Actin mRNA Research Articles

Trichosanthin-induced specific changes of cytoskeleton configuration were associated with the decreased expression level of actin and tubulin genes in apoptotic Hela cells

Trichosanthin (TCS) possesses a broad spectrum of biological and pharmacological activities, including anti-cancer activities through apoptosis pathway. However, little is known about the effects of TCS on the cytoskeleton configuration and expression of actin and tubulin genes in Hela cell apoptosis. In the present study, apoptotic cytoskeleton structures were observed by confocal immunofluorescence microscopy, absolute amounts of actin and tubulin subunit mRNAs were determined by quantitative real-time PCR assays (QRT-PCR). Our results showed that the execution phase of cell apoptosis was a highly coordinated process of cellular reorganization, depolymerized microfilaments (MFs) accumulated in the coarsened cytoplasm and apoptotic bodies, followed by the formation of a ring microtubule (MT) structure beneath the plasma membrane. Importantly, apoptosis occurred by a suppression of actin and tubulin subunit gene expression. In particular, a rapid decrease in the amounts of γ-actin mRNA preceded that of β-actin; α- and β-tubulin mRNAs were subsequently down-regulated in the later stage of Hela cell apoptosis. These results suggested that the execution of Hela cell apoptosis induced by TCS accompanied the specific changes of cytoskeleton configuration and, significantly, decreased the expression level of actin and tubulin subunit genes in different stages.

ENOS Deficient Mice Develop Progressive Cardiac Hypertrophy with Altered Cytokine and Calcium Handling Protein Expression

Although studies have shown that endothelial nitric oxide synthase (eNOS) homozygous knockout mice (eNOS-/-) develop left ventricular (LV) hypertrophy, well compensated at least to 24 wks, uncertainty still exists as to the cardiac functional and molecular mechanistic consequences of eNOS deficiency at later time-points. To bridge the gap in existent data, we examined whole hearts from eNOS-/- and age-matched wild-type (WT) control mice ranging in age from 18 to 52 wks for macroscopic and microscopic histopathology, LV mRNA and protein expression using RNA Dot blots and Western blots, respectively, and LV function using isolated perfused work-performing heart preparations. Heart weight to body weight (HW/BW in mg/g) ratio increased significantly as eNOS-/- mice aged (82.2%, P < 0.001). Multi-focal replacement fibrosis and myocyte degeneration/death were first apparent in eNOS-/- mouse hearts at 40 wks. Progressive increases in LV atrial natriuretic factor (ANF) and alpha-skeletal actin mRNA levels both correlated significantly with increasing HW/BW ratio in aged eNOS-/- mice (r = 0.722 and r = 0.648, respectively; P < 0.001). At 52 wks eNOS-/- mouse hearts exhibited basal LV hypercontractility yet blunted beta adrenergic receptor (betaAR) responsiveness that coincided with a significant reduction in the LV ratio of phospholamban to sarcoplasmic reticulum Ca2+-ATPase-2a protein levels and was preceded by a significant upregulation in LV steady-state mRNA and protein levels of the 28 kDa membrane-bound form of tumor necrosis factor-alpha. We conclude that absence of eNOS in eNOS-/- mice results in a progressive concentric hypertrophic cardiac phenotype that is functionally compensated with decreased betaAR responsiveness, and is associated with a potential cytokine-mediated alteration of calcium handling protein expression.

Expression of c-Fos-LacZ transgene after LiCl administration.

