The art and design of genetic screens: filamentous fungi

In the 1940s, screens for metabolic mutants of the filamentous fungus Neurospora crassa established the fundamental, one-to-one relationship between a gene and a specific protein, and also established fungi as important genetic organisms. Today, a wide range of filamentous species, which represents a billion years of evolutionary divergence, is used for experimental studies. The developmental complexity of these fungi sets them apart from unicellular yeasts, and allows the development of new screens that enable us to address biological questions that are relevant to all eukaryotes.     

A brief history of vertebrate functional morphology

The discipline of functional morphology grew out of a comparativeanatomical tradition, its transformation into a modern experimentalscience facilitated largely by technological advances. Earlymorphologists, such as Cuvier, felt that function was predictablefrom organismal form, to the extent that animals and plantsrepresented perfect adaptations to their habits. However, anatomyalone could not reveal how organisms actually performed theiractivities. Recording techniques capable of capturing fast motionwere first required to begin to understand animal movement.Muybridge is most famous for his pioneering work in fast photographyin the late 19th century, enabling him to "freeze" images ofeven the fastest horse at a full gallop. In fact, contemporarykinematic analysis grew directly out of the techniques Muybridgedeveloped. Marey made perhaps an even greater contribution toexperimental science through his invention of automatic apparatifor recording events of animal motion. Over the first half ofthe 20th century, scientists developed practical methods torecord activity patterns from muscles of a living, behavinghuman or animal. The technique of electromyography, initiallyused in clinical applications, was co-opted as a tool of organismalbiologists in the late 1960s. Comparative anatomy, kinematicanalysis and electromyography have for many years been the mainstayof vertebrate functional morphology; however, those interestedin animal form and function have recently begun branching outto incorporate approaches from experimental biomechanics andother disciplines (see accompanying symposium papers), and functionalmorphology now stands at the threshold of becoming a truly integrative,central field in organismal biology.     

Blockade of EphA receptor tyrosine kinase activation inhibits vascular endothelial cell growth factor-induced angiogenesis

Angiogenesis is a multistep process involving a diverse array of molecular signals. Ligands for receptor tyrosine kinases (RTKs) have emerged as critical mediators of angiogenesis. Three families of ligands, vascular endothelial cell growth factors (VEGFs), angiopoietins, and ephrins, act via RTKs expressed in endothelial cells. Recent evidence indicates that VEGF cooperates with angiopoietins to regulate vascular remodeling and angiogenesis in both embryogenesis and tumor neovascularization. However, the relationship between VEGF and ephrins remains unclear. Here we show that interaction between EphA RTKs and ephrinA ligands is necessary for induction of maximal neovascularization by VEGF. EphA2 RTK is activated by VEGF through induction of ephrinA1 ligand. A soluble EphA2-Fc receptor inhibits VEGF-, but not basic fibroblast growth factor-induced endothelial cell survival, migration, sprouting, and corneal angiogenesis. As an independent, but complementary approach, EphA2 antisense oligonucleotides inhibited endothelial expression of EphA2 receptor and suppressed ephrinA1- and VEGF-induced cell migration. Taken together, these data indicate an essential role for EphA receptor activation in VEGF-dependent angiogenesis and suggest a potential new target for therapeutic intervention in pathogenic angiogenesis.     

A novel missense mutation (S18N) in the 5′ non-HMG box region of the SRY gene in a patient with partial gonadal dysgenesis and his normal male relatives

Mutations in the sex-determining region of the Y chromosome (the SRY gene) have been reported in low frequency in patients with 46,XY gonadal dysgenesis. We investigated 21 Brazilian 46,XY sex-reversed patients, who presented either complete or partial gonadal dysgenesis or embryonic testicular regression syndrome. Using Southern blotting, polymerase chain reaction, denaturing gradient gel electrophoresis and direct sequencing, we analyzed deletions and point mutations in the SRY gene. We found a missense mutation at codon 18 upstream of the 5′ border of the HMG box of the SRY gene in one patient with partial gonadal dysgenesis. This variant sequence was also found in DNA obtained from blood and sperm cells of his father and in blood cells of his normal brother. The S18N mutation was not found in 50 normal males, ruling out the possibility of a common polymorphism. We identified a novel familial missense mutation (S18N) in the 5’ non-HMG box of the SRY gene in 1 of 21 patients with 46,XY sex reversal. Received: 6 May 1997 / Accepted: 2 October 1997     

DEVELOPMENTAL STUDIES IN PORPHYRA. I. BLADE DIFFERENTIATION IN PORPHYRA PERFORATA AS EXPRESSED BY MORPHOLOGY,ENZYMATIC DIGESTION,AND PROTOPLAST REGENERATION1

Four areas containing different cell morphologies were mapped on Porphyra blades and five different cell types (i.e. tapered with long extensions, large and vacuolated, vegetative and dividing, and reproductive: males and females) were identified in them. Tissues from these areas were dissociated, and protoplasts and single cells were isolated from the dissociated tissue of each distinct region. Regeneration rates of the isolated cells and protoplasts (isolates) varied depending on their morphological type. Regeneration rates were lowest in cultured isolates from the area just above the holdfast (ca. 1 %) and increased gradually to over 80% in isolates from areas of vegetative and reproductive regions away from the holdfast. Four distinct morphological patterns were observed among the regenerating plants. Cells isolated from vegetative areas developed into leafy plants while in liquid culture, and into calli when grown on solid medium. Isolates from reproductive areas developed into either a long thin or short thick filamentous plant. Those from ripe patches of carposporangia developed into thin conchocelis filaments, while isolates from non-differentiated cells bordering the ripe reproductive patches developed into thick filaments resembling the morphology of conchosporangial branches. The blade of Porphyra appears simple as it consists of a single cell layer; however, it is complex both morphologically and physiologically.     

