Priyadarsini Kumar

Nesprin-3 regulates endothelial cell morphology, perinuclear cytoskeletal architecture, and flow-induced polarization

Nesprin-3, a protein that links intermediate filaments to the nucleus, plays a role in vascular endothelial cell (EC) function. Nesprin-3 regulates EC morphology, perinuclear cytoskeletal organization, centrosome–nuclear connectivity, and flow-induced cell polarization and migration.

Trophoblast Stem Cells: Models for Investigating Trophectoderm Differentiation and Placental Development

The placenta is an ephemeral organ containing diverse populations of trophoblasts that are all derived from the embryonic trophectoderm but have morphological, functional, and molecular diversity within and across species. In hemochorial placentation, these cells play especially important roles, interfacing with and modifying the cells of the maternal decidua. Within the rapidly growing placenta, it has been shown that there are trophoblast stem cells well characterized in the mouse and postulated but not well understood in primates. This review will discuss the characteristics of candidates for human and nonhuman primate trophoblast stem cells, present the diverse methods of their generation, and propose future prospects for experimental systems in which they can shed light on developmental and pathophysiological processes in human pregnancy.

Mice Deficient in CHRNA7, a Subunit of the Nicotinic Acetylcholine Receptor, Produce Sperm with Impaired Motility

Abstract In this study we investigate the role of the CHRNA7 subunit (also known as the alpha7 subunit) of the nicotinic acetylcholine receptor in mouse sperm function. We confirm by reverse-transcription-polymerase chain reaction the expression in adult mouse testis of Chrna7 mRNA and demonstrate the subunits presence in mouse sperm by immunoblot. Alpha-bungarotoxin binds a range of nicotinic acetylcholine receptor subunits, including the CHRNA7 subunit. Localization studies using a fluorescent alpha-bungarotoxin-tetramethyl-rhodamine conjugate revealed specific binding sites on the midpiece of mouse sperm with fainter alpha-bungarotoxin binding on the remainder of the flagellum. Mice engineered with a double-null disruption of the Chrna7 gene displayed only faint fluorescence on the midpiece, suggesting that the CHRNA7 contributed the majority of the observed alpha-bungarotoxin binding sites. The location of alpha-bungarotoxin binding suggested that nicotinic acetylcholine receptors may play an ionotropic role in sperm motility. Sperm from Chrna7−/− mice display no difference in number, morphology, viability or spontaneous acrosome reaction rate compared with Chrna7+/+ sperm. Studies using computer-assisted sperm analysis indicate the motility of Chrna7−/− sperm is significantly impaired. This impairment is characterized by significantly reduced swimming velocities, failure to maintain vigorous swimming, and lower levels of hyperactivated swimming patterns in Chrna7−/− sperm compared with Chrna7+/+ sperm. This is the first genetic evidence that sperm nicotinic acetylcholine receptors are important for maintenance of normal sperm motility.

A Role for the Human Sperm Glycine Receptor/Cl− Channel in the Acrosome Reaction Initiated by Recombinant ZP3

Abstract Previously, we have demonstrated an essential role for the neuronal glycine receptor (GlyR) in the acrosome reaction (AR) of mouse and porcine sperm initiated by the egg zona pellucida (ZP). In the present study, we have demonstrated presence of the GlyR in human sperm by immunoprecipitation and Western blot analysis, investigated the potential of a recombinant human ZP3 (rhZP3) preparation as an alternative research tool to solubilized human ZP, and shown that the human sperm GlyR is essential to the human AR initiated by rhZP3. Additionally, we have been able to demonstrate that rhZP3 possesses biological activity, because it is able to rapidly stimulate the AR in capacitated human sperm and its action is blocked by the addition of pertussis toxin. Moreover, spectrofluorometric studies using fura-2-loaded human sperm have shown that rhZP3 triggers a peak-and-plateau rise in intracellular Ca2+ levels similar to that seen with solubilized mammalian ZP. These results suggest that the actions of rhZP3 and solubilized ZP are elicited via the same signal transduction pathways. Furthermore, incubation of human sperm with an antibody directed against the α1 subunit of the human spinal cord GlyR or with 50 nM strychnine caused significant inhibition in the rhZP3-initated AR. Finally, studies using fura-2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the Ca2+ influx associated with addition of rhZP3. These results further support the view that rhZP3 and the ZP work through the same mechanisms, show that the GlyR is involved in rhZP3-initiated AR, and suggest that the GlyR may also play a role in the early signal transduction cascades associated with ZP-initiated AR in vivo.

Multiplicity of the β Form of the cAMP-dependent Protein Kinase Inhibitor Protein Generated by Post-translational Modification and Alternate Translational Initiation

Two distinct species of the thermostable inhibitor of the cAMP-dependent protein kinase, PKIα and PKIβ, exist that are the products of separate genes. The PKIβ form, as first isolated from rat testis, is a 70-amino acid protein, but the genomic sequence suggested that an alternate form might exist, arising as a consequence of alternate translational initiation. This species, now termed PKIβ-78, has been synthesized by bacterial expression, demonstrated to be equipotent with PKIβ-70, and also now demonstrated to occur in vivo. By Western blot analyses, six additional species of PKIβ are also evident in tissues. Two of these represent the phospho forms of PKIβ-78 and PKIβ-70. The other four represent phospho and dephospho forms of two higher molecular mass PKIβ species. These latter forms are currently termed PKIβ-X and PKIβ-Y, awaiting the full elucidation of their molecular identity. In adult rat testis and cerebellum, PKIβ-70, PKIβ-X, and PKIβ-Y constitute 39, 23, and 32% and 15, 29, and 54% of the total tissue levels, respectively. In adult rat testis, 35–42% of each of these three species is present as a monophospho form, whereas no phosphorylation of them is evident in cerebellum. PKIβ-78 is present at much lower levels in both rat testis and cerebellum (∼6 and 2% of the total, respectively) and almost entirely as a monophospho species. PKIβ-78, like PKIβ-70, is a high affinity and specific inhibitor of the cAMP-dependent protein kinase. PKIβ-Y and PKIβ-X, in contrast, also significantly inhibit the cGMP-dependent protein kinase.

