Chylomicron-chylomicron remnant clearance by liver and bone marrow in rabbits. Factors that modify tissue-specific uptake

The metabolism of [14C]cholesterol- and [3H]retinol-labeled chylomicrons obtained from canine thoracic duct or rabbit mesenteric lymph was investigated in normal fasted rabbits. Typically, 70-80% of the chylomicrons injected into the rabbits were cleared from the plasma in 20 min, and their uptake was accounted for principally by the liver and the bone marrow. Surprisingly, the bone marrow was a major site of uptake; the uptake ranged from about half that of the liver to a nearly equal amount. The importance and specificity of chylomicron-chylomicron remnant uptake by the bone marrow were established by demonstrating that (a) bone marrow throughout the body accumulated these lipoproteins, (b) the level of uptake was consistent regardless of how the values were calculated or how the chylomicrons were prepared, (c) the uptake represented specific binding, and (d) radiolabeled intestinal lipoproteins induced in vivo delivered cholesterol and retinol to the marrow. Electron microscopic examination of the rabbit bone marrow established that perisinusoidal macrophages uniquely accounted for the uptake of the chylomicrons. Whereas liver cleared a variety of both triglyceride-rich lipoproteins (chylomicrons, chylomicron remnants, and very low density lipoproteins) and cholesterol-rich lipoproteins (beta-very low density lipoproteins and high density lipoproteins containing apolipoprotein E), bone marrow uptake appeared to be restricted to the triglyceride-rich lipoproteins. More chylomicron remnants (generated in a hepatectomized rabbit) were cleared by the liver than by the bone marrow, and the addition of excess apolipoprotein E to chylomicrons resulted in their preferential uptake by the liver. The role of chylomicron-chylomicron remnant delivery of lipids or lipid-soluble vitamins to rabbit bone marrow is open to speculation, and whether triglyceride-rich lipoprotein uptake occurs to a significant extent in the bone marrow of humans remains to be determined.     

Role of endogenous gibberellins in germination of melon (Cucumis melo) seeds

The effect of the plant growth retardants ancymidol. mefluidide and uniconazole on germination of two melon accessions differing in their ability to germinate at 14°C was examined. The accessions were the cold sensitive Noy Yizre'el and the cold tolerant Persia 202. The three growth retardants were able to delay the germination of intact Noy Yizre'el seeds, but did not affect that of intact Persia 202 seeds. On the other hand germination of decoated seeds of both accessions was unaffected by these inhibitors at normal oxygen concentration, but was inhibited at 5% oxygen. When gibberellin-like activity was measured by a dwarf rice biological assay following HPLC fractionation, it was found that seeds of Persia 202 contained much more gibberellin-like activity than Noy Yizre'el seeds. Among the extracted compounds several endogenous gibberellins were identified by combined gas chromatography-mass spectrometry (GC-MS). They included GA₄, GA₂₀, GA₁ and GA₃ in Noy Yizre'el and GA₃₄, GA₂₀, GA₁ and GA₈ in Persia 202. It is suggested that the better germination of intact Persia 202 seeds, compared to Noy Yizre'el seeds at low temperature and low oxygen concentration, is due to a higher endogenous level of GA and a better seed coat permeability to oxygen.     

Monitoring the stability of crosslinked protein crystals biotemplates: a feasibility study

Protein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein molecules in the crystal is a complementary 3D array of voids made of interconnected cavities and exhibiting highly ordered porosity. The permeability of the porosity of chemically crosslinked enzyme protein crystals to low molecular weight solutes, was used for enzyme mediated organic synthesis and size exclusion chromatography. This permeability might be extended to explore new potential applications for protein crystals, for example, their use as bio-templates for the fabrication of novel, nano-structured composite materials. The quality of composites obtained from "filling" of the ordered voids in protein crystals and their potential applications will be strongly dependent upon an accurate preservation of the order in the original protein crystal 3D array during the "filling" process. Here we propose and demonstrate the feasibility of monitoring the changes in 3D order of the protein array by a step-by-step molecular level monitoring of a model system for hydrogel bio-templating by glutaraldehyde crosslinked lysozyme crystals. This monitoring is based on step-by-step comparative analysis of data obtained from (i) X-ray crystallography: resolution, unit cell dimensions and B-factor values and (ii) fluorescence decay kinetics of ultra-fast laser activated dye, impregnated within these crystals. Our results demonstrated feasibility of the proposed monitoring approach and confirmed that the stabilized protein crystal template retained its 3D structure throughout the process.     

