Altered Lipid Droplet Dynamics in Hepatocytes Lacking Triacylglycerol Hydrolase Expression

Lipid droplets (LDs) form from the endoplasmic reticulum (ER) and grow in size by obtaining triacylglycerols (TG). Triacylglycerol hydrolase (TGH), a lipase residing in the ER, is involved in the mobilization of TG stored in LDs for the secretion of very-low-density lipoproteins. In this study, we investigated TGH-mediated changes in cytosolic LD dynamics. We have found that TGH deficiency resulted in decreased size and increased number of LDs in hepatocytes. Using fluorescent fatty acid analogues to trace LD formation, we observed that TGH deficiency did not affect the formation of nascent LDs on the ER. However, the rate of lipid transfer into preformed LDs was significantly slower in the absence of TGH. Absence of TGH expression resulted in increased levels of membrane diacylglycerol and augmented phospholipid synthesis, which may be responsible for the delayed lipid transfer. Therefore, altered maturation (growth) rather than nascent formation (de novo synthesis) may be responsible for the observed morphological changes of LDs in TGH-deficient hepatocytes.

The effect of two different oral antiseptics on dental plaque formation (De Novo Biofilm) and on gingival inflammation

Introduction. Since the discovery that periodontal diseases are caused by microbial plaque the interest of many scientists has been focused on oral antiseptics. There are very few mouthrinses with oral antiseptic effect originally designed in our country. One of these is Ozosept? solution. Objective. This study evaluated the effect of Ozosept? solution (phenol compound) on the oral hygiene and gingival inflammation, in comparison with Hibidex DAP? solution (chlorhexidine digluconate). Methods. Two groups, each of 21 persons, which did not significantly differ concerning Silness-L?e plaque index (PI) and L?e-Sillnes gingival index (GI), used one of the studied oral antiseptic for a 15-day period. Oral hygiene was maintained by subjects' habitual home methods, and no technique of professionally advised brushing was performed during the experimental period. Results. At the end of the study, PI and GI scores were lowered to a statistically high significance in both groups of participants in comparison to the indexes at the beginning of the study. At the end of the study, PI and GI scores did not significantly differ between the two analyzed groups. No side effects, which were recorded in the Hibidex DAP? group (tooth and filling staining 9.5% and 4.74% respectively, transitory tongue numbness 28.6% and reduced taste sensation 14.3%), were registered in the group of subjects using Ozosept? solution. Conclusion. It is concluded that Ozosept? solution is effective in the control of dental plaque - biofilm accumulation and gingival inflammation, and produces no side effects related to chlorhexidine digluconate usage.

Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins

Many proteins found in mineralized tissues have been proposed to function as regulators of the mineralization process, either as nucleators or inhibitors of hydroxyapatite (HA) formation. We have studied the HA-nucleating and HA-inhibiting properties of proteins from bone [osteocalcin (OC), osteopontin (OPN), osteonectin (ON) and bone sialoprotein (BSP)], dentine [phosphophoryn (DPP)] and calcified cartilage [chondrocalcin (CC)] over a wide range of concentrations. Nucleation of HA was studied with a steady-state agarose gel system at sub-threshold [Ca]×[PO4] product. BSP and DPP exhibited nucleation activity at minimum concentrations of 0.3 μg/ml (9 nM) and 10 μg/ml (67 nM) respectively. OC, OPN, ON and CC all lacked nucleation activity at concentrations up to 100 μg/ml. Inhibition of HA formation de novo was studied with calcium phosphate solutions buffered by autotitration. OPN was found to be a potent inhibitor of HA formation [IC50 = 0.32 μg/ml (0.01 μM)] whereas OC was of lower potency [IC50 = 6.1 μg/ml (1.1 μM)]; BSP, ON and CC all lacked inhibitory activity at concentrations up to 10 μg/ml. The effect of OPN on HA formation de novo is mainly to inhibit crystal growth, whereas OC delays nucleation. These findings are consistent with the view that BSP and DPP may play roles in the initiation of mineralization in bone and dentine respectively. OPN seems to be the mineralized tissue protein most likely to function in the inhibition of HA formation, possibly by preventing phase separation in tissue fluids of high supersaturation.

Investigation of microtubule assembly and organization accompanying tension-induced neurite initiation

Pulling on the margin of embryonic chick sensory neurons induces neurite formation de novo. We find that these neurites contain microtubules within minutes after the application of tension and apparently normal microtubule arrays within 10–20 min. We wished to determine whether these microtubules reflected existing microtubules that were reorganized, e.g. pulled into the neurite by the applied forces, or whether they reflected primarily new assembly of tubulin. We investigated tension-induced neurite initiation in the presence of 4 nM vinblastine, a concentration that poisons net microtubule assembly but does not depolymerize extant polymers, thus separating new assembly from movements of existing microtubules. We find that vinblastine seriously compromises the ability of chick sensory neurons to initiate neurites in response to tension. The few poisoned neurites that did form were abnormal in several respects. In contrast to unpoisoned cells, poisoned neurites were prone to stretching and breaking while pulling, as though they lacked normal structural support. Indeed, poisoned neurites possessed only short microtubule fragments. We conclude that the microtubule array seen in tension-induced neurites reflects primarily new microtubule assembly, rather than existing microtubules that were reorganized to invade the neurite. This implies that tension applied to unpoisoned chick sensory neurons rapidly stimulates new microtubule assembly concomitant with neurite initiation. Examination of the tension-induced microtubules shows that both their spatial pattern and their acetylation are similar to that reported for normal growth cone-mediated neurites.

The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin

The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and ‘half-maximal concentration’ values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.

Phosphatidylglycerol of rat lung Intracellular sites of formation de novo and acyl species pattern in mitochondria, microsomes and surfactant

The subcellular site of phosphatidylglycerol (PG) formation for lung surfactant has not been convincingly clarified. To approach this problem we analysed the acyl species pattern of lung PG in mitochondria, microsomes and surfactant by h.p.l.c. separation of its 1,2-diacyl-3-naphthylurethane derivatives. Both mitochondrial and microsomal PG proved identical with surfactant PG, containing the major species 1-palmitoyl-2-oleoyl-PG and 1,2-dipalmitoyl-PG. The fatty acid composition of mitochondrial PG differs markedly from that of diphosphatidylglycerol. This may be taken as an indication that mitochondrial PG is synthesized on purpose to form surfactant, rather than being only the precursor of diphosphatidylglycerol. In vitro, sn-[U-14C]glycerol 3-phosphate incorporation into PG of mitochondria or microsomes occurs in the presence of CTP, ATP and CoA but independently of the supply of exogenous lipoidic precursors. Although the rate in vitro of autonomous PG synthesis, and the endogenous PG content, are higher in mitochondria than in microsomes, it is assumed that both subcellular fractions are involved in PG formation for surfactant.

Formation de novo and development of neuromuscular junctions in vitro

The purpose of this study has been to examine, by means of isolation in vitro, the conditions under which neuromyal junctions develop. More than a hundred years ago Doyère (1840) observed in the water-bear Milnesium tardigradum that the nerve fibers terminate in characteristic eminences of muscle fibers. This observation, made on an arthropod, stimulated research into the connexion between muscle and nerve fiber, and resulted in abandonment of the older concept that motor nerves after having formed loops around the muscles return to the central nervous system as sensory pathways. Although investigations during the intervening century have identified and characterized morphologically the ‘motor end plate’ (as it was designated by Krause, 1863), and have revealed microscopic and fine-structural differences between types of motor endings, there is as yet no general agreement on the respective roles played by nerve, muscle and ambient influences in the development of the neuromuscular junction.