Regression of blood vessels precedes cartilage differentiation during chick limb development

We have previously investigated distinct areas of vascular regression in the developing vascular system of the chick limb bud. Avascular areas appear in a characteristic spatial and temporal pattern, and are correlated with the position of developing cartilage. In the present study, we examined limb-bud sections which had been double labeled for endothelial cells and developing cartilage in order to determine the relationship between the appearance of cartilage and the disappearance of capillaries. Endothelial cells, which specifically take up acetylated low-density lipoprotein (acLDL), were labeled by intravenously injecting fluorescent acLDL (DiIacLDL) into chick embryos at Hamburger and Hamilton stages 26-30. Avascular zones, which correspond to the developing digits, were clearly visible within the fluorescently labeled distal vasculature. The same sections were labeled with monoclonal antibodies specific for cartilage. We found that progressing avascularity in the digital regions was followed by increased staining for cartilage antigens in the same areas. Zones of avascularity always developed earlier than morphologically and immunologically detectable cartilage in all planes of section and were always larger than the areas of cartilage. These results demonstrate that blood vessels disappear in predictable areas prior to the overt differentiation of cartilage.     

The distribution of the Hb Constant Spring gene in Southeast Asian populations

Summary The distribution of the hemoglobin Constant Spring (Hb CS) gene in eight populations in Southeast Asia (including Assam) was determined using oligonucleotide hybridization. Hb CS was absent in two Assamese populations with a high prevalence of Hb E. The Hb CS gene frequency was 0.033 in northern Thailand and near 0.01 in central Thailand and Cambodia. High frequencies, between 0.05 and 0.06, were observed in northeastern Thailand. The present data and a similar study in Laotians suggest that the Lao-speaking populations of the Mekong River basin in northeastern Thailand and Laos have the highest frequencies of the Hb CS gene in Southeast Asia.     

Purification of acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase from Catharanthus roseus leaves

The enzyme acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase which terminates vindoline biosynthesis has been isolated from Catharanthus roseus leaves, further characterized and purified to homogeneity by three step column chromatography and subsequent preparative isoelectric focusing. Kinetic properties concerning the enzyme reaction are discussed. Five multiple forms of the acetyl-transferase could be observed, each consisting of two subunits. This enzyme is now the best characterized of the enzymes involved in vindoline biosynthesis.Abbreviations DTE dithiothreitol - EDTA ethylenediamine-tetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid - IEF isoelectric focusing - KP_i potassium phosphate - M_r rel.molecular mass - PEG polyethylene glycol - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

In goodeid teleosts, prolonged embryonic development takes place within the ovarian cavity. Apposed maternal and embryonic epithelia interface via a nutritive liquid (embryotrophe) and facilitate aplacental matrotrophy. The role of the internal ovarian epithelium (IOE) in providing proteins for the embryotrophe has been studied using transmission electron-microscopic examinations of both the resting and the active ovarian lining, and isoelectric focusing of embryotrophe and maternal blood serum. The simple IOE is apparently composed of only one, filament-containing cell-type. In the non-gravid ovary these cells are cuboidal to columnar in shape, and are either compact and electron-dense or oedematous and light. During gestation, swelling of the ovarian connective tissue gives rise to dovetailing of the IOE with the subjacent capillary plexus. Part of the IOE overlying the capillaries becomes stretched, resulting in a thin endothelium-like demarcation. The nuclei and the bulk of the cytoplasm are usually recessed between the meshes of the protruding capillary network. The blood-embryotrophe pathway is thus reduced in places, to less than one m. The active form of the IOE contains a well-developed vacuolar apparatus composed of small vesicles, vacuoles, multivesicular bodies, and a few lysosomes. Elements of the RER are sparsely distributed throughout the cytoplasm. Endocytotic activity is observable at the apical and basolateral plasma membrane. Isoelectric focusing of both serum and embryotrophe produces numerous bands each between pI 4–8, which reveal many homologies. The intensity of corresponding bands varies considerably. It is concluded that the cells of the IOE provide a transport pathway for serum-derived macromolecular substances rather than produce proteinaceous secretions.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Differential localization of leu-enkephalin and hyperglycemic hormone in axon terminals of the sinus gland of the crabsCarcinus maenas,eriocheir sinensis,andUca pugilator

Summary Leu-enkephalin containing secretory granules were demonstrated in axon terminals of immunogoldlabeled electron-microscopic sections of the sinus gland of three brachyuran crustaceans. These granules have a diameter of 120+-15 nm and differ in electron density from those located in adjacent terminals containing hyperglycemic or molt-inhibiting hormone. These neurohormones do not show co-localization with leu-enkephalin. The cross-reactivity of leu-enkephalin antiserum with met-enkephalin is less than 1%. The sinus glands of the three species examined show no immunoreactivity for FMRF-amide. A modulatory activity of endogenous enkephalin by paracrine mechanisms is suggested.     

