Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. (2004) J. Biol. Chem. 279, 39757-39766). In this study we have investigated the putative functional role of MTP in the initial lipidation of apoB:1000 in stable transformants of McA-RH7777 cells. Inhibition of MTP lipid transfer activity by 0.1 microm BMS-197636 and 5, 10, and 20 microm of BMS-200150 had no detectable effect on the synthesis, lipidation, and secretion of apoB:1000-containing particles. Under identical experimental conditions, the synthesis, lipidation, and secretion of endogenous apoB100-containing particles in HepG2 and parental untransfected McA-RH7777 cells were inhibited by 86-94%. BMS-200150 at 40 microm nearly abolished the secretion of endogenous apoB100-containing particles in HepG2 and parental McA-RH cells but caused only 15-20% inhibition in the secretion of apoB: 1000-containing particles. This modest decrease was attributable to the nonspecific effect of a high concentration of this compound on hepatic protein synthesis, as reflected in a similar (20-25%) reduction in albumin secretion. Suppression of MTP gene expression in stable transformants of McA-RH7777 cells by micro-interfering RNA led to 60-70% decrease in MTP mRNA and protein levels, but it had no detectable effect on the secretion of apoB:1000. Our results provide a compelling argument that the initial addition of phospholipids to apoB:1000 and initiation of apoB-containing lipoprotein assembly occur independently of MTP lipid transfer activity.     

Charged amino acid residues 997-1000 of human apolipoprotein B100 are critical for the initiation of lipoprotein assembly and the formation of a stable lipidated primordial particle in McA-RH7777 cells

We previously demonstrated that a portion, or perhaps all, of the residues between 931 and 1000 of apolipoprotein (apo) B100 are required for the initiation of apoB-containing particle assembly. Based on our structural model of the first 1000 residues of apoB (designated as apoB:1000), we hypothesized that this domain folds into a three-sided lipovitellin-like "lipid pocket" via a hairpin-bridge mechanism. We proposed that salt bridges are formed between four tandem charged residues 717-720 in the turn of the hairpin bridge and four tandem complementary residues 997-1000 located at the C-terminal end of the model. To identify the specific motif within residues 931 and 1000 that is critical for apoB particle assembly, apoB:956 and apoB:986 were produced. To test the hairpin-bridge hypothesis, the following mutations were made: 1) residues 997-1000 deletion (apoB:996), 2) residues 717-720 deletion (apoB:1000Delta717-720), and 3) substitution of charged residues 997-1000 with alanines (apoB:996 + 4Ala). Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. 1) ApoB:956 did not form stable particles and was secreted as large lipid-rich aggregates. 2) ApoB:986 formed both a lipidated particle that was denser than HDL(3) and large lipid-rich aggregates. 3) Compared with wild-type apoB:1000, apoB:1000Delta717-720 displayed the following: (i) significantly diminished capacity to form intact lipidated particles and (ii) increased propensity to form large lipid-rich aggregates. 4) In striking contrast to wild-type apoB:1000, (i) apoB:996 and apoB:996 + 4Ala were highly susceptible to intracellular degradation, (ii) only a small proportion of the secreted proteins formed stable HDL(3)-like lipoproteins, and (iii) a majority of the secreted proteins formed large lipid-rich aggregates. We conclude that the first 1000 amino acid residues of human apoB100 are required for the initiation of nascent apoB-containing lipoprotein assembly, and residues 717-720 and 997-1000 play key roles in this process, perhaps via a hairpin-bridge mechanism.     

In vivo bone regeneration using a bioactive nanocomposite scaffold and human mesenchymal stem cells

Cell and Tissue Banking - Due to the osteoconductive role of bioceramics, use of these bioactive nanocomposite scaffolds that can maintain their structural integrity during bone tissue...     

MEK7-dependent activation of p38 MAP kinase in keratinocytes

Previous studies suggest that a PKC/Ras/MEKK1 cascade regulates involucrin (hINV) gene expression in human epidermal keratinocytes. MEK7, which is expressed in epidermis, has been identified as a member of this cascade (Efimova, T., LaCelle, P., Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395 and Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601-1607). However, the kinase that functions downstream of MEK7 has not been identified. Our present studies show that MEK7 expression in keratinocytes markedly activates p38alpha and modestly activates JNK. Activation of p38 MAPK by MEK7 is a novel finding, as previous reports have assigned MEK7 as a JNK regulator. We also demonstrate that this regulation is physiologically important, as the p38alpha- and JNK-dependent activities regulate hINV promoter activity and expression of the endogenous hINV gene.     

