Keratocan-deficient mice display alterations in corneal structure

Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera-/- cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera-/- cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera-/- mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision.     

Phylogeography of the pantropical sea urchin Tripneustes: contrasting patterns of population structure between oceans

To understand how allopatric speciation proceeds, we need information on barriers to gene flow, their antiquity, and their efficacy. For marine organisms with planktonic larvae, much of this information can only be obtained through the determination of divergence between populations. We evaluated the importance of ocean barriers by studying the mitochondrial DNA phylogeography of Tripneustes, a pantropical genus of shallow water sea urchin. A region of cytochrome oxidase I (COI) was sequenced in 187 individuals from locations around the globe. The COI phylogeny agreed with a previously published phylogeny of bindin that barriers important to the evolution of Tripneustes are: (1) the cold water upwelling close to the tip of South Africa, (2) the Isthmus of Panama, (3) the long stretch of deep water separating the eastern from the western Atlantic, and (4) the freshwater plume of the Orinoco and the Amazon rivers between the Caribbean and the coast of Brazil. These barriers have previously been shown to be important in at least a subset of the shallow water marine organisms in which phylogeography has been studied. In contrast, the Eastern Pacific Barrier, 5000 km of deep water between the central and the eastern Pacific that has caused the deepest splits in other genera of sea urchins, is remarkably unimportant as a cause of genetic subdivision in Tripneustes. There is also no discernible subdivision between the Pacific and Indian Ocean populations of this genus. The most common COI haplotype is found in the eastern, central, and western Pacific as well as the Indian Ocean. Morphology, COI, and bindin data agree that T. depressus from the eastern Pacific and T. gratilla from the western Pacific are, in fact, the same species. The distribution of haplotype differences in the Indo-Pacific exhibits characteristics expected from a sea urchin genus with ephemeral local populations, but with high fecundity, dispersal, and growth: there is little phylogenetic structure, and mismatch distributions conform to models of recent population expansion on a nearly global scale. Yet, comparisons between local populations produce large and significant F(ST) values, indicating nonrandom haplotype distribution. This apparent local differentiation is only weakly reflected in regional divergence, and there is no evidence of isolation by distance in correlations between F(ST) values and either geographical or current distance. Thus, Tripneustes in the Indo-Pacific (but not in the Atlantic) seems to be one large metapopulation spanning two oceans and containing chaotic, nonequilibrium local variation, produced by the haphazard arrival of larvae or by unpredictable local extinction.     

Heterogeneous selection at specific loci in natural environments in Arabidopsis thaliana

Genetic variation for quantitative traits is often greater than that expected to be maintained by mutation in the face of purifying natural selection. One possible explanation for this observed variation is the action of heterogeneous natural selection in the wild. Here we report that selection on quantitative trait loci (QTL) for fitness traits in the model plant species Arabidopsis thaliana differs among natural ecological settings and genetic backgrounds. At one QTL, the allele that enhanced the viability of fall-germinating seedlings in North Carolina reduced the fecundity of spring-germinating seedlings in Rhode Island. Several other QTL experienced strong directional selection, but only in one site and seasonal cohort. Thus, different loci were exposed to selection in different natural environments. Selection on allelic variation also depended upon the genetic background. The allelic fitness effects of two QTL reversed direction depending on the genotype at the other locus. Moreover, alternative alleles at each of these loci caused reversals in the allelic fitness effects of a QTL closely linked to TFL1, a candidate developmental gene displaying nucleotide sequence polymorphism consistent with balancing selection. Thus, both environmental heterogeneity and epistatic selection may maintain genetic variation for fitness in wild plant species.     

