Hepatic processing and biliary secretion of the cholesteryl esters from beta very-low-density lipoproteins in the rat

beta-Migrating very-low-density lipoproteins (beta-VLDL) are cholesteryl-ester-enriched lipoproteins which accumulate in the serum of cholesterol-fed animals or patients with type III hyperlipoproteinemia. In the rat, beta-VLDL are rapidly cleared by the liver and parenchymal liver cells form the major site for uptake. In this investigation, beta-VLDL were labeled with [3H]cholesteryl esters and the hepatic intracellular transport of these esters was followed. 2 min after injection, the major part of the [3H]cholesteryl esters is already associated with the liver and a significant proportion is recovered in endosomes. Up to 25 min after injection, an increase in radioactivity in the lysosomal compartment is noticed. This radioactivity initially represents cholesteryl esters, while from 25 min onward, radioactivity is mainly present in unesterified cholesterol. Between 45 min and 90 min after beta-VLDL injection, specific transfer of unesterified [3H]cholesterol to the endoplasmic reticulum is observed, while by 3 h the majority is located in this fraction. The appearance of radioactivity in the bile was rather slow as compared to the rapid initial uptake and processing, and up to 5 h after injection only 10% of the injected dose had reached the bile (mainly as bile acids). 72 h after injection, the amount of the injected radioactivity recovered in the bile had increased to 50%. Chloroquine treatment of the rats inhibited the hydrolysis of the cholesteryl esters and the appearance of radioactivity in the bile was retarded. It is concluded that beta-VLDL are rapidly processed by parenchymal liver cells and that the cholesteryl esters from beta-VLDL are hydrolyzed in the lysosomal compartment. Unesterified cholesterol remains associated with the endoplasmic reticulum for a prolonged time, although ultimately the majority will be secreted into the bile as bile acids. The effective operation of this pathway will prevent extrahepatic accumulation of cholesteryl esters from beta-VLDL, while the prolonged residence time of unesterified cholesterol in the endoplasmic reticulum might be important for regulation of low-density lipoprotein (LDL) receptors in liver and thus for LDL levels in the blood.     

Pre-strained epimuscular connections cause muscular myofascial force transmission to affect properties of synergistic EHL and EDL muscles of the rat

BACKGROUND: Myofascial force transmission occurs between muscles (intermuscular myofascial force transmission) and from muscles to surrounding nonmuscular structures such as neurovascular tracts and bone (extramuscular myofascial force transmission). The purpose was to investigate the mechanical role of the epimuscular connections (the integral system of inter- and extramuscular connections) as well as the isolated role of extramuscular connections on myofascial force transmission and to test the hypothesis, if such connections are prestrained. METHOD OF APPROACH: Length-force characteristics of extensor hallucis longus (EHL) muscle of the rat were measured in two conditions: (I) with the neighboring EDL muscle and epimuscular connections of the muscles intact: EDL was kept at a constant muscle tendon complex length. (II) After removing EDL, leaving EHL with intact extramuscular connections exclusively. RESULTS: (I) Epimuscular connections of the tested muscles proved to be prestrained significantly. (1) Passive EHL force was nonzero for all isometric EHL lengths including very low lengths, increasing with length to approximately 13% of optimum force at high length. (2) Significant proximodistal EDL force differences were found at all EHL lengths: Initially, proximal EDL force = 1.18 +/- 0.11 N, where as distal EDL force = 1.50 +/- 0.08 N (mean +/- SE). EHL lengthening decreased the proximo-distal EDL force difference significantly (by 18.4%) but the dominance of EDL distal force remained. This shows that EHL lengthening reduces the prestrain on epimuscular connections via intermuscular connections; however; the prestrain on the extramuscular connections of EDL remains effective. (II) Removing EDL muscle affected EHL forces significantly. (1) Passive EHL forces decreased at all muscle lengths by approximately 17%. However, EHL passive force was still non-zero for the entire isometric EHL length range, indicating pre-strain of extramuscular connections of EHL. This indicates that a substantial part of the effects originates solely from the extramuscular connections of EHL. However, a role for intermuscular connections between EHL and EDL, when present, cannot be excluded. (2) Total EHL forces included significant shape changes in the length-force curve (e.g., optimal EHL force decreased significantly by 6%) showing that due to myofascial force transmission muscle length-force characteristics are not specific properties of individual muscles. CONCLUSIONS: The pre-strain in the epimuscular connections of EDL and EHL indicate that these myofascial pathways are sufficiently stiff to transmit force even after small changes in relative position of a muscle with respect to its neighboring muscular and nonmuscular tissues. This suggests the likelihood of such effects also in vivo.     

