Strain-induced Proliferation Requires the Phosphatidylinositol 3-Kinase/AKT/Glycogen Synthase Kinase Pathway

The intestinal epithelium is repetitively deformed by shear, peristalsis, and villous motility. Such repetitive deformation stimulates the proliferation of intestinal epithelial cells on collagen or laminin substrates via ERK, but the upstream mediators of this effect are poorly understood. We hypothesized that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3β (GSK-3β) were phosphorylated by 10 cycles/min strain at an average 10% deformation, and pharmacologic blockade of these molecules or reduction by small interfering RNA (siRNA) prevented the mitogenic effect of strain in Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that AKT2 but not AKT1 is required for GSK-3β phosphorylation and the strain mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera including the PH domain and hinge region of AKT2 and the catalytic domain and C-tail of AKT1 prevented strain activation of GSK-3β, but overexpression of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the catalytic domain and C-tail of AKT2 did not. These data delineate a role for PI3K, AKT2, and GSK-3β in the mitogenic effect of strain. PI3K is required for both ERK and AKT2 activation, whereas AKT2 is sequentially required for GSK-3β. Furthermore, AKT2 specificity requires its catalytic domain and tail region. Manipulating this pathway may prevent mucosal atrophy and maintain the mucosal barrier in conditions such as ileus, sepsis, and prolonged fasting when peristalsis and villous motility are decreased and the mucosal barrier fails.Mechanical forces are part of the normal intestinal epithelial environment. Numerous different forces deform these cells including shear stress from endoluminal chyme, bowel peristalsis, and villous motility (1, 2). During normal bowel function the mucosa is subjected to injury that must be repaired to maintain the mucosal barrier (3, 4). Deformation patterns of the bowel are altered in conditions such as prolonged fasting, post-surgical ileus, and sepsis states, resulting in profoundly reduced mucosal deformation. When such states are prolonged, proliferation slows, the mucosa becomes atrophic, and bacterial translocation may ensue as the mucosal barrier of the gut breaks down (5–7).In vitro, repetitive deformation is trophic for intestinal epithelial cells (8) cultured on type I or type IV collagen or laminin. Human Caco-2 intestinal epithelial cells (9), non-transformed rat IEC-6 intestinal epithelial cells (10), and primary human intestinal epithelial cells isolated from surgical specimens (11) proliferate more rapidly in response to cyclic strain (12) unless substantial quantities of fibronectin are added to the media or matrix (11) to mimic the acute phase reaction of acute or chronic inflammation and injury. Cyclic strain also stimulates proliferation in HCT 116 colon cancer cells (13) and differentiation of Caco-2 cells cultured on a collagen substrate (9). This phenomenon has also been observed in vivo (14). Thus, repetitive deformation may help to maintain the normal homeostasis of the gut mucosa under non-inflammatory conditions. Previous work in our laboratory has implicated Src, focal adhesion kinase, and the mitogen-activated protein kinase (MAPK)² extracellular signal-related kinase (ERK) in the mitogenic effect of strain (10). Although p38 is also activated in Caco-2 cells subjected to cyclic strain on a collagen matrix, its activity is not required for the mitogenic effect of strain (12).Although often the PI3K/AKT pathway is thought of as a parallel pathway to the MAPK, this is not always the case. Protein kinase C isoenzymes differentially modulate thrombin effect on MAPK-dependent retinal pigment epithelial cell (RPE) proliferation, and it has been shown that PI3K or AKT inhibition prevented thrombin-induced ERK activation and RPE proliferation (15).PI3K, AKT, and glycogen synthase kinase (GSK), a downstream target of AKT (16), have been implemented in intestinal epithelial cell proliferation in numerous cell systems not involving strain (17–19) including uncontrolled proliferation in gastrointestinal cancers (20–22). Mechanical forces activate this pathway as well. PI3K and AKT are required for increased extracellular pressure to stimulate colon cancer cell adhesion (23), although the pathway by which pressure stimulates colon cancer cells in suspension differs from the response of adherent intestinal epithelial cells to repetitive deformation (24), and GSK is not involved in this effect.³ Repetitive strain also stimulates vascular endothelial cell proliferation via PI3K and AKT (25, 26), whereas respiratory strain stimulates angiogenic responses via PI3K (27). We, therefore, hypothesized that the PI3K/AKT/GSK axis would be involved in the mitogenic effects of repetitive deformation on a collagen matrix.To test this hypothesis, we used the Flexcell apparatus to rhythmically deform Caco-2 intestinal epithelial cells. IEC-6 cells were used to confirm key results. A frequency of 10 cycles per min was used, which is similar in order of magnitude to the frequency that the intestinal mucosa might be deformed by peristalsis or villous motility in vivo (28, 29). Mechanical forces such as repetitive deformation are likely cell-type and frequency-specific, as different cell types respond to different frequencies. Vascular endothelial cells respond to frequencies of 60–80 cycles/min (25), whereas intestinal epithelial cells may actually decrease proliferation in response to frequencies of 5 cycles/min (30). We characterized PI3K, AKT, and GSK phosphorylation with strain, blocked these molecules pharmacologically or by siRNA, and delineated the specificity of the AKT effect using isozyme-specific siRNA and transfection of AKT1/2 chimeras. We also characterized the interaction of this pathway with the activation of ERK by strain, which has previously been implicated in the mitogenic response (12).     

