Small regulatory RNAs in mammals

Mammalian cells harbor numerous small non-protein-coding RNAs, including small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs, which regulate gene expression at many levels including chromatin architecture, RNA editing, RNA stability, translation, and quite possibly transcription and splicing. These RNAs are processed by multistep pathways from the introns and exons of longer primary transcripts, including protein-coding transcripts. Most show distinctive temporal- and tissue-specific expression patterns in different tissues, including embryonal stem cells and the brain, and some are imprinted. Small RNAs control a wide range of developmental and physiological pathways in animals, including hematopoietic differentiation, adipocyte differentiation and insulin secretion in mammals, and have been shown to be perturbed in cancer and other diseases. The extent of transcription of non-coding sequences and the abundance of small RNAs suggests the existence of an extensive regulatory network on the basis of RNA signaling which may underpin the development and much of the phenotypic variation in mammals and other complex organisms and which may have different genetic signatures from sequences encoding proteins.

Hypermethylation of the 5 ' CpG island of the gene encoding the serine protease Testisin promotes its loss in testicular tumorigenesis

The Testisin gene (PRSS21) encodes a glycosylphosphatidylinositol (GPI)-linked serine protease that exhibits testis tissue-specific expression. Loss of Testisin has been implicated in testicular tumorigenesis, but its role in testis biology and tumorigenesis is not known. Here we have investigated the role of CpG methylation in Testisin gene inactivation and tested the hypothesis that Testisin may act as a tumour suppressor for testicular tumorigenesis. Using sequence analysis of bisulphite-treated genomic DNA, we find a strong relationship between hypermethylation of a 385 bp 50 CpG rich island of the Testisin gene, and silencing of the Testisin gene in a range of human tumour cell lines and in 100% (eight/eight) of testicular germ cell tumours. We show that treatment of Testisin-negative cell lines with demethylating agents and/or a histone deacetylase inhibitor results in reactivation of Testisin gene expression, implicating hypermethylation in Testisin gene silencing. Stable expression of Testisin in the Testisin-negative Tera-2 testicular cancer line suppressed tumorigenicity as revealed by inhibition of both anchorage-dependent cell growth and tumour formation in an SCID mouse model of testicular tumorigenesis. Together, these data show that loss of Testisin is caused, at least in part, by DNA hypermethylation and histone deacetylation, and suggest a tumour suppressor role for Testisin in testicular tumorigenesis.

P4502C18 catalyzes the metabolic bioactivation of phenytoin

The safe clinical use of phenytoin (PHT) is compromised by a drug hypersensitivity reaction, hypothesized to be due to bioactivation of the drug to a protein-reactive metabolite. Previous studies have shown PHT is metabolized to the primary phenol metabolite, HPPH, then converted to a catechol which then autoxidizes to produce reactive quinone. PHT is known to be metabolized to HPPH by cytochromes P450 (P450s) 2C9 and 2C19 and then to the catechol by P450s 2C9, 2C19, 3A4, 3A5, and 3A7. However, the role of many poorly expressed or extrahepatic P450s in the metabolism and/or bioactivation of PHT is not known. The aim of this study was to assess the ability of other human P450s to catalyze PHT metabolism. P450 2C18 catalyzed the primary hydroxylation of PHT with a k(cat) (2.46 +/- 0.09 min(-1)) more than an order of magnitude higher than that of P450 2C9 (0.051 +/- 0.004 min(-1)) and P450 2C19 (0.054 +/- 0.002 min(-1)) and K-m (45 +/- 5 mu M) slightly greater than those of P450 2C9 (12 +/- 4 mu M) and P450 2C19 (29 +/- 4 mu M). P450 2C18 also efficiently catalyzed the secondary hydroxylation of PHT as well as covalent drug-protein adduct formation from both PHT and HPPH in vitro. While P450 2C18 is expressed poorly in the liver, significant expression has been reported in the skin. Thus, P450 2C18 may be important for the extrahepatic tissue-specific bioactivation of PHT in vivo.

Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms

The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5'-truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclear-localized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles.

Discovery of cyclotide-like protein sequences in graminaceous crop plants: Ancestral precursors of circular proteins?

Cyclotides are peptides from plants of the Rubiaceae and Violaceae families that have the unusual characteristic of a macrocylic backbone. They are further characterized by their incorporation of a cystine knot in which two disulfides, along with the intervening backbone residues, form a ring through which a third disulfide is threaded. The cyclotides have been found in every Violaceae species screened to date but are apparently present in only a few Rubiaceae species. The selective distribution reported so far raises questions about the evolution of the cyclotides within the plant kingdom. In this study, we use a combined bioinformatics and expression analysis approach to elucidate the evolution and distribution of the cyclotides in the plant kingdom and report the discovery of related sequences widespread in the Poaceae family, including crop plants such as rice ( Oryza sativa), maize ( Zea mays), and wheat ( Triticum aestivum), which carry considerable economic and social importance. The presence of cyclotide-like sequences within these plants suggests that the cyclotides may be derived from an ancestral gene of great antiquity. Quantitative RT-PCR was used to show that two of the discovered cyclotide-like genes from rice and barley ( Hordeum vulgare) have tissue-specific expression patterns.

Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome

Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did flat contain ail apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-rnRNA contamination during library construction, or alternatively represent nonspecific transcriptional noise. Here we Show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts Of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide Stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.

Genome-wide analysis of mammalian promoter architecture and evolution

Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a welldefined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.

Mitochondrial dysmorphology in the neuroepithelium of rat embryos following a single dose of maternal hyperthermia during gestation

Hyperthermia is teratogenic to human and animal embryos and induces mainly anomalies of the nervous system. However, the teratogenic mechanism is poorly understood. Mammalian embryos are known to switch from anaerobic to aerobic metabolism around the time of neural tube closure. This critical event might be sensitive to hyperthermia. The objective of the present study was to evaluate the ultrastructural changes of the mitochondria of the neuroepithelium (NE) of rat embryos following maternal exposure to hyperthermia. Pregnant rats were heat stressed for an hour on gestation day (GD) 9 and embryos were examined by electron microscopy on GD 10. NE presented extensive apoptosis. Intercellular junctions were weakened and copious cellular debris projected into the ventricle. The mitochondria were of diverse size and shape. Most of them were swollen and had short cristae and electron dense matrix. Hydropic changes were also observed in numerous mitochondria. Lipid-laden mitochondria were found in the apical portions of neuroblasts. The mesenchyme (ME) of heat-treated embryos showed paucity of cells and only as frequent apoptosis as the controls. Their mitochondria also showed changes similar to those of the NE. Additionally extensive lipid accumulation was observed in and in the vicinity of mitochondria, often surrounded by short strands of endoplasmic reticulum. Whereas mitochondrial pathology was associated with profound apoptosis in the NE, growth restriction and lipid accumulation accompanied mitochondrial changes in the ME. The results of this study indicate that the embryonic response to maternal heat shock is tissue-specific and morphologically distinct in this species.

Design, Synthesis, Potency and Cytoselectivity of Anticancer Agents Derived by Parallel Synthesis from alpha-Aminosuberic Acid

