A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules

To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network.These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed.     

Viral silencing suppressors and cellular proteins partner with plant RRP6-like exoribonucleases

Virus Genes - RNA silencing and RNA decay are functionally interlaced, regulate gene expression and play a pivotal role in antiviral responses. As a counter-defensive strategy, many plant and...     

The 10-item Remembered Relationship with Parents (RRP10) scale: two-factor model and association with adult depressive symptoms

BACKGROUND: Dysfunctional parenting styles are associated with poor mental and physical health. The 10-item Remembered Relationship with Parents (RRP(10)) scale retrospectively assesses Alienation (dysfunctional communication and intimacy) and Control (overprotection by parents), with an emphasis on deficiencies in empathic parenting. We examined the 2-factor structure of the RRP(10) and its relationship with adult depression. METHODS: 664 respondents from the general population (48% men, mean age 54.6+/-14.2 years) completed the RRP(10), Parental Bonding Instrument (PBI), and Beck Depression Inventory. RESULTS: The Alienation and Control dimensions of the RRP(10) displayed a sound factor structure, good internal consistency (Cronbach's alpha=0.83-0.86), and convergent validity against the PBI scales. No significant gender differences were found on the RRP(10) scales. Stratifying by RRP(10) dimensions showed that respondents high in Alienation and Control, for both father (33.3% vs. 14.5%, p<0.0001) and mother (42% vs. 12.9%, p<0.0001) items, experienced the highest levels of depressive symptoms compared with respondents low in Alienation and Control. While scoring high on Alienation or Control alone was also significantly and independently associated with depressive symptoms, scoring high on both Alienation and Control was most strongly connected with depressive symptoms for both father (OR=2.48, p<0.004) and mother (OR=5.34, p<0.0001) items. LIMITATIONS: Cross-sectional study design. CONCLUSIONS: The RRP(10) is a reliable and valid measure of remembered parental Alienation and Control. High Alienation and Control were independently related to increased risk of depressive symptoms. Given the brevity of the RRP(10), it can easily be used in epidemiological/clinical research on the link between the remembered relationship with parents and mental/physical health.     

Functional characterisation and subcellular localisation of HCN1 channels in rabbit retinal rod photoreceptors

Gating of voltage-dependent conductances in retinal photoreceptors is the first step of a process leading to the enhancement of the temporal performance of the visual system. The molecular components underlying voltage-dependent gating in rods are presently poorly defined. In the present work we have investigated the isoform composition and the functional characteristics of hyperpolarisation-activated cyclic nucleotide-gated channels (HCN) in rabbit rods. Using immunocytochemistry we show the expression in the inner segment and cell body of the isoform 1 (HCN1). Electrophysiological investigations show that hyperpolarisation-activated currents ( I _h) can be measured only from the cell regions where HCN1 is expressed. Half-activation voltage (–75.0 ± 0.3 mV) and kinetics ( t _1/2 of 101 ± 8 ms at –110 mV and 20 °C) of the I _h in rods are similar to those of the macroscopic current carried by homomeric rabbit HCN1 channels expressed in HEK 293 cells. The homomeric nature of HCN1 channels in rods is compatible with the observation that cAMP induces a small shift (2.3 ± 0.8 mV) in the half-activation voltage of I _h. In addition, the observation that within the physiological range of membrane potentials, cAMP does not significantly affect the gain of the current-to-voltage conversion, may reflect the need to protect the first step in the processing of visual signals from changes in cAMP turnover.     

The scope of receptor editing and its association with autoimmunity

Random assembly of antibody variable (V), diversity (D) and joining (J) gene segments creates a vast repertoire of antigen receptors, including autoreactive ones. Three ways that are known to reduce autoreactivity in the B-cell compartment include clonal deletion, functional inactivation and receptor editing, a mechanism involving a change in antigen receptor specificity through continued V(D)J recombination. New data suggest that editing can efficiently eliminate autoreactivity, yet, in an autoimmune context, secondary antibody gene rearrangements might also contribute to autoimmunity.     

Genetic polymorphism in intron 6 of the LMP7 gene in Japanese and its association with sarcoidosis

Genetic polymorphism in intron 6 of the LMP7 gene was investigated using polymerase chain reaction-restriction fragment length polymorphism in 90 unrelated healthy Japanese controls and 66 Japanese patients with sarcoidosis. Four alleles, including two new ones recently identified in Koreans, LMP7*C and LMP7*D, were found in the Japanese population. The frequency of LMP7*C in the sarcoidosis patients was higher than in the healthy controls. However, this difference might be explained by a secondary association with HLA-DRB1*08 in the HLA-DRB1 gene, which is thought to be the gene primarily responsible for susceptibility to sarcoidosis.     

