Subcellular compartmentalization of mRNAs

The importance of the cytoskeleton in protein synthesis was studied in differentiating L6 rat myoblasts. Soluble and cytoskeletal fractions obtained after gentle, non-ionic detergent lysis of myoblasts and myotubes were analysed for the presence of ribosomes and mRNPs. The role of the cytoskeleton in compartmentalization of specific mRNPs in myoblasts and myotubes was investigated. -- mRNPs in polysomal arrays were primarily associated with the cytoskeleton. In addition, the cytoskeletal fraction also contained mRNA in the form of free mRNPs. Therefore, the association of mRNA with the cytoskeleton did not seem to depend on the presence of ribosomes for attachment. Furthermore, analysis of specific mRNAs in the various subcellular fractions of myoblasts and myotubes revealed differences in the distribution pattern of these mRNAs. -- The effects of depolymerizing the microfilaments with cytochalasin B in myoblasts, was investigated. Treatment of myoblasts with cytochalasin B did not result in movement of ribosomes or specific mRNPs from the cytoskeletal fraction to the soluble fraction. This indicates that in L6 myoblasts, ribosomes and mRNPs are not associated with microfilaments. In addition, it was observed that cytochalasin B inhibited the incorporation of precursor into RNA and not protein. The effect on RNA synthesis, however, was due to an inhibition of uptake of precursor uridine, which was found to be reversible

Effect of IL-10 and anti-IL-10 antibody (α IL-10) on in vitro MGC formation.

<p>Figure shows representative Leishman stained pictures of MGC formation and fusion rate of monocytes obtained from healthy controls (n=5) incubated with autologous culture supernatants. (A) unstimulated culture supernatant (Med-SN) (B) Med-SN + IL-10 (C) ConA stimulated culture supernatant (ConA-SN) (D) ConA-SN + IL-10 (E) ConA-SN + α IL-10 (F) ConA-SN + IL-10 + α IL-10 (G) Graph showing effect of IL-10 and anti-IL-10 on percent fusion rate of in vitro MGC formation. Comparability of groups was analyzed by Mann–Whitney U-test. A Bonferroni–Holm procedure was used to correct for multiple comparisons between groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.</p

Cytokine analysis of culture supernatants: Culture supernatants obtained from peripheral mononuclear cells of patients (Pt) and controls (C) incubated with ConA (ConA-SN) and PPD (PPD-SN).

<p>Control wells were seeded with cells without any antigen/mitogen (Med-SN). The levels of (A) IL-2 , (B) TNF-α, (C) IL-10 (D) TGF-β and (E) IL-4 were analysed by ELISA. Comparability of groups was analyzed by Mann–Whitney U-test. A Bonferroni–Holm procedure was used to correct for multiple comparisons between groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.</p

Effect of IL-4 and anti-IL-4 antibody (α IL-4) on in vitro MGC formation.

<p>Figure shows representative Leishman stained pictures of MGC formation and fusion rate of monocytes obtained from healthy controls (n=5) incubated with autologous culture supernatants. (A) unstimulated culture supernatant (Med-SN) (B) Med-SN + IL-4 (C) Med-SN + IL-4 + αIL-4 (D) ConA stimulated culture supernatant (ConA-SN) (E) ConA-SN + IL-4 (F) ConA-SN + α IL-4 (G) ConA-SN + IL-4 + α IL-4 (H) Graph showing effect of IL-4 and anti-IL-4 on percent fusion rate of in vitro MGC formation. Comparability of groups was analyzed by Mann–Whitney U-test. A Bonferroni–Holm procedure was used to correct for multiple comparisons between groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.</p