PKCα tumor suppression in the intestine is associated with transcriptional and translational inhibition of cyclin D1

Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCα in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCα or PKCδ in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCα and δ levels were generally reduced in colon carcinoma cell lines, PKCβII was elevated and PKCε showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCα downregulated cyclin D1 by two independent mechanisms. PKCα expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCδ had modest and variable effects on cyclin D1 steady state levels and failed to restore responsiveness to PKC agonists. Notably, PKCα expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCδ had only minor effects. Loss of PKCα and effects of its re-expression were independent of the status of the APC/β-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCα is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the frequent loss of this isozyme during intestinal carcinogenesis

Growth of Hyperthermophilic Archaeon Pyrococcus furiosus on Chitin Involves Two Family 18 Chitinases

Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiA(BD)), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiB(BD) [11 kDa]), followed by a catalytic domain (ChiB(cat) [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media

Ultrasound-Mediated Gene Delivery with Cationic Versus Neutral Microbubbles: Effect of DNA and Microbubble Dose on In Vivo Transfection Efficiency

<p><b>Objective</b>: To assess the effect of varying microbubble (MB) and DNA doses on the overall and comparative efficiencies of ultrasound (US)-mediated gene delivery (UMGD) to murine hindlimb skeletal muscle using cationic versus neutral MBs.</p><p><b>Materials and Methods:</b> Cationic and control neutral MBs were characterized for size, charge, plasmid DNA binding, and ability to protect DNA against endonuclease degradation. UMGD of a codon optimized firefly luciferase (Fluc) reporter plasmid to endothelial cells (1 MHz, 1 W/cm&#178;, 20% duty cycle, 1 min) was performed in cell culture using cationic, neutral, or no MBs. <i>In vivo</i> UMGD to mouse hindlimb muscle was performed by insonation (1 MHz, 2 W/cm&#178;, 50% duty cycle, 5 min) after intravenous administration of Fluc combined with cationic, neutral, or no MBs. Gene delivery efficiency was assessed by serial <i>in vivo</i> bioluminescence imaging. Efficiency of <i>in vivo </i>UMGD with cationic versus neutral MBs was systematically evaluated by varying plasmid DNA dose (10, 17.5, 25, 37.5, and 50 &#181;g) while maintaining a constant MB dose of 1x10⁸ MBs and by changing MB dose (1x10⁷, 5x10⁷, 1x10⁸, or 5x10⁸ MBs) while keeping a constant DNA dose of 50 &#181;g.</p><p><b>Results: </b>Cationic and size-matched control neutral MBs differed significantly in zeta potential with cationic MBs being able to bind plasmid DNA (binding capacity of 0.03 pg/MB) and partially protect DNA from nuclease degradation while neutral MBs could not. Cationic MBs enhanced UMGD compared to neutral MBs as well as no MB and no US controls both in cell culture (<i>P</i> &#60; 0.001) and <i>in vivo</i> (<i>P &#60; </i>0.05). Regardless of MB type, <i>in vivo</i> UMGD efficiency increased dose-dependently with DNA dose and showed overall maximum transfection with 50 &#181;g DNA. However, there was an inverse correlation (&#961; = -0.90; <i>P</i> = 0.02) between DNA dose and the degree of enhanced UMGD efficiency observed with using cationic MBs instead of neutral MBs. The delivery efficiency advantage associated with cationic MBs was most prominent at the lowest investigated DNA dose (7.5-fold increase with cationic versus neutral MBs at a DNA dose of 10 &#181;g; <i>P</i> = 0.02) compared to only a 1.4-fold increase at a DNA dose of 50 &#181;g (<i>P</i> &#60; 0.01). With increasing MB dose, overall <i>in vivo </i>UMGD efficiency increased dose-dependently with a maximum reached at a dose of 1x10⁸ MBs with no further significant increase with 5x10⁸ MBs (<i>P</i> = 0.97). However, compared to neutral MBs, cationic MBs enhanced UMGD efficiency the most at low MB doses. Relative enhancement of UMGD efficiency using cationic over neutral MBs decreased from a factor of 27 for 1x10⁷ MBs (<i>P</i> = 0.02) to a factor of 1.4 for 1x10⁸ MBs (<i>P</i> &#60; 0.01) and no significant difference for 5x10⁸ MBs.</p><p><b>Conclusions:</b> Cationic MBs enhance UMGD to mouse skeletal muscle relative to neutral MBs but this is dependent on MB and DNA dose. The enhancement effect of cationic MBs on UMGD efficiency is more evident when lower doses of MBs or DNA are used, whereas the advantage of cationic MBs over neutral MBs is substantially reduced in the presence of excess MBs or DNA.</p

Transcriptional Analysis of Biofilm Formation Processes in the Anaerobic, Hyperthermophilic Bacterium Thermotoga maritima

Thermotoga maritima, a fermentative, anaerobic, hyperthermophilic bacterium, was found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters during chemostat cultivation on maltose-based media at 80°C. A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations. Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in sessile cells (6% of the genome), over a third of which were initially annotated as hypothetical proteins in the T. maritima genome. Among the previously annotated genes in the T. maritima genome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli, and Staphylococcus epidermidis). Most notably, T. maritima biofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones. These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells. Significant up-regulation of several β-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat. The reasons for increased β-glycosidase levels are unclear but are likely related to the processing of biofilm-based polysaccharides. In addition to revealing insights into the phenotype of sessile T. maritima communities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments