Caries experience and treatment needs of five-year-old caucasian nursery school children in Pretoria.

The caries experience of five-year-old caucasian children at various sites in the world at the present time could be termed moderate to low, and a declining tendency has been noted in the past decade. The aim of this study was to determine the current status and trends of caries experience of 5-year-old caucasian nursery school children in Pretoria. The results from this study, conducted in 1987, showed that 50.9 per cent of 5-year-olds were free of caries experience (dmft = 0) and that the mean dmft was low, viz. 1.97. A comparison of these results with figures published in 1979 indicates that a notable reduction in caries experience has taken place during a period of eight years, the proportion of 5-year-olds free of caries experience having been 40 per cent in 1979, with a mean dmft of 2.9. The most significant change during this period was the decrease in the decayed component of the dmft.

Decreased fucose incorporation in cell surface carbohydrates is associated with inhibition of invasion.

Invasion of malignant MO4 cells into embryonic chick heart fragments in an organ culture assay was arrested for at least 7 days when the temperature was lowered to 28 degrees C. Prolonged culturing of MO4 cells at 28 degrees C on tissue culture substrates showed no recuperation of fucose incorporation into cell surface glycopeptides. However, invasion was restored after 10 days of organ culture in confrontation with chick heart tissue at 28 degrees C. A histoautoradiographic study showed that the regained capability to invade was accompanied by an increase in fucose labeling of the MO4 cells in the invading areas. At 28 degrees C the incorporation of [3H]fucose into total cell protein was drastically reduced, whereas [3H]leucine incorporation as a measure for protein synthesis was less affected. Cell surface glycopeptides, metabolically labeled with either fucose or glucosamine at 28 degrees C, showed a time-dependent decrease in the incorporation of fucose but not of glucosamine and no changes in overall size distribution. Low temperature did not reduce fucosyltransferase activity but the relative accumulation of fucose-1-P suggested inhibited conversion towards GDP-fucose. Moreover, mouse L cells which were incapable of invading chick heart tissue appeared also deficient in fucose incorporation, owing to low levels of fucosyltransferase activity. According to the results, fucosylation of surface carbohydrates may be required for invasive capacity and restored in MO4 cells invading at 28 degrees C by metabolic cooperation with the host tissue.

Establishment and characterization of long-term primary mouse urothelial cell cultures.

Long-term mouse urothelial cell cultures were routinely established from explants of neonatal mouse bladders. Foci of proliferating cells could be observed one week after the initiation of the explant cultures. These persisted throughout the culture period and up to one year. Expression of keratin proteins confirmed the epithelial nature of the cultured cells. Morphologic analysis of nuclei sorted after DNA flow cytometry revealed a population of DNA-tetraploid and octoploid cells with large nuclei and prominent nucleoli in addition to a DNA-diploid cell population. Both cell populations showed DNA replicative activity as reflected by bromodeoxyuridine incorporation studies and mitotic activity. These long-term primary mouse urothelial cell cultures may prove useful for studies on urothelial cell kinetics and bladder carcinogenesis.

Modification of cell surface carbohydrates and invasive behavior by an alkyl lysophospholipid.

The effect of the alkyl lysophospholipid racemic-1-O-octadecyl-2-O-methyl glycero-3-phosphocholine on the expression of cell surface carbohydrates of four matched pairs of normal and malignant cells was studied using chromatographic techniques. After treatment with alkyl lysophospholipid, glycopeptides proteolytically derived from normal and malignant cells displayed a shift in the size distribution profiles obtained by gel filtration. These drug-induced changes in molecular weight distribution were expressed most strongly in untransformed cells and resembled the carbohydrate alterations found after their malignant transformation. Desialylation abolished the effect of alkyl lysophospholipid, thus suggesting an increased amount of sialic acid in the surface carbohydrates of drug-treated cells. Chromatography of glycopeptides on concanavalin A-Sepharose, Ricinus communis agglutinin I-agarose, and Bio-Gel P-4 columns excluded a higher degree of branching but suggested addition of extra terminal sialic acid residues as the major cause of the observed alterations. Alkyl lysophospholipid stimulated glycoprotein sialylation of normal cells to the level observed in malignant cells, thus inducing a "malignant-like" surface phenotype. The drug-induced carbohydrate changes in normal chick heart tissue prevented its being invaded by tumor cells when tested in an organotypic assay. The alkyl lysophospholipid thus appears to modulate in a nontoxic fashion the expression of surface molecules implicated in various cellular interactions including invasiveness.

Mitogenic and antimitogenic effects of cholera toxin-mediated cyclic AMP levels in 3T3 cells.