Conditioned taste aversion (CTA) occurs after pairing of a novel taste with a toxin (e.g. sucrose with LiCl). c-Fos is expressed, e.g. in the amydala, during CTA acquisition, but other transcription factors contribute to CTA learning. To aid in the identification of c-Fos-positive cells for future transcriptional profiling, we characterized β -galactosidase expression in transgenic mice carrying a c-fos-lacZ fusion gene. Mice were injected with LiCl (40 ml/kg, 0.15 M, i.p.) or saline and perfused 1, 3, 6 and 9 h later. Tissue sections were processed for c-Fos immunohistochemistry and X-gal staining in the amygdala. There was a high density of both c-Fos- and X-gal-positive cells in the cortex and hippocampus. LiCl increased c-Fos and X-gal staining in the amygdala: c-Fos levels were low in the lateral nucleus (<20 cells/section), but increased rapidly in the central nucleus (631±97% at 1 h) and slowly in the basolateral amygdala (185±29% at 6 h). To confirm c-Fos expression in X-gal-positive cells, we examined microdisseccted X-gal-positive cells of the amygdala, cortex and hippocampus using laser capture microdissection (LCM) and RT-PCR. Following LiCl injection, tissue sections (8 μm thick) were processed for X-gal staining using DEPC-treated reagents to preserve RNA. Approximately 30 individual X-gal-positive cells were collected from each brain region by LCM for each RT-PCR reaction. Both β -actin and c-Fos mRNA were found in the X-gal-positive cells from each brain region. The expression of other immediate-early genes within X-gal-positive cells will also be examined. Support: NIDCD03198.

Importance of inhibitor of DNA binding/differentiation 2 in hepatic stellate cell differentiation and proliferation

In liver fibrosis, activated hepatic stellate cells (HSC) are transformed into myofibroblasts. Helix-loop-helix (HLH) transcriptional factors such as MyoD regulate the differentiation of myocytes, and the inhibitor of DNA binding/differentiation (Id) family comprises dominant negative HLH transcriptional regulators that inhibit differentiation and promote cell proliferation. In the present study, we investigated how the Id family proteins regulate HSC. In primary rat HSC, inhibitor of DNA binding/differentiation (Id)2 and alpha-smooth muscle actin (alpha-SMA) mRNA expression increased 4 days after isolation. Next we established Id2 expressing HSC (HSC-T6-Id2-green fluorescent protein (GFP)) using HSC-T6 cells with retrovirus that expressed GFP-tagged Id2. HSC-T6-Id2-GFP increased cell proliferation with cyclin D1 expression. In contrast, alpha-SMA expression wassuppressed. Real-time reverse transcription-polymerase chain reaction analysis showed Id2 induction significantly suppressed alpha-SMA, collagen-1, matrix metalloproteinase (MMP)-2, and MMP-9 mRNA (P < 0.05) but had no effect on tissue inhibitor of metalloproteinase or transforming growth factor-beta1 levels. These findings suggest Id2, an HLH transcriptional regulator, plays an important regulatory role in the proliferation and differentiation of HSC.

Efficient RNA polyuridylation by noncanonical poly(A) polymerases.

Nuclear poly(A) polymerase (PAP) polyadenylates nascent mRNAs, promoting their nuclear export, stability, and translation, while the related cytoplasmic polymerase GLD-2 activates translation of deadenylated mRNAs. Here we characterize the biochemical activity of fission yeast Schizosaccharomyces pombe Cid1, a putative cytoplasmic PAP implicated in cell cycle checkpoint controls. Surprisingly, Cid1 has robust poly(U) polymerase activity in vitro, especially when isolated in native multiprotein complexes. Furthermore, we found that upon S-phase arrest, the 3' ends of actin mRNAs were posttranscriptionally uridylated in a Cid1-dependent manner. Finally, Hs2 (ZCCHC6), a human ortholog of Cid1, shows similar activity. These data suggest that uridylation of mRNA forms the basis of an evolutionarily conserved mechanism of gene regulation.

Induction of actin gene expression in the mosquito midgut by blood ingestion correlates with striking changes of cell shape

Ingestion of a blood meal by the female mosquito Anopheles gambiae (L., Diptera: Culicidae), results in a dramatic distention of the midgut epithelium. Here, we report that these events correlate with a transient increase of actin mRNA and protein abundance. The newly synthesized actin may provide a pool of actin protein needed to remodel epithelial cell cytoarchitecture. We also document changes in midgut epithelial cell morphology. Upon blood ingestion, the columnar cells flatten accompanied by the loss of microvilli on the lumenal side and the unfolding of the labyrinth on the basal side. These changes correlate with the large increase of epithelial surface area needed to accommodate the blood meal. Actin gene expression, actin synthesis and cell morphology all return to the pre-feeding state by 24 h after blood intake.

