Regulation of Myosin Heavy Chain Expression during Rat Skeletal Muscle Development In Vitro

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and ∼10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN–nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

In vitro motility from recombinant dynein heavy chain.

The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain.

Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.

Tertiary structure of an amyloid immunoglobulin light chain protein: a proposed model for amyloid fibril formation.

An immunoglobulin light chain protein was isolated from the urine of an individual (BRE) with systemic amyloidosis. Complete amino acid sequence of the variable region of the light chain (VL) protein established it as a kappa I, which when compared with other kappa I amyloid associated proteins had unique residues, including Ile-34, Leu-40, and Tyr-71. To study the tertiary structure, BRE VL was expressed in Escherichia coli by using a PCR product amplified from the patient BRE's bone marrow DNA. The PCR product was ligated into pCZ11, a thermal-inducible replication vector. Recombinant BRE VL was isolated, purified to homogeneity, and crystallized by using ammonium sulfate as the precipitant. Two crystal forms were obtained. In crystal form I the BRE VL kappa domain crystallizes as a dimer with unit cell constants isomorphous to previously published kappa protein structures. Comparison with a nonamyloid VL kappa domain from patient REI, identified significant differences in position of residues in the hypervariable segments plus variations in framework region (FR) segments 40-46 (FR2) and 66-67 (FR3). In addition, positional differences can be seen along the two types of local diads, corresponding to the monomer-monomer and dimer-dimer interfaces. From the packing diagram, a model for the amyloid light chain (AL) fibril is proposed based on a pseudohexagonal spiral structure with a rise of approximately the width of two dimers per 360 degree turn. This spiral structure could be consistent with the dimensions of amyloid fibrils as determined by electron microscopy.

Characterization and localization of the cytoplasmic dynein heavy chain in Aspergillus nidulans.

Migration of nuclei throughout the mycelium is essential for the growth and differentiation of filamentous fungi. In Aspergillus nidulans, the nudA gene, which is involved in nuclear migration, encodes a cytoplasmic dynein heavy chain. In this paper we use antibodies to characterize the Aspergillus cytoplasmic dynein heavy chain (ACDHC) and to show that the ACDHC is concentrated at the growing tip of the fungal mycelium. We demonstrate that four temperature-sensitive mutations in the nudA gene result in a striking decrease in ACDHC protein. Cytoplasmic dynein has been implicated in nuclear division in animal cells. Because the temperature-sensitive nudA mutants are able to grow slowly with occasional nuclei found in the mycelium and are able to undergo nuclear division, we have created a deletion/disruption nudA mutation and a tightly downregulated nudA mutation. These mutants exhibit a phenotype very similar to that of the temperature-sensitive nudA mutants with respect to growth, nuclear distribution, and nuclear division. This suggests that there are redundant backup motor proteins for both nuclear migration and nuclear division.

Characterization of IGH locus breakpoints in multiple myeloma indicates a subset of translocations appear to occur in pregerminal center B cells.

Translocations in myeloma are thought to occur solely in mature B cells in the germinal center through class switch recombination (CSR). We used a targeted captured technique followed by massively parallel sequencing to determine the exact breakpoints in both the immunoglobulin heavy chain (IGH) locus and the partner chromosome in 61 presentation multiple myeloma samples. The majority of samples (62%) have a breakpoint within the switch regions upstream of the IGH constant genes and are generated through CSR in a mature B cell. However, the proportion of CSR translocations is not consistent between cytogenetic subgroups. We find that 100% of t(4;14) are CSR-mediated; however, 21% of t(11;14) and 25% of t(14;20) are generated through DH-JH recombination activation gene-mediated mechanisms, indicating they occur earlier in B-cell development at the pro-B-cell stage in the bone marrow. These 2 groups also generate translocations through receptor revision, as determined by the breakpoints and mutation status of the segments used in 10% and 50% of t(11;14) and t(14;20) samples, respectively. The study indicates that in a significant number of cases the translocation-based etiological events underlying myeloma may arise at the pro-B-cell hematological progenitor cell level, much earlier in B-cell development than was previously thought.

A global expression-based analysis of the consequences of the t(4;14) translocation in myeloma

Purpose: Our purpose in this report was to define genes and pathways dysregulated as a consequence of the t(4;14) in myeloma, and to gain insight into the downstream functional effects that may explain the different prognosis of this subgroup.Experimental Design: Fibroblast growth factor receptor 3 (FGFR3) overexpression, the presence of immunoglobulin heavy chain-multiple myeloma SET domain (IgH-MMSET) fusion products and the identification of t(4;14) breakpoints were determined in a series of myeloma cases. Differentially expressed genes were identified between cases with (n = 55) and without (n = 24) a t(4;14) by using global gene expression analysis.Results: Cases with a t(4;14) have a distinct expression pattern compared with other cases of myeloma. A total of 127 genes were identified as being differentially expressed including MMSET and cyclin D2, which have been previously reported as being associated with this translocation. Other important functional classes of genes include cell signaling, apoptosis and related genes, oncogenes, chromatin structure, and DNA repair genes. Interestingly, 25% of myeloma cases lacking evidence of this translocation had up-regulation of the MMSET transcript to the same level as cases with a translocation.Conclusions: t(4;14) cases form a distinct subgroup of myeloma cases with a unique gene signature that may account for their poor prognosis. A number of non-t(4;14) cases also express MMSET consistent with this gene playing a role in myeloma pathogenesis.

