After hMSH2 and hMLH1- what next? Analysis of three-generational, population-based, early-onset colorectal cancer families

The aim of our study was to examine the role of genetic factors on early-onset colorectal cancer after excluding the impact of germline mutations in the two major mismatch repair genes. A total of 131 incident probands, under 45 years at diagnosis of a first primary colorectal cancer selected from the Victorian Cancer Registry, and their first-and second-degree relatives, were interviewed. Germline DNA from all 12 probands with a family history meeting the modified Amsterdam Criteria for Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and a random sample of 31 of the remaining probands was screened for mutations in hMSH2 and hMLH1 via manual sequencing. Germline mutations were identified in 6 of the 131 probands (5%), all from the "HNPCC" families. Of the remaining 125 probands, 51 (41%) reported at least one first-or second-degree relative with colorectal cancer with an excess of colorectal cancer in first-degree relatives (SMR = 2.7, 95% CI = 1.7-4.1, p < 0.001). The lifetime risk to age 70 for first-degree relatives was 8.0% (5.0-12.8%), compared to the Victorian population risk of 3.2% (p = 0.01). The best fitting major gene model was a recessively-inherited risk of 98% to age 70 (95% CI = 24-100%) carried by 0.17% of the population and would explain 15% of all colorectal cancer in cases with a diagnosis before age 45. Early-onset colorectal cancer is strongly familial even after excluding families found to be segregating a mutation in either of the 2 major mismatch repair genes. There is evidence for a role of yet to be identified genes associated with a high recessively-inherited risk of colorectal cancer.

Differential effects of exercise on insulin-signaling gene expression in human skeletal muscle.

Skeletal muscle insulin sensitivity is enhanced after acute exercise and short-term endurance training. We investigated the impact of exercise on the gene expression of key insulin-signaling proteins in humans. Seven untrained subjects (4 women and 3 men) completed 9 days of cycling at 63 ± 2% of peak O₂ uptake for 60 min/day. Muscle biopsies were taken before, immediately after, and 3 h after the exercise bouts (on days 1 and 9). The gene expression of insulin receptor substrate-2 and the p85α subunit of phosphatidylinositol 3-kinase was significantly higher 3 h after a single exercise bout, although short-term training ameliorated this effect. Gene expression of insulin receptor and insulin receptor substrate-1 was not significantly altered at any time point. These results suggest that exercise may have a transitory impact on the expression of insulin receptor substrate-2 and phosphatidylinositol 3-kinase; however, the predominant actions of exercise on insulin sensitivity appear not to reside in the transcriptional activation of the genes encoding major insulin-signaling proteins.

Exercise, diet and skeletal muscle gene expression

Skeletal muscle, as a consequence of its mass and great capacity for altered metabolism, has a major impact on whole-body metabolic homeostasis and is capable of remarkable adaptation in response to various physiological stimuli, including exercise and dietary intervention. Exercise-induced increases in skeletal muscle mRNA levels of a number of genes have been reported, due to transcriptional activation and/or increased mRNA stability. The cellular adaptations to exercise training appear to be due to the cumulative effects of transient increases in gene transcription after repeated exercise bouts. The relative importance of transcriptional (mRNA synthesis) and translational (mRNA stability or translational efficiency) mechanisms for the training-induced increases in skeletal muscle protein abundance remains to be fully elucidated. Dietary manipulation, and the associated alterations in nutrient availability and hormone levels, can also modify skeletal muscle gene expression, although fewer studies have been reported. A major challenge is to understand how exercise and diet exert their effects on gene and protein expression in skeletal muscle. In relation to exercise, potential stimuli include stretch and muscle tension, the pattern of motor nerve activity and the resultant calcium transients, the energy charge of the cell and substrate availability, oxygen tension and circulating hormones. These are detected by various cellular signaling mechanisms, acting on a range of downstream targets and a wide range of putative transcription factors. A key goal in the years ahead is to identify how alterations at the level of gene expression are coupled to the changes in skeletal muscle phenotype. It is clear that gene expression, although representing a specific site of regulation, is only one step in a complex cascade from the initial stimulus to the final phenotypic adaptation and integrated physiological response.

