A Note on the Log‐Rank Test in Life Table Analysis with Correlated Observations

Survival data consisting of independent sets of correlated failure times may arise in many situations. For example, we may take repeated observations of the failure time of interest from each patient or observations of the failure time on siblings, or consider the failure times on littermates in toxicological experiments. Because the failure times taken on the same patient or related family members or from the same litter are likely correlated, use of the classical log‐rank test in these situations can be quite misleading with respect to type I error. To avoid this concern, this paper develops two closed‐form asymptotic summary tests, that account for the intraclass correlation between the failure times within patients or units. In fact, one of these two test includes the classical log‐rank test as a special case when the intraclass correlation equals 0. Furthermore, to evaluate the finite‐sample performance of the two tests developed here, this paper applies Monte Carlo simulation and notes that they can actually perform quite well in a variety of situations considered here.     

HOP/OB1/NECC1 promoter DNA is frequently hypermethylated and involved in tumorigenic ability in esophageal squamous cell carcinoma

Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2'-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a) expression status, (b) methylation status, and (c) functional involvement in cancer cell lines. (a) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPalpha and HOPbeta, respectively), and HOPalpha and HOPbeta were both down-regulated in ESCC independently. (b) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPbeta silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPalpha silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer.     

Purple acid phosphatase-like sequences in prokaryotic genomes and the characterization of an atypical purple alkaline phosphatase from Burkholderia cenocepacia J2315

Purple acid phosphatases (PAP) are a group of dimetallic phosphohydrolase first identified in eukaryotes. Bioinformatics analysis revealed 57 prokaryotic PAP-like sequences in the genomes of 43 bacteria and 4 cyanobacteria species. A putative PAP gene (BcPAP) from the bacteria Burkholderia cenocepacia J2315 was chosen for further studies. Synteny analysis showed that this gene is present as an independent gene in most of the members of the genus Burkholderia. The predicted 561 a.a. polypeptide of BcPAP was found to harbour all the conserved motifs of the eukaryotic PAPs and an N-terminal twin-arginine translocation signal. Expression and biochemical characterization of BcPAP in Escherichia coli revealed that this enzyme has a relatively narrow substrate spectrum, preferably towards phosphotyrosine, phosphoserine and phosphoenolpyruvate. Interestingly, this enzyme was found to have a pH optimum at 8.5, rather than an acidic optima exhibited by eukaryotic PAPs. BcPAP contains a dimetallic ion centre composed of Fe and Zn, and site-directed mutagenesis confirmed that BcPAP utilizes the invariant residues for metal-ligation and catalysis. The enzyme is secreted by the wild type bacteria and its expression is regulated by the availability of orthophosphate. Our findings suggest that not all members in the PAP family have acidic pH optimum and broad substrate specificity.     

Identification and Characterization of RBEL1 Subfamily of GTPases in the Ras Superfamily Involved in Cell Growth Regulation