Synthesis and antitubercular activity of substituted phenylmethyl- and pyridylmethyl amines

A total of 42 benzyl- and pyridylmethyl amines were synthesized either by reductive amination of aromatic/heteroaromatic aldehydes with amines or by conjugate addition of amines to the cinnamates followed by reduction of the ester group with lithium aluminium hydride to the respective propanolamines. All the synthesized compounds were evaluated against both avirulent and virulent strains of Mycobacterium tuberculosis. Many of the compounds exhibited MIC as low as 1.56 μg/mL. Few of potent compounds were also evaluated against clinical isolates of MDR TB and found to be active at one or other concentrations with MIC as low as 3.12 μg/mL.     

Subcellular localization and biological actions of activated RSK1 are determined by its interactions with subunits of cyclic AMP-dependent protein kinase

Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.     

Altered expression and glycosylation of plasma proteins in rheumatoid arthritis

Altered glycosylation of plasma proteins has been directly implicated in the pathogenesis of rheumatoid arthritis (RA). The present study investigated the changes in the Concanavalin-A (Con-A)-bound plasma proteins in the RA patients in comparison to that of the healthy controls. Two proteins (MW ∼32 kDa and ∼62 kDa) showed an alteration in expression while an altered monosaccharide profile (high mannose) was observed in the ∼62 kDa protein in the samples collected from RA patients. The 2-dimensional polyacrylamide gel electrophoresis analysis of the Con-A-bound plasma samples showed a large number of protein spots, a few of which were differentially expressed in the RA patients. Some unidentified proteins were detected in the RA patients which were absent in the control samples. The present study, therefore, enunciates the role of carbohydrates as well as that of the acute phase response in the disease pathogenesis.     

An efficient method for homologous gene reconstitution in Cryptococcus gattii using URA5 auxotrophic marker

Cryptococcus gattii (Cg) is an emerging pathogen of both healthy and immunocompromised patients worldwide. Understanding the molecular genetic basis of virulence and physiology of this pathogen will be critical for defining its pathogenic mechanisms. The purine biosynthetic gene, URA5 encoding orate phosphorybosyltransferase (OPRTase), has been successfully used as a selectable marker for gene disruption by transformation and homologous recombination in Cg. Here, we report the characterization of ura5 auxotrophy and URA5 reversion phenomenon at the molecular, genetic, and structural levels, and use of ura5→URA5 reversion as a tool for reconstitution of gene of interest and auxotrophic marker to their native loci. We identified a single mutation of GG₁₂₈T→GAT with substitution of glycine to aspartic acid at amino acid position 43 resulting in ura5 auxotrophy. The ura5→URA5 reversion on CSM lacking uracil (CSM-U) was found to be a rare phenomenon with a reversion frequency of 0.000002%, and sequence analysis of URA5 from all the reverted strains revealed mutation of GA₁₂₈T→GGT back to its ancestral state. The URA5 allele in the reverted strains was fully functional, as demonstrated by the excellent growth of these strains on medium lacking uracil, as well as by the ability of this allele to efficiently transform ura5 mutant to restore prototrophy. The deduced Cg URA5 protein modeled on the known crystal structures of OPRTase from Salmonella typhimurium (1LH0_A, 1STO) and from Escherichia coli (1ORO_A) indicated that the glycine 43 of Cg URA5 was situated on a conserved loop, and it’s substitution to more globose aspartic acid may have resulted in URA5 inactivation in auxotrophic strain. The advantages of this approach for the generation of a reconstituted strain are (1) that it restores the functionality of the native URA5, (2) that it eliminates an additional biolistic delivery of exogenous URA5, and (3) that it allows easy selection of reconstituted strains with homologous integration. This strategy was successfully used for the generation of Cg can2+CAN2/URA5 homologous reconstituted strains, which grew in ambient air to the wild-type level while can2 mutant exhibited severe growth defect under similar conditions. Srinivas D. Narasipura and Ping Ren contributed equally to this work.     

Synthesis and in vitro antiviral activities of 3′-fluoro (or chloro) and 2′,3′-difluoro 2′,3′-dideoxynucleoside analogs against hepatitis B and C viruses

Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) lead to serious liver diseases worldwide. Co-infection with HBV and HCV is very common and is associated with increased risk of liver pathogenesis, liver cancer, and liver failure. Several 5-substituted 3′-fluoro (or chloro) (1–4, 6, 7, 17–19) and 2′,3′-difluoro 2′,3′-dideoxynucleosides (15 and 16) were synthesized and evaluated for in vitro antiviral activities against duck hepatitis B virus (DHBV), human hepatitis B virus, and hepatitis C virus. Of these compounds 4, 7, 17, and 19 demonstrated moderate anti-HBV activity, and 2, 4, 7, 8, and 19 were weak inhibitors of HCV. Although 5-iodo derivative (7) was most inhibitory against HCV, it exhibited a reduction in cellular RNA levels in Huh-7 cells. The 5-hydroxymethyl-3′-fluoro-2′,3′-dideoxyuridine (4) and 1-(3-chloro-2,3-dideoxy-β-d-erythro-pentofuranosyl)-5-fluorouracil (19) provided the most inhibition of both viruses without cytotoxicity.     