Uptake and metabolism of enterolactone and enterodiol by human colon epithelial cells

The enterolignans enterolactone and enterodiol are phytoestrogens that are formed from plant lignans by microorganisms in the human colon. Enterolignans circulate in plasma as conjugates. We hypothesized that conjugation of enterolignans takes place in colon epithelial cells, and studied the time course of uptake and metabolism of enterolactone and enterodiol in three human colon epithelial cell lines. In addition, the conjugates were identified by mass spectrometry with accurate mass measurement (LC/QTOFMS/MS). Intracellular levels of conjugated enterolactone and enterodiol in HT29 cells rose immediately after starting the exposure. This was accompanied by a rapid decrease in free enterolactone and enterodiol in the exposure medium of HT29 and (un)differentiated CaCo-2 but not of CCD841CoTr cells. Conjugation and excretion of enterolactone and enterodiol was complete within 8 h, except for enterodiol in CaCo-2 cells ( approximately 48 h). Enterolactone appears to be more rapidly metabolized and/or excreted than enterodiol, and also the appearance of conjugated enterolactone in medium is less affected by the presence of enterodiol than vice versa. Total (free plus conjugated) enterolignan concentrations remained constant throughout the experiments. Three conjugates were identified in exposure medium of HT29 cells: enterolactone-sulfate, enterolactone-glucuronide, and enterodiol-glucuronide.Taken together, our data suggest that phase II metabolism of enterolactone and enterodiol already may take place during uptake in the colon and that colon epithelial cells may be responsible for this metabolism.     

Muscle force is determined also by muscle relative position: isolated effects

Effects on force of changes of the position of extensor digitorum longus muscle (EDL) relative to surrounding tissues were investigated in rat. Connective tissue at the muscle bellies of tibialis anterior (TA), extensor hallucis longus (EHL) and EDL was left intact, to allow myofascial force transmission. The position of EDL muscle was altered, without changing EDL muscle-tendon complex length, and force exerted at proximal and distal tendons of EDL as well as summed force exerted at the distal tendons of TA and EHL muscles (TA+EHL) were measured. Proximal and distal EDL forces as well as distal TA+EHL force changed significantly on repositioning EDL muscle. These muscle position-force characteristics were assessed at two EDL lengths and two TA+EHL lengths. It was shown that changes of muscle force with length changes of a muscle is the result of the length changes per se, as well as of changes of relative position of parts of the muscle. It is concluded that in addition to length, muscle position relative to its surroundings co-determines isometric muscle force.     

Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering

Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.     

Postnatal development of mucosa-associated lymphoid tissues in chickens

Summary The postnatal development of chicken mucosa-associated lymphoid tissues of the eyes, lungs, and intestines were investigated with monoclonal antibodies specific for either all leucocytes, B lymphocytes, mononuclear phagocytes, IgM, IgG, or IgA. Attention has been paid to the relation of lymphoid infiltrates with their surrounding mucosae, the segregation into B-cell and T-cell areas, development of germinal centers, and secretory immunoglobulins. Abudant secretory IgM and IgA was detected in the epithelium of the Harderian glands in the orbits, even though they lacked large leucocyte infiltrates with germinal centers. Lymphoid tissues in the mucosae of lungs and intestines developed separate B-cell and T-cell areas. The proventriculus, Meckel's diverticulum, and Peyer's patches generally contained germinal centers from 12 weeks of age on. Because chickens as young as 2 weeks old had germinal centers in bronchus-associated lymphoid tissue and cecal tonsils, these areas were probably highly stimulated by antigens. Isotype-specific monoclonal antibodies were used to detect IgM-, IgG-, and IgA-bearing follicular cells in the same germinal center.     

Characteristics of iron release from isolated heavy and light endosomes

1. Endosomes were isolated from K 562 cells after 3 min after the endocytosis of a single cohort of transferrin molecules. 2. The change in 125I/59Fe ratio of heavy and light endosomes, relative to that of the transferrin used and 59Fe from light endosomes. 3. Incubation of heavy and light endosomes with PBS or PIH showed equal ATP specific iron release from both heavy and light endosomes, but in the presence of a NADH/NAD+ redox couple iron release from light endosomes was reduced. 4. Incubation of heavy and light endosomes with PIH and NEM did not completely abolish ATP specific iron release from heavy and light endosomes.     

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

Introduction

Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors.

Methods

Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ).

Results

The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections.

Conclusion

We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.     

Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system.