Effect of elevated lipid concentrations on human skeletal muscle gene expression

Dietary fatty acids regulate the abundance and activity of various proteins involved in the regulation of fat oxidation by functioning as regulators of gene transcription. To determine whether the transcription of key lipid metabolic proteins necessary for fat metabolism within human skeletal muscle are regulated by acute elevations in circulating free fatty acid (FFA) concentrations, 7 healthy men underwent 3 randomized resting infusions of Intralipid (20%) with heparin sodium, saline and heparin sodium, or saline only for 5 hours. These infusions significantly elevated plasma FFA concentrations by 15-fold (to 1.67 ± 0.13 mmol/L) in the Intralipid infusion trial, with modest elevations observed in the saline and heparin sodium and saline alone infusion groups (0.67 ± 0.09 and 0.49 ± 0.087 mmol/L, P < .01 both vs Intralipid infusion). Analysis of messenger RNA (mRNA) concentration demonstrated that pyruvate dehydrogenase kinase isoform 4 (PDK4) mRNA, a key negative regulator of glucose oxidation, was increased in all trials with a 24-fold response after Intralipid infusion, 15-fold after saline and heparin infusion, and 9-fold after saline alone. The PDK4 increases were not significantly different between the 3 trials. The mRNA concentration of the major uncoupling protein within skeletal muscle, uncoupling protein 3, was not elevated in parallel to the increased plasma FFA as similar (not, vert, similar2-fold) increases were evident in all trials. Additional genes involved in lipid transport (fatty acid translocase/CD36), oxidation (carnitine palmitoyltransferase I), and metabolism (1-acylglycerol-3-phosphate O-acyltransferase 1, hormone-sensitive lipase, and peroxisomal proliferator-activated receptor-γ coactivator-1α) were not altered by increased circulating FFA concentrations. The present data demonstrate that of the genes analyzed that encode proteins that are key regulators of lipid homeostasis within skeletal muscle, only the PDK4 gene is uniquely sensitive to increasing FFA concentrations after increased plasma FFA achieved by intravenous lipid infusion.

Microarray analysis for gene expression that involves trace metals

Microarray plays a major role to identify the over- and under-expressed genes. It is a well-known fact that trace elements in our body play a major role in the metabolic processes of all living organisms. In this paper, the microarray studies related to major trace metals are reviewed. This review forms the basis for the converged effort to locate the genes that are either defective and destabilise the concentration of the trace metals or influenced by the changed concentration of the trace metals that are needed for proper functions of the human body, at different parts of the body.

Role of the ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C_max) and area under the plasma concentration-time curve (AUC) with increasing dosage. The time to C_max (T_max) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P_app) based on TSB disappearance from the lumen (P_lumen) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (P_blood). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P_app values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC_0-24hr in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.

Copper depletion down-regulates expression of the Alzheimer's Disease amyloid-ß precursor protein gene

Alzheimer's disease is characterized by the accumulation of amyloid-ß peptide, which is cleaved from the amyloid-ß precursor protein (APP). Reduction in levels of the potentially toxic amyloid-ß has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the regulation of the APP gene. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for APP and amyloid-ß in copper homeostasis. We hypothesized that metals and in particular copper might alter APP gene expression. To test the hypothesis, we utilized human fibroblasts overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. MNK deletion fibroblasts have high intracellular copper, whereas MNK overexpressing fibroblasts have severely depleted intracellular copper. We demonstrate that copper depletion significantly reduced APP protein levels and down-regulated APP gene expression. Furthermore, APP promoter deletion constructs identified the copper-regulatory region between -490 and +104 of the APP gene promoter in both basal MNK overexpressing cells and in copper-chelated MNK deletion cells. Overall these data support the hypothesis that copper can regulate APP expression and further support a role for APP to function in copper homeostasis. Copper-regulated APP expression may also provide a potential therapeutic target in Alzheimer's disease.