Developmental Changes of Col3a1 mRNA Expression in Muscle and Their Association with Intramuscular Collagen in Pigs

Eighty-four castrated boars including Laiwu Black (LW) (weight 30–90 kg, n = 6) and Lulai Black (LL) (weight 40–100 kg, n = 6) were used to study the developmental changes of collagen type ? alpha 1 ( Col3a1) mRNA expression in the muscle and their association with intramuscular collagen (IMC). The muscle total RNA was extracted to determine the abundance of Col3a1 mRNA using relative quantitative RT-PCR with ?-actin mRNA as the internal standard. The results indicated that the developmental patterns of muscle Col3a1 mRNA in LW and LL pigs were similar. The abundance of Col3a1 mRNA increased with body weight, but decreased a little at 70 kg and 80 kg phases for LW and LL, respectively. On the whole, the expression level of Col3a1 mRNA in muscle of LW was higher than that of LL ( P < 0.05). Correlation analysis showed that the expression of Col3a1 mRNA in muscle was positively correlated with total and insoluble IMC, but was negatively correlated with IMC solubility for LW pigs ( P < 0.01) and LL pigs ( P < 0.05), respectively. These results suggest that the muscle Col3a1 gene expression is affected by body weight and genotype and has important effect on IMC content and characteristics.

The adaptor Grb7 links netrin-1 signaling to regulation of mRNA translation

We previously reported a novel biological activity of Netrin-1 in translational stimulation of kappa opioid receptor (KOR). We now identify Grb7 as a new RNA-binding protein that serves as the molecular adaptor for transmitting Netrin-1 signals, through focal adhesion kinase (FAK), to the translation machinery. Grb7 binds specifically to the first stem loop of kor mRNA 5'-UTR through a new RNA-binding domain located in its amino terminus. Upon binding to its capped, target mRNA, Grb7 blocks the recruitment of eIF4E, rendering mRNA untranslatable. The RNA-binding and translation-repressive activity is reduced by FAK-mediated hyperphosphorylation on two tyrosine residues of its carboxyl terminus. This study reports an adaptor protein Grb7 that transmits the stimulating signals of Netrin-1 to the translational machinery to rapidly regulate mRNA translation.

Abelson interacting protein 1 (Abi-1) is essential for dendrite morphogenesis and synapse formation

Synaptogenesis and synaptic plasticity depend crucially on the dynamic and locally specific regulation of the actin cytoskeleton. We identified an important component for controlled actin assembly, abelson interacting protein-1 (Abi-1), as a binding partner for the postsynaptic density (PSD) protein ProSAP2/Shank3. During early neuronal development, Abi-1 is localized in neurites and growth cones; at later stages, the protein is enriched in dendritic spines and PSDs, as are components of a trimeric complex consisting of Abi-1, Eps8 and Sos-1. Abi-1 translocates upon NMDA application from PSDs to nuclei. Nuclear entry depends on abelson kinase activity. Abi-1 co-immunoprecipitates with the transcription factor complex of Myc/Max proteins and enhances E-box-regulated gene transcription. Downregulation of Abi-1 by small interfering RNA results in excessive dendrite branching, immature spine and synapse morphology and a reduction of synapses, whereas overexpression of Abi-1 has the opposite effect. Data show that Abi-1 can act as a specific synapto-nuclear messenger and is essentially involved in dendrite and synapse formation.

