Detection of CaMV gene I and gene VI protein products in vivo using antisera raised to COOH-terminal β-galactosidase fusion proteins

Specific antisera were prepared to the inclusion body protein (gene VI product) and the gene I product of cauliflower mosaic virus (CaMV). Translational fusions between the lacZ gene and gene VI or gene I were constructed by cloning the relevant DNA fragments into the expression vectors pUR290, pUR291 or pUR292. Large amounts of fusion protein were synthesized when the inserted DNA fragment was in frame with the lacZ gene of the expression vector. These fusion proteins were used to raise specific antisera to gene VI and gene I proteins of CaMV. Antiserum to the gene VI product detected a range of proteins in crude extracts and in a subcellular fraction enriched for virus inclusion bodies. This range of proteins was further shown to be related to gene VI by Staphylococcus aureus V8 partial proteolysis. Antiserum to the gene I product detected viral specific proteins of 46, 42 and 38 K in preparations of CaMV replication complexes from infected plants but not in any other subcellular fraction.     

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.     

Ultrasonographic determination of ovarian follicular. Development in superovulated heifers pretreated with FSH-P at the beginning of the estrous cycle

On Day 3 of the estrous cycle (estrus = Day 0), dairy heifers were given either 10 mg i.m. FSH-P (FSH-P primed; n = 9) or a saline vehicle (saline primed; n = 9). On Day 10, all heifers were superovulated with FSH-P (total = 27.7 mg i.m.) in declining doses over 5 d. Heifers were inseminated artificially at estrus. From Day 2 until estrus, the number and size of follicles >2 mm were monitored daily by ultrasonography. The mean (+/- SEM) number of corpora lutea (CL) (6.2 +/- 1.5 vs 10.7 +/- 0.9; P<0.05) and the mean number of recovered embryos and unfertilized ova (3.6 +/- 1.7 vs 8.4 +/- 2.2; P<0.05) were lower in FSH-P-primed than in saline-primed heifers. Prior to initiation of superovulation, follicles >10 mm appeared on Days 6 to 7 in saline-primed heifers but only on Days 8 to 10 in FSH-P-primed heifers (P<0.05). Also, until Day 10, the mean number of follicles 4 to 6 mm and 7 to 10 mm was higher (P<0.05) in FSH-P-primed than in saline-primed heifers. After initiation of the superovulatory treatment (Day 10 to estrus), saline-primed heifers had a greater and faster increase in the mean number of follicles >10 mm (P<0.02) than FSH-P-primed heifers did. Depletion in the number of follicles 2 to 3 mm (P<0.001) between Day 10 and estrus and in the number of follicles 4 to 6 mm (P<0.05) between Day 12 and estrus occurred in both groups of heifers. Decreased superovulatory response and embryo recovery in FSH-P-primed heifers may have been due to the presence of large follicles (>10 mm) prior to the initiation of the superovulatory treatment which reduced the ability of small follicles to grow into larger size classes during superovulatory treatment.     

A protein induced by NGF in PC12 cells is stored in secretory vesicles and released through the regulated pathway.

We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a-beta-galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters.