IIA Form Research Articles

Purse-string contraction guides mechanical gradient-dictated heterogeneous migration of epithelial monolayer.

Mechanical heterogeneity has been recognized as an important role in mediating collective cell migration, yet the related mechanism has not been elucidated. Herein, we fabricate heterogeneous stiffness gradients by leveraging microelastically-patterned hydrogels with varying periodic distance. We observe that a decrease in the periodic distance of the mechanical heterogeneity is accompanied by an overall increase in the velocity and directionality of the migrating monolayer. Moreover, inhibition of ROCK- and myosin ⅡA- but not Rac1-mediated contraction reduces monolayer migration on the mechanically heterogeneous substrates. Furthermore, we find that F-actin and myosin ⅡA form purse-string at the leading edge on the mechanically heterogeneous substrates. Together, these findings not only show that the orientational cell-cell contraction promotes collective cell migration under the mechanical heterogeneity, but also demonstrate that the mechanosensation arising from large-scale cell-cell interactions through purse-string formation mediated cell-cell orientational contraction can feed back to regulate the reorganization of epithelial tissues. STATEMENT OF SIGNIFICANCE: By detecting the links between heterogenous rigidity and collective cell migration behavior at the molecular level, we reveal that collective cell migration in the mechanical heterogeneity is driven by ROCK- and myosin-ⅡA-dependent cytoskeletal tension. We confirm that cytoskeletal tension across the epithelial tissue is holistically linked through F-actin and myosin-ⅡA, which cooperate to form purse-string structures for modulating collective tissue behavior on the exogenous matrix with mechanical heterogeneity. Mechanical heterogeneity initiates tissue growth, remodelling, and morphogenesis by orientating cell contractility. Therefore, tensional homeostasis across large-scale cell interactions appears to be necessary and sufficient to trigger collective tissue behavior. Overall, these findings shed light on the role of mechanical heterogeneity in tissue microenvironment for reorganization and morphogenesis.

Uncovering photo-excited intramolecular charge transfer and ESIPT mechanism for 5,5′-(9,9-dioctyl-9H-fluorene-2,7-diyl) bis(2-benzo[d]thiazol-2-yl) phenol compound

In view of their facile synthetic routes and remarkable photo-induced stabilities, excited state intramolecular proton-transfer (ESIPT)-vibrant luminous materials have drawn more and more attention. In this work, we mainly focus on the photo-induced excitation behaviours, including excited state hydrogen bonding interactions and ESIPT reaction of a new 5,5′-(9,9-dioctyl-9H-fluorene-2,7-diyl)-bis(2-benzo[d]thiazol-2-yl) phenol molecule theoretically. To be consistent with the previous experiment, we name this molecule as IIa. Using molecular electrostatic potential (MEP), we explored the dual intramolecular hydrogen bonding interactions in IIa form. Then on the level of chemical geometries and infrared (IR) spectra, we confirmed that the double hydrogen bonds should be enhanced upon photoexcitation. Probing into charge reorganisation, we found that hydrogen proton does prefer to be attracted by enhanced electronic densities around N3 and N6. Combining potential energy surfaces (PESs) and electronic spectra, the excited state intramolecular single proton-transfer (ESISPT) behaviour could be verified for IIa although it owns two hydrogen bonds.

Patient-Derived Skeletal Dysplasia Induced Pluripotent Stem Cells Display Abnormal Chondrogenic Marker Expression and Regulation byBMP2andTGFβ1