Chemotherapy in the last century was characterized by cytotoxic drugs that did not discriminate between cancerous and normal cell types and were consequently accompanied by toxic side effects that were often dose limiting. The ability of differentiating agents to selectively kill cancer cells or transform them to a nonproliferating or normal phenotype could lead to cell- and tissue-specific drugs without the side effects of current cancer chemotherapeutics. This may be possible for a new generation of histone deacetylase inhibitors derived from amino acids. Structure-activity relationships are now reported for 43 compounds derived from 2-aminosuberic acid that kill a range of cancer cells, 26 being potent cytotoxins against MM96L melanoma cells (IC50 20 nM-1 mu M), while 17 were between 5- and 60-fold more selective in killing MM96L melanoma cells versus normal (neonatal foreskin fibroblasts, NFF) cells. This represents a 10- to 100-fold increase in potency and up to a 10-fold higher selectivity over previously reported compounds derived from cysteine (J. Med. Chem. 2004, 47, 2984). Selectivity is also an underestimate, because the normal cells, NFF, are rarely all killed by the drugs that also induce selective blockade of the cell cycle for normal but not cancer cells. Selected compounds were tested against a panel of human cancer cell lines (melanomas, prostate, breast, ovarian, cervical, lung, and colon) and found to be both selective and potent cytotoxins (IC50 20 nM-1 mu M). Compounds in this class typically inhibit human histone deacetylases, as evidenced by hyperacetylation of histones in both normal and cancer cells, induce expression of p21, and differentiate surviving cancer cells to a nonproliferating phenotype. These compounds may be valuable leads for the development of new chemotherapeutic agents.

Tissue Doppler imaging for evaluation of myocardial function in patients with diabetes mellitus

Purpose of review Heart failure and diabetes mellitus are frequently associated, and diabetes appears to potentiate the clinical presentation of heart failure related to other causes. The purpose of this review is to examine recent advances in the application of tissue Doppler imaging for the assessment of diabetic heart disease. Recent findings Recent studies have documented that both myocardial systolic and diastolic abnormalities can be identified in apparently healthy patients with diabetes and no overt cardiac dysfunction. Interestingly, these are disturbances of longitudinal function, with compensatory increases of radial function-suggesting primary involvement of the subendocardium, which is a hallmark of myocardial ischemia. Despite this, there is limited evidence that diabetic microangiopathy is responsible-with reduced myocardial blood volume rather than reduced resting flow, and at least some evidence suggesting a normal increment of tissue velocity with stress. Finally, a few correlative studies have shown association of diabetic myocardial disease with poor glycemic control, while angiotensin converting enzyme inhibition may be protective. Summary Tissue Doppler imaging (and the related technique of strain rate imaging) appears to be extremely effective for the identification of subclinical LV dysfunction in diabetic patients It is hoped that the recognition of this condition will prompt specific therapy to prevent the development of overt LV dysfunction.

Low level microsatellite instability may be associated with reduced cancer specific survival in sporadic stage C colorectal carcinoma

Background: Colorectal cancers (CRCs) may be categorised according to the degree of microsatellite instability (MSI) exhibited, as MSI-high (MSI-H), MSI-low (MSI-L), or microsatellite stable (MSS). MSI-H status confers a survival advantage to patients with sporadic CRC. Aims: To determine if low levels of MSI are related to the clinicopathological features and prognosis of sporadic stage C CRC. Patients: A total of 255 patients who underwent resection for sporadic stage C CRC were studied. No patient received chemotherapy. Minimum follow up was five years. Methods: DNA extracted from archival malignant and non-malignant tissue was amplified by polymerase chain reaction using a panel of 11 microsatellites. MSI-H was defined as instability at greater than or equal to40% of markers, MSS as no instability, and MSI-L as instability at >0% but,40% of markers. Patients with MSI-H CRC were excluded from analysis as they have previously been shown to have better survival. Results: Thirty three MSI-L and 176 MSS CRCs were identified. There was no difference in biological characteristics or overall survival of MSI-L compared with MSS CRC but MSI-L was associated with poorer cancer specific survival (hazard ratio 2.0 (95% confidence interval 1.1-3.6)). Conclusions: Sporadic MSI-L and MSS CRCs have comparable clinicopathological features. Further studies are required to assess the impact of MSI-L on prognosis.

