Siddhartha S. Jana

Identification and Characterization of Nonmuscle Myosin II-C, a New Member of the Myosin II Family

A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a Vmax of 0.55 + 0.18 s–1, which was half-maximally activated at an actin concentration of 16.5 + 7.2 μm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 μm/s in the absence of tropomyosin and at 0.072 + 0.019 μm/s in the presence of tropomyosin in an in vitro motility assay.

Sustained Activation of Mitogen-Activated Protein Kinases and Activator Protein 1 by the Hepatitis B Virus X Protein in Mouse Hepatocytes In Vivo

ABSTRACT Transcriptional activation of diverse cellular genes by the X protein (HBx) of hepatitis B virus (HBV) has been suggested as one of the mechanisms for HBV-associated hepatocellular carcinoma. However, such functions of HBx have been studied using transformed cells in culture and have not been examined in the normal adult hepatocytes, a natural host of HBV. Using an efficient hepatocyte-specific virus-based gene delivery system developed in our laboratory earlier, we studied the HBx action in vivo. We demonstrate that following virosome-mediated delivery of HBx DNA, a large population (>50%) of hepatocytes express the HBx protein in a dose-dependent manner, which induces a significant increase in the activity of extracellular signal-regulated kinases (ERKs) in the livers of HBx-transfected mice. Inhibition of HBx-induced ERK activation following intravenous administration of PD98059, a mitogen-activated protein kinase kinase kinase (MEK) inhibitor, confirmed the requirement for MEK in the activation of ERKs by HBx. Induction of ERK activity by HBx was sustained for up to 30 days. Interestingly, sustained activation of c-Jun N-terminal kinases (JNKs) for up to 30 days was also noted. Such constitutive ERK and JNK activation as a consequence of continued HBx expression also led to sustained stimulation of further downstream events, such as increased levels of c-Jun and c-Fos proteins along with the persistent induction of activator protein 1 binding activity. Taken together, our data suggest a critical role of these molecules in HBx-mediated cell transformation.

Ablation of Nonmuscle Myosin II-B and II-C Reveals a Role for Nonmuscle Myosin II in Cardiac Myocyte Karyokinesis

Ablation of nonmuscle myosin (NM) II-B and II-C in mice results in a defect in cardiac myocyte karyokinesis. More than 90% of the double knockout cardiac myocytes demonstrate defects in chromatid segregation and mitotic spindle formation. The requirement for NM II in karyokinesis is demonstrated in both the intact heart and in HL-1 atrial myocytes in culture.

A specific isoform of nonmuscle myosin II-C is required for cytokinesis in a tumor cell line.

Nonmuscle myosin IIs play an essential role during cytokinesis. Here, we explore the function of an alternatively spliced isoform of nonmuscle myosin heavy chain (NMHC) II-C, called NMHC II-C1, in the A549 human lung tumor cell line during cytokinesis. NMHC II-C1 contains an insert of 8 amino acids in the head region of NMHC II-C. First, we show that there is a marked increase in both the mRNA encoding NMHC II-C1 and protein in tumor cell lines compared with nontumor cell lines derived from the same tissue. Quantification of the amount of myosin II isoforms in the A549 cells shows that the amounts of NMHC II-A and II-C1 protein are about equal and substantially greater than NMHC II-B. Using specific siRNAs to decrease NMHC II-C1 in cultured A549 cells resulted in a 5.5-fold decrease in the number of cells at 120 h, whereas decreasing NMHC II-A with siRNA does not affect cell proliferation. This decreased proliferation can be rescued by reintroducing NMHC II-C1 but not NMHC II-A or II-B into A549 cells, although noninserted NMHC II-C does rescue to a limited extent. Time lapse video microscopy revealed that loss of NMHC II-C1 leads to a delay in cytokinesis and prolongs it from 2 to 8-10 h. These findings are consistent with the localization of NMHC II-C1 to the intercellular bridge that attaches the two dividing cells during the late phases of cytokinesis. The results suggest a specific function for NMHC II-C1 in cytokinesis in the A549 tumor cell line.

β-Amino Acid and Amino-Alcohol Conjugation of a Nonsteroidal Anti-Inflammatory Drug (NSAID) Imparts Hydrogelation Displaying Remarkable Biostability, Biocompatibility, and Anti-Inflammatory Properties

A well-known nonsteroidal anti-inflammatory drug (NSAID), namely, naproxen (Np), was conjugated with β-alanine and various combinations of amino alcohols and l-alanine. Quite a few bioconjugates, thus synthesized, were capable of gelling pure water, NaCl solution (0.9 wt %), and phosphate-buffered saline (PBS) (pH 7.4). The hydrogels were characterized by rheology and electron microscopy. Hydrogelation was probed by FT-IR and temperature-variable (1)H NMR studies. Single-crystal X-ray diffraction (SXRD) of a nonhydrogelator and a hydrogelator in the series established a useful structure-property (gelation) correlation. MTT assay of the hydrogelators in the mouse macrophage RAW 264.7 cell line showed excellent biocompatibility. The prostaglandin E2 (PGE2) assay of the hydrogelators revealed their anti-inflammatory response, which was comparable to that of the parent NSAID naproxen sodium (Ns).