Recently, we reported the identification of a novel gene named RBEL1 (Rab-like protein 1) and characterized its two encoded isoforms, RBEL1A and RBEL1B, that function as novel GTPases of Ras superfamily. Here we report the identification of two additional splice variants of RBEL1 that we have named RBEL1C and -D. All four RBEL1 isoforms (A, B, C, and D) have identical N termini harboring the Rab-like GTPase domains but contain variable C termini. Although all isoforms can be detected in both cytoplasm and nucleus, RBEL1A is predominantly cytoplasmic, whereas RBEL1B is mostly nuclear. RBEL1C and -D, by contrast, are evenly distributed between the cytoplasm and nucleus. Furthermore, all four RBEL1 proteins are also capable of associating with cellular membrane. The RBEL1 proteins also exhibit a unique nucleotide-binding potential and, whereas the larger A and B isoforms are mainly GTP-bound, the smaller C and D variants bind to both GTP and GDP. Furthermore, a regulatory region at amino acid position 236–302 immediately adjacent to the GTP-binding domain is important for GTP-binding potential of RBEL1A, because deletion of this region converts RBEL1A from predominantly GTP-bound to GDP-bound. RBEL1 knockdown via RNA interference results in marked cell growth suppression, which is associated with morphological and biochemical features of apoptosis as well as inhibition of extracellular signal-regulated kinase phosphorylation. Taken together, our results indicate that RBEL1 proteins are linked to cell growth and survival and possess unique biochemical, cellular, and functional characteristics and, therefore, appear to form a novel subfamily of GTPases within the Ras superfamily.The Ras superfamily is known to comprise five structurally distinct subfamilies of small GTPases, including Ras, Rho, Rab, Sar1/Arf, and Ran, and each subfamily of these GTPases possess distinct functions in the regulation of a variety of cellular processes such as cell proliferation, cell differentiation, cytoskeletal organization, protein transport, and trafficking (1–4). The Ras subfamily of GTPases (N-, H-, and K-Ras) function predominantly in relaying signals from receptors at the plasma membrane and modulating cell signaling pathways that regulate cell proliferation, differentiation, and survival (5). Ran GTPase, on other hand, is a key regulator of nucleocytoplasmic transport that regulates protein transport across the nuclear pore complex (6, 7). The Rab subfamily is the largest subfamily among the Ras superfamily and contains more than 60 members. The key functions of the Rab GTPases are to regulate protein exocytic and endocytic pathways and modulate intracellular protein transport/trafficking (8–13).In general, the Ras superfamily GTPases cycle between an active GTP-bound state and an inactive GDP-bound state. There are five N-terminal motifs involved in the binding and hydrolysis of GTP that are highly conserved among all GTPases: G1 (GXXXXGK(S/T)), G2 (T), G3 (DXXG), G4 ((N/T)(K/Q)XD), and G5 (EXSAX). Each sequence has particular functions involved in binding nucleotides (GTP or GDP) and facilitating hydrolysis (4, 14, 15). In general, the intrinsic GTPase activity (converting GTP to GDP) and exchange of GDP for GTP are slow processes for these GTPases and thus require regulatory proteins such as GTPase-activating proteins and GDP/GTP exchange factors to facilitate these processes (16–18).For the last two decades, the Ras superfamily has been a major focus in the cancer field as many of the members are either mutated or dysregulated in cancer. The founding members of the Ras superfamily, H-Ras and K-Ras, were first identified as viral oncogenes (1, 4). Later studies demonstrated that mutations of the Ras proteins (H-, N-, and K-Ras) occur frequently in human cancers, and the mutations identified are mostly clustered within the GTP-binding domains of the proteins thus locking Ras proteins in a GTP-bound configuration. GTP-bound Ras is constitutively active; it constantly activates its effector proteins to transduce cell proliferative signals (1, 4). Unlike Ras subfamily genes, mutations occurring in Rab and Rab-like genes are less common, yet alterations in gene expression of a number of Rab genes have been reported in multiple human malignancies. For example, Rab25 overexpression has been linked to prostate cancer progression (19). Rab2 overexpression has been found in lung adenomas and adenocarcinomas (20). In addition, alterations in Rab gene expression have also been linked to cancer drug resistance. For instance, resistance to the anticancer drug doxorubicin in MCF-7 cells has been linked with reduced expression of Rab6C, and introduction of exogenous Rab6C restores drug sensitivity (21).We have recently reported the identification two novel Ras superfamily GTPases, RBEL1A and RBEL1B (22). RBEL1A and RBEL1B are two splice variants of the RBEL1 gene and are highly homologous to the Rab and Ran GTPases within their N-terminal GTP-binding domains (22). Our studies show that both RBEL1A and -B predominantly bind to GTP. A single point mutation (T57N) in the GTP-binding domain of RBEL1A and -B abolishes their ability to bind to both GTP and GDP. Both RBEL1A and RBEL1B localize in the nucleus as well as in the cytosol. Whereas RBEL1A is predominantly cytosolic, RBEL1B is primarily nuclear. Interestingly, our studies also suggested that nucleotide (GTP or GDP)-binding could be important for the nuclear distribution of RBEL1B, because the nucleotide binding-deficient mutant form (T57N) of RBEL1B did not reside in the nucleus but rather became largely cytosolic (22).In our continuous efforts to fully elucidate the function of RBEL1, we have identified two additional splice variants that we have named RBEL1C and RBEL1D. Here we report further characterization of all four RBEL1 splice variants in terms of their GTPase activities, subcellular localizations, regulations, and potential functions. Our results indicate that RBEL1 GTPases, although sharing some common features with other Ras superfamily members, also harbor unique characteristics that are significantly different from other Ras superfamily GTPases. Based on our findings, we suggest that RBEL1 proteins appear to form a novel subfamily of GTPases within the Ras superfamily.     

