Bioengineered Internal Anal Sphincter Derived From Isolated Human Internal Anal Sphincter Smooth Muscle Cells

BACKGROUND & AIMS: The internal anal sphincter (IAS) is a specialized circular smooth muscle that maintains rectoanal continence. In vitro models are needed to study the pathophysiology of human IAS disorders. We bioengineered sphincteric rings from human IAS smooth muscle cells (SMC) and investigated their response to cholinergic stimulation as well as investigated whether protein kinase C (PKC) and Rho kinase signaling pathways remain functional. METHODS: 3-Dimensional bioengineered ring (3DBR) model of the human IAS was constructed from isolated human IAS SMC obtained from surgery. Contractile properties and force generation in response to acetylcholine, PKC inhibitor calphostin-C, Rho/ROCK inhibitor Y-27632, permeable Rho/ROCK inhibitor c3-exoenzyme, and PKC activator PdBU was measured. RESULTS: The human IAS 3DBR has the essential characteristics of physiologically functional IAS; it generated a spontaneous myogenic basal tone, and the constructs were able to relax in response to relaxants and contract in response to contractile agents. The constructs generated dose-dependent force in response to acetylcholine. Basal tone was significantly reduced by calphostin-C but not with Y-27632. Acetylcholine-induced force generation was also significantly reduced by calphostin-C but not with Y-27632. PdBU generated force that was equal in magnitude to acetylcholine. Thus, calphostin-C inhibited PdBU-induced force generation, whereas Y-27632 and c3 exoenzyme did not. CONCLUSIONS: These data indicate that basal tone and acetylcholine-induced force generation depend on signaling through the PKC pathway in human IAS; PKC-mediated force generation is independent of the Rho/ROCK pathway. This human IAS 3DBR model can be used to study the pathophysiology associated with IAS malfunctions

Cytocentric measurement for regenerative medicine

Any Regenerative Medicine (RM) business requires reliably predictable cell and tissue products. Regulatory agencies expect control and documentation. However, laboratory tissue production is currently not predictable or well-controlled. Before conditions can be controlled to meet the needs of cells and tissues in culture for RM, we have to know what those needs are and be able to quantify them. Therefore, identification and measurement of critical cell quality attributes at a cellular or pericellular level is essential to generating reproducible cell and tissue products. Here, we identify some of the critical cell and process parameters for cell and tissue products as well as technologies available for sensing them. We also discuss available and needed technologies for monitoring both 2D and 3D cultures to manufacture reliable cell and tissue products for clinical and non-clinical use. As any industry matures, it improves and standardizes the quality of its products. Cytocentric measurement of cell and tissue quality attributes are needed for RM

Direct association of calponin with specific domains of PKC-α

Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated Mg-ATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKC-α in the membrane. In the present study, we have determined the domain of PKC-α involved in direct association with calponin. In vitro binding assay was carried out by incubating glutathione S-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC-α. Calponin was found to bind directly to the full-length PKC-α. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC-α. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4, and C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these in vitro bindings mimic the in vivo associations, and in vivo binding assay was performed by transfecting colonic smooth muscle cells with His-tagged proteins of individual domains and different combinations of domains of PKC-α. Coimmunoprecipitation of calponin with His-tagged truncated forms of PKC-α showed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKC-α and with C2-C3-C4 in cells in the resting state, and this association increased upon stimulation with acetylcholine. These data suggest that calponin bound to fragments that may mimic the active form of PKC-α and that the functional association of PKC-α with calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells

Over-expression of parathion hydrolase of <i>Flavobacterium balustinum </i>in <i>E. coli:</i> Purification and characterization of His-tagged parathion hydrolase

82-86The organophosphorus pesticide degrading (opd) gene was cloned downstream to the transcriptional and translational signals of expression vectors pTrc99A and pET32A. The resulting recombinant expression plasmids pNH2 and pHH2 were introduced into E. coli JM105 and E. coli BL21 respectively. On induction the E. coli cells containing pNH2 produced high levels of parathion hydrolase. A 60 kD fusion protein was produced in E. coli cultures containing recombinant plasmid pHH2. The molecular mass of the fusion protein coincided with the molecular mass of 40 kD parathion hydrolase and 20 kD N-terminal His tag encoded by the vector. Further, the fusion protein was purified using Ni-column and the N-terminal Histag was removed by digesting it with thrombin. The resulting protein folded properly in presence of Zn2+ ions, and showed parathion hydrolase activity

Localisation of identical organophosphorus pesticide degrading <i>(opd) </i>genes on genetically dissimilar indigenous plasmids of soil bacteria: PCR amplification, <span style="font-size:14.0pt;line-height:115%;font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";color:black;mso-ansi-language:EN-IN; mso-fareast-language:EN-IN;mso-bidi-language:HI" lang="EN-IN">cloning and sequencing of <i>opd </i>gene from <i>Flavobacterium balustinum</i></span>

774-779Plasmid borne organophosphorus pesticide degrading (opd) gene of Flavobacterium balustinum has been amplified using polymerase chain reaction (PCR) and the resulting PCR product (1.25 Kb) was cloned in pUC18. Further, a detailed restriction map was determined to PCR product and subcloned as overlapping restriction fragments. The nucleotide sequence was determined for all subclones to obtain complete sequence of PCR amplified fragment. The sequence showed 98% similarity to opd genes cloned from other soil bacteria isolated from diversified geographical regions. The protein sequence predicted from the nucleotide sequence was almost identical to parathion hydrolase, a triesterase involved in hydrolysis of triester bond found in variety of op-pesticides. The signal sequence of parathion hydrolase contained recently discovered twin arginine transport (tat) motif. It appears that tat motif plays a critical role in membrane targeting of parathion hydrolase.</span