A Functional Genomics Analysis of the B56 Isoforms of Drosophila Protein Phosphatase 2A

Members of the B56 family of protein phosphatase 2A (PP2A) regulatory subunits play crucial roles in Drosophila cell survival. Distinct functions of two B56 subunits were investigated using a combination of RNA interference, DNA microarrays, and proteomics. RNA interference-mediated knockdown of the B56-1 subunit (PP2A-B') but not the catalytic (mts) or B56-2 subunit (wdb) of PP2A resulted in increased expression of the apoptotic inducers reaper and sickle. Co-knockdown of B56-1 with reaper, but not with sickle, reduced the apoptosis caused by depletion of the B56 subunits. Two-dimensional gel electrophoresis and mass spectrometry identified proteins modified in cells depleted of PP2A subunits. These included generation of caspase-dependent cleavage products, increases in protein abundance, and covalent modifications. Results suggested that up-regulation of the ribosome-associated protein stubarista can serve as a sensitive marker of apoptosis. Up-regulation of transcripts for multiple glutathione transferases and other proteins suggested that loss of PP2A affected pathways involved in the response to oxidative stress. Knockdown of PP2A elevated basal JNK activity and substantially decreased activation of ERK in response to oxidative stress. The results reveal that the B56-containing isoform of PP2A functions within multiple signaling pathways, including those that regulate expression of reaper and the response to oxidative stress, thus promoting cell survival in Drosophila.

The Anti-rheumatic Gold Salt Aurothiomalate Suppresses Interleukin-1β-induced Hyaluronan Accumulation by Blocking HAS1 Transcription and by Acting as a COX-2 Transcriptional Repressor

Gold compounds are among the oldest disease-modifying drugs and are still widely used today for treating rheumatoid arthritis. Despite decades of use, little is known about the mode of action of this class of drugs. Here we have demonstrated that aurothiomalate (AuTM) suppresses hyaluronan accumulation by blocking interleukin (IL)-1beta-induced hyaluronan synthase-1 transcription. We have further demonstrated that, in fibroblast-like synoviocytes (FLSs), AuTM acts as a specific COX-2 transcriptional repressor in that IL-1beta-induced COX-2 transcription is blocked, whereas COX-1 transcription and translation is unaffected. As a consequence, PGE2 levels released by FLS are dose-dependently reduced in cells exposed to AuTM. Of similar importance is the demonstration that AuTM does block NFkappaB-DNA interaction. In addition, two other transcription factors implicated in inflammatory events, namely AP-1 and STAT3, are blocked as well. The effect on NFkappaB likely explains the inhibition of COX-2 as well as that of HAS1, as both are genes that depend on the activation of NFkappaB. Interestingly, AuTM does not interfere with IL-1beta-induced IkappaB alpha degradation, in most cases a prerequisite for subsequent NFkappaB activation. Furthermore, evidence is presented that, in FLS, AuTM blocks NFkappaB-DNA interaction neither by binding to NFkappaB binding sites nor by interacting with activated NFkappaB proteins. Taken together, AuTM treatment of FLS blocks two of the most important proinflammatory events that are associated with rheumatoid arthritis. AuTM blocks the release of PGE2 and prevents the activation of NFkappaB, therefore blocking IL-1beta-induced hyaluronan accumulation and likely a series of other pro-inflammatory NFkappaB-dependent genes.

Performance of a Foot-and-Mouth Disease virus Reverse Transcription-Polymerase Chain Reaction with Amplification Controls between Three Real-Time Instruments

The foot-and-mouth disease virus (FMDV) is a member of the picornavirus family, possessing an 8-kb single-stranded RNA genome of positive polarity. It is highly contagious among several livestock species and can lead to severe economic consequences, as evidenced by the UK outbreak in 2001. The usage of real-time polymerase chain reaction has facilitated rapid detection of FMDV. Several real-time PCR instruments are available with various capabilities, such as portability and high sample volume analysis. Primers and a dual-labeled TaqMan probe were optimized to detect a single, highly conserved 88-bp segment of the FMDV 3D (RNA polymerase) gene. To increase the confidence of the RT-PCR result, a positive amplification control was synthesized to detect potential false-positive results due to contamination if a wild-type virus is used as positive control. In addition, a preventative measure against false-negative results was developed in which endogenous beta actin mRNA is coamplified by RT-PCR. Assay performance was compared on the LightCycler1.2 (Roche), the SmartCyclerII (Cepheid), and the SDS 7900HT (ABI). These assays successfully identified the FMDV genome and beta actin mRNA from several sources of infected nasal and oral swabs, as well as probang samples.

The effect of cholecyst-derived extracellular matrix on the phenotypic behaviour of valvular endothelial and valvular interstitial cells

Cholecyst-derived extracellular matrix (CEM) is a novel, proteinaceous biomaterial, derived from the porcine cholecyst, which may have potential applications as a scaffold in the area of heart valve tissue engineering. In this study the potential of CEM to support the proliferation of valvular endothelial cells (VECs) and valvular interstitial cells (VICs), while maintaining their phenotypic mRNA synthesis, protein expression and morphology was assessed by biochemical assays, electron microscopy, immunostaining and reverse-transcriptase polymerase chain reaction. VICs and VECs were isolated from the porcine aortic valve and techniques were developed for the isolation of CEM for cell culture. VECs and VICs cultured on CEM adhered and proliferated, maintaining their phenotypic morphology. VECs synthesised von Willebrand factor mRNA and endothelial nitric oxide synthase (eNOS) mRNA and expressed eNOS and VICs synthesised alpha-smooth muscle actin ( αSMA) mRNA and expressed αSMA. Cellular area fraction of VICs expressing αSMA was 87.7±6.8% and cellular area fraction of VECs expressing eNOS was 93.8±9.3%. Findings of this study support the hypothesis that CEM is a potential biomaterial for tissue engineered heart valve scaffold design.

Molecular and cellular defects of skeletal muscle in an animal model of acute quadriplegic myopathy

Muscle denervation and concomitant high-dose dexamethasone treatment in rodents produces characteristic pathologic features of severe muscle atrophy and selective myosin heavy filament (MyHC) depletion, identical to those seen in acute quadriplegic myopathy (AQM), also known as critical illness myopathy. We tested the hypothesis that defective pre-translational processes contribute to the atrophy and selective MyHC depletion in this model. We examined the effects of combined glucocorticoid-denervation treatment on MyHC and actin mRNA populations; we also studied mRNA expression of the myogenic regulatory factors (MRFs), primary transcription factors for MyHC. Adult female rats were subjected to proximal sciatic denervation followed by high-dose dexamethasone (DD) treatment (5 mg/kg body weight daily) for 7 days. Disease controls included rats treated with denervation alone (DN) or dexamethasone alone (DX). At 1 week the plantaris atrophied by approximately 42% in DD muscles. DD treatment resulted in selective MyHC protein depletion; actin protein concentration was not significantly changed. Despite an increase in total RNA concentration in DN and DD muscles, MyHC and actin mRNA concentrations were significantly decreased in these muscles. MyHC mRNA showed a significantly more extensive depletion relative to actin mRNA in DD muscles. Glucocorticoid treatment did not influence a denervation-induced increase in the mRNA expression of the MRFs. We conclude that a deleterious interaction between glucocorticoid and denervation treatments in skeletal muscle is responsible for pre-translational defects that reduce actin and MyHC mRNA substrates in a disproportionate fashion. The resultant selective MyHC depletion contributes to the severe muscle atrophy.

Sequential Modifications in Class II Transactivator Isoform 1 Induced by Lipopolysaccharide Stimulate Major Histocompatibility Complex Class II Transcription in Macrophages

By presenting antigenic peptides on major histocompatibility complex class (MHC) II determinants to CD4(+) T cells, macrophages help to direct the establishment of adaptive immunity. We found that in these cells, lipopolysaccharide stimulates the expression of MHC II genes via the activation of Erk1/2, which is mediated by Toll-like receptor 4. Erk1/2 then phosphorylates the serine at position 357, which is located in a degron of CIITA isoform 1 that leads to its monoubiquitylation. Thus modified, CIITA isoform 1 binds P-TEFb, which mediates the elongation of RNA polymerase II and co-transcriptional processing of nascent transcripts. This induction leads to the expression of MHC II genes. Subsequent polyubiquitylation results in the degradation of CIITA isoform 1. Thus, the signaling cascade from Toll-like receptor 4 to CIITA isoform 1 represents one connection between innate and adaptive immunity in macrophages.