Skeletal dysplasias (SDs) are caused by abnormal chondrogenesis during cartilage growth plate differentiation. To study early stages of aberrant cartilage formation in vitro, we generated the first induced pluripotent stem cells (iPSCs) from fibroblasts of an SD patient with a lethal form of metatropic dysplasia, caused by a dominant mutation (I604M) in the calcium channel gene TRPV4. When micromasses were grown in chondrogenic differentiation conditions and compared with control iPSCs, mutant TRPV4-iPSCs showed significantly (P<0.05) decreased expression by quantitative real-time polymerase chain reaction of COL2A1 (IIA and IIB forms), SOX9, Aggrecan, COL10A1, and RUNX2, all of which are cartilage growth plate markers. We found that stimulation with BMP2, but not TGFβ1, up-regulated COL2A1 (IIA and IIB) and SOX9 gene expression, only in control iPSCs. COL2A1 (Collagen II) expression data were confirmed at the protein level by western blot and immunofluorescence microscopy. TRPV4-iPSCs showed only focal areas of Alcian blue stain for proteoglycans, while in control iPSCs the stain was seen throughout the micromass sample. Similar staining patterns were found in neonatal cartilage from control and patient samples. We also found that COL1A1 (Collagen I), a marker of osteogenic differentiation, was significantly (P<0.05) up-regulated at the mRNA level in TRPV4-iPSCs when compared with the control, and confirmed at the protein level. Collagen I expression in the TRPV4 model also may correlate with abnormal staining patterns seen in patient tissues. This study demonstrates that an iPSC model can recapitulate normal chondrogenesis and that mutant TRPV4-iPSCs reflect molecular evidence of aberrant chondrogenic developmental processes, which could be used to design therapeutic approaches for disorders of cartilage.

Characterization of Three Full-length Human Nonmuscle Myosin II Paralogs

Nonmuscle myosin IIs (NM IIs) are a group of molecular motors involved in a wide variety of cellular processes including cytokinesis, migration, and control of cell morphology. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene. These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. Most previous studies on NM II used tissue-purified protein or expressed fragments of the molecule, which presents potential drawbacks for characterizing individual paralogs of the intact protein in vitro. To circumvent current limitations and approach their native properties, we have successfully expressed and purified the three full-length human NM II proteins with their light chains, using the baculovirus/Sf9 system. The enzymatic and structural properties of the three paralogs were characterized. Although each NM II is capable of forming bipolar filaments, those formed by IIC tend to contain fewer constituent molecules than those of IIA and IIB. All paralogs adopt the compact conformation in the presence of ATP. Phosphorylation of the regulatory light chain leads to assembly into filaments, which bind to actin in the presence of ATP. The nature of interactions with actin filaments is shown with different paralogs exhibiting different actin binding behaviors under equivalent conditions. The data show that although NM IIA and IIB form filaments with similar properties, NM IIC forms filaments that are less well suited to roles such as tension maintenance within the cell.

Molecular properties and fibril ultrastructure of types II and XI collagens in cartilage of mice expressing exclusively the α1(IIA) collagen isoform

Until now, no biological tools have been available to determine if a cross-linked collagen fibrillar network derived entirely from type IIA procollagen isoforms, can form in the extracellular matrix (ECM) of cartilage. Recently, homozygous knock-in transgenic mice (Col2a1+ex2, ki/ki) were generated that exclusively express the IIA procollagen isoform during post-natal development while type IIB procollagen, normally present in the ECM of wild type mice, is absent. The difference between these Col2a1 isoforms is the inclusion (IIA) or exclusion (IIB) of exon 2 that is alternatively spliced in a developmentally regulated manner. Specifically, chondroprogenitor cells synthesize predominantly IIA mRNA isoforms while differentiated chondrocytes produce mainly IIB mRNA isoforms. Recent characterization of the Col2a1+ex2 mice has surprisingly shown that disruption of alternative splicing does not affect overt cartilage formation. In the present study, biochemical analyses showed that type IIA collagen extracted from ki/ki mouse rib cartilage can form homopolymers that are stabilized predominantly by hydroxylysyl pyridinoline (HP) cross-links at levels that differed from wild type rib cartilage. The findings indicate that mature type II collagen derived exclusively from type IIA procollagen molecules can form hetero-fibrils with type XI collagen and contribute to cartilage structure and function. Heteropolymers with type XI collagen also formed. Electron microscopy revealed mainly thin type IIA collagen fibrils in ki/ki mouse rib cartilage. Immunoprecipitation and mass spectrometry of purified type XI collagen revealed a heterotrimeric molecular composition of α1(XI)α2(XI)α1(IIA) chains where the α1(IIA) chain is the IIA form of the α3(XI) chain. Since the N-propeptide of type XI collagen regulates type II collagen fibril diameter in cartilage, the retention of the exon 2-encoded IIA globular domain would structurally alter the N-propeptide of type XI collagen. This structural change may subsequently affect the regulatory function of type XI collagen resulting in the collagen fibril and cross-linking differences observed in this study.