The Epstein-Barr virus and its association with human cancers.

The Epstein-Barr virus (EBV) has been linked to the development of a variety of human malignancies, including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, some T cell lymphomas, post-transplant lymphoproliferative disease, and more recently, certain cancers of the stomach and smooth muscle. This review summarizes these associations and in particular the role of the viral latent genes in the transformation process.     

The Epstein-Barr virus and its association with human cancers.

The Epstein-Barr virus (EBV) has been linked to the development of a variety of human malignancies, including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, some T cell lymphomas, post-transplant lymphoproliferative disease, and more recently, certain cancers of the stomach and smooth muscle. This review summarizes these associations and in particular the role of the viral latent genes in the transformation process.     

Prediction of the archaeal exosome and its connections with the proteasome and the translation and transcription machineries by a comparative-genomic approach

By comparing the gene order in the completely sequenced archaeal genomes complemented by sequence profile analysis, we predict the existence and protein composition of the archaeal counterpart of the eukaryotic exosome, a complex of RNAses, RNA-binding proteins, and helicases that mediates processing and 3'->5' degradation of a variety of RNA species. The majority of the predicted archaeal exosome subunits are encoded in what appears to be a previously undetected superoperon. In Methanobacterium thermoautotrophicum, this predicted superoperon consists of 15 genes; in the Crenarchaea, Sulfolobus solfataricus and Aeropyrum pernix, one and two of the genes from the superoperon, respectively, are relocated in the genome, whereas in other Euryarchaeota, the superoperon is split into a variable number of predicted operons and solitary genes. Methanococcus jannaschii partially retains the superoperon, but lacks the three core exosome subunits, and in Halobacterium sp., the superoperon is divided into two predicted operons, with the same three exosome subunits missing. This suggests concerted gene loss and an alteration of the structure and function of the predicted exosome in the Methanococcus and Halobacterium lineages. Additional potential components of the exosome are encoded by partially conserved predicted small operons. Along with the orthologs of eukaryotic exosome subunits, namely an RNase PH and two RNA-binding proteins, the predicted archaeal exosomal superoperon also encodes orthologs of two protein subunits of RNase P. This suggests a functional and possibly a physical interaction between RNase P and the postulated archaeal exosome, a connection that has not been reported in eukaryotes. In a pattern of apparent gene loss complementary to that seen in Methanococcus and Halobacterium, Thermoplasma acidophilum lacks the RNase P subunits. Unexpectedly, the identified exosomal superoperon, in addition to the predicted exosome components, encodes the catalytic subunits of the archaeal proteasome, two ribosomal proteins and a DNA-directed RNA polymerase subunit. These observations suggest that in archaea, a tight functional coupling exists between translation, RNA processing and degradation, (apparently mediated by the predicted exosome) and protein degradation (mediated by the proteasome), and may have implications for cross-talk between these processes in eukaryotes.     

Analysis of exosome release and its prognostic value in human colorectal cancer

A significant proportion of extracellular nucleic acids in plasma circulate highly protected in tumor‐specific exosomes, but it is unclear how the release of exosomes is modulated in carcinogenesis. We quantified by cytometry exosomes in plasma of 91 colorectal cancer patients to evaluate their potential as a tumor indicator and their repercussions on diagnosis and prognosis. We examined the involvement of TSAP6, a TP53‐regulated gene involved in the regulation of vesicular secretion, in levels of circulating exosomes in plasma of colorectal patients and in HCT116 TP53‐(wild‐type and null) human colorectal cancer cell lines. The fraction of exosomes in cancer patients was statistically higher than in healthy controls (mean rank = 53.93 vs. 24.35). High levels of exosomes in plasma of patients correlated with high levels of carcino‐embryonic antigen (P = 0.029) and with poorly differentiated tumors (P = 0.039) and tended to have shorter overall survival than patients with low levels (P = 0.056). Release of exosomes did not correlate with TSAP6 expression; and regulation of TSAP6 by TP53 was not shown either in tumor samples or in HCT116 cell lines. Although it was not suggested that the TP53/TSAP6 pathway regulates the release of exosomes into the plasma of colorectal cancer patients, the level of circulating exosomes may be used as a tumor indicator, because it correlates with poor prognosis parameters and shorter survival. © 2011 Wiley Periodicals, Inc.     