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

Phylogenetic relationships among nine scallop species (Bivalvia : Pectinidae) inferred from nucleotide sequences of one mitochondrial and three nuclear gene regions

Current knowledge of the evolutionary relationships among scallop species (Mollusca: Bivalvia: Pectinidae) in the Indo-Pacific region is rather scanty. To enhance the understanding of the relationships within this group, phylogenies of nine species of scallops with the majority from coastal regions of Thailand, were reconstructed by maximum parsimony, maximum likelihood, and Bayesian methods using sequences of the 16S rRNA of the mitochondrial genome, and a fragment containing the ITS1, 5.8S and ITS2 genes of the nuclear DNA. The trees that resulted from the three methods of analysis were topologically identical, however, gained different levels of support at some nodes. Nine species were clustered into two major clades, corresponding to two subfamilies (Pectininae and Chlamydinae) of the three currently recognized subfamilies within Pectinidae. Overall, the relationships reported herein are mostly in accordance with the previous molecular studies that used sequences of the mtDNA cytochrome oxidase subunit I, and the classification system based on microsculpture of shell features and morphological characteristics of juveniles. Levels of divergences were different among genes (i.e., the 5.8S gene showed the lowest levels of nucleotide divergence at all levels, whereas the 16S rRNA showed the highest level of variation within species, and ITS2 gene revealed the highest level of divergence at higher levels).

Histone modifications and skeletal muscle metabolic gene expression

1.      Skeletal muscle oxidative function and metabolic gene expression are co-ordinately downregulated in metabolic diseases such as insulin resistance, obesity and Type 2 diabetes. Altering skeletal muscle metabolic gene expression to favour enhanced energy expenditure is considered a potential therapy to combat these diseases.

2.      Histone deacetylases (HDACs) are chromatin-remodelling enzymes that repress gene expression. It has been shown that HDAC4 and 5 co-operatively regulate a number of genes involved in various aspects of metabolism. Understanding how HDACs are regulated provides insights into the mechanisms regulating skeletal muscle metabolic gene expression.

3.      Multiple kinases control phosphorylation-dependent nuclear export of HDACs, rendering them unable to repress transcription. We have found a major role for the AMP-activated protein kinase (AMPK) in response to energetic stress, yet metabolic gene expression is maintained in the absence of AMPK activity. Preliminary evidence suggests a potential role for protein kinase D, also a Class IIa HDAC kinase, in this response.

4.      The HDACs are also regulated by ubiquitin-mediated proteasomal degradation, although the exact mediators of this process have not been identified.

5.      Because HDACs appear to be critical regulators of skeletal muscle metabolic gene expression, HDAC inhibition could be an effective therapy to treat metabolic diseases.

6.      Together, these data show that HDAC4 and 5 are critical regulators of metabolic gene expression and that understanding their regulation could provide a number of points of intervention for therapies designed to treat metabolic diseases, such as insulin resistance, obesity and Type 2 diabetes.

The platelet glycoprotein Ia/IIa gene polymorphism C807T/G873A: a novel risk factor for retinal vein occlusion

Retinal vein occlusion (RVO) is associated with hyperhomocysteinaemia and the antiphospholipid syndrome—disorders known to contribute to both arterial and venous thrombosis. In both of these conditions and RVO, platelet activation occurs. Aspirin, not warfarin, is the most effective antithrombotic agent in RVO and, taken together, these observations suggest an important role for platelets in this common ocular thrombotic condition. Platelet glycoprotein Ia/IIa (GpIa/IIa) is an adhesion molecule mediating platelet–collagen interactions and is key to the initiation of thrombosis. Recently, the cellular density of this molecule was shown to be determined by two silent, linked polymorphisms (C807T/G873A) within the GpIa/IIa gene. There is evidence that some of the resulting genotypes are associated with thrombo-embolic disease. This study therefore aimed to establish the prevalence of the GpIa/IIa polymorphisms and the three commonest hereditary thrombophilic disorders (prothrombin gene G20210A (PT) mutation, Factor V Leiden (FVL), and the thermolabile methylene tetrahydrofolate reductase C677T (MTHFR) mutation) in patients with RVO and normal controls. The GpIa/IIa polymorphisms and thrombophilic abnormalities were all identified using the polymerase chain reaction.

