Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps)

Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples.     

Phylogenetic relationships and host-plant evolution within the basal clade of Halictidae (Hymenoptera, Apoidea)

Bees are among the most important pollinators of angiosperm plants. Many bee species show narrow host‐plant preferences, reflected both in behavioral and morphological adaptations to particular attributes of host‐plant pollen or floral morphology. Whether bee host‐plant associations reflect co‐cladogenesis of bees and their host plants or host‐switches to unrelated host plants is not clear. Rophitinae is a basal subfamily of Halictidae in which most species show narrow host‐plant preferences (oligolecty). We reconstructed the phylogenetic relationships among the rophitine genera using a combination of adult morphology (24 characters) and DNA sequence data (EF‐1α, LW rhodopsin, wingless; 2700 bp total). The data set was analyzed by parsimony, maximum likelihood and Bayesian methods. All methods yielded highly congruent results. Using the phylogeny, we investigated the pattern of host‐plant association as well as the historical biogeography of Rophitinae. Our biogeographical analysis suggests a number of dispersal/vicariance events: (1) a basal split between North America and South America (most likely a dispersal from South America to North America), and (2) at least two subsequent interchanges between North America and Eurasia (presumably via the northern hemisphere land bridges). Our analysis of host‐plant associations indicates that Rophitinae specialized on a closely related group of angiosperm orders in the Euasterid I clade (mainly Gentianales, Lamiales and Solanales). However, there is little evidence of cocladogenesis between bees and plants and strong evidence of host switches to unrelated host plants. Based on our phylogenetic results we describe two new tribes of Rophitinae: Conanthalictini new tribe (including the genus Conanthalictus) and Xeralictini new tribe (including Xeralictus and Protodufourea). © The Willi Hennig Society 2007.     

Australian Lasioglossum + Homalictus form a monophyletic group: resolving the "Australian enigma"

The bee genus Lasioglossum includes > 1,000 species of bees distributed on all continents except Antarctica. Lasioglossum is a major component of the bee fauna in the Holarctic, Ethiopian, and Asian regions and is an important group for investigating the evolution of social behavior in bees. Given its cosmopolitan distribution, the historical biogeography of the genus is of considerable interest. We reconstructed phylogenetic relationships among the subgenera and species within Lasioglossum s.s., using DNA sequence data from a slowly evolving nuclear gene, elongation factor-1 alpha. The entire data set includes > 1,604 aligned nucleotide sites (including three exons plus two introns) for 89 species (17 outgroups plus 72 ingroups). Parsimony and maximum likelihood analyses provide strong evidence that the primarily Indoaustralian subgenera (Homalictus, Chilalictus, Parasphecodes) form a monophyletic group. Bootstrap support for the Australian clade ranged from 73% to 77%, depending on the method of analysis. Monophyly of the Australian Lasioglossum suggests that a single colonization event (by way of Southeast Asia and New Guinea) gave rise to a lineage of > 350 native Indoaustralian bees. We discuss the implications of Australian monophyly for resolving the "Australian enigma"--the similarity in social behavior among the Australian halictine bees relative to that of Holarctic groups.     

Vascular endothelial growth factor stimulates preantral follicle growth in the rat ovary

The regulation of preantral follicle growth in mammals is poorly understood. The availability of an adequate vascular supply to provide endocrine and paracrine signals may be important during the early states of follicle growth as well as the later states of follicle selection and dominance. The objective of the present study was to investigate whether vascular endothelial growth factor (VEGF) plays a role in preantral follicular development in the rat ovary. Immature (age, 21 days) Sprague-Dawley rats were injected with 500 ng of VEGF in saline or 50 microg of diethylstilbestrol (DES) in oil under the bursa of one ovary. The contralateral ovary was injected with a corresponding volume of vehicle. Rats were killed 48 h later, and the ovaries were removed and analyzed histologically. Intrabursal administration of VEGF significantly increased the number of primary and small secondary, but not of large secondary, preantral follicles in the ovary, similar to the effect of DES (P < 0.05). The VEGF stimulated preantral follicle growth in a time- and dose-dependent manner. Subcutaneous DES administration increased the number of primary and secondary follicles, and both s.c. and intrabursal estrogen administration stimulated VEGF protein expression in the rat ovary. These data indicate that VEGF stimulates preantral follicular development in the rat ovary, is regulated by estrogen, and may be one of the factors that participate in the regulation of early follicle growth in the rat.     

Two groups control light-induced schiff base deprotonation and the proton affinity of asp(85) in the Arg(82)His mutant of bacteriorhodopsin

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.     

125I-luteinizing hormone (LH) binding to soluble receptors from the primate (Macaca mulatta) corpus luteum: effects of ethanol exposure

In vitro exposure to alcohols unmasks additional binding sites for gonadotropin in cell/membrane preparations of the corpus luteum of rhesus monkeys. In the current study, we compared the effects of ethanol on gonadotropin receptors solubilized from macaque luteal membranes to those on receptors associated with the lipid bilayer. Treatment with 1% Triton X-100 for 30 min at 4C, followed by precipitation with polyethylene glycol, resulted in recovery of 50% more binding sites for 125I-human luteinizing hormone (hLH) than were available in particulate preparations (p less than 0.05). However, the soluble receptors displayed a 3-fold lower affinity for 125I-hLH (p less than 0.05). Conditions which enhanced LH binding to particulates, i.e., 1-8% ethanol at 25C, decreased specific 125I-hLH binding to soluble receptors. Steady-state LH binding to soluble receptors during incubation at 4C was half of that observed at 25C. The presence of 8% ethanol at 4C restored LH binding to levels observed in the absence of ethanol at 25C. Thus, LH binding sites in the primate corpus luteum can be effectively solubilized with Triton X-100. The different binding characteristics of particulate and soluble receptors, including the response to ethanol exposure, suggest that the lipid environment in the luteal membrane modulates the availability and affinity of gonadotropin receptors.