Primary nasopharyngeal mucoepidermoid carcinoma in Japanese patients: two case reports with histochemical and immunohistochemical analysis and a review of the literature

Mucoepidermoid carcinoma is a common neoplasm of the salivary glands. Salivary gland-type neoplasms are very rare in the nasopharynx, and there are only few reports on mucoepidermoid carcinoma of the nasopharynx. Two additional cases of mucoepidermoid carcinoma arising from the nasopharynx are reported here. Case 1: the patient was a 57-year-old Japanese woman who had bloody sputum. Case 2: the patient was a 51-year-old Japanese woman who underwent resection of a nasopharyngeal tumor. Histologically, both tumors were also low-grade mucoepidermoid carcinomas with clear cells. Histochemically, the gland-like nests and mucous cells were positive for mucin staining. Immunohistochemically, the lesions were positive for cytokeratins (CKs), p63, and MUC1, but negative for alpha-smooth muscle actin and EBER mRNA. The Ki-67 labeling indices of the two tumors were 10.4% and 4.3%, respectively. The two present cases and a review of the English literature indicate that salivary gland-type neoplasms arising from the nasopahrynx are extremely rare. The prognosis of salivary gland-type carcinomas of the nasopharynx is still unknown.

IL7 Administration in Humans Results in Preferential Expansion of Naive and Memory CD4+ & CD8+ T Cells with a Relative Decrease in Regulatory T-Cells (T-Regs).

Interleukin-7 (IL7) is a multifunctional cytokine with critical and non-redundant roles in T-cell development, T-lymphopoiesis and peripheral T-cell homeostasis. We report preliminary results of the first phase I study of recombinant human IL7, “CYT 99-007” (rhIL7). Adults with incurable malignancy (11 men and 3 women), 20–71 years old (median 48) received escalating doses (3, 10, 30 or 60 μg/kg/dose) subcutaneously every other day for 2 weeks.IL7 was overall well tolerated with no Maximum Tolerated Dose yet reached. Dose-dependent and age independent biological effects consistent with murine and non-human primate pre-clinical models were observed in all patients receiving 10 μg/Kg/dose or more: enlargement of spleen and lymph nodes (but not thymus) by CT scan and marked proliferation and expansion of T-cells subsets. Increases in CD4+ and CD8+ T-cells were most prominent in naïve T-cells (CD27+CD45RA+), including Recent Thymic Emigrants (RTE) (CD4+/CD45RA+/CD31+) with a mean 6-fold expansion, and in memory T-cells (CD27+CD45RA−) but less so in effector T-cells (CD27−). Furthermore, IL7 therapy resulted in a decline in the relative frequency of T-regs (50–80% decline in FoxP3 mRNA copies normalized to Actin mRNA copies in sorted CD4+ T-cells).Although the magnitude of biological effects was variable, the kinetics were similar in all T-cell subsets and at all effective doses. After one week of therapy at 30μg/ Kg /dose, 30–70% of naïve CD4+ and CD8+ T-cells were in cycle (Ki-67+) and expressed elevated levels of Bcl-2. Bcl-2 up-regulation (maintained at week 2) and high proliferation rates resulted in T-cell expansion (mean of 6- and up to 14-fold in some individuals at the 30μg dose) persisting one to several weeks after treatment. Consistent with animal data, we observed IL7 receptor α-chain (IL7Rα) down-regulation: both IL7Rα mean fluorescent intensity by flow cytometry (CD127) and mRNA copy numbers declined more than 50% at one week. By the end of treatment, proliferation rates were halved and down to baseline by week 3, coincident with recovery of IL7Rα expression after cessation of treatment. The T-cell increase was primarily due to IL7 induced peripheral expansion. There was no dilution effect of T-Cell Receptor Excision Circles (TREC) numbers per 105 CD4+ sorted cells which suggests cell recruitment or expansion, consistent with preclinical data but does not exclude a thymic contribution.rhIL7 is safe at biologically active doses in humans and induces an expansion of naïve (including RTE) and memory T-cells. In contrast to IL2, IL7's role in T-regs homeostasis appears to be minimal. rhIL7 may prove clinically valuable in adoptive immunotherapy as an immuno-restorative agent in conditions of disease (HIV) or chemotherapy induced T-cell depletion or as an adjunct to tumor vaccination for immune response modulation.