Tautomers and polymorphs of pyrazolinone derivatives, generated from reactions of arylhydrazines, ArNHNH2, with 1,3-dicarbonyl compounds

Abstract From reactions of arylhydrazines with R2COCH2CONHPh or R2COCH2CO2Me, different tautomeric pyrazolinone forms, 1-aryl-3-R2-4,5-dihydro-1H-pyrazol-5-ones (IIa), 1-aryl-3-R2-1H-pyrazol-5-ols (IIb) and 2-aryl-5-R2-1,2-dihydro-3H-pyrazol-3-ones (IIc)], can be isolated. The crystal structures of monoclinic- and triclinic-3-phenyl-1-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-5-one (IIa forms), tautomeric 1-(4-methylphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-one (IIa form), and 1-(4-methylphenyl)-3-phenyl-1H-pyrazol-5-ol (IIb form), as well as1-(4-methoxyphenyl)-3-phenyl-1H-pyrazo-5-ol (IIb form) and 2-(4-methoxyphenyl)-5-methyl-1,2-dihydro-3H-pyrazol-3-one (IIc form) are reported. The data suggest thatthe most electron withdrawing aryl group [3-nitrophenyl] generates IIa forms, while the most electron releasing aryl group [4-methoxyphenyl] generates IIb and IIc forms and that the aryl group with the intermediate electronic effect [4-methylphenyl] provides both IIa and IIb forms. This trend is supported by the reported isolations of 1-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-one and 1-(2,4-nitrophenyl)-3-methyl-4,5-dihydro-1H-pyrazol-5-one [both IIa-forms] with electron-withdrawing chloro and nitro groups respectively and by the isolation of IIb and co-crystallised IIb/IIc forms from use of phenylhydrazine as the reactant.

Quantification of type II procollagen splice forms using alternative transcript-qPCR (AT-qPCR)

During skeletal development, the onset of chondrogenic differentiation is marked by expression of the α1(II) procollagen (Col2a1) gene. Exon 2 of Col2a1 codes for a cysteine-rich von Willebrand factor C-like domain. Chondroprogenitors express the exon 2-containing IIA and IID splice forms by utilizing adjacent 5′ splice sites separated by 3 base pairs. There is a shift to expression of the shorter, exon 2-lacking IIB splice form with further differentiation. Alternative splicing analysis of Col2a1 splice forms has often relied upon semi-quantitative PCR, using a single set of PCR primers to amplify multiple splice forms. We show that this widely used method is inaccurate due to mismatched amplification efficiency of different-sized PCR products. We have developed the TaqMan®-based AT-qPCR (Alternative Transcript-qPCR) assay to more accurately quantify alternatively spliced mRNA, and demonstrate the measurement of Col2a1 splice form expression in differentiating ATDC5 cells in vitro, and in wild type mouse embryonic and postnatal cartilage in vivo. The AT-qPCR assay is based on the use of a multiple-amplicon standard (MAS) plasmid, containing a chemically synthesized cluster of splice site-spanning PCR amplicons, to quantify alternative splice forms by standard curve-based qPCR. The MAS plasmid designed for Col2a1 also contained an 18S rRNA amplicon for sample normalization, and an amplicon corresponding to a region spanning exon 52–53 to measure total Col2a1 mRNA. In mouse E12.5 to P70 cartilages, we observed the expected switch between the IIA and IIB splice forms; no IID or IIC splice products were observed. However, in the ATDC5 cultures, predominant expression of the IIA and IID splice forms was found at all times in culture. Additionally, we observed that the sum of the IIA, IIB and IID splice forms comprises only a small fraction of Col2a1 transcripts containing the constitutive exon 52–53 junction. We conclude from our results that the majority of ATDC5 cells in the assay described in this study remained as chondroprogenitors during culture in standard differentiation conditions, and that additional Col2a1 transcripts may be present. The validity of this novel AT-qPCR assay was confirmed by demonstrating the expected Col2a1 isoform expression patterns in vivo in developing mouse cartilage. The ability to measure true levels of procollagen type II splice forms will provide better monitoring of chondrocyte differentiation in other model systems. In addition, the AT-qPCR assay described here could be applied to any gene of interest to detect and quantify known and predicted alternative splice forms and can be scaled up for high throughput assays.

