Restoring avian wing digits

The precise identification of the digits of the avian wing is of importance in evolutionary studies. If the digits are numbered two, three and four, this has been taken to suggest that birds are not descended directly from dinosaurs. If the digits are numbered one, two and three, dinosaur origins become more plausible. Studies of the development of the avian wing have failed to resolve this dilemma. However, in some instances, it is possible to deduce information about evolutionary morphologies by manipulating development experimentally. We grafted beads loaded with fibroblast growth factor 4 into the distal tip of chick wing buds at times when the apical ectodermal ridge is regressing. The consequence was that the cartilage structure conventionally labelled ''element 5'' increased dramatically in size and acquired a digit-like morphology in some instances. Corresponding changes in soft tissue morphology were also observed. We conclude that it may be possible to resolve the issue of avian digit homology by the induction of experimental atavisms of this kind.     

ANTIALGAL ACTIVITY OF SOME SIMPLE PHENOLS1

The antialgal activity of a number of simple phenols was examined for their effect on the growth of 7 species of unicellular marine algae. The 3 knoiun algal phenols, 5-bromo-3,4-dihydroxybenzaldehyde; 2,3-dibromo-4,5-dihydroxybenzylalcohol, and 3,4-dihy-droxyphenylethylamine, were highly toxic as were other ortho dihydroxy compounds. Monohydroxy compounds were notably less toxic. Skeletonema costatum and Olisthodiscus sp. were the most sensitive organisms examined and Dunaliella tertiolecta was the most resistant. Possible ecological implications of these results are discussed.     

The androgen-dependent mouse seminal vesicle secretory protein IV: characterization and complementary deoxyribonucleic acid cloning

Seminal vesicle secretory protein IV of a mouse has been isolated, and the cDNA coding for its mRNA has been cloned and sequenced. The 556-nucleotides encode 16 amino acid signal peptides and 92 residues of mature protein. Considerable homology between mouse and rat SVS IV cDNA was found. In the leader peptide and 3'-noncoding region there is 92% and 85% homology, respectively. The other regional homologies are 86% for the first 12, 68.5% for the last 35, and 40% for the middle 44 amino acids. The expression of mouse SVS IV mRNA is under the control of androgen. Administration of testosterone to castrated mice resulted in induction of the mRNA level to 50% of the mature male in 96 h of hormone treatment. Secretion of the protein after testosterone injection follows a similar pattern.     

Larval habitat duration and size at metamorphosis in frogs

The relationship between size at metamorphosis and adult size was studied in 12 closely-related species of frog from Malawi (Central Africa). These species of frogs breed in water of different durations, and occupy different habitats as adults. We could demonstrate no correlation between size at metamorphosis and size of adults when frogs were divided into groups on the basis of occupying similar habitats as adults, but when frogs were divided into groups on the basis of similar duration of larval habitat we demonstrated a strong correlation between size at metamorphosis and adult size. Thus we suggest that duration of the larval habitat is a major determinant of size at metamorphosis, with species which breed in the more temporary habitats metamorphosing at smaller size than species which breed in more permanent habitats, but which are of similar size as adults. Such manipulation of the life cycle appears to be adaptive since it results in individuals becoming independent of water earlier when the likelyhood of early loss of larval habitat is high.     

Identification of a novel growth factor with transforming activity secreted by individual chick embryos

Previously we have developed a microassay for anchorage independent growth (AIG) of fibroblasts in soft agar, which can detect very small quantities of transforming growth factors (TGFs). Using this assay, we have shown that small pieces of dissected chick embryo tissue will stimulate AIG of both NR6 and NRK 49f cells, and that this property can be used to map production of growth factors with transforming activity in individual early embryos. We now show that this activity can be transferred to conditioned medium (CM) prepared using chick embryo tissue. Using two cell lines with differential responsiveness to TGFs, and by coincubating normal and heat-treated CM with trypsin, Con-A and neutralising antibodies, we show that CM contains at least two different growth factors with transforming activity. One of these is heat-stable, and stimulates colony formation in NRK 49f cells in the presence of EGF, but not in its absence. This activity corresponds to a TGF beta-like molecule. The other component is a heat-labile glycoprotein, which has TGF alpha-like properties, but does not appear to behave like known TGFs with these properties. It therefore appears to be a novel growth factor. Both activities are present from the intermediate primitive streak stage of development.     

Characteristics of estrogen-induced peroxidase in mouse uterine luminal fluid.