The steatohepatitic variant of hepatocellular carcinoma and its association with underlying steatohepatitis

Steatohepatitis and metabolic syndrome are increasingly recognized as important risk factors for development of hepatocellular carcinoma. We have recently described a histologic subtype of hepatocellular carcinoma termed steatohepatitic hepatocellular carcinoma, which shows features resembling steatohepatitis in the nonneoplastic liver. The present study is undertaken to assess the association between the steatohepatitic hepatocellular carcinoma variant and underlying steatohepatitis and features of metabolic syndrome. We examined all hepatocellular carcinomas diagnosed on resections and explant specimens over a 3.5-year period at our institution. Tumors were classified as either conventional hepatocellular carcinoma or steatohepatitic hepatocellular carcinoma variant based on their predominant histopathologic pattern. The underlying chronic liver disease in each case was determined. The steatohepatitic hepatocellular carcinoma variant represented 13.5% (16/118) of cases. All but one case of steatohepatitic hepatocellular carcinoma occurred in patients with underlying steatohepatitis. Steatohepatitic hepatocellular carcinoma was diagnosed in 35.7% of patients with either nonalcoholic steatohepatitis or alcoholic liver disease compared with 1.3% of patient with other chronic liver diseases (P < .0001). The steatohepatitic hepatocellular carcinoma group had a significantly higher number of metabolic syndrome risk factors (2.44 versus 1.48, P = .01) and a higher percentage of patients with at least 3 metabolic syndrome components (50% versus 22.5%, P = .02). Immunohistochemically, there were diffuse loss of cytoplasmic CK8/18 and increased numbers of activated hepatic stellate cells within steatohepatitic hepatocellular carcinoma, in a pattern identical to that seen in steatohepatitis in nonneoplastic liver. Hepatocellular carcinomas showing a "steatohepatitic" histologic phenotype are strongly associated with underlying steatohepatitis and metabolic syndrome. This association further supports a possible role of steatohepatitis in human hepatocarcinogenesis.     

Partial characterisation of murine huntingtin and apparent variations in the subcellular localisation of huntingtin in human, mouse and rat brain

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by the expansion of a CAG repeat in a gene coding for a protein of unknown function. We have raised a polyclonal antibody against a 12 amino acid peptide (residues 2110-2121 of human huntingtin) which specifically recognises huntingtin on Western blots of human, rat and mouse brain. We have characterised huntingtin expression in the mouse. The protein was detected on Western blots of all mouse tissues examined, with the highest expression seen in brain. Human, mouse and rat brain were fractionated by differential centrifugation and discontinuous Percoll gradients. The fractions were analysed by Western blotting for huntingtin and synaptophysin (a synaptic vesicle localised protein). In mouse brain, huntingtin was localised in the soluble S3 fraction; in rat brain it was localised in the soluble S3 fraction and also in the membrane P2 and P3 fractions; in both normal and HD- affected human brain, huntingtin was membrane bound with a distribution essentially the same as that of synaptophysin. These observed differences in the subcellular localisation of huntingtin between mouse and human brain are important in the context of mouse models for HD.      

Red蛋白抗血清的制备及其亚细胞定位研究

目的:在大肠杆菌中分别表达3种Red蛋白,并制备兔抗Red蛋白的抗体。方法:从λ噬菌体基因组中,通过PCR分别扩增gam、bet和exoDNA的全长序列。将PCR产物克隆入非融合表达载体pDH2中,构建Red蛋白的高表达工程菌。通过温敏诱导表达3种Red蛋白(Bet、Exo和Gam),用PACTE薄层扫描检测目的蛋白含量。用3种Red蛋白分别免疫家兔制备抗血清,并以Westernblot鉴定抗体的效价和特异性。结果:大肠杆菌中表达的Bet、Exo和Gam蛋白,分别占菌体总蛋白量的40.3%、49.2%和73.4%。3种抗血清的效价约为1∶2000。Westernblot分析显示抗血清具有较好的特异性。结论:成功地获得Bet、Exo和Gam3种蛋白,并分别制备了相应的抗血清。使用这3种抗血清,检测了3种蛋白在真核细胞中的表达和定位,为研究Red蛋白的同源重组奠定了基础。     

The T cell receptor gene and its association with human diseases.