Functional and Protein Processing Insights from a Truncation Mutation in Subunit III of Cytochrome c Oxidase from Rhodobacter sphaeroides

A human mitochondrial DNA mutation in the Cytochrome c Oxidase (COX) subunit III (SIII) gene causes a truncation after its three n-terminal transmembrane helices and results phenotypically in severe lactic acidosis episodes. We created the equivalent mutation at position I115 in SIII of R. sphaeroides COX (I115stop). Truncated SIII enzyme was expressed and purified, and SDS-PAGE showed an absence of full length SIII and a doublet band of lower molecular weight which was immunoreactive to a SIII site-specific antibody. MALDI-TOF determined these peptide masses to be 12919 m/z and 11462 m/z, results consistent with a I115 truncation in SIII (12915 m/z) and subsequent proteolytic processing after F101 (11461 m/z). Wildtype COX subunit II is known to undergo protease processing in vivo, yielding different forms of the subunit (IIA, IIC). SDS-PAGE and MALDI-TOF showed higher levels of the less-processed IIC form in I115stop mutant preparations as compared to wildtype, which had higher levels of the IIA form. Functional assays show the I115stop mutant has a maximal electron transfer activity that is approximately 30% of wildtype (480±90 e-/s∗mol versus 1670±180 e-/s∗mol) and exhibits suicide inactivation similar to a form of the enzyme lacking SIII altogether (I/IIOX). The first three helices of SIII putatively contain conserved lipid binding sites, so the electron assays were then conducted in the presence of exogenous lipids. The I115stop mutant exhibited a greater stimulation of activity due to lipid than I/IIOX (23% versus 5%). Additionally, protection from suicide inactivation by lipid was 2.4 fold greater in the I115stop mutant than I/IIOX. Taken together, the results indicate that the truncation mutation alters native subunit II c-terminal processing, and they support the hypothesis that SIII is involved in functional lipid binding.

DNA Double Helix Unwinding Triggers Transcription Block-Dependent Apoptosis: A Semiquantitative Probe of the Response of ATM, RNAPII, and p53 to Two DNA Intercalators

We have previously shown the binding modes of two DNA interacting analogues (1)a {3-(4-methyl-piperazin)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} and (3)a {3-(3-dimethylamino-propylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} with the DNA double helix. In this study, we have determined the notably different DNA damage signal pathway elicited by (1)a and (3)a due to the different extents to which they unwind the DNA double helix. First, we have identified that ataxia-telangiectasia-mutated (ATM) protein kinase can respond to DNA double helix unwinding caused by both (1)a and (3)a. In addition, the amount of ATM activation is consistent with the degree to which the DNA double helix was unwound. Consequently, we used (1)a and (3)a to semiquantitatively probe the response of RNA polymerase II (RNAPII) and p53 toward DNA double helix unwinding in vivo. By means of flow cytometry, immunocytochemistry, ChIP, quantitative real-time polymerase chain reaction, and Western blot analyses, we measured the level of p53 and RNAPII phosphorylation, in addition to the dynamics of the RNAPII distribution along the c-Myc gene. These results provided novel evidence for the impact of subtle DNA structural changes on the activity of RNAPII and p53. Moreover, DNA double helix conformational damage-dependent apoptosis was studied for the first time. These results indicated that (1)a can induce transcriptional blockage following a shift of the unphosphorylated IIa form of RNAPII to the phosphorylated IIo form, while (3)a is unable to induce the same effect. Subsequently, p53 accumulation and phosphorylation events occur that lead to apoptosis in the case of (1)a exposure. This suggests that the transcriptional blockage is also correlated to the degree of double helix unwinding. Furthermore, we found that the degree of DNA conformational damage determines whether or not apoptosis occurs through transcriptional blockage. Under our experimental conditions, ATM does not participate in the downstream events even when it has been activated. Thus, p53-mediated apoptosis may be independently triggered by transcriptional blockage.