Isolates of Candida albicans that differ in virulence for mice elicit strain-specific antibody-mediated protective responses

Three distinct isolates of Candida albicans were used to establish systemic and oral infections in inbred mice that are genetically resistant or susceptible to tissue damage. Patterns of infection differed significantly between both yeasts and mouse strains. Systemic infection conferred significant protection against re-challenge with the homologous, but not the heterologous yeast; however, the protective effect was more evident in the tissue-susceptible CBA/CaH mice than in the resistant BALB/c strain. In contrast, oral infection induced protection against both homologous and heterologous oral challenge, although this was significant only in the CBA/CaH mice. CBA/CaH mice produced antibodies of both IgG1 and IgG2a subclasses, whereas BALB/c mice produced predominantly IgG1. Western blotting demonstrated considerable differences between epitopes recognised by serum antibodies from mice of both strains after immunisation with each of the three yeasts. Thus, different strains of yeast show considerable specificity in antibody responses elicited by either systemic or oral infection. (c) 2005 Elsevier SAS. All rights reserved.

Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen-specific CD8(+) T-cell function in Hodgkin lymphoma patients

In Hodgkin lymphoma (HL), the malignant Hodgkin Reed-Sternberg (HRS) cells constitute only 0.5% of 10% of the diseased tissue. The surrounding cellular infiltrate is enriched with T cells that are hypothesized to modulate antitumor immunity. We show that a marker of regulatory T cells, LAG-3, is strongly expressed on infiltrating lymphocytes present in proximity to HRS cells. Circulating regulatory T cells (CD4(+) CD25(hi) CD45 ROhi, CD4(+) CTLA4(hi), and CD4(+) LAG-3(hi)) were elevated in HL patients with active disease when compared with remission. Longitudinal profiling of EBV-specific CD8(+) T-cell responses in 94 HL patients revealed a selective loss of interferon-gamma expression by CD8(+) T cells specific for latent membrane proteins 1 and 2 (LMP1/2), irrespective of EBV tissue status. Intratumoral LAG-3 expression was associated with EBV tissue positivity, whereas FOXP3 was linked with neither LAG-3 nor EBV tissue status. The level of LAG-3 and FOXP3 expression on the tumor-infiltrating lymphocytes was coincident with impairment of LMP1/2-specific T-cell function. In vitro pre-exposure of peripheral blood mono-nuclear cells to HRS cell line supernatant significantly increased the expansion of regulatory T cells and suppressed LMP-specific T-cell responses. Deletion of CD4(+) LAG-3(+) T cells enhanced LMP-specific reactivity. These findings indicate a pivotal role for regulatory T cells and LAG-3 in the suppression of EBV-specific cell-mediated immunity in HL.

Are echo techniques comparable for measurement of left ventricular dysynchrony? Comparison of real time 3D and tissue Doppler echocardiography in patients with ischaemic cardiomyopathy

Tissue Doppler (TD) assessment of dysynchrony (DYS) is established in evaluation for bi-ventricular pacing. Time to regional minimal volume by real-time 3D echo (3D) has been applied to DYS. 3D offers simultaneous assessment of all segments and may limit errors in localization of maximum delay due to off-axis images.We compared TD and 3D for assessment of DYS. 27 patients with ischaemic cardiomyopathy (aged 60±11 years, 85% male) underwent TD with generation of regional velocity curves. The interval between QRS onset and maximal systolic velocity (TTV) was measured in 6 basal and 6 mid-cavity segments. Onthe same day,3Dwas performed and data analysed offline with Q-Lab software (Philips, Andover, MA). Using 12 analogous regional time-volume curves time to minimal volume (T3D)was calculated. The standard deviation (S.D.) between segments in TTV and T3D was calculated as a measure ofDYS. In 7 patients itwas not possible to measureT3D due to poor images. In the remaining 20, LV diastolic volume, systolic volume and EF were 128±35 ml, 68±23 ml and 46±13%, respectively. Mean TTV was less than mean T3D (150±33ms versus 348±54 ms; p < 0.01). The intrapatient range was 20–210ms for TTV and 0–410ms for T3D. Of 9 patients (45%) with significantDYS (S.D. TTV > 32 ms), S.D. T3D was 69±37ms compared to 48±34ms in those without DYS (p = ns). In DYS patients there was concordance of the most delayed segment in 4 (44%) cases.Therefore, different techniques for assessing DYS are not directly comparable. Specific cut-offs for DYS are needed for each technique.