Beneficial effect of glatiramer acetate (Copaxone) on immune modulation of experimental hepatic fibrosis

While CD8 subsets activate hepatic fibrosis, natural killer (NK) cells exhibit antifibrotic activity. Glatiramer acetate (GA) is an immune modulator for multiple sclerosis. We assessed the potential impact of GA on mouse hepatic fibrogenesis. Hepatic fibrosis was induced in C57BL/6 mice by intraperitoneal administration of carbon tetrachloride (CCl(4)) for 6 wk. During the last 2 wk, animals were also treated with either GA (200 mu/day ip) or medium and compared with naive and fibrotic mice (8 animals/group). GA markedly attenuated fibrosis without altering reactive oxygen species production. By morphometric measurement of Sirius red-stained tissue sections, the relative fibrosis area decreased from 5.28 +/- 0.32% (mean +/- SE) in the untreated CCl(4) group to 2.01 +/- 0.28% in CCl(4)+GA-treated animals, compared with 0.38 +/- 0.07% in naive mice. alpha-Smooth muscle actin immunoblotting and mRNA expression revealed a similar pattern. Serum aminotransferase and Ishak-Knodell necroinflammatory score were markedly elevated, to the same extent, in both CCl(4)-treated groups. Fibrosis induction was associated with significant increase in CD8 subsets and decrease in CD4 T cells. After GA treatment, however, NK content, CD4(+)CD25(+)FoxP3(+) cells, hepatic expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and apoptosis of hepatic stellate cells were all increased. Serum interleukin (IL)-10 levels markedly rose, whereas IL-4 fell. In vitro activation of human hepatic stellate cells cocultured with hepatitis C virus-derived peripheral blood lymphocytes decreased when lymphocytes were preincubated with GA before coculture. In an animal model of hepatic fibrosis, GA has an antifibrotic effect associated with decreased CD8 cells and reduced serum IL-4 levels and increased NK cells, CD4(+)CD25(+)FoxP3(+) cells, TRAIL, and elevated serum IL-10 levels.

Effect of age on skeletal muscle myofibrillar mRNA abundance: Relationship to myosin heavy chain protein synthesis rate

Age-related changes in myosin heavy chain (MHC) phenotype impact both the quantity and functional character of skeletal muscle. The present study was undertaken to examine the hypothesis that age-related changes in MHC mRNA abundance underlie alterations in protein synthesis rates and content. We measured the abundance of mRNA for MHC isoforms (MHC I, MHC IIa, IIx) and actin by RT-PCR in 6 young (mean ± SE; 29 ± 3) and 12 elderly (73 ± 1 yr; P < 0.01) volunteers and examined their association to MHC protein synthesis rates and their respective protein products. We found no differences between young and elderly volunteers in MHC isoform or actin transcript levels. Because of the absence of age effects, data were pooled for correlation analyses. Although total MHC mRNA levels were not related to MHC fractional synthesis rates, the relative abundance of MHC I and MHC IIa mRNA were positively ( r = 0.450; P = 0.06) and negatively ( r = −0.493; P < 0.05) associated with MHC protein synthesis rates, respectively. MHC mRNA levels were positively correlated to their respective protein products (range of r-values: 0.551–0.727; P < 0.05 to P < 0.01), but were not related to skeletal muscle IGF-I mRNA abundance, circulating IGF-I or markers of immune activation. Our results argue against the notion that changes in MHC protein synthesis rates with age are related to altered MHC mRNA abundance, although our findings do suggest that possibility that individual variability in MHC protein synthesis rates is related to the relative abundance of MHC I versus MHC IIa transcripts.

Caffeine Targets TOR Complex I and Provides Evidence for a Regulatory Link between the FRB and Kinase Domains of Tor1p

Caffeine Targets TOR Complex I and Provides Evidence for a Regulatory Link between the FRB and Kinase Domains of Tor1p