Postnatal D2-CAM/N-CAM Sialylation State Is Controlled by a Developmentally Regulated Golgi Sialyltransferase

Abstract: Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [¹⁴C]sialic acid was found to be associated with the high-molecular-weight [>200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro*

Abstract. Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [³H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G₀/G₁, S, and G₂/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [³H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [³H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G₂ tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells

We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obtained by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.     

Cloning and sequencing of the gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough

Abstract The gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough (148 amino acid residues), the first flavoprotein for which a three-dimensional structure has been determined, was cloned with the use of two synthetic oligonucleotides, designed to recognize the coding sequence for amino acid residues 11–19 and 98–103, respectively. The two oligonucleotides were used to screen a library of 900 λ-clones of the D. vulgaris chromosome. A single clone, λFL1, reacting with both probes was isolated. The entire structural gene for flavodoxin is contained in the 15 kb insert of λFL1 as found by nucleic acid sequencing. The codon usage in the flavodoxin gene is strongly biased towards G or C in the third codon position. A table in which codon usage information from all genes of D. vulgaris sequenced to date is combined is presented and should facilitate further gene cloning with oligonucleotide probes.     

Differentiation-Dependent Sialylation of Individual Neural Cell Adhesion Molecule Polypeptides During Postnatal Development

The postnatal sialylation of individual neural cell adhesion molecule (N-CAM) polypeptides by a developmentally regulated sialyltransferase in Golgi-enriched fractions isolated from rat brain is described. The 120-kilodalton polypeptide of N-CAM was found to be sialylated at each developmental age examined. This was in contrast to the 140- and 180-kilodalton N-CAM polypeptides which were only sialylated until postnatal day 10 and from postnatal day 12, respectively. Immunoblotting procedures demonstrated that all N-CAM polypeptides were expressed in the Golgi fractions at each developmental stage examined. The heavily sialylated "embryonic" form of N-CAM was found to be reexpressed at postnatal days 10 and 12, a time coincident with extensive fibre outgrowth. The "embryonic" form of N-CAM incorporated similar amounts of [14C]sialic acid into its constituent polypeptides reflecting the difference in sialic acid to protein ratio, as this form of N-CAM was virtually undetectable in the immunoblots of postnatal material.     

The biological activity of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid in the rat vaginal smear assay

The biological activity of a series of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid has been evaluated in the vaginal smear assay carried out in vitamin A-deficient rats. These analogs were designed to probe the role of the 13-cis isomer in the actions of the parent all-trans-retinoic acid by blocking the interconversion of these two compounds. Although relatively less active, the potency of some of the cyclopropyl analogs suggests that 13-cis-retinoic acid is a fully active metabolite of all-trans-retinoic acid. Since 13-cis-retinoic acid represents a small percentage of the retinoic acid metabolites, the physiological significance of this activity is still unclear. Possible reasons for the reduced activity of the cyclopropyl analogs, as well as an aromatic analog of retinoic acid, are discussed.     

Evidence for a correlation between ambient cholesterol levels and soluble plasma sialyltransferase enzyme activity

While soluble forms of the sialyltransferase (sialyl-T) enzyme have been detected in significant quantities in serum, the exact source(s) of the enzyme, or the factors controlling its secretion are poorly understood. In this study, we have examined the relationship between ambient plasma cholesterol concentrations and sialyl-T activities and also levels of constituent plasma sialoglycoproteins (SGP). There was an inverse relationship between levels of the 2,6 sialyl-T enzyme and both total plasma cholesterol and HDL, although no such relationship was observed for the 2,3 enzyme. While there was no correlation between total cholesterol and the levels of plasma SGPs, there was an inverse relationship between the HDL component and 2,3 SGPs.     

Redox and flavin-binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough).

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3-4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild-type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N-terminal methionine residue. Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is -143 mV (25 degrees C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is -440 mV. The effects of pH on the observed potentials were examined; E2 varies linearly with pH (slope = -59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox-linked protonation of the flavodoxin hydroquinone. D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25 degrees C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd = 0.72 microM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25 degrees C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1 = -262 mV, E2 = -193 mV; 25 degrees C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.     

On the defence strategy of Physa fontinalis (L.), a freshwater pulmonate snail

Summary Physa fontinalis (L.) gives a characteristic, chemically mediated escape response when stimulated by the majority of British leeches and flatworms. The snail responds rapidly and consistently to contact with all the molluskivorous leeches but also to three species which may be considered harmless. However, no response was given to Erpobdella octoculata, the most abundant and widespread of the harmless leeches. The flatworms generally evoked less strong reactions. The adaptive significance of the pattern of responsiveness is discussed. A weaker shell-shaking response is elicited in conspecifics and it is shown that this antisocial behaviour leads to a relatively spaced-out dispersion pattern. A possible adaptive advantage is the reduction of risk of detection by shell-crushing fish predators, to which the snails are otherwise extremely vulnerable.