Caenorhabditis elegans WNK–STE20 pathway regulates tube formation by modulating ClC channel activity

WNK kinases are a small group of unique serine/threonine protein kinases that are conserved among multicellular organisms. Mutations in WNK1-4 cause pseudohypoaldosteronism type II-a form of hypertension. WNKs have been linked to the STE20 kinases and ion carriers, but the underlying molecular mechanisms by which WNKs regulate cellular processes in whole animals are unknown. The Caenorhabditis elegans WNK-like kinase WNK-1 interacts with and phosphorylates germinal centre kinase (GCK)-3--a STE20-like kinase--which is known to inactivate CLH-3, a CIC chloride channel. The wnk-1 or gck-3 deletion mutation causes an Exc phenotype, a defect in the tubular extension of excretory canals. Expression of the activated form of GCK-3 or the clh-3 deletion mutation can partly suppress wnk-1 or gck-3 defects, respectively. These results indicate that WNK-1 controls the tubular formation of excretory canals by activating GCK-3, resulting in downregulation of CIC channel activity.

Missense and nonsense mutations in the alternatively-spliced exon 2 ofCOL2A1cause the ocular variant of Stickler syndrome

Stickler syndrome type I (STL1) is a phenotypically heterogeneous disorder characterized by ocular and extraocular features. It is caused by null-allele mutations in the COL2A1 gene that codes for procollagen II. COL2A1 precursor mRNA undergoes alternative splicing, resulting in two isoforms, a long form including exon 2 (type IIA isoform) and a short form excluding exon 2 (type IIB isoform). The short form is predominantly expressed by differentiated chondrocytes in adult cartilage, and the long form in chondroprogenitor cells during early development and in the vitreous of the eye, which is the only adult tissue containing procollagen IIA. Recent evidence indicates that due to the tissue-specific expression of these two isoforms, premature termination codon mutations in exon 2 cause Stickler syndrome with minimal or no extraocular manifestations. We describe here two mutations in exon 2 of COL2A1 in three patients with predominantly ocular Stickler syndrome: Cys64Stop in two patients, and a novel structural mutation, Cys57Tyr, in one patient. RT-PCR of total lymphoblast RNA from one patient with the Cys64Stop mutation revealed that only the normal allele of the IIA form was present, indicating that the mutation resulted either in complete loss of the allele by nonsense-mediated mRNA decay or by skipping of exon 2 via nonsense-mediated altered splicing, resulting in production of the type IIB isoform. The results of COL2A1 minigene expression studies suggest that both Cys64Stop and Cys57Tyr alter positive cis regulatory elements for splicing, resulting in a lower IIA:IIB ratio.

Evaluation of Maximal Respiratory Pressures in Myasthenia gravis

We assess the prognosis of mild forms of myasthenia gravis (MG) by maximal respiratory pressures (MRP) and single fiber electromyography (SFEMG). Fifty MG patients (12 form I, 21 form IIa and 17 form IIb) are valued by MRP [maximal expiratory pressure (MEP) and maximal inspiratory pressure (MIP)] and SFEMG, and are followed-up clinically. We have found in form I patients developing form IIa and form IIa worsening to form IIb, MEP and MIP mean relative values significantly lower than the rest. Inversely, IIb form patients improving to IIa form display MIP mean relative values higher than the rest; no difference appears with MEP. A reduction under 50% of fifth-percentile implies clinical deterioration in forms I and IIa, while its surpassing in IIb form suggests a tendency to improvement. No evident differences are found by SFEMG. MRP allow the follow-up of MG patients and could warn us of a clinical prognosis.

Type II Collagen Accumulation in Overlying Dermo-Epidermal Junction of Pilomatricoma Is Mediated by Bone Morphogenetic Protein 2 and 4

Pilomatricoma consists of the cells differentiating towards hair matrix cells. Immunohistochemical study revealed the deposition of type II collagen in the overlying dermo-epidermal junction (DEJ) of this benign tumor. Proalpha(1)(II) mRNA was detected by RT-PCR in the overlying epidermal layer but not in the dermal layer prepared from the lesional skin of pilomatricoma. The neutral salt-soluble proteins extracted from the tumor of pilomatricoma induced proalpha(1)(II) mRNA in the cultured human keratinocytes but not in the cultured dermal fibroblasts. Bone morphogenetic protein 2 or 4 (BMP2 or 4) was immunohistochemically detected in some shadow cells of pilomatricoma. Recombinant BMP2 and BMP4 were found to induce proalpha(1)(II) mRNA concentration dependently in the cultured human keratinocytes but not in the cultured fibroblasts. Proalpha(1)(II) mRNA induced by BMP2 and in cultured keratinocytes contained exon 2, indicating that the mRNA species is non-chondrogenic type IIA form. The results strongly suggest that BMP2 or 4 expressed in pilomatricoma is responsible for the induction of proalpha(1)(II) collagen mRNA in the overlying epidermal cells resulting in the deposition of type II collagen in the DEJ. When human keratinocytes were cultured on type II collagen substratum in vitro, the cell proliferation was accelerated at the early period of culture but was inhibited at the late period of culture, whereas the cell proliferation was persistently accelerated by type I or IV collagen substratum. Type II collagen deposition in the DEJ may potentially exert profound effects on keratinocyte proliferation and differentiation.

Cisplatin-induced ubiquitination of RNA polymerase II large subunit and suppression of induction by 7-hydroxystaurosporine (UCN-01)

Exposure of cells to DNA-damaging agents induces hyperphosphorylation of the C-terminal domain (CTD) of mammalian RNA polymerase II (RNAP II) large subunit (LS); the hyperphosphorylated RNAP II is then ubiquitinated. The purpose of this study was to verify that cisplatin-induced RNAP II ubiquitination is transcription dependent in living cells and to determine whether 7-hydroxystaurosporine (UCN-01) inhibits the ubiquitination induced by cisplatin. Cisplatin at clinically achievable concentrations (2.5-10 micro M) induced the ubiquitination of RNAP II in exponentially growing A2780 human ovarian tumor cells; the effect was drug-dose and exposure-time dependent. Such induction, however, was not observed in colcemid-selected mitotic cells. When detergent extraction was applied, the ubiquitinated RNAP II was recovered in the detergent-insoluble fraction, indicating that the protein was tightly bound to DNA. In an in vitro transcription reaction that consists of nuclear extracts and an immobilized DNA template containing a site-specific cisplatin lesion, the elongating RNAP II that was stalled at a cisplatin lesion site on the template was targeted by ubiquitins. Together, our results indicate that the ubiquitination is associated with transcription-coupled repair. We previously showed that the Ser/The kinase-inhibitor UCN-01 inhibits nucleotide excision repair. Here, we further determined the effect of UCN-01 on the phosphorylation and ubiquitination of RNAP II LS in a whole-cell system. Immunoblotting results showed that UCN-01 suppressed the cisplatin-induced ubiquitination and the cisplatin-induced shift from the hypophosphorylated IIa to the hyperphosphorylated IIo, without affecting the basal levels of the IIo and IIa forms of the RNAP II CTD, suggesting that UCN-01 acts by suppressing cisplatin-mediated induction of the one or more kinases that is responsible for the conversion of the IIo that is important for ubiquitination.

Heterotic enhançon

The enhancon mechanism is studied in the heterotic string theory. We consider the N_L=0 winding strings with momentum (NS1-W*) and the Kaluza-Klein dyons (KK5-NS5*). The NS1-W* and KK5-NS5* systems are dualized to the D4-D0* and D6-D2* systems, respectively, under the d=6 heterotic/IIA S-duality. The heterotic form has a number of advantages over the type IIA form. We study these backgrounds and obtain the enhancon radii by brane probe analysis. The results are consistent with S-duality.

Level of the RNA polymerase II in the fission yeast stays constant but phosphorylation of its carboxyl terminal domain varies depending on the phase and rate of cell growth.