Peroxidase activity in the uterine luminal fluid of mice treated with diethylstilbestrol was measured by the guaiacol assay and also by the formation of 3H2O from [2-3H]estradiol. In the radiometric assay, the generation of 3H2O and 3H-labeled water-soluble products was dependent on H2O2 (25 to 100 microM), with higher concentrations being inhibitory. Tyrosine or 2,4-dichlorophenol strongly enhanced the reaction catalyzed either by the luminal fluid peroxidase or the enzyme in the CaCl2 extract of the uterus, but decreased the formation of 3H2O from [2-3H]estradiol by lactoperoxidase in the presence of H2O2 (80 microM). NADPH, ascorbate, and cytochrome c inhibited both luminal fluid and uterine tissue peroxidase activity to the same extent, while superoxide dismutase showed a marginal activating effect. Lactoferrin, a major protein component of uterine luminal fluid, was shown not to contribute to its peroxidative activity, and such an effect by prostaglandin synthase was also ruled out. However, it was not possible to exclude eosinophil peroxidase, brought to the uterus after estrogen stimulation, as being the source of peroxidase activity in uterine luminal fluid.     

Occurrence of Prorocentrum lima,a DSP toxin-producing species from the Atlantic coast of Canada

A species of Prorocentrum (Dinophyta, Prorocentrales), isolated from a phytoplankton net sample from the Atlantic coast of Nova Scotia, has been brought into unialgal culture. The sample was collected at an aquaculture site immediately following an incident of diarrhetic shellfish poisoning (DSP) due to the consumption of contaminated mussels. This clonal isolate has been identified as P. lima, based on its morphological characteristics. Analysis of the culture extract, using high performance liquid chromatography (HPLC) with fluorescence detection, indicated the presence of the DSP toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX-1).     

Substrate specificity of N-acetylglucosamine 1-phosphate transferase activity in Chinese hamster ovary cells.

The assembly pathway of the oligosaccharide chains of asparagine-linked glycoproteins in mammalian cells begins with the formation of GlcNAc-PP-dolichol in a reaction catalysed by the enzyme N-acetylglucosamine 1-phosphate transferase. We have investigated the efficiency of two lipid substrates for the transferase activity in an in vitro assay using Chinese hamster ovary (CHO) cell membranes as an enzyme source. Experiments were carried out with varying concentrations of dolichyl phosphate or its precursor, polyprenyl phosphate. We determined that enzyme activity was optimal at pH 9, where the enzyme exhibited a 3-fold higher Vmax and a 2-fold lower Km for the dolichol substrate. At pH 7.4, the Km and Vmax differences between the two lipids were 10-fold. Under all assay conditions tested, we found that GlcNAc-PP-lipid was the only product formed. We conclude from these results that dolichyl phosphate rather than polyprenyl phosphate is the preferred substrate for the transferase enzyme in CHO cells. This observation is significant in light of the fact that we have previously isolated CHO glycosylation mutants which fail to convert polyprenol into dolichol, and hence utilize polyprenyl derivatives for glycosylation reactions. Thus, these results contribute to our understanding of the glycosylation defects in the mutant cell lines.     

Preparation and activity of carbonic anhydrase immobilized on porous silica beads and graphite rods

Techniques for the immobilization of bovine carbonic anhydrase (BCA) on porous silica beads and graphite are presented. Surface coverage on porous silica beads was found to be 1.5 x 10(-5) mmol BCA/m(2), and on graphite it was 1.7 x 10(-3) mmol BCA/m(2) nominal surface area. Greater than 97% (silica support) and 85% (graphite support) enzyme activity was maintained upon storage of the immobilized enzyme for 50 days in pH 8 buffer at 4 degrees C. After 500 days storage, the porous silica bead immobilized enzyme exhibited over 70% activity. Operational stability of the enzyme on silica at 23 degrees C and pH 8 was found to be 50% after 30 days. Catalytic activity expressed as an apparent second-order rate constant K'(Enz) for the hydrolysis of p-nitrophenyl acetate (p-NPA) catalyzed by BCA immobilized on silica beads and graphite at pH 8 and 25 degrees C is 2.6 x 10(2) and 5.6 x 10(2) M(-1)s(-1) respectively. The corresponding K(ENZ) value for the free enzyme is 9.1 x 10(2) M(-1)s(-1). Activity of the immobilized enzyme was found to vary with pH in such a manner that the active site pK, on the porous silica bead support is 6.75, and on graphite it is 7.41. Possible reasons for a microenvironmental influence on carbonic anhydrase pK(a), are discussed. Comparison with literature data shows that the enzyme surface coverage on silica beads reported here is superior to previously reported data on silica beads and polyacrylamide gels and is comparable to an organic matrix support. Shifts in BCA-active site pK(a) values with support material, a lack of pH dependent activity studies in the literature, and differing criteria for reporting enzyme activity complicate literature comparisons of activity; however, immobilized BCA reported here generally exhibits comparable or greater activity than previous reports for immobilized BCA.