The genetic organization and protein structure of the T cell receptor (TCR)/CD3 complex are currently under investigation, and recent work has provided information about its assembly, expression and function. This article reviews what is currently known about the structure and assembly of the TCR/CD3 complex. The TCR chains are members of the immunoglobulin gene superfamily and are generated by combinatorial associations of V, J, D, and C genes. The presence of certain gene rearrangements within these chains has been associated with lymphoproliferative disorders, autoimmune disease and immunodeficiencies. TCR rearrangements can be useful in the diagnosis of lymphoproliferative disorders and in the identification of patients who will subsequently relapse, once in remission. With respect to autoimmune disease, the possibility now exists of immunotherapy with TCR designer polypeptides to prevent disease. In patients with primary immunodeficiencies secondary to defects in expression of the TCR, the possibility of somatic gene therapy now exists. As more information unfolds about the role that TCR gene rearrangements have in various diseases, new insights are gained into better diagnosis and treatment.     

KIAA0280的亚细胞定位及在缺血脑组织中的表达

目的观察大鼠急性局灶性脑缺血时缺血组织与非缺血组织中KIAA0280蛋白的表达及亚细胞定位。方法应用免疫组织化学法测定KIAA0280在大鼠急性脑缺血后在缺血与非缺血区表达的差异,利用激光共聚焦扫描显微镜检测KIAA0280的亚细胞定位。结果免疫组织化学结果证实了KIAA0280在大鼠急性局灶性脑缺血后表达的差异,激光共聚焦扫描显微镜证实KIAA0280在大鼠皮层细胞。胶质细胞胞浆和细胞膜上均出现表达。KIAA0280在脑缺血后明显上调表达。结论KIAA0280与蛋白质合成和信号传递有密切关系,为新的脑缺血相关蛋白质,对其功能深入研究将对诊断和治疗脑缺血缺氧疾病奠定基础。     

Bcl-xL overexpression and its association with the progress of tongue carcinoma

Apoptosis-related protein B-cell lymphoma-extra large (Bcl-xL) has a crucial role in the control of cell death through its inhibition of apoptosis. This study was designed to investigate the expression of Bcl-xL in relation to the development of tongue carcinoma and whether it has potential as a marker for the clinical diagnosis of tongue carcinoma and as a therapeutic target to evaluate the dynamic of tongue carcinoma progression. A statistical analysis of 100 cases oral tongue carcinoma tissue specimens were performed using pathological grading and clinical TNM staging, and 14 cases corresponding non-tumor tissues as control. The changes in Bcl-xL mRNA expression between different pathological grades and clinical TNM stages of tissue were analyzed by RT-PCR. Additionally, immunohistochemical SP method and Western blot assays were employed to detect changes in Bcl-xL protein expression in different tongue carcinoma tissues. The results showed the expression of Bcl-xL was significantly higher in tongue carcinoma tissues than in normal tongue tissues and was positively associated with the degree of differentiation and the clinical TNM staging, but negatively correlated with the degree of malignancy of the tumor. There was higher expression of Bcl-xL in oral tongue squamous cell carcinoma (OTSCC) tissues compared with oral tongue adenocarcinoma (OTA) tissues, but Bcl-xL expression in tissue with lymph node metastasis was significantly higher than that without lymph node metastasis. Thus, Bcl-xL overexpression may be closely related to the dynamic of the pathogenesis and development of tongue carcinoma. It may be a useful marker for clinical diagnosis and an aid to evaluating the efficacy of therapeutics in tongue carcinoma.     

Positive illusions and its association with cardiovascular functions

The relationship between positive illusions (or self-enhancement) and cardiovascular functions was investigated using Asian samples in two studies. In phase 1 of Study 1, a generalized self-enhancement index was created for 241 participants using a paired word association memory task, a facial emotion recognition task, and a reading test. 122 participants subsequently volunteered for a second phase in this study where their ambulatory cardiovascular functions were measured throughout a single waking day. In Study 2, a priming procedure experimentally induced self-enhancement (n = 35) and self-effacement (n = 37) and the participants' cardiovascular arousal and perceived control for a mental arithmetic task were measured. Self-enhancement predicted lower cardiovascular functions for both studies. In Study 1, self-enhancement assessed at phase 2 was a significant predictor while self-enhancement measured at phase 1 was not. In Study 2, the relationship between self-enhancement and vascular reactivity was partially mediated by perceived control. The findings indicate that the relationship between self-enhancement and cardiovascular stress response, which has implications for cardiovascular health, (i) is relevant for Asian populations, (ii) is not just correlational but potentially causal, and (iii) is partly mediated by an increase in perceived control for vascular reactivity.