The RNA polymerase II of the fission yeast Schizosaccharomyces pombe consists of 12 Rpb subunits, of which four (Rpb1, Rpb2, Rpb3 and Rpb11) form the assembly and catalytic core and five (Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) are shared among RNA polymerases I, II and III. The intracellular levels of three RNA polymerase forms should be interrelated, but the control of RNA polymerase formation remains mostly unknown. To reveal the physiological role and the synthesis control of each Rpb subunit, the intracellular levels of the Rpb proteins were examined in S. pombe growing at various phases under various conditions. Results indicate that the intracellular concentrations of the Rpb proteins stay constant at levels characteristic of the rate and phase of cell growth, and the relative level between the 12 subunits also remains constant, together implying that the intracellular concentration of RNA polymerase II stays constant, as in the case of prokaryotes. As an attempt to gain insights into the activity control of RNA polymerase II, we also analysed the phosphorylation level of the carboxyl-terminal domain (CTD) of the largest subunit Rpb1. Phosphorylated forms of Tyr1 and Thr4 within 29 repeats of the YSPTSPS heptapeptide were detected in both slow-migrating IIo and fast-migrating IIa forms of Rpb1 on SDS-PAGE (polyacrylamide gel electrophoresis). However, phosphorylated Ser2 and Ser5 were identified only in the IIo form, indicating that Ser phosphorylation contributes to the conformational change in CTD. The phosphorylation levels of Ser, Thr and Tyr all vary depending on the cell culture conditions. The intracellular level of RNA polymerase II stays constant, but the amount engaged in transcription cycle varies depending on the culture conditions, as estimated from the sites and levels of phosphorylation of Rpb1 CTD.

Alternative Splicing of Type II Procollagen Gene in the Dedifferentiation of Rat Epiphyseal Chondrocytes Serially Cultured in Monolayer

Type II procollagen is synthesized in two forms (IIA and IIB), generated by the alternative splicing of its precursor mRNA. The purpose of this study was to determine whether alternative splicing of type II procollagen pre-mRNA occurs during the dedifferentiation of chondrocytes in monolayer culture. We serially cultured rat epiphyseal chondrocytes for eight weeks in monolayer, and investigated the expression patterns of the two forms of type II procollagen transcripts according to the duration of the culture period. Reverse transcription-polymerase chain reaction and northern analysis revealed that both transcripts peaked on the day of cell isolation, but the expression level of IIB form was much higher than that of IIA form. The expression of IIA form decreased gradually, whereas the expression of IIB form decreased so rapidly that the ratio of type IIB and type IIA transcripts was reversed in the second week. In situ hybridization showed that all cells expressed type IIA mRNA throughout the culture period. The signal intensity for type IIA transcript decreased gradually in accordance with the results of RT-PCR and northern analysis. In situ hybridization of type IIB mRNA showed different results from in situ hybridization of type IIA mRNA. Like type IIA transcript, the freshly isolated chondrocytes and the cells in the first week showed positive signals for type IIB transcript. However, in the second week, positive signal was detected neither in polygonal cells nor large fibroblastic cells. Only a few cells of small size were weakly positive. From the third to the eighth week, no positive signal was detected. These findings indicate that alternative splicing of type II procollagen gene does occur and is regulated during the dedifferentiation of chondrocytes in monolayer culture.

Alternative Splicing of Type II Procollagen Gene in the Dedifferentiation of Rat Epiphyseal Chondrocytes Serially Cultured in Monolayer

Type II procollagen is synthesized in two forms (IIA and IIB), generated by the alternative splicing of its precursor mRNA. The purpose of this study was to determine whether alternative splicing of type II procollagen pre-mRNA occurs during the dedifferentiation of chondrocytes in monolayer culture. We serially cultured rat epiphyseal chondrocytes for eight weeks in monolayer, and investigated the expression patterns of the two forms of type II procollagen transcripts according to the duration of the culture period. Reverse transcription-polymerase chain reaction and northern analysis revealed that both transcripts peaked on the day of cell isolation, but the expression level of IIB form was much higher than that of IIA form. The expression of IIA form decreased gradually, whereas the expression of IIB form decreased so rapidly that the ratio of type IIB and type IIA transcripts was reversed in the second week. In situ hybridization showed that all cells expressed type IIA mRNA throughout the culture period. The signal intensity for type IIA transcript decreased gradually in accordance with the results of RT-PCR and northern analysis. In situ hybridization of type IIB mRNA showed different results from in situ hybridization of type IIA mRNA. Like type IIA transcript, the freshly isolated chondrocytes and the cells in the first week showed positive signals for type IIB transcript. However, in the second week, positive signal was detected neither in polygonal cells nor large fibroblastic cells. Only a few cells of small size were weakly positive. From the third to the eighth week, no positive signal was detected. These findings indicate that alternative splicing of type II procollagen gene does occur and is regulated during the dedifferentiation of chondrocytes in monolayer culture.

Type IIA procollagen in development of the human intervertebral disc: regulated expression of the NH(2)-propeptide by enzymic processing reveals a unique developmental pathway.