Our results show that the frequency of the GpIa/IIa polymorphisms was similar in our normal control population to previously published series. Patients with RVO, however, had only a 10% (4/40) frequency of the lowest risk subtype (CC/GG) compared to 37.5% (15/40) in the control group—P 0.0039. The incidence of the PT, FVL, and MTHFR thrombophilic mutations was not different between the two groups, but interestingly none of the 7/40 RVO cases with a PT, FVL, or MTHFR mutation had the low-risk GpIa/IIa genotype while all but one of the controls did—P<0.05. Thus, 17.5% of RVO patients harboured more than one prothrombotic abnormality. The principal difference between the RVO and control group was the very high incidence of the intermediate-risk GpIa/IIa subtype (CT/GA)—82.5 vs 50%, P<0.05.

These results suggest a major role for GpIa/IIa polymorphisms in the pathogenesis of RVO.

Expression of the male reproduction-related gene (Mar-Mrr) in the spermatic duct of the giant freshwater prawn, Macrobrachium rosenbergii

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, Macrobrachium rosenbergii, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the Mar-Mrr gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The Mar-Mrr gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated.

The Bassian Isthmus and the major ocean currents of southeast Australia influence the phylogeography and population structure of a southern Australian intertidal barnacle Catomerus polymerus (Darwin)

Southern Australia is currently divided into three marine biogeographical provinces based on faunal distributions and physical parameters. These regions indicate eastern and western distributions, with an overlap occurring in the Bass Strait in Victoria. However, studies indicate that the boundaries of these provinces vary depending on the species being examined, and in particular on the mode of development employed by that species, be they direct developers or planktonic larvae dispersers. Mitochondrial DNA sequence analysis of the surf barnacle Catomerus polymerus in southern Australia revealed an east–west phylogeographical split involving two highly divergent clades (cytochrome oxidase I 3.5 ± 0.76%, control region 6.7 ± 0.65%), with almost no geographical overlap. Spatial genetic structure was not detected within either clade, indicative of a relatively long-lived planktonic larval phase. Five microsatellite loci indicated that C. polymerus populations exhibit relatively high levels of genetic divergence, and fall into four subregions: eastern Australia, central Victoria, western Victoria and Tasmania, and South Australia. F_ST values between eastern Australia (from the eastern mitochondrial DNA clade) and the remaining three subregions ranged from 0.038 to 0.159, with other analyses indicating isolation by distance between the subregions of western mitochondrial origin. We suggest that the east–west division is indicative of allopatric divergence resulting from the emergence of the Bassian land-bridge during glacial maxima, preventing gene flow between these two lineages. Subsequently, contemporary ecological conditions, namely the East Australian, Leeuwin, and Zeehan currents and the geographical disjunctions at the Coorong and Ninety Mile Beach are most likely responsible for the four subregions indicated by the microsatellite data.

Association of differential gene expression with imatinib mesylate and omacetaxine mepesuccinate toxicity in lymphoblastoid cell lines

Background

Imatinib mesylate is currently the drug of choice to treat chronic myeloid leukemia. However, patient resistance and cytotoxicity make secondary lines of treatment, such as omacetaxine mepesuccinate, a necessity. Given that drug cytotoxicity represents a major problem during treatment, it is essential to understand the biological pathways affected to better predict poor drug response and prioritize a treatment regime.
Methods

We conducted cell viability and gene expression assays to determine heritability and gene expression changes associated with imatinib and omacetaxine treatment of 55 non-cancerous lymphoblastoid cell lines, derived from 17 pedigrees. In total, 48,803 transcripts derived from Illumina Human WG-6 BeadChips were analyzed for each sample using SOLAR, whilst correcting for kinship structure.
Results

Cytotoxicity within cell lines was highly heritable following imatinib treatment (h2 = 0.60-0.73), but not omacetaxine treatment. Cell lines treated with an IC20 dose of imatinib or omacetaxine showed differential gene expression for 956 (1.96%) and 3,892 transcripts (7.97%), respectively; 395 of these (0.8%) were significantly influenced by both imatinib and omacetaxine treatment. k-means clustering and DAVID functional annotation showed expression changes in genes related to kinase binding and vacuole-related functions following imatinib treatment, whilst expression changes in genes related to cell division and apoptosis were evident following treatment with omacetaxine. The enrichment scores for these ontologies were very high (mostly >10).
Conclusions

Induction of gene expression changes related to different pathways following imatinib and omacetaxine treatment suggests that the cytotoxicity of such drugs may be differentially tolerated by individuals based on their genetic background.