The effect of time-controlled exposures to cholera toxin (CT) on intracellular levels of cyclic AMP (cAMP) and on the proliferative response of serum-stimulated 3T3 cells was investigated. Continuous exposure to CT caused up to 8-fold raises in cAMP content and inhibited DNA replication by delaying G1-S transition and by reducing the fraction of cells committed to DNA replication. In contrast, short exposures to CT during G0-G1 transition increased the fraction of cells responding to serum stimulation and potentiated the serum-induced morphological changes in the cell monolayer. A short exposure during late G1 phase, however, inhibited the onset of DNA synthesis but had little effect on ongoing DNA replication. The results indicate that cAMP has diverse and opposite effects on two defined restriction points in cell cycle control. Cyclic AMP was positively involved in the acquisition of the state of competence by quiescent cells (G0-G1 transition) but antagonistic on the onset of DNA replication (G1-S transition) in committed cells. The observations reconcile a number of controversial conclusions regarding the role of cAMP in cell cycle control.

Specific inhibition of human natural killer cell-mediated cytotoxicity by sialic acid and sialo-oligosaccharides.

We have tried to identify carbohydrate structures involved in recognition and/or lysis of K562 target cells by human natural killer (NK) cells. Inhibition studies were performed with mono-, di- and trisaccharides, and with glycopeptides and glycoproteins of known carbohydrate composition. When tested with various monosaccharides, lysis of K562 cells was inhibited only by N-acetylneuraminic acid (NeuAc). Di- and trisaccharides and glycopeptides containing NeuAc or N-glycolylneuraminic acid (NeuGc) all inhibited NK cell-mediated lysis. Among the non-sialylated carbohydrates tested, only Gal beta(1----3)GalNAcol was effective. The inhibitory capacity of sialylated compounds appeared to be dependent on the linkage type of the sialic acid residue; carbohydrates containing alpha(2----6)-linked sialic acids were more potent inhibitors than their alpha(2----3) isomers. Also the sugar to which the sialic acid residue was attached was of importance, NeuAc alpha(2----6)GalNAcol being more effective than NeuAc alpha(2----6)Gal beta 1----R (where R = glucose or oligosaccharide-peptide). Sialylated compounds and free sialic acid had minor or no effects on cell-mediated cytotoxicity by allo-sensitized cytotoxic T lymphocytes. The conjugation of target cells and NK effector cells was not inhibited by carbohydrates that effectively blocked the cytolytic response. These results may indicate that cell-surface carbohydrates containing alpha(2----6)-linked sialic acid are crucial structures in a post-binding event in NK-cell-mediated lysis.

Effect of cancer-related and drug-induced alterations in surface carbohydrates on the invasive capacity of mouse and rat cells.

The effect of alterations in cell surface carbohydrates on invasion of mouse and rat cells into embryonic chick heart fragments in organ culture was studied. Matching pairs of malignant and nonmalignant cells, including all categories of carcinogenic induction (i.e., viral, chemical, or oncogenic), were compared for their alterations in cell surface carbohydrates and invasive behavior. Glycopeptides derived from the surface of malignant cells expressed cancer-related changes in carbohydrate composition, demonstrated by gel filtration chromatography as a shift in size distribution in comparison with those from nonmalignant counterparts. This phenotypic property strictly correlated with the acquisition of the invasive capacity. Morphological transformation of cells without simultaneous alteration in surface carbohydrates was, however, insufficient for invasion. To test the possible mechanistic role of altered surface carbohydrates in the invasive capacity of cells, the surface molecules of noninvasive cells were modified by incubation with an alkyl-lysophospholipid (racemic 1-O-octadecyl-2-O-methyl glycero-3-phosphocholine). Alkyl-lysophospholipid induced an increase in surface sialylation resembling the changes found in malignant and invasive cells. After pretreatment with alkyl-lysophospholipid, morphologically transformed but nonmalignant and noninvasive cells became able to invade chick heart tissue. These findings indicate that alterations in cell surface carbohydrates, induced by entirely different mechanisms, endowed cells with invasive capacity.

Effect of temperature on invasion of MO4 mouse fibrosarcoma cells in organ culture.

Invasion by MO4 mouse fibrosarcoma cells into fragments of embryonic chick heart or lung in organ culture was studied histologically and ultrastructurally at various temperatures between 12 and 40 degrees C. Invasion was absent for at least 7 days at or below temperatures of 29 degrees C. Invasion was invariably observed at or above 30.5 degrees C. Differences in invasion between 29 and 30.5 degrees C could not be ascribed to differences in growth, migration, or microtubule assembly/disassembly of MO4 cells. Neither could they be explained through differences in the attachment of MO4 cells to the heart fragments. Possible explanations for the absence of invasion at lower temperature are: altered resistance of the extracellular matrix in heart or lung fragments, and deficient expression of fucosylated glycoproteins at the surface of MO4 cells. A population of MO4 cells plated from the parent line and adapted to grow at 28 degrees C (MO(4)28 cell line) did not differ in invasiveness from the parent MO4 cells. We conclude that the temperature dependence of invasion in organ culture might indicate as yet unexplored aspects of the mechanisms of tumour invasion.

