Spatial and temporal gene expression patterns occur during corm development.

We investigated gene expression patterns that occur during taro corm development. Two-dimensional gel electrophoresis identified several different prevalent proteins that accumulate during corm development. Microsequencing studies indicated that some of these proteins are related to taste-modifying proteins, such as curculin and miraculin, and proteins found in other storage organs, such as sporamin and the Kunitz trypsin inhibitor. A curculin-encoding cDNA clone, designated as TC1, was identified that corresponds to a highly prevalent 1-kb corm mRNA. The TC1 mRNA accumulates during corm development, is more prevalent in corm apical than basal regions, and is either absent, or present at low concentrations, in other vegetative organs such as the leaf and root. In situ hybridization experiments showed that the TC1 mRNA is highly concentrated in corm storage parenchyma cells and is absent, or present in reduced concentrations, in other corm cells and tissues. Our results show that corm development is associated with the differentiation of specialized cells and tissues, and that these differentiation events are coupled with the temporal and spatial expression of corm-specific genes.     

Molecular cloning and characterization of prolactin-like protein C complementary deoxyribonucleic acid.

In this report, we describe the isolation and characterization of a full length cDNA clone for rat prolactin-like protein C (PLP-C) and describe the expression of PLP-C mRNA in the developing rat placenta. Nucleotide sequence analysis of the PLP-C cDNA clone predicted a mature protein of 238 amino acids, including a 30-amino acid signal sequence. The predicted PLP-C amino acid sequence contains seven cysteine residues, three tryptophan residues, and two putative N-linked glycosylation sites. Six of the cysteine residues in PLP-C are located in positions homologous to the cysteines of pituitary prolactin (PRL). Additional sequence similarities with pituitary PRL and other members of the rat placental PRL family are evident. The PLP-C gene was localized to rat chromosome 17. Northern blot analysis showed that the PLP-C cDNA clone specifically hybridized to a 1.0-kilobase mRNA. PLP-C mRNA was first detectable between days 13 and 14 of gestation, peaked by day 18 of gestation, and remained elevated until term. In situ hybridization analysis indicated that PLP-C mRNA was specifically expressed by spongiotrophoblast cells and some trophoblast giant cells in the junctional zone region of rat chorioallantoic placenta.     

Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator.

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.     

An A alpha Ser-434 to N-glycosylated Asn substitution in a dysfibrinogen, fibrinogen Caracas II, characterized by impaired fibrin gel formation.

We have identified a unique N-glycosylated Asn substitution for a Ser at position 434 of the A alpha chain of an abnormal fibrinogen designated fibrinogen Caracas II. This dysfibrinogen was characterized by impaired fibrin monomer aggregation. Since there were 4 Thr residues immediately following the mutation, a new Asn-X-Thr/Ser-type consensus sequence, Asn-Thr-Thr arose for N-glycosylation of the Asn. The extra oligosaccharide was found to consist mainly of a disialylated biantennary structure comprising 81.9%, while a neutral and a monosialylated biantennary oligosaccharide represented only 3.6% and 14.5%, respectively. The mutation resides in the carboxyl-terminal region of the A alpha chain, which could fold back to form an extra small globular region located near the central region of the molecule (Erickson, H.P., and Fowler, W.E. (1983) Ann. N. Y. Acad. Sci. 408, 146-163; Weisel, H.P., Stauffacher, C.V., Bullitt, E., and Cohen, C. (1985) Science 230, 3124-3133). Therefore, the participation of this region, referred to as an additional central domain or an alpha domain, in fibrin gel formation is strongly implicated.     

Paraguayan pharmacies and the sale of pseudo-abortifacients

This study was conducted in 1985 in Asunción, Paraguay, 6 years after the closure of the state supported family planning services. Data from national surveys in 1977 and 1987 permit a comparison of sources of contraceptive supplies before and after the elimination of government support for family planning. The purchase of pseudo-abortifacients from private pharmacies was used as an indication of induced abortion. After the loss of government clinics, it is suggested that some women turned to pharmacists to obtain pseudo-abortifacients when faced with unwanted pregnancy. There is an indication of increased pseudo-abortifacient use, particularly among unmarried women and those from poorer neighbourhoods.     

Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily

The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions.     

Aerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture.

Klebsiella pneumoniae NCTC 418 is able to convert 2-ketogluconate intracellularly to 6-phosphogluconate by the combined action of an NADPH-dependent 2-ketogluconate reductase and gluconate kinase. Synthesis of the former enzyme was maximal under 2-ketogluconate-limited growth conditions. An instantaneous transition to a 2-ketogluconate-excess condition resulted in an acceleration of catabolism of this carbon source, accompanied by complete inhibition of biosynthesis. It is suggested that the cause of this inhibition resides in depletion of the NADPH pool due to the high rate at which NADPH is oxidized by 2-ketogluconate reductase.     

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.     

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions.

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.