Homeobox Genes, Retinoic Acid and the Development and Evolution of Dual Body Plans in the Ascidian Herdmania curvata

Ascidians, along with other urochordates, are the most evolutionarydistant group from vertebrates to display definitive chordate-specificcharacters, such as a notochord, dorsal hollow nerve cord, pharynxand endostyle. Most solitary ascidians have a biphasic lifehistory that has partitioned the development of these charactersbetween a planktonic microscopic tadpole larva (notochord anddorsal nerve cord) and a larger sessile adult (pharynx and endostyle).Very little is known of the molecular axial patterning processesoperating during ascidian postlarval development. Two axialpatterning homeobox genes Otx and Cdx are expressed in a spatiallyrestricted manner along the ascidian anteroposterior axis duringembryogenesis and postlarval development (i.e., metamorphosis).Comparisons of these patterns with those of homologous cephalochordateand vertebrate genes suggest that the novel ascidian biphasicbody plan was not accompanied by a deployment of these genesinto new pathways but by a heterochronic shift in tissue-specificexpression. Studies examining the role of all-trans retinoicacid (RA) in axial patterning in chordates also contribute toour understanding of the role of homeobox genes in the developmentof larval and adult ascidian body plans. Our studies demonstratethat RA does not regulate axial patterning in the developingascidian larval neuroaxis in a manner homologous to that foundin vertebrates. Although RA may regulate the expression of someascidian homeobox genes, ectopic application of RA does notappear to alter the morphology of the larval CNS. However, treatmentwith similar or lower concentrations of RA, have a profoundeffect on postlarval development and the juvenile body plan.These changes are correlated to a dramatic reduction of Otxexpression. Through these RA-induced effects we infer that whileRA may regulate the expression of some homeobox genes duringembryogenesis it has a far more dramatic impact on postlarvaldevelopment where regulative processes predominate.     

Isolation of brain tubulin by affinity chromatography

An affinity chromatographic procedure for the isolation of tubulin from brain is described. The yield is good and the method rapid and gentle. Criteria of purity are colchicine binding, SDS acrylamide gel electrophoresis, and velocity sedimentation.     

The distance dependence prediction of the Janzen-Connell hypothesis: a meta-analysis

The Janzen-Connell hypothesis explains the maintenance of tropical diversity through the interacting effects of parent-centered dispersal patterns and distance- and density-dependent propagule survival. These effects were thought to support regular spacing of species within tropical forest, enhancing diversity. One of the predictions of the hypothesis is that seed and seedling survival should improve with increased parental distance. Although there are many independent tests of this hypothesis for individual species, there are few synthetic studies that have brought these data together to test its validity across species. This paper reports the results of a meta-analysis of the effect of distance on enhancing propagule survival, employing an odds-ratio effect size metric. We found no general support for the distance-dependent prediction of the hypothesis, and conclude that further testing to explore this hypothesis as a diversity-maintaining mechanism is unnecessary. However, we did find that distance from parent slightly reduces survivorship in the temperate zone, as contrasted with the tropics, and we saw stronger evidence in support of the hypothesis for seedlings than for seeds. The phenomenon of enhanced propagule survival with distance from the parent may be important for the population biology of particular species, but it is not a general phenomenon across communities, life history stages or life forms.     

Photomonomerization of pyrimidine dimers by indoles and proteins.

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wave-lengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases.Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the ¹⁴C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed.Although indoles are able to split the dimers in RNA, they fail to photo-reactivate u.v.-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactivate viral RNA when irradiated at 313 nm, possibly because of side reactions.     

Systems for Maintenance of Urinary Sterility After Prostatectomy

Closed drainage is recommended for all patients after prostatectomy where hemostasis has been adequate. Although closed drainage can maintain sterility of the bladder, thereby fostering healing and reducing infectious complications, such drainage is not insisted upon at most hospitals because of the inconveniences associated with it. However, when closed drainage was used in 25 consecutive cases of transurethral resection, infection was reduced to 25 per cent (in contrast to the 85 to 100 per cent encountered with open drainage).The ideal closed system should incorporate:1. Fixed tubing to prevent contamination where the catheter joins the tubing and where the tubing is attached to the container;2. An aseptic method of emptying;3. A device to prevent reflux of the potentially contaminated urine in the container into the bladder;4. Free urinary flow from bladder to container; and5. Portability for the patient and convenience for the staff.A system is proposed that incorporates these features. Particularly effective are a fixed drip chamber with vents at the site of attachment of the tubing to the bag and a protected spigot for emptying.     

Detection of Biosignatures by Geomatrix-Assisted Laser Desorption/Ionization (GALDI) Mass Spectrometry

Identification of mineral-associated biosignatures is of significance for retrieving biochemical information from geological records here on Earth and for detecting signs of life on other planets, such as Mars. An investigation using laser desorption Fourier transform mass spectrometry was conducted to determine whether geomatrix-assisted laser desorption/ionization (GALDI) can be used to detect amino acids (e.g., histidine, threonine, and cysteine) and small proteins (e.g., gramicidin S) associated with mineral phases and whether the geomatrix impacts detection. Iron oxide (Fe₂ O ₃ ) and sodium chloride (NaCl) were investigated as clean chemical analogues of hematite and halite, respectively, which have both been detected on the surface of Mars. Samples were prepared by 2 methods: (1) application of analyte solution to the geomatrix surface with subsequent drying; and (2) physical mixing of analyte and geomatrix. Amino acids incorporated within NaCl by physical mixing yielded a better signal-to-noise ratio than those that were applied to the surface of a NaCl pellet. The composition of the geomatrix had an influence on the detection of biomolecules. Peaks corresponding to the cation-attached biomolecular ions were observed for the NaCl prepared samples. However, no biomolecular ion species were observed in samples using Fe ₂ O ₃ as geomatrix. Instead, only minor peaks that may correspond to ions derived from fragments of the biomolecules were obtained.     

Hemps, a novel EGF-like protein, plays a central role in ascidian metamorphosis

All chordates share several characteristic features including a dorsal hollow neural tube, a notochord, a pharynx and an endostyle. Unlike other chordate taxa, ascidians have a biphasic life-history with two distinct body plans. During metamorphosis, the larval nerve cord and notochord degenerate and the pharyngeal gill slits and endostyle form. While ascidians, like other marine invertebrates, metamorphose in response to specific environmental cues, it remains unclear how these cues trigger metamorphosis. We have identified a novel gene (Hemps) which encodes a protein with a putative secretion signal sequence and four epidermal growth factor (EGF)-like repeats which is a key regulator of metamorphosis in the ascidian Herdmania curvata. Expression of Hemps increases markedly when the swimming tadpole larva becomes competent to undergo metamorphosis and then during the first 24 hours of metamorphosis. The Hemps protein is localised to the larval papillae and anterior epidermis of the larva in the region known to be required for metamorphosis. When the larva contacts an inductive cue the protein is released, spreading posteriorly and into the tunic as metamorphosis progresses. Metamorphosis is blocked by incubating larvae in anti-Hemps antibodies prior to the addition of the cue. Addition of recombinant Hemps protein to competent larvae induces metamorphosis in a concentration-dependent manner. A subgroup of genes are specifically induced during this process. These results demonstrate that the Hemps protein is a key regulator of ascidian metamorphosis and is distinct from previously described inducers of this process in terrestrial arthropods and aquatic vertebrates.     

Age-dependent myelin degeneration and proteolysis of oligodendrocyte proteins is associated with the activation of calpain-1 in the rhesus monkey

Myelin provides important insulating properties to axons allowing for propagation of action potentials over large distances at high velocity. Disruption of the myelin sheath could therefore contribute to cognitive impairment, such as that observed during the normal aging process. In the present study, age-related changes in myelin, myelin proteins and oligodendrocyte proteins were assessed in relationship to calpain-1 expression and cognition in the rhesus monkey. Isolation of myelin fractions from brain white matter revealed that as the content of the intact myelin fraction decreased with age, there was a corresponding increase in the floating or degraded myelin fraction, suggesting an increased breakdown of intact myelin with age. Of the myelin proteins examined, only the myelin-associated glycoprotein decreased with age. Levels of the oligodendrocyte-specific proteins 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin/oligodendrocyte-specific protein (MOSP) increased dramatically in white matter homogenates and myelin with age. Age-related increases in degraded CNPase also were demonstrable in white matter in association with increases in activated calpain-1. Degraded CNPase was also detectable in myelin fractions, with only the floating fraction containing activated calpain-1. The increases in the activated enzyme in white matter were much greater than those found in myelin fractions suggesting a source other than the myelin membrane for the marked overexpression of activated calpain-1 with age. In addition, CNPase was demonstrated to be a substrate for calpain in vitro. In summary, changes in myelin and oligodendrocyte proteins occur with age, and they appear to have a significant relationship to cognitive impairment. The overexpression of CNPase and MOSP suggests new formation of myelin by oligodendrocytes, which may occur in response to myelin degradation and injury caused by proteolytic enzymes such as calpain.