The RAVE complex is essential for stable assembly of the yeast V-ATPase

Vacuolar proton-translocating ATPases are composed of a peripheral complex, V(1), attached to an integral membrane complex, V(o). Association of the two complexes is essential for ATP-driven proton transport and is regulated post-translationally in response to glucose concentration. A new complex, RAVE, was recently isolated and implicated in glucose-dependent reassembly of V-ATPase complexes that had disassembled in response to glucose deprivation (Seol, J. H., Shevchenko, A., and Deshaies, R. J. (2001) Nat. Cell Biol. 3, 384-391). Here, we provide evidence supporting a role for RAVE in reassembly of the V-ATPase but also demonstrate an essential role in V-ATPase assembly under other conditions. The RAVE complex associates reversibly with V(1) complexes released from the membrane by glucose deprivation but binds constitutively to cytosolic V(1) sectors in a mutant lacking V(o) sectors. V-ATPase complexes from cells lacking RAVE subunits show serious structural and functional defects even in glucose-grown cells or in combination with a mutation that blocks disassembly of the V-ATPase. RAVE small middle dotV(1) interactions are specifically disrupted in cells lacking V(1) subunits E or G, suggesting a direct involvement for these subunits in interaction of the two complexes. Skp1p, a RAVE subunit involved in many different signal transduction pathways, binds stably to other RAVE subunits under conditions that alter RAVE small middle dotV(1) binding; thus, Skp1p recruitment to the RAVE complex does not appear to provide a signal for V-ATPase assembly.     

A new method for the study of velopharyngeal function using gated magnetic resonance imaging

The purpose of this project was to assess the feasibility of imaging the velopharynx of adult volunteers during repetitive speech, using gated magnetic resonance imaging (MRI). Although a number of investigators have used conventional MRI in the study of the human vocal tract, the mismatch between the lengthy time necessary to acquire sufficiently detailed images and the rapidity of movement of the vocal tract during speech has forced investigators to acquire images either while the subject is at rest or during sustained utterances. The technique used here acquired a portion of each image during repetitive utterances, building the full image over multiple utterance cycles. The velopharyngeal portal was imaged on a 1.5-Tesla GE Signa LX 8.2 platform with gated fast spoiled gradient echo protocol. An external 1-Hertz trigger was fed to the cardiac gate. Subjects synchronized utterance of consonant-vowel syllables to a flashing light synchronized with the external trigger. Each acquisition of 30 phases per second at a single-slice location took 22 to 29 seconds. Four consonant-vowel syllables (/pa/, /ma/, /sa/, and /ka/) were evaluated. Subjects vocalized throughout the acquisition, beginning 5 to 6 seconds beforehand to establish a regular rhythm. Imaging of the velopharyngeal portal was performed for sagittal, velopharyngeal axial (aligned perpendicular to the "knee" of the velum), axial, and coronal planes. Volumes were obtained by sequential acquisition of six to 10 slices (each with 30 phases) in the axial or sagittal planes during repetition of the /pa/ syllable. Spatiotemporal volumes of the single-slice data were sectioned to provide time-motion images (analogous to M-mode echocardiograms). Three-dimensional dynamic volume renderings of palate motion were displayed interactively (Vortex; CieMed, Singapore). A method suitable for the collection and visualization of four-dimensional information regarding monosyllabic speech using gated MRI was developed. These techniques were applied to a population of adult volunteer subjects with no history of speech problems and two patients with a history of cleft lip and palate. The techniques allowed good real-time visualization of velopharyngeal anatomy during its entire range of motion and was also able to image pathology-specific anatomic differences in the subjects with cleft lip and cleft palate. These methods may be applicable to a wide spectrum of problems in speech physiology research and for clinical decision-making regarding surgery for speech and outcomes analysis.     

A review of in vitro gamete maturation and embryo culture and potential impact on future animal biotechnology

This review considers the relationship of in vitro gamete maturation and embryo culture to the future development of animal biotechnology. The areas reviewed are oocyte maturation in vitro and embryo culture and their importance for successful in vitro embryo production. The rapidly developing area of spermatogonial cell transplantation and culture is also reviewed. The scientific milestones leading to the development of each area, the problems and prospects for future development and the possible significance of major advances in each area are discussed.     

