Life-history correlates of the evolution of live bearing in fishes

Selection for live bearing is thought to occur when the benefits of increasing offspring survival exceed the costs of reduced fecundity, mobility and the increased metabolic demands of carrying offspring throughout development. We present evidence that live bearing has evolved from egg laying 12 times in teleost (bony) fishes, bringing the total number of transitions to 21 to 22 times in all fishes, including elasmobranchs (sharks and rays). Live bearers produce larger offspring than egg layers in all of 13 independent comparisons for which data were available. However, contrary to our expectation there has not been a consistent reduction in fecundity; live bearers have fewer offspring in seven out of the 11 available comparisons. It was predicted that live bearers would have a larger body size, as this facilitates accommodation of developing offspring. This prediction was upheld in 14 out of 20 comparisons. However, this trend was driven by elasmobranchs, with large live bearers in seven out of eight comparisons. Thus, while the evolution of live bearing in elasmobranchs is correlated with predicted increases in offspring size and adult size, teleost live bearers do not have such a consistent suite of life-history correlates. This suggests that constraints or selection pressures on associated life histories may differ in live-bearing elasmobranchs and teleost fishes.     

Blood flow dynamics after photodynamic therapy with verteporfin in the RIF-1 tumor

In the present study, the effects of photodynamic therapy (PDT) with verteporfin on tumor blood flow and tumor regrowth were compared as verteporfin distributed in different compartments within the RIF-1 tumor. Tissue distribution of verteporfin was examined by fluorescence microscopy, and blood flow measurements were taken with a laser Doppler system. It was found that, at 15 min after drug administration, when verteporfin was mainly confined within the vasculature, PDT induced a complete arrest of blood flow by 6 h after treatment. PDT treatment at a longer drug-light interval (3 h), which allowed the drug to diffuse to the tumor interstitium, caused significantly less flow decrease, only to 50% of the initial flow in 6 h. A histological study and Hoechst 33342 staining of functional tumor vasculature confirmed the primary vascular damage and the decrease in tumor perfusion. The regrowth rate of tumors treated with 15-min interval PDT was 64% of that of the control group. However, when tumors were treated with 3-h interval PDT, the regrowth rate was not significantly different from that of the control, indicating that only the 15-min interval PDT caused serious damage to the tumor vascular bed. These results support the hypothesis that temporal pharmacokinetic changes in the distribution of the photosensitizer between the tumor parenchyma and blood vessels can significantly alter the tumor target of PDT.     

Progress towards single-molecule DNA sequencing: a one color demonstration

Single molecules of fluorescently labeled nucleotides were detected during the cleavage of individual DNA fragments by a processive exonuclease. In these experiments, multiple (10-100) strands of DNA with tetramethyl rhodamine labeled dUMP (TMR-dUMP) incorporated into the sequence were anchored in flow upstream of the detection region of an ultra sensitive flow cytometer. A dilute solution of Exonuclease I passed over the microspheres. When an exonuclease attached to a strand, processive digestion of that strand began. The liberated, labeled bases flowed through the detection region and were detected at high efficiency at the single-molecule level by laser-induced fluorescence. The digestion of a single strand of DNA by a single exonuclease was discernable in these experiments. This result demonstrates the feasibility of single-molecule DNA sequencing. In addition, these experiments point to a new and practical means of arriving at a consensus sequence by individually reading out identical sequences on multiple fragments.     

Comparative study of the relationship between monomer structure and reactivity for two polyhydroxyalkanoate synthases

Using organically synthesized hydroxyalkanoate coenzyme A thioesters, the activities of two short-chain polyhydroxalkanoate (PHA) synthases were investigated--Ralstonia eutropha PHA synthase (a type I PHA synthase) and Ectothiorhodospira shaposhnikovii PHA synthase (a type III synthase). The results indicate that the two synthases have similar activities towards most of the monomers tested. 3-Hydroxybutyryl CoA was found to be the most efficient substrate for both synthases. Changes in the side-chain length of the monomers affect monomer reactivity, with shortening of the side-chain length having the more severe effect. Hydrophobicity in the side chain appears to play an important role in the catalytic reaction. The configuration and the position of the hydroxyl group also affect the reactivity of a monomer. Monomers with the [S] configuration can not be recognized by either synthase. Moving the hydroxyl group from the beta carbon to the alpha carbon has a much more severe effect on the reactivity of the monomer than moving the hydroxyl group to the gamma carbon. The results demonstrate that the in vitro system can be used to prepare entirely novel polymers that may not be obtainable from living cells because of metabolic restrictions.     

Microgravity culture reduces apoptosis and increases the differentiation of a human colorectal carcinoma cell line

Our hypothesis is that rotation increases apoptosis in standard tissue culture medium at shear stresses of greater than approximately 0.3 dyn/cm2. Human MIP-101 poorly differentiated colorectal carcinoma cells were cultured for 6 d in complete medium in monolayers, on Teflon-coated nonadherent surfaces (static three-dimensional [3D]) or in rotating 3D cultures either in microgravity in low-earth orbit (3D microg) or in unit gravity on the ground (3D 1g). Apoptosis (determined morphologically), proliferation (by MIB1 staining), and the expression of epidermal growth-factor receptor (EGF-R), TGF-alpha, or TGF-beta were assessed by immunohistochemistry, while the expression of the differentiation marker carcinoembryonic antigen (CEA) was assessed on Western blots. Over the course of 6 d, static 3D cultures displayed the highest rates of proliferation and lowest apoptosis. This was associated with high EGF-R, TGF-alpha, and TGF-beta expression which was greater than that of a monolayer culture. Both rotated 3D lg and 3D microg cultures displayed lower expression of EGF-R, TGF-alpha, or TGF-beta and proliferation than that of monolayer or static 3D cultures. However, rotated 3D microg displayed significantly less apoptosis and greater CEA expression than rotated 3D 1g cultures. When rotated cultures of MIP-101 cells were grown uncler static conditions for another 3 d, proliferation increased and apoptosis decreased. Thus, rotation appears to increase apoptosis and decrease proliferation, whereas static 3D cultures in either unit or microgravity have less apoptosis, and reduced rotation in microgravity increases CEA expression.