The immune inhibitory receptor LAIR-1 is highly expressed by plasmacytoid dendritic cells and acts complementary with NKp44 to control IFNα production

Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells endowed with the capacity of producing large amounts of IFNα. Here we show that the Leukocyte-Associated Ig-like Receptor-1 (LAIR-1) is abundantly expressed on pDCs (the highest expression among all leukocytes) and its cross-linking inhibits IFNα production in response to Toll-like receptor ligands. Remarkably, LAIR-1 expression in pDCs is down-regulated in the presence of interleukin (IL)-3, thus indicating coordinated functions with NKp44, another pDC inhibitory receptor, which is conversely induced by IL-3. Nevertheless, the expression of NKp44 in pDCs isolated from secondary lymphoid organs, which is thought to be influenced by IL-3, is not coupled to a decreased expression of LAIR-1. Interestingly, pDCs isolated from peripheral blood of systemic lupus erithematosus (SLE) patients express lower levels of LAIR-1 while displaying slight but consistent expression of NKp44, usually undetectable on pDCs derived from healthy donors. Using sera derived from SLE patients, we show that LAIR-1 and NKp44 display synergistic inhibitory effects on IFNα production by interleukin IL-3 cultured pDCs stimulated with DNA immunocomplexes. In conclusion, our results indicate that the inhibitory function of LAIR-1 may play a relevant role in the mechanisms controlling IFNα production by pDCs both in normal and pathological innate immune responses.     

Prenatal stress in rat causes long-term spatial memory deficit and hippocampus MRI abnormality: differential effects of postweaning enriched environment

Prenatal stress (PS) can cause long-term hippocampus alternations in structure and plasticity in adult offspring. Enriched environment (EE) has an effect in rescuing a variety of neurological disorders. Pregnant dams were left undisturbed (prenatal control, PC) or restrained 6h per day from days 14 to 21 (prenatal stress, PS). Control and prenatal stressed offspring rats were subjected to a standard rearing environment (SE) or an EE on postnatal days 22-120 (PC/SE PC/EE, PS/SE, and PS/EE; n=5, each group). At ～4 months of age, all rats underwent Morris water maze test and brain MRI examination. Hippocampi were then dissected for biochemical analyses, including, Western blot for NMDA receptor (NR) subunits and synaptophysin and RT-PCR forβ1 integrin and tissue-plasminogen activator (t-PA). MRI showed all 5 rats in the PS/SE group and 5 in the PS/EE group exhibited increased signals in bilateral hippocampus and increased T2 time in the PS/SE group. Exposure to EE treatment on postnatal days 22-120 counteracted the deficit in spatial memory and increased NR1 protein expression, but it did not affect the rate of high signals and increased T2 time, decreased NR2, synaptophysin, β1 integrin and t-PA mRNA expressions in PS adult offspring. The results of this study indicate PS in rats causes long-term spatial memory deficits and gross hippocampus pathology. Postnatal EE treatment has differential benefits in terms of spatial learning, signaling molecules, and gross hippocampus pathology.     

MHC-independent genetic factors control the magnitude of CD4+ T cell responses to amyloid-β peptide in mice through regulatory T cell-mediated inhibition

Accumulation of amyloid-β peptide (Aβ) is considered the triggering factor of pathogenic lesions in Alzheimer's disease (AD), and vaccines targeting Aβ are promising therapeutic options. However, the occurrence of meningoencephalitides attributed to T cell responses in 6% of Aβ-immunized patients underscores the need for a better understanding of T cell responses to Aβ. We characterized the parameters controlling the magnitude of Aβ-specific CD4(+) T cell responses in mice. T cell responsiveness to Aβ1-42 was highly heterogeneous between mouse strains of different H-2 haplotypes, with SJL/J (H-2(s)) mice displaying a strong response, mainly specific for Aβ10-24, and C57BL/6 (H-2(b)) mice displaying a weak response to Aβ16-30. Surprisingly, C57BL/6 mice congenic for the H-2(s) haplotype (B6.H-2(S)), which display a "permissive" MHC class II allele for presentation of the immunodominant Aβ10-24 epitope, showed a very weak CD4(+) T cell response to Aβ, suggesting that MHC-independent genes downmodulate Aβ-specific CD4(+) T cell responses in C57BL/6 background. Vaccine-induced CD4(+) T cell responses to Aβ were significantly enhanced in both C57BL/6 and B6.H-2(S) mice upon depletion of regulatory T cells (Tregs), whereas Treg-depleted SJL/J mice displayed unaltered Aβ-specific T cell responses. Finally, Treg depletion in C57BL/6 transgenic APPPS1 mice, a mouse model of AD, results in enhanced vaccine-induced CD4(+) T cell responses in AD compared with wild-type animals. We concluded that the magnitude of Aβ-specific CD4(+) T cell responses is critically controlled in both physiological and pathological settings by MHC-independent genetic factors that determine the overall potency of Aβ-specific Treg responses.