A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

Discovery and Characterization of a Potent and Specific Peptide Ligand Targeting Endothelial Progenitor Cells and Endothelial Cells for Tissue Regeneration

Endothelial progenitor cells (EPCs) and endothelial cells (ECs) play a vital role in endothelialization and vascularization for tissue regeneration. Various EPC/EC targeting biomolecules have been investigated to improve tissue regeneration with limited success often due to their limited functional specificity and structural stability. One-bead one-compound (OBOC) combinatorial technology is an ultrahigh throughput chemical library synthesis and screening method suitable for ligand discovery against a wide range of biological targets, such as integrins. In this study, using primary human EPCs/ECs as living probes, we identified an αvβ3 integrin ligand LXW7 discovered by OBOC combinatorial technology as a potent and specific EPC/EC targeting ligand. LXW7 overcomes the major barriers of other functional biomolecules that have previously been used to improve vascularization for tissue regeneration and possesses optimal stability, EPC/EC specificity, and functionality. LXW7 is a disulfide cyclic octa-peptide (cGRGDdvc) containing unnatural amino acids flanking both sides of the main functional motif; therefore it will be more resistant to proteolysis and more stable in vivo compared to linear peptides and peptides consisting of only natural amino acids. Compared with the conventional αvβ3 integrin ligand GRGD peptide, LXW7 showed stronger binding affinity to primary EPCs/ECs but weaker binding to platelets and no binding to THP-1 monocytes. In addition, ECs bound to the LXW7 treated culture surface exhibited enhanced biological functions such as proliferation, likely due to increased phosphorylation of VEGF receptor 2 (VEGF-R2) and activation of mitogen-activated protein kinase (MAPK) ERK1/2. Surface modification of electrospun microfibrous PLLA/PCL biomaterial scaffolds with LXW7 via Click chemistry resulted in significantly improved endothelial coverage. LXW7 and its derivatives hold great promise for EPC/EC recruitment and delivery and can be widely applied to functionalize various biological and medical materials to improve endothelialization and vascularization for tissue regeneration applications.

Micrococcus luteus mediated dual mode synthesis of gold nanoparticles: Involvement of extracellular α-amylase and cell wall teichuronic acid

In the present study we have utilized the bioreductive potential of Micrococcus luteus for the synthesis of gold nanoparticles. Biochemical and physiological analysis indicate that the biosynthesized GNPs were achieved by dual mode, involving extracellular α-amylase and cell wall teichuronic acid (TUA) of M. luteus. The biosynthetic potential of both α-amylase and TUA, after isolation from bacterium, was examined. Under optimum conditions, these biomolecules reduces Au(3+) into Au(0) and the resulting GNPs were found to be stable for 1 month. The synthesized GNPs were characterized by UV-VIS spectrometry, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS). Results demonstrated that the synthesized GNPs were found to be monodispersive and spherical in shape with an average size of ∼6 nm and ∼50 nm for α-amylase and teichuronic acid, respectively. These findings suggest that M. luteus can be exploited as a potential biosource for the eco-friendly synthesis of gold nanoparticles.

Expressed Phosphorylase b Kinase and Its αγδ Subcomplex as Regulatory Models for the Rabbit Skeletal Muscle Holoenzyme

Understanding the regulatory interactions among the 16 subunits of the (alphabetagammadelta)(4) phosphorylase b kinase (PhK) complex can only be achieved through reconstructing the holoenzyme or its subcomplexes from the individual subunits. In this study, recombinant baculovirus carrying a vector containing a multigene cassette was created to coexpress in insect cells alpha, beta, gamma, and delta subunits corresponding to rabbit skeletal muscle PhK. The hexadecameric recombinant PhK (rPhK) and its corresponding alphagammadelta trimeric subcomplex were purified to homogeneity with proper subunit stoichiometries. The catalytic activity of rPhK at pH 8.2 and its ratio of activities at pH 6.8 versus pH 8.2 were comparable to those of PhK purified from rabbit muscle (RM PhK), as was the hysteresis (autoactivation) in the rate of product formation at pH 6.8. Both the rPhK and alphagammadelta exhibited only a very low Ca(2+)-independent activity and a Ca(2+)-dependent activity similar to that of the native holoenzyme with [Ca(2+)](0.5) of 0.4 microM for the RM PhK, 0.7 microM for the rPhK, and 1.5 microM for the alphagammadelta trimer. The RM PhK, rPhK, and alphagammadelta subcomplex were also all activated through self-phosphorylation. Using cross-linking and limited proteolysis, the alpha-gamma intersubunit contacts previously observed within the intact RM PhK complex were also observed within the recombinant alphagammadelta subcomplex. Our results indicate that both the rPhK and alphagammadelta subcomplex are promising models for future structure-function studies on the regulation of PhK activity through intersubunit contacts, because both retained the regulatory properties of the enzyme purified from skeletal muscle.

Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery

BACKGROUND In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). METHODS PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). RESULTS PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3-5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. DISCUSSION PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.