Type II collagen can be synthesized in two forms generated by alternative splicing of the precursor mRNA. Type IIA procollagen, which contains a cysteine-rich domain in the NH(2)-propeptide (exon 2), is produced by precartilage and noncartilage epithelial and mesenchymal cells, and type IIB procollagen, without the cysteine-rich domain, is characteristic of chondrocytes. Mice lacking type II collagen fail to develop intervertebral discs. We have previously shown that the human intervertebral disc and notochord synthesize primarily the type IIA form of procollagen. Therefore, we investigated the distribution of type IIA procollagen during early disc development in humans. By processes of radioactive in situ hybridization and fluorescence immunohistochemistry, we localized mRNA and protein of type IIA procollagen, type I collagen, and type III collagen in fetal intervertebral disc specimens ranging from day 42 (embryonic stage 17) to day 101 (week 14.5) of gestation. Antibodies to the three distinct domains of type IIA procollagen: the NH(2)-propeptide, the fibrillar domain, and the COOH-propeptide were used. The earliest stage of developing intervertebral disc (42 days, stage 17) was characterized by diffuse synthesis of types I and III collagens in the dense zone (intervertebral area) and synthesis of type IIA procollagen by the chondrocyte progenitor cells surrounding the disc. The notochord cells synthesized and deposited into the notochordal sheath all three fibrillar collagens. By 54 days (stage 22), the developing disc was clearly divided into three regions: 1.) the outer annulus, characterized by synthesis and deposition of types I and III collagens; 2.) the inner annulus, characterized by synthesis and deposition of type IIA collagen containing the NH(2)-propeptide but devoid of the COOH-propeptide (pN-procollagen); and 3.) the notochord, the cells of which synthesized and deposited of all three fibrillar collagens. In later stages of fetal development (72-101 days), a change in type IIA procollagen processing was observed in the cells of the inner annulus: even though these cells continued to synthesize type IIA procollagen, they deposited into the extracellular matrix (ECM) only the processed fibrillar domain, with the NH(2)-propeptide removed. This finding indicates that there is a developmentally regulated change in the processing of type IIA procollagen NH(2)-propeptide in the cells of the inner annulus. This mechanism is in contrast to previously shown developmental regulation of the cysteine-rich domain of the NH(2)-propeptide by alternative splicing of the precursor mRNA. Although the cells of the inner annulus have been identified as chondrocytes, based on their shape and synthesis of characteristic ECM components, they appear to represent a distinct developmental pathway characterized by their synthesis and differential processing of type IIA procollagen. This developmental pattern may prove important for disc regeneration.

UV light-induced degradation of RNA polymerase II is dependent on the Cockayne’s syndrome A and B proteins but not p53 or MLH1

It has been hypothesized that the degradation of the largest subunit of RNA polymerase II (polIILS) is required for transcription-coupled repair (TCR) of UV light-induced transcription-blocking lesions. In this study we further investigated the mechanism of UV-induced degradation of polIILS using cell lines with specific defects in TCR or in the recovery of RNA synthesis. It was found that the hypophosphorylated IIa form of polIILS rapidly decreased following UV-irradiation in all cell lines tested. Inhibition of proteasome activity resulted in an increase of the hyperphosphorylated IIo form of polIILS in UV-irradiated cells, while inhibition of CTD-kinases resulted in the retention of the IIa form. In UV-irradiated Cockayne’s syndrome cells, which are defective in TCR, the levels of the IIo form increased in a similar manner as when proteasome inhibitors were added to UV-irradiated normal cells. In contrast, TCR-deficient HCT116 cells, which lack the mismatch repair protein MLH1, showed proficient degradation of polIILS as did cells with deficiencies in the recovery of RNA synthesis following UV-irradiation due to defective p53. Furthermore, we found that proteasome function was important for the recovery of mRNA synthesis even in TCR-deficient HCT116 cells. Our results suggest that proteasome-mediated degradation of polIILS is preceded by phosphorylation of the C-terminal domain of polIILS and requires the CS-A and CS-B but not MLH1 or p53 proteins. Furthermore, our results suggest that following UV-irradiation, the degradation of polIILS is required for the efficient recovery of mRNA synthesis but not for TCR per se.