Metamorphosis of the marine gastropod Phestilla Sibogae Bergh (nudibranchia: aeolidacea). I. Light and electron microscopic analysis of larval and metamorphic stages

The structure and fate of transitory larval organs (velum, shell, operculum, retractor muscles, part of the epidermis) of Phestilla sibogae Bergh were studied before, during, and after metamorphosis with both light and electron microscopy to elucidate the morphology of these organs and the mechanisms by which they are lost.Loss of the velar lobes is the first morphological sign of metamorphosis, and involves selective dissociation and subsequent ingestion of the ciliated velar cells; the remaining aggregate of supportive cells is apparently incorporated into cephalic epidermis. Attachment of the larval body to shell and operculum is primarily at sites of retractor muscle insertions; once the velum is gone, the attachment between shell and larval body is lost and the shell is cast off as the visceral organs exit through the shell aperture. Merger of visceral and cephalopedal elements results in flattening of the postlarval body and reorientation of internal organs. Simultaneously, a rapid spreading of epipodial epidermis over the lateral, dorsal, and posterior sides of the body produces the definitive integument. The squamous cells which comprise the larval perivisceral epidermis are pushed ahead of the definitive epidermis and are seen shortly after the shell is cast as a constricted aggregate of cells on the posterior end of the body. Autolysis of the left and right retractor muscles begins during metamorphosis and no trace of them is left after 24 to 48 h. The metapodial mucous glands which hypertrophy before metamorphosis are also lost within 48 h following exit of the post larva from the shell. Metamorphosis produces a detorsion caused in part by muscular action and in part by continuing growth and development.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.     

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

Genetic polymorphims of estrogen receptor alpha −397 PvuII (T>C) and −351 XbaI (A>G) in a portuguese population: prevalence and relation with breast cancer susceptibility

Estrogen receptor alpha (ERα), that mediates the biologic effects of estrogen in estrogen-sensitive tissues like breast, is genetically polymorphic. To evaluate the association between ?397 PvuII (T>C) and ?351 XbaI (A>G) restriction fragment length polymorphisms (RFLPs) in intron 1 of ERα gene and susceptibility of breast cancer, we undertook a case–control study in BRCA1 185delAG and 5382insC/BRCA2 6174delT negative Portuguese women. The study population consisted of 107 patients with histological diagnosis of breast cancer and 121 women with no history of breast cancer. Genomic DNA was extracted from blood samples and genotyping analyses were performed by PCR–RFLP. XbaI polymorphism was associated with a significant reduced risk of breast cancer for carriers of the x allele in homozygozity (OR 0.178; 95 % CI 0.070–0.456; P < 0.001) or heterozigozity (OR 0.223; 95 % CI 0.089–0.561; P = 0.001). The PvuII polymorphism was associated with a non-significantly reduced risk. The combined analysis of PvuII and XbaI polymorphisms revealed none synergistic effect of the two genotypes, except for simultaneous carriers of pp and xx genotypes, that have a reduced risk of breast cancer (OR 0.226; 95 % CI 0.049–1.035; P = 0.044). The combination of PvuII and XbaI genotypes into haplotypes showed that carriers of two copies of the px (ppxx) haplotype had a reduced risk of breast cancer (OR 0.405; 95 % CI 0.194–0.843; P = 0.014), compared with PX (PPXX + PPXx + PpXX + PpXx) haplotypes. PvuII and XbaI polymorphisms were in linkage disequilibrium both in cases (D = 0.044, r² = 0.049, X² = 5.216, P = 0.022) and controls (D = 0.090, r² = 0.139, X² = 16.819, P < 0.001), but not in the entire sample population analyzed as a whole (D = 0.087, r² = 0.0076, X² = 1.733, P = 0.188). In conclusion, in this case–control study we found that ERα gene XbaI polymorphism may modify individual susceptibility for breast cancer in this population.     

Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals.

The conversion of substrate, heptenitol, to product, beta-1-C-methyl, alpha-D-glucose-1-phosphate (heptulose-2-P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5-dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer. In the Laue diffraction experiments, data to 2.8 A resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800-ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed. Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 A resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose-2-P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 A resolution to establish the fractional occupancies of the enzyme-substrate and enzyme-product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal.(ABSTRACT TRUNCATED AT 250 WORDS)