the analysis of blood spatter on surfaces in the reconstruction of crime scenes relies on the assumption that blood droplets are spherical when they strike the surface. This paper explores the effects of their shape on the reconstruction of trajectories from their impact pattern, and reports a theoretical analysis of the lifetime of droplet oscillations. Oscillations damp quickly in blood droplets due to the viscosity. The analysis provides ranges of velocities and distances from the point of droplet projection within which it is unreliable to assume the droplets are spherical when they stain a surface. Non-spherical droplet stains predict incorrect positioning of the droplet projection point. Experimental data are presented to show that the estimates apply in practice.

Evolutionary conservation of ten microsatellite loci in four species of Felidae.

Short tandem repeat polymorphisms (STRP), or microsatellites, are widespread among vertebrate genomes and are useful in gene mapping and population studies due to a high level of length polymorphism. We describe here the isolation, characterization, and PCR amplification of 10 microsatellite loci from the domestic cat, Felis catus. The flanking primer sequences were conserved among other Felidae species, and amplification products demonstrated abundant polymorphism in puma, lion, cheetah, and domestic cat. The cheetah sample exhibited the lowest level of polymorphism for these loci among felid species.

Reconnection of severed nerve axons with polyethylene glycol.

Severed medial giant axons in crayfish can be rejoined in vitro with polyethylene glycol (PEG) to produce axoplasmic continuity and through transmission of action potentials. Severed axon-like processes of a mammalian neuroblastoma/glioma cell line seem to be rejoined to the cell body using PEG in tissue culture. Our data suggest that PEG might be used to rejoin severed axons in vivo in various organisms.

Haptoglobin typing in Zimbabwe.

The value of haptoglobins as a method of typing negroid blood in Zimbabwe has been evaluated. three hundred sixty-nine blood samples from negroid people were examined using gradient polyacrylamide gel electrophoresis. The method provides good differentiation of all phenotypes including the 2-1M variant. In addition data on haptoglobin types of animals, both domestic and wild, are presented.