Induction of micronuclei by 153Sm-EDTMP in peripheral blood lymphocytes of patients with bone metastases.

The purpose of this study was to evaluate the degree of cytological radiation damage to peripheral blood lymphocytes induced by 153Sm-EDTMP applied for palliation of metastatic bone pain. Blood samples from 16 patients (46-82 years old), 10 without previous radiotherapy and 6 with previous radiotherapy, were collected before and one hour after the administration of a mean activity of 41.7+/-5.8 MBq/kg of 153Sm-EDTMP. Then the lymphocytes were cultured for cytokinesis block micronucleus (MN) assay. The number of MNper binucleated cells (BC) in patients without previous radiotherapy before the treatment was of 0.030 (+/- 0.016) and after one hour 0.035 (+/- 0.013), although we could find inter individual differences. The basal MN/BC of the patients with no previous radiotherapy was similar to the controls. The increment in the percentage of BC with MN was similar in patients with and without previous radiotherapy. The observed mean of MN/BC is equivalent to a dose range of 0.05 to 0.10 Gy of 153Sm-EDTMP in vitro. The relatively low frequency of lymphocyte with micronuclei after the exposure to 153Sm-EDTMP supported the contention that radiation damage in lymphocytes of patients with painful bone metastases is minimal.     

Anti-CEA monoclonal antibody: technetium-99m labeling and the validation process of a scintigraphic animal model with a non-cellular antigenic implant.

Animal models are currently used to verify the biodistribution of different radiopharmaceuticals before its clinical application in Nuclear Medicine; however, there may be some limitations. The utilization of labelled anti-tumor monoclonal antibodies (MoAb) in experimental models often requires implant of human antigens (usually a cellular implant), which cannot be achieved in immunocompetent animals. Our purpose was to label an anti-CEA MoAb with technetium-99m (99Tc) and to validate a simplified animal model using a noncellular antigenic implant. MoAb was directly labelled with 99mTc, after reduction with 2-mercaptoethanol. Labeling efficiency was checked by ascending chromatography and immunoreactive fraction was measured in plastic wells sensitized with the antigen. Radiopharmaceutical biodistribution was evaluated by dissection and scintigraphy in 5 mice groups; following the subcutaneous administration of Al(OH)3, CEA adsorbed Al(OH)2 and a control group evaluation. Labeling efficiency was 94+/-3%, which showed to be stable for 24 hr, with immunoreactive fraction above 50%. Invasive biodistribution evaluation showed prolonged blood retention, hepatic and renal uptake. A significant increase in uptake was observed in scintigraphic studies of animals with CEA-adsorbed Al(OH)3 implants compared with the other groups (p<0.05). The non-cellular antigenic implant model simplifies the pre-clinical evaluation of labelled MoAb.     

A NOVEL TECHNIQUE FOR PREPARATION OF AXENIC CULTURES OF SYMBIODINIUM (PYRROPHYTA) THROUGH SELECTIVE DIGESTION BY AMOEBAE1

The marine amoeba Trichosphaerium Am-I-7 was used as a tool for preparing unialgal axenic cultures of nondigestible Symbiodinium and Porphyridium species. The resistance of these unicellular algae to the amoebal digestive enzymes, and the differential digestion of bacteria, protozoans, and other algae, resulted in cleansed cells of Symbiodinium and Porphyridium that remained in the amoebal food vacuoles. During multiple fission, the amoeba evacuated its food vacuoles and released the trapped and intact algae, which were then successfully cultured. This method of cleaning was especially useful with algal species that were sensitive to antibiotics or other germicidal agents.     

ONTOGENETIC VARIATION IN PATTERNS OF DEVELOPMENTAL AND FUNCTIONAL INTEGRATION IN SKULLS OF SIGMODON FULVIVENTER

Patterns of variation and covariation within populations can influence how characters respond to natural selection and random genetic drift and so constrain the ability of natural selection to modify the phenotype. We examined several potential developmental and functional explanations of character covariation throughout ontogeny using known-age samples of the cotton rat (Sigmodon fulviventer) to identify the causes of covariation and to assess the variability of patterns of covariation throughout postnatal growth. Competing developmental and functional models were fit to samples of orofacial and neurocranial measures by confirmatory factor analysis and evaluated for their ability to reconstruct observed variance-covariance matrices. Samples of successive ages were simultaneously fit to a common model to test the hypothesis that the patterns of developmental and functional integration were invariant between ages. Orofacial characters derived from the same branchial-arch primordium covary early in ontogeny. Subsequently, there is a repatterning of integration that may reflect a transition from developmental to functional sources of integration. Neurocranial characters exhibit even more variation in patterns of covariation: initially, characters appear to comprise a single integrated unit; before puberty, they appear to respond to localized bone growth; after puberty, they form separate calvarial and basicranial components. This ontogenetic variation in patterns of covariation suggests that developmental constraints are transient and flexible and that the consequences of selection may depend upon the age at which it acts.