Target organ-specific inactivation of drug metabolizing enzymes in kidney of hamsters treated with estradiol

Chronic treatment of hamsters with estradiol for several months has previously been shown to decrease the specific content of cytochrome P450 in the kidney, a target of hormonal carcinogenesis, but not in liver. The reason for this decrease in metabolic enzyme activity is unknown and has been examined in this investigation. We now report that the decrease in specific content of renal cytochrome P450 by 73% in response to estradiol was not affected by co-treatment with tamoxifen for 1 month. The subcutaneous infusion of 250 μg/day estradiol for 7 days lowered renal cytochrome P450 by 71% from control values and was therefore used for further mechanistic studies. This treatment decreased renal activities of estradiol 2- or 4-hydroxylase by 77 to 80%, of 7-ethoxycoumarin-O-deethylase by 66% of control values, respectively, and completely eliminated aryl hydrocarbon hydroxylase activities, whereas liver enzymes remained unaffected. After 7 days of infusion of estradiol, fluorescent products of lipid peroxidation were more than doubled in hamster kidney but remained unchanged in liver. The possibility of enzyme destruction by binding of estradiol 2,3-quinone to metabolizing enzymes was investigatedin vitro. In the presence of 2-hydroxyestradiol, cumene hydroperoxide, and microsomes, conditions known to favor the oxidation of the steroid to quinone, the binding of catechol estrogen metabolite to microsomal protein increased 60 fold over control values in the absence of cofactor. Purified rat liver cytochrome P450c also oxidized 2-hydroxyestradiol to 2,3-estradiol quinone. The rate of oxidation was linear for the first 2–3 min, but thereafter decreased with time. Under these incubation conditions, irreversible binding of catechol estrogen metabolite to cytochrome P450c increased for the first 2–3 min and then remained at this plateau level. It was concluded that enzyme destruction by a reactive estrogen metabolite or by lipid peroxides may be a major reason for the organ-specific decrease in cytochrome P450 enzymes in kidneys of estrogen-treated hamsters.     

Mutagenicity of azo dyes in the Salmonella/microsome assay using in vitro and in vivo activation

The mutagenicity of 6 azo dyes, including direct black 38 (DB38), direct black 19 (DB19), direct brown 95 (DB95), solvent yellow 3 (SY3), trypan blue (TPB), and food black 2 (FB2), was examined in the Salmonella/microsome assay. The effect of chemical azo reduction (dithionite) and in vivo metabolism on the mutagenicity of the dyes was also studied. In vivo azo-dye metabolites were isolated from the urine of rats intubated with dyes by XAD-2 column chromatography. Urinary metabolites from all the treated animals, except animals treated with FB2, induced frame-shift mutations in strains TA1538 and TA98 in the presence of liver S9 activation. The control urine did not increase the incidence of revertants in strains TA1538 and TA98. Thus, XAD-2 chromatography can be used to isolate genotoxic metabolites from the urine of animals intubated with azo dyes.     

Late enzymes of vindoline biosynthesis

From differentiated plants of Catharanthus roseus (L.) G. Don we have isolated a specific enzyme of the vindoline biosynthetic pathway catalysing the S-adenosylmethionine-dependent methylation of 11-O-demethyl-17-O-deacetyl-vindoline. The enzyme we named S-adenosyl-L-methionine : 11-O-demethyl-17-O-deacetylvindoline 11-O-methyltransferase. This transferase exhibits a high substrate specificity. Obviously the O-methylation at C-11 precedes the O-acetylation at the C-17 position during the biosynthesis of vindoline.A second enzyme was detected which hydrolyses the acetyl function of vindoline. The distribution of this acetylesterase in C. roseus plants demonstrates that the enzyme is not specifically associated with the vindoline distribution in the plant material. Most probably this enzyme plays no essential role in the biosynthesis of vindoline.     

Late enzymes of vindoline biosynthesis. Acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyl-transferase

From differentiated plants of Catharanthus roseus (L.) G. Don, a specific enzyme was isolated and named acetyl-CoA : 17-O-deacetylvindoline 17-O-acetyltransferase, acting on the biosynthetic formation of the Aspidosperma type alkaloid vindoline.The enzyme shows a high selectivity towards different substrates. The acetyl-CoA-dependent transferase also catalyses the reverse reaction by hydrolysis of the 17-O-acetyl group of vindoline in the presence of free CoA. This enzyme is localized only in vindoline-containing plant parts, but was so far not detectable in cell suspension cultures of C. roseus. The enzyme allows the synthesis of labelled vindoline with high specific activity, applicable for instance as tracer for radioimmunoassays of vindoline.