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

The Myosin Heavy Chain specific A4.1025 antibody discriminates different cardiac segments in ancient groups of gnathostomes: Morphological and evolutionary implications

The pan-Myosin Heavy Chain (pan-MyHC) marker MF20 have been reported to show similar, homogeneous signal in the myocardial segments of the heart of teleosts and tetrapods. However, in an ongoing study of the myocardial structure of the dogfish (Scyliorhinus canicula; Chondrichthyes), we observed differential immunostaining of the cardiac segments using another pan-MyHC, the A4.1025 antibody. In order to investigate the relevance of this finding for better understanding of the morphology and evolution of the vertebrate heart, we performed immunohistochemistry, slot blot and western blot in several species of chondrichthyans, actinopterygians and mammals using the above mentioned antibodies. In the dogfish heart, A4.1025 and MF20 specifically recognized MyHC isoforms, although with different degree of affinity. MF20 reactivity was homogeneous and high in all the myocardial segments. However, A4.1025 reactivity was heterogeneous. It was high in the sinus venosus (external layer), atrium and atrioventricular region, low in the ventricle and conus arteriosus, and null in the internal layer of the sinus venosus. A heterogeneous pattern of A4.1025 immunoreactivity was also detected in two other elasmobranchs, a holocephalan, a polypteryform and an acipenseriform. In all of these species, MF20 immunoreactivity was homogeneous. In addition, both markers showed a homogeneous immunoreactivity pattern in teleosts and mammals. Our results indicate that in the hearts of ancient gnathostomes, in all of which a conspicuous conus arteriosus exists, one or more MyHC isoforms with low affinity for A4.1025 show segment-specific distributions. Thus, A4.1025 appears to be an appropriated marker to identify the cardiac segments and their boundaries. We propose that the segmentspecific distribution of MyHC isoforms may generate a particular type of myocardial contractility associated with the presence of a conus arteriosus.

High levels of BACH2 associated with lower levels of BCL2 transcript abundance in t(14;18)(q21;q34) translocation positive non-Hodgkin’s lymphoma

The t(14;18)(q21;q34) BCL2 translocation is a common genetic alteration in follicular and diffuse large B-cell lymphoma. However, it is not invariably associated with BCL2 gene overexpression due to undefined mechanisms that regulate expression from the proximal immunoglobulin heavy-chain (IgH) promoter. The BACH2 transcriptional repressor is able to modulate activity of this promoter. Here we have shown that, in tumor samples with BCL2 translocation, those with high levels of BACH2 had significantly lower BCL2 transcript abundance compared to those with low levels of BACH2. This indicates that BACH2 may be partially responsible for regulation of BCL2 expression from the t(14;18)(q21;q34) translocation.

IgM, IgD and IgY and their expression pattern in the Chinese soft-shelled turtle Pelodiscus sinensis

Three Ig isotypes, IgM, IgD, and IgA, were previously known in reptiles. Here, in this report we describe IgM, IgD and a novel immunoglobulin heavy-chain isotype upsilon (IgY) in Chinese soft-shelled turtle (Pelodiscus sinensis). The IgM and IgY constant domains are characteristically similar to their counterparts described in other vertebrates. The expression of IgM and IgD were detected at mRNA level early during embryonic development, and their expression increased during further development. However, the IgY expression was not detected in larval turtles until 90 days after hatching-out. The increase in the transcription of these three Ig molecules was analyzed by using real-time PCR in spleen, kidney and blood following the injection of inactivated Aeromonas hydrophila. The primary increase in the expression of these three Igs was observed I week after the first injection, although not statistically significant, and the second injection 2 weeks after the first injection provoked a significant increase in the expression of these Igs, revealing a pattern of primary and secondary antibody response in the turtle. The present study represents the first report on reptile IgY and the pattern of IgM, IgD and IgY transcription in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Cloning and expression of glucose regulated protein 78 (GRP78) in Fenneropenaeus chinensis

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35A degrees C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25A degrees C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.

T-cell-rich histiocyte-rich B-cell lymphoma of parotid gland

We describe a malignant lymphoma of the parotid gland arising in a patient with Sjögren's syndrome. Diagnosis was established on needle biopsy which showed a mixed population of lymphoid cells. Immunohistochemistry revealed B-lymphoid cells, T-lymphoid cells and histiocytes. Clonal immunoglobulin heavy chain gene rearrangement was demonstrated using the polymerase chain reaction. Within the confines of the small biopsy, the lesion qualifies for the designation T-cell-rich histiocyte-rich B-cell lymphoma. The value of molecular techniques in the diagnosis of malignant lymphoma on limited tissue samples is highlighted by this case.

Absence of Melatonin Induces Night-Time Hepatic Insulin Resistance and Increased Gluconeogenesis Due to Stimulation of Nocturnal Unfolded Protein Response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-alpha serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. (Endocrinology 152: 1253-1263, 2011)