GluR2 knockdown reveals a dissociation between [Ca2+]i surge and neurotoxicity

Reduction in GluR2 subunit expression and subsequent increases in AMPA receptor mediated Ca(2+) currents were postulated to exacerbate glutamate neurotoxicity following seizures or global ischemia. To directly test the effects of shifting the GluR1/GluR2 subunit ratio on excitotoxicity, GluR2 antisense deoxyoligonucleotides (AS-ODNs) were applied to dissociated hippocampal cultures for 1-8 days. The GluR1/GluR2 protein ratio was examined immunohistochemically and by Western blotting. [Ca(2+)](i) concentrations were determined by ratiometric imaging of Fura 2-loaded cells. The cultures were exposed to glutamate, AMPA, NMDA or kainic acid (KA) 3 days after GluR2 knockdown and cell viability was determined 1 day later by MTT reduction assay or Trypan blue exclusion. Although GluR2 AS-ODNs increased the GluR1/GluR2 protein ratio in a time dependent manner, neurons and glia appeared healthy and MTT reduction values were similar to untreated and sense controls. Basal [Ca(2+)](i) levels were unchanged but [Ca(2+)](i) was selectively increased by agonist stimulation of AMPA receptors. Unexpectedly, delayed neurotoxicity was attenuated at saturating doses of glutamate while little difference in cell viability was observed at lower doses or with the other excitotoxins at any concentration. Therefore, there was a dissociation between rises in AMPA receptor-mediated Ca(2+) influx and neurotoxicity despite marked decreases in GluR2 but not GluR1 immunoreactivity. It is proposed that a modification of AMPA receptor stochiometry that raises agonist-stimulated Ca(2+) influx during an excitotoxic insult may have eventual neuroprotective effects.     

Dendritic spines and long-term plasticity

A recent flurry of time-lapse imaging studies of live neurons have tried to address the century-old question: what morphological changes in dendritic spines can be related to long-term memory? Changes that have been proposed to relate to memory include the formation of new spines, the enlargement of spine heads and the pruning of spines. These observations also relate to a more general question of how stable dendritic spines are. The objective of this review is to critically assess the new data and to propose much needed criteria that relate spines to memory, thereby allowing progress in understanding the morphological basis of memory.     

Identification of novel cell-adhesion molecules in peripheral nerves using a signal-sequence trap

The development and maintenance of myelinated nerves in the PNS requires constant and reciprocal communication between Schwann cells and their associated axons. However, little is known about the nature of the cell-surface molecules that mediate axon-glial interactions at the onset of myelination and during maintenance of the myelin sheath in the adult. Based on the rationale that such molecules contain a signal sequence in order to be presented on the cell surface, we have employed a eukaryotic-based, signal-sequence-trap approach to identify novel secreted and membrane-bound molecules that are expressed in myelinating and non-myelinating Schwann cells. Using cDNA libraries derived from dbcAMP-stimulated primary Schwann cells and 3-day-old rat sciatic nerve mRNAs, we generated an extensive list of novel molecules expressed in myelinating nerves in the PNS. Many of the identified proteins are cell-adhesion molecules (CAMs) and extracellular matrix (ECM) components, most of which have not been described previously in Schwann cells. In addition, we have identified several signaling receptors, growth and differentiation factors, ecto-enzymes and proteins that are associated with the endoplasmic reticulum and the Golgi network. We further examined the expression of several of the novel molecules in Schwann cells in culture and in rat sciatic nerve by primer-specific, real-time PCR and in situ hybridization. Our results indicate that myelinating Schwann cells express a battery of novel CAMs that might mediate their interactions with the underlying axons.     

Interactions between human and carp (Cyprimus carpio) low density lipoproteins (LDL) and LDL receptors

1. We have compared the concentration and chemical composition of carp and human plasma lipoproteins and studied their interaction with human fibroblast LDL receptors. 2. The main lipoproteins in carp are of high density (HDL) in contrast to low density lipoproteins (LDL) in human. 3. Carp lipoproteins are devoid of apolipoprotein (apo) E, a major ligand for interaction with LDL receptors in mammals. 4. Carp very low density lipoproteins (VLDL) and LDL but not HDL nor apoA-I cross react with human LDL in their interaction with LDL receptors on human cultured fibroblasts. 5. Carp liver membranes possess high affinity receptors that are saturable and have calcium dependent ligand specificity (apoB and apoE) similar to human LDL receptor. Carp VLDL and LDL but not HDL nor its major apolipoprotein complexed to L-alpha-phosphatidylcholine dimyristoyl (apoA-I-DMPC) competed with the specific binding of human LDL to this receptor.     

Origin and transport of the A-I and arginine-rich apolipoproteins in mesenteric lymph of rats.

Transport of apolipoprotein A-I and argininerich apolipoprotein in mesenteric lymph was examined in rats given constant intraduodenal infusions of saline, glucose in saline, or emulsified fat. Lymph flow in all groups was constant from 5 to 50 hr after beginning the infusions. Lymphatic transport of triglycerides was about 20-fold greater and transport of apoprotein A-I was about twofold greater in fat-infused rats than in the other two groups. In each group transport of apoprotein A-I bore a significant positive relationship to transport of triglycerides. Lymphatic transport of the arginine-rich apoprotein was only 6-12% of that of apoprotein A-I and was more closely related to lymphatic transport of total protein than to that of triglycerides. In fat-infused rats given [(3)H]lysine intraduodenally, about two-thirds of the (3)H in the chylomicron proteins was in apoprotein A-I and only about 1% was in the arginine-rich apoprotein. Estimated specific activity of chylomicron proteins was highest for apoprotein A-I and apoprotein A-IV, and lowest for the arginine-rich apoprotein and proteins of low molecular weight (mainly C apoproteins). In fat-infused rats given constant intravenous infusions of radioiodinated high density lipoproteins from blood plasma, the specific activity of apoprotein A-I in lymph chylomicrons was only about 5% of that of apoprotein A-I in blood high density lipoproteins, indicating that more than 90% of the apoprotein A-I in chylomicrons was synthesized in the intestine. From these and other data it is concluded that both the intestine and liver are significant sources of apoprotein A-I whereas only the liver synthesizes significant amounts of the arginine-rich apoprotein.