Simultaneous reconstruction of the secondary bilateral cleft lip and nasal deformity: Abbé flap revisited

The purpose of this retrospective study was to review the method of using the Abbé flap for correction of secondary bilateral cleft lip deformity in selected patients with tight upper lip, short prolabium, lack of acceptable philtral column and Cupid's bow definition, central vermilion deficiency, irregular lip scars, and associated nasal deformity. A total of 39 patients with the bilateral cleft lip nasal deformity received Abbé flap and simultaneous nasal reconstruction during a period of 6 years. Mean patient age at the time of the operation was 19.1 years, and ranged from 6.6 to 38.5 years. The average follow-up period was 1.8 years. Fourteen patients had prior orthognathic operations. The Abbé flap was designed 13 to 14 mm in length and 8 to 9 mm in width and contained full-thickness tissue from the central lower lip, with a slightly narrow reverse-V caudal end. The prolabium, including the scars and central vermilion, was excised. Lengthening procedures of the upper lip segments were performed if vertical deficiency existed. Part of the prolabial skin was preserved and mobilized for columellar elongation, if indicated. Open rhinoplasty was carried out with or without cartilage graft for columella and nasal tip reconstruction. Reduction of the alar width and nostrils was achieved by a Z-plasty or excision of scar tissue at the nostril floor. The Abbé flap was then transposed cephalad, insetting into the median defect and sutured in layers. The results demonstrated no flap problems or perioperative complications. Seven patients needed further minor revisions on the nose and/or lip. Laser treatment was used to improve the lip scars in three patients. The patients were satisfied with the final outcome and found the lower lip scars acceptable. In conclusion, the described technique of Abbé flap and simultaneous rhinoplasty is an effective reconstructive method for select patients with bilateral cleft lip and nasal deformity.     

Role of sphingosine-1 phosphate in the enhancement of endothelial barrier integrity by platelet-released products

In vitro and in vivo evidence indicates that circulating platelets affect both vascular integrity and hemostasis. How platelets enhance the permeability barrier of the vascular endothelium is not well understood. We measured the effect of isolated human platelets on human pulmonary artery endothelial cell (EC) barrier integrity by monitoring transmonolayer electrical resistance. EC barrier function was significantly increased by the addition of platelets ( approximately 40% maximum, 2.5 x 106 platelets/ml). Platelet supernatants, derived from 2.5 x 106 platelets/ml, reproduced the barrier enhancement and reversed the barrier dysfunction produced by the edemagenic agonist thrombin, which implicates a soluble barrier-promoting factor. The barrier-enhancing effect of platelet supernatants was heat stable but was attenuated by either charcoal delipidation (suggesting a vasoactive lipid mediator) or pertussis toxin, implying involvement of a Gialpha-coupled receptor signal transduction pathway. Sphingosine-1-phosphate (S1P), a sphingolipid that is released from activated platelets, is known to ligate G protein-coupled EC differentiation gene (EDG) receptors, increase EC electrical resistance, and reorganize the actin cytoskeleton (Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, and English D. J Clin Invest 108: 689-701, 2001). Infection of EC with an adenoviral vector expressing an antisense oligonucleotide directed against EDG-1 but not infection with control vector attenuated the barrier-enhancing effect of both platelet supernatants and S1P. These results indicate that a major physiologically relevant vascular barrier-protective mediator produced by human platelets is S1P.     

Morphological Observations on Brevipalpus phoenicis (Acari: Tenuipalpidae) Including Comparisons with B. californicus and B. obovatus

The genus Brevipalpus has over 300 species worldwide. The three most important agricultural pest species in the genus, Brevipalpus californicus (Banks), B. obovatus Donnadieu, and B. phoenicis (Geijskes), have been consistently confused and misidentified for more than 50 years. The present study provides a discussion of the characters and character states used to separate these mites. Low-temperature scanning electron microscopy and traditional light microscopy techniques were used to illustrate the subtle morphological differences between these three species. Morphology of the dorsal propodosoma, opisthosoma, and leg chaetotaxy of all three species was examined and compared. The number of dorsal setae, the number of solenidia (omega) on tarsus II, and dorsal cuticular patterns were the most important characters in the identification of Brevipalpus species. B. phoenicis is similar to B. californicus in having two omega on tarsus leg II and different from B. obovatus which has only one omega on tarsus leg II and similar to B. obovatus in having only one pair of F setae (f3), but differing from B. californicus which has two pairs of F setae (f2-3). The dorsal opisthosomal and propodisomal cuticular patterns frequently used to distinguish between these three species are useful but one must be aware that age, feeding, and mounting techniques can affect the appearance of these characters.