An alternatively spliced isoform of non-muscle myosin II-C is not regulated by myosin light chain phosphorylation.

We report a novel isoform of non-muscle myosin II-C (NM II-C), NM II-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC20) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1, which contains 8 amino acids inserted into loop 1, are dependent on MLC20 phosphorylation for both actin-activated MgATPase activity and in vitro motility ( Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-22775 ). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC20 phosphorylation for full activity similar to HMM II-C2. These constitutively active C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells.

Targeted cytosolic delivery of hydrogel nanoparticles into HepG2 cells through engineered Sendai viral envelopes.

Hydrogel nanoparticles of cross‐linked polyvinylpyrrolidone (PVP‐NP) (35–50 nm in diameter) containing fluoresceinated dextran (FITC‐Dx) were encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F‐virosomes1). Incubation of these loaded F‐virosomes with human hepatoblastoma cells (HepG2) in culture resulted in membrane‐fusion‐mediated delivery of NPs to the cell cytoplasm, as inferred from the ability of cells to internalize FITC‐Dx loaded PVP‐NP (PVPf‐NP) in the presence of azide (an inhibitor of the endocytotic process). Introduction of PVPf‐NP into the HepG2 cells was assured by selective accumulation of FITC fluorescence in the cytosolic compartment. The structural integrity of the internalized PVPf‐NP was also confirmed by fluorescence microscopy and ultracentrifugation analysis. The potential usefulness of PVP‐NP‐mediated cytosolic release of water soluble drugs both in vitro and in vivo has been established for the first time.

Site-specific covalent attachment of heme proteins on self-assembled monolayers

Naturally occurring hemin cofactor has been functionalized to introduce two terminal alkyne groups. This modified hemin has been successfully covalently attached to mixed self-assembled monolayers of alkanethiols and azide-terminated alkanethiols on gold electrodes using a CuI-catalyzed 1,3-cycloaddition reaction. However these hemin-modified electrodes could not be used to reconstitute apomyoglobin on gold electrodes owing to the hydrophobicity of the alkane thiol self-assembled monolayer. Modification of existing techniques allowed covalent attachment of alkyne-terminated electroactive species onto mixed monolayers of azidothiols and carboxylatoalkanethiols on electrodes using the same CuI-catalyzed 1,3-cycloaddition reaction. Apomyoglobin could be reconstituted using the hemin covalently attached to these hydrophilic electrodes. The electrochemical data, UV–vis absorption data, surface-enhanced resonance Raman spectroscopy data, and atomic force microscopy data indicate the presence of these modified myoglobin proteins on these electrodes. The direct attachment of the heme cofactor of these modified myoglobin proteins to the electrode allows fast electron transfer to the heme center from the electrode and affords efficient O2-reducing bioelectrodes under physiological conditions.Graphical Abstract

An internal segment (residues 58–119) of the hepatitis B virus X protein is sufficient to activate MAP kinase pathways in mouse liver

The human hepatitis B virus X protein (HBx) is known as a dual‐specificity transactivator stimulating the transcriptional machinery in the nucleus and signal transduction pathways in the cytoplasm. HBx‐induced activation of mitogen‐activated protein kinase (MAPK) signaling cascades is considered to play an important role in hepatitis B virus‐mediated hepatocarcinogenesis. Herein, we have identified the regions of HBx that are crucial for activating such signaling cascades in vivo. A truncated mutant incorporating regions C–E (amino acids 58–140) was as effective as the full‐length HBx in activating MAPKs and enhancing activator protein‐1 binding activity. While deletion of region C (amino acids 58–84) or D (amino acids 85–119) led to a drastic loss of function, region E (amino acids 120–140) was dispensable for the activation of signaling cascades. Overall, these findings provide the first evidence for the requirement of domain 58–119 of HBx in transmitting mitogenic signals to the nucleus in vivo.

Role of Red-Ox Cycle in Structural Oscillations and Solvation Dynamics in the Mitochondria of a Live Cell

Structural oscillations and solvation dynamics in the mitochondria of a live cell are studied by time-resolved microscopy using a covalent fluorescence probe. We compared the dynamics in a human breast cancer cell (MCF-7) with that in a normal breast cell MCF-10A. The probe, CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin), binds with the free thiol groups. In MCF-10A cell, CPM binds with the discrete mitochondria. In MCF-7, CPM labels the clustered mitochondria in the peri-nuclear region. Location of the CPM in the mitochondria is confirmed by colocalization with a mitochondria-tracker dye. The red-ox cycle in the mitochondria causes periodic fluctuation in the microenvironment in the discrete mitochondria. This is manifested in fluctuations in fluorescence intensity of CPM bound to mitochondria. The magnitude of oscillation is much less for CPM bound to the clustered mitochondria (in which the red-ox cycle is inefficient) in the cancer cell (MCF-7). In both of the cells (MCF-10A and MCF-7) CPM bound to thiol-containing proteins in mitochondria exhibits ultraslow response with average solvation time (⟨τs⟩) of 850 and 1400 ps in MCF-10A and MCF-7, respectively.