Dedifferentiation of leaf explants and cytotoxic activity of an aqueous extract of cell cultures of Lantana camara L.

Several secondary metabolites are present in Lantana camara L. as its leaves serve as reservoirs for various bioactive compounds. Callus cultures of L. camara were induced from leaf discs incubated on Murashige and Skoog medium supplemented with 5 μM 6-benzyladenine, 1 μM 2,4-dichlorophenoxyacetic acid, and 1 μM α-naphthalene acetic acid (NAA). An aqueous extract (0.23%), obtained from these calli (50 g dry mass), had an apparent cytotoxic effect on HeLa cells with an IC₅₀ value of 1,500 μg/ml in 36 h. A dose-time dependent activity of the extract was established wherein higher dosage exhibited increased activity; however, over time cell necrosis was observed.     

p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1��: ROLE IN THE REGULATION OF PKA AND RSK1 ACTIVITIES*

Previously we showed that the inactive form of p90 ribosomal S6 kinase 1 (RSK1) interacts with the regulatory subunit, PKARIα, of protein kinase A (PKA), whereas the active RSK1 interacts with the catalytic subunit (PKAc) of PKA. Herein, we demonstrate that the N-terminal kinase domain (NTK) of RSK1 is necessary for interactions with PKARIα. Substitution of the activation loop phosphorylation site (Ser-221) in the NTK with the negatively charged Asp residue abrogated the association between RSK1 and PKARIα. This explains the lack of an interaction between active RSK1 and PKARIα. Full-length RSK1 bound to PKARIα with an affinity of 0.8 nm. The NTK domain of RSK1 competed with PKAc for binding to the pseudosubstrate region (amino acids 93–99) of PKARIα. Overexpressed RSK1 dissociated PKAc from PKARIα, increasing PKAc activity, whereas silencing of RSK1 increased PKAc/PKARIα interactions and decreased PKAc activity. Unlike PKAc, which requires Arg-95 and -96 in the pseudosubstrate region of PKARIα for their interactions, RSK1/PKARIα association requires all four Arg residues (Arg-93–96) in the pseudosubstrate site of PKARIα. A peptide (Wt-PS) corresponding to residues 91–99 of PKARIα competed for binding of RSK1 with PKARIα both in vitro and in intact cells. Furthermore, peptide Wt-PS (but not control peptide Mut-PS), by dissociating RSK1 from PKARIα, activated RSK1 in the absence of any growth factors and protected cells from apoptosis. Thus, by competing for binding to the pseudosubstrate region of PKARIα, RSK1 regulates PKAc activity in a cAMP-independent manner, and PKARIα by associating with RSK1 regulates its activation and its biological functions.     

Temporal phase relation of circadian neural oscillations as the basis of testicular maturation in mice: A test of a coincidence model

To study the underlying mechanism of gonadal growth during the attainment of puberty and to test a coincidence model, 7 experimental groups of 2-week-old male mice, Mus musculus, were administered the serotonin precursor, 5-hydroxytryptophan, followed by the dopamine precursor, l-dihydroxyphenylalanine at hourly intervals of 6, 7, 8, 9, 10, 11 and 12 h (5 mg/100 g body weight per day for 13 days). At 11 days post-treatment, a suppression of gonadal activity was seen in the 7-h mice and a maximum suppression in the 8-h mice, along with a significantly increased degree of gonadal development in the 12-h mice, as compared with the controls. In addition to its known regulation of seasonal gonadal cycles, the relative position of two circadian neural oscillations may also affect the rate of gonadal development during the attainment of puberty in mice. Moreover, the present study provides an experimental paradigm to test the coincidence model of circadian oscillations.     

Bioremediation through microbes: systems biology and metabolic engineering approach

Today, environmental pollution is a serious problem, and bioremediation can play an important role in cleaning contaminated sites. Remediation strategies, such as chemical and physical approaches, are not enough to mitigate pollution problems because of the continuous generation of novel recalcitrant pollutants due to anthropogenic activities. Bioremediation using microbes is an eco-friendly and socially acceptable alternative to conventional remediation approaches. Many microbes with a bioremediation potential have been isolated and characterized but, in many cases, cannot completely degrade the targeted pollutant or are ineffective in situations with mixed wastes. This review envisages advances in systems biology (SB), which enables the analysis of microbial behavior at a community level under different environmental stresses. By applying a SB approach, crucial preliminary information can be obtained for metabolic engineering (ME) of microbes for their enhanced bioremediation capabilities. This review also highlights the integrated SB and ME tools and techniques for bioremediation purposes.