Increased production of apolipoprotein B and its lipoproteins by oleic acid in Caco-2 cells

The production of lipids, apolipoproteins (apo), and lipoproteins induced by oleic acid has been examined in Caco-2 cells. The rates of accumulation in the control medium of 15-day-old Caco-2 cells of triglycerides, unesterified cholesterol, and cholesteryl esters were 102 +/- 8, 73 +/- 5, and 11 +/- 1 ng/mg cell protein/h, respectively; the accumulation rates for apolipoproteins A-I, B, C-III, and E were 111 +/- 9, 53 +/- 4, 13 +/- 1, and 63 +/- 4 ng/mg cell protein/h, respectively. Whereas apolipoproteins A-IV and C-II were detected by immunoblotting, apoA-II was absent in most culture media. In contrast to an early production of apolipoproteins A-I and E occurring 2 days after plating, the apoB expression appeared to be differentiation-dependent and was not measurable in the medium until the sixth day post-confluency. In the control medium, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL), and lipid-poor very high density lipoproteins (VHDL) accounted for 12%, 46%, 18%, and 24% of the total lipid and apolipoprotein contents, respectively. The triglyceride-rich VLDL contained mainly apoE (75%) and apoB (23%), while the protein moiety of LDL was composed of apoB (59%), apoE (20%), apoA-I (15%), and apoC-III (6%). The cholesterol-rich HDL contained mainly apoA-I (69%) and apoE (27%). In the control medium, major portions of apolipoproteins B and C-III (93-97%) were present in LDL, whereas the main parts of apoA-I (92%) and apoE (76%) were associated with HDL and VHDL. Oleate increased the production of triglycerides 10-fold, cholesteryl esters 7-fold, and apoB 2- to 4-fold. There was also a moderate increase (39%) in the production of apoC-III but no significant changes in those of apolipoproteins A-I and E. These increases were reflected mainly in a 55-fold elevation in the concentration of VLDL, and a 2-fold increase in the level of LDL; there were no significant changes in HDL and VHDL. VLDL contained the major parts of total neutral lipids (74-86%), apoB (65%), apoC-III (81%) and apoE (58%). In the presence of oleate, the VLDL, LDL, HDL, and VHDL accounted for 76%, 15%, 3%, and 6% of the total lipoproteins, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hedgehog signalling as an antagonist of ageing and its associated diseases

Hedgehog is an important morphogenic signal that directs pattern formation during embryogenesis, but its activity also remains present through adult life. It is now becoming increasingly clear that during the reproductive phase of life and beyond it continues to direct cell renewal (which is essential to combat the chronic environmental stress to which the body is constantly exposed) and counteracts vascular, osteolytic and sometimes oncological insults to the body. Conversely, down-regulation of hedgehog signalling is associated with ageing-related diseases such as type 2 diabetes, neurodegeneration, atherosclerosis and osteoporosis. Hence, in this essay we argue that hedgehog signalling is not only important at the start of life, but also constitutes an important anti-geriatric influence, and that enhanced understanding of its properties may contribute to developing rational strategies for healthy ageing and prevention of ageing-related diseases. Also watch the Video Abstract.     

Effect of preservation of human semen sample at 4–6 and 25 °C on sperm motility

Sperm motility is the result of transverse movements that exist along its tail. It plays an important role in male fertility. The aim of this study was to evaluate the influence of keeping washed normozoospermic semen samples at 4–6 and 25 °C on the motility of spermatozoa. 26 semen samples of normozoospermic were washed twice in modified Ham’s F10 medium. Then, thirteen of the semen samples were kept in refrigerator (4–6 °C) and the remaining samples were stored in incubator (25 °C) for 12 days. On the 0 (immediately after sampling as control group), 1st, 2nd, 5th, 7th and the 12th days, the percentage of fast progressive (grade a), slow progressive (grade b), non-progressive (grade c) and immotile (grade d) sperm cells were calculated for each temperature. The data obtained from this study showed that the percentages of a, b and c grades of motile spermatozoa were significantly decreased (p?<?0.001) during 12 days at the both temperatures but reduction of these percentages has a gentle slope at 4–6 °C. There was no motile sperm after 12 days of storage. This study suggests that motile spermatozoa could be retrieved up to 7 days after the storage of washed normozoospermic men semen samples at 4–6 and 25 °C. Also, there were no motile sperm cells 12 days after sampling.     

Race differences in the prevalence of the factor V Leiden mutation in Kuwaiti nationals

Factor V Leiden mutation (FVL; G1691A) is an established risk factor for venous thromboembolic disorders. FVL was reported with high prevalence in Caucasians (1–15%) but was absent in non-Caucasians like Africans and Asians. Studies reported FVL in 5–27% of Arabs and non-Arabs living in the Middle Eastern countries northern to the Arabian Peninsula, but was almost absent in Arabs in the Arabian Peninsula itself. Kuwait is an Arabic country present on the northern border of the Arabian Peninsula, and Kuwaitis are originally from Saudi Arabia (Southern to Kuwait and within the Arabian Peninsula) or from Iran and Iraq (northern to Kuwait and the Arabian Peninsula). This study was conducted to study FVL in Kuwaitis in relation to their origin. Real-time PCR was performed on DNA samples of 285 apparently healthy Kuwaitis using specially designed primers and probes for FVL. There were 109 Kuwaitis of Iranian origin, 71 of Iraqi origin and 105 of Saudi origin. FVL was present in 7 and 5 Kuwaitis of Iranian and Iraqi origin, respectively. None of the Kuwaitis of Saudi origin had the mutation. Prevalence of FVL in Kuwaitis of Iranian (6.42%) and Iraqi (7.04%) origin were statistically different from prevalence in Kuwaitis of Saudi (0%) origin (P-value < 0.05). No difference was found between females and males (P-value > 0.6). In conclusion, FVL is present in Kuwaitis of Iranian or Iraqi origin only. Therefore, testing and providing genetic consultation for FVL may be needed in those Kuwaitis only which should save time, cost and efforts. However, this assumption should be confirmed by other studies and on larger number of cases.