Depressor effects, drug concentration and cholinesterase activities in the brain after administration of paraoxon in the cat.

After simultaneous administration of paraoxon into both the left and right vertebral artery (4-8 micrograms) or into a femoral vein (150-550 micrograms.kg-1) of the anaesthetized cat, dose-dependent drug concentrations are measured in various brain regions. These amounts induce inhibition of brain acetylcholinesterase activity and dose-dependent depressor effects. Although paraoxon is rapidly eliminated from the CNS, enzyme activity remains at a low level. After central application of paraoxon into both vertebral arteries no detectable amounts are found in the hypothalamus and acetylcholinesterase activity in that brain region is not or only slightly affected. Curves representing the relationship between the decrease in blood pressure and the concentration of paraoxon or enzyme inhibition in the medulla oblongata are steep. Also, a steep dose-response curve for the depressor response to paraoxon is found. A reduction of brain enzyme activity to about 35% has no influence on blood pressure. However, inhibition by 65-100% induces dose-dependent depressor effects. The results support earlier findings that the depressor response to paraoxon is mediated by a central mechanism. This site of action is probably located within the medulla oblongata region.

Quantitative determination of the cholinesterase inhibitor physostigmine in brain tissue samples using reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic method, based on a dynamic cation-exchange system was used for the determination of physostigmine in brain tissue extracts. The precision and detection limit of the method as well as the extraction efficiency were established. The distribution of physostigmine over several parts of the brain after intravertebral application is reported.

Expression of chronic myeloid leukaemia-associated changes in membrane glycopeptides in somatic cell hybrids.

Hybrid cell lines, derived from fusion of rodent cells with peripheral leukocytes from patients with chronic myeloid leukaemia (CML) and from normal donors, were assayed for the present of CML-associated changes in the carbohydrate moieties of membrane glycoproteins. Expression of this characteristic phenotype did occur in several hybrid clones derived from fusions both with leukaemic and normal human leukocytes. But, expression was only observed when tumourigenic rodent cells were used as fusion partners, and was not observed after fusion with non-tumourigenic rodent cells. Chromosome analysis of the hybrid cells did not reveal a single human chromosome- or chromosomal aberration (e.g. the Philadelphia chromosome)-bearing factor(s) responsible for this expression. The phenotypic expression observed could be due to dissociation of certain human genes from their regulator(s), as a result of chromosome loss in interspecific cell hybrids.

The assay of fentanyl and its metabolites in plasma of patients using gas chromatography with alkali flame ionisation detection and gas chromatography-mass spectrometry.

Fentanyl was determined using gas chromatography (GC) and alkali flame ionisation detection (AFID), in the plasma of patients who had received a high single dose (up to 60 microgram/kg body weight). The relative standard deviation is 6% for 11 ng/ml while the calculated detection limit is 3.3 ng of fentanyl per 1 ml of plasma. The concentration of fentanyl in patients ranged from 40 to 3 ng/ml of plasma. The concentration of fentanyl in patients ranged from 40 to 3 ng/ml of plasma in the first hour after administration. In the plasma of patients treated with fentanyl two metabolites could be detected and identified using GC-AFID and GC-MS.

Selective effects of trypsinization on established and tumour-derived mouse 3T3 cells.

Explanted tumour cells are much more sensitive to the deleterious effects of routine trypsinization than are the parent 3T3 or SV40 transformed established cell lines. This differential sensitivity causes the disappearance of tumour-derived cells when grown in co-culture with untransformed 3T3 cells and accounts in some tumor explants for the emergence of trypsin-resistant varient cells which have lost tumour-specific properties.

Determination of the metabolites of bezitramide in urine. I. Acidic metabolite.

Two methylation methods are compared in relation to the determination of low levels (less than microgram/ml) of the acidic metabolite of bezitramide in human urine. It was necessary to use alkali flame ionisation detector, which specifically detects nitrogen-containing compounds. Several difficulties associated with the use of this detector are described.

The regional localisation of a new potent centrally acting antihypertensive agent R 28935 and its less active threo-isomer R29814 in the cat brain.

Systemic administration of the centrally acting antihypertensive agent R 28935 to cats resulted in a long lasting decrease of mean arterial pressure (+/-30%) whereas the same dose of the threo-isomer R 29814 was ineffective. The antihypertensive activity was due to the unaltered drug. In spite of an identical log P, pKa, dose and a comparable plasma level, the concentration of R 28935 in all the brain areas tested was about twice that of the threo-isomer, suggesting a stereoselective uptake and/or binding of R 28935.