Interrelationship between family history of alcoholism and generational status in the prediction of alcohol dependence in US Hispanics.

BACKGROUND: Both a family history of alcoholism and migration-related factors like US v. foreign nativity increase the risk for developing alcohol use disorders in Hispanic Americans. For this study, we integrated these two lines of research to test whether the relationship between familial alcoholism and alcohol dependence changes with successive generations in the United States. METHOD: Data were from the waves 1 and 2 National Epidemiologic Survey on Alcohol and Related Conditions (NESARC). Subjects self-identified Hispanic ethnicity (N = 4122; n = 1784 first, n = 1169 second, and n = 1169 third or later generation) and reported ever consuming ⩾12 drinks in a 1-year period. A family history of alcoholism was assessed in first- and second-degree relatives. Analyses predicting the number of alcohol dependence symptoms were path models. RESULTS: Alcohol dependence symptoms were associated with a stronger family history of alcoholism and later generational status. There was a significant interaction effect between familial alcoholism and generational status; the relationship of familial alcoholism with alcohol dependence symptoms increased significantly with successive generations in the United States, more strongly in women than men. Acculturation partially mediated the interaction effect between familial alcoholism and generational status on alcohol dependence, although not in the expected direction. CONCLUSIONS: Familial alcoholism interacted with generational status in predicting alcohol dependence symptoms in US Hispanic drinkers. This relationship suggests that heritability for alcoholism is influenced by a higher-order environmental factor, likely characterized by a relaxing of social restrictions on drinking.

Controlling the kinetic chain length of the crosslinks in photo-polymerized biodegradable networks.

Biodegradable polymer networks were prepared by photo-initiated radical polymerization of methacrylate functionalized poly(D,L-lactide) oligomers. The kinetic chains formed in this radical polymerization are the multifunctional crosslinks of the networks. These chains are carbon-carbon chains that remain after degradation. If their molecular weight is too high these poly(methacrylic acid) chains can not be excreted by the kidneys. The effect of the photo-initiator concentration and the addition of 2-mercaptoethanol as a chain transfer agent on the molecular weight of the kinetic chains was investigated. It was found that both increasing the initiator concentration and adding 2-mercaptoethanol decrease the kinetic chain length. However, the effect of adding 2-mercaptoethanol was much larger. Some network properties such as the glass transition temperature and the swelling ratio in acetone are affected when the kinetic chain length is decreased.

Aquaculture facilities promote populational stability throughout seasons and increase medusae size for the invasive jellyfish Cassiopea andromeda.

Cassiopea jellyfish have successfully invaded several marine ecosystems worldwide. We investigated if Cassiopea andromeda grows larger (umbrella size) and if their populations are more stable in shrimp farms than in mangroves in the Brazilian coast. Our results show that jellyfish abundance is higher in the shrimp farm during the rainy season and in the mangrove during dry season. The population is stable during both seasons in the shrimp farm, but unstable in the mangroves, as jellyfish are absent during rainy season. Shrimp farm-associated jellyfish are three times larger than those in the mangroves, regardless of season. We recorded the largest (49.2 cm of umbrella diameter) ever C. andromeda individual in the shrimp farm. Unlike the mangroves, the shrimp farm provides environmental intra-annual stability that promotes jellyfish growth and population persistence. Therefore, C. andromeda populations can be seasonally dynamic and artificial environments such as aquaculture facilities may facilitate the invasion process.

A complex medical phenotype in a patient with triplication of 2q12.3 to 2q13 characterized with oligonucleotide array CGH.

We report an adult female with a left polycystic kidney, patent ductus arteriosus, left streak ovary, bicornuate uterus and deafness who presented with infertility. She has an intrachromosomal triplication of bands 2q12.3 to 2q13, with inversion of the central segment, which arose de novo from a paternal interchomosomal event. The triplication contains 68 known genes within the 7.28 Mb of DNA between base pairs 107,140,721 and 114,416,131. All intrachromosomal triplications are rare and, while partial duplications of 2q have been previously described, this patient is a unique surviving case of a triplication of proximal 2q.

A pseudoautosomal gene in man.

The X/Y homologous gene MIC2 was shown to exchange between the sex chromosomes, thus demonstrating that it is a pseudoautosomal gene in man. MIC2 recombines with the sex-determining gene(s) TDF at a frequency of 2 to 3 percent. It is the most proximal pseudoautosomal locus thus far described and as such is an important marker for use in studies directed towards the isolation of TDF.

Frame-shift deletions in patients with Duchenne and Becker muscular dystrophy.

Duchenne muscular dystrophy (DMD) and its less severe form Becker muscular dystrophy (BMD) are allelic disorders. It has been suggested that in the mutations involving BMD, the translational reading frame of messenger RNA is maintained and a smaller, though partially functional, protein is produced. In order to test this, the exon-intron boundaries of the first ten exons of the DMD gene were determined, and 29 patients were analyzed. In a number of BMD patients (mild and severe BMD), the reading frame of messenger RNA was not maintained. On the basis of these findings, a model for reinitiation from an internal start codon is suggested.

Peroxynitrite Generation and Increased Heterotrophic Capacity Are Linked to the Disruption of the Coral-Dinoflagellate Symbiosis in a Scleractinian and Hydrocoral Species.

Ocean warming is one of the greatest global threats to coral reef ecosystems; it leads to the disruption of the coral-dinoflagellate symbiosis (bleaching) and to nutrient starvation, because corals mostly rely on autotrophy (i.e., the supply of photosynthates from the dinoflagellate symbionts) for their energy requirements. Although coral bleaching has been well studied, the early warning signs of bleaching, as well as the capacity of corals to shift from autotrophy to heterotrophy, are still under investigation. In this study, we evaluated the bleaching occurrence of the scleractinian coral Mussismillia harttii and the hydrocoral Millepora alcicornis during a natural thermal stress event, under the 2015-2016 El Niño influence in three reef sites of the South Atlantic. We focused on the link between peroxynitrite (ONOO-) generation and coral bleaching, as ONOO- has been very poorly investigated in corals and never during a natural bleaching event. We also investigated the natural trophic plasticity of the two corals through the use of new lipid biomarkers. The results obtained first demonstrate that ONOO- is linked to the onset and intensity of bleaching in both scleractinian corals and hydrocorals. Indeed, ONOO- concentrations were correlated with bleaching intensity, with the highest levels preceding the highest bleaching intensity. The time lag between bleaching and ONOO- peak was, however, species-specific. In addition, we observed that elevated temperatures forced heterotrophy in scleractinian corals, as Mu. harttii presented high heterotrophic activity 15 to 30 days prior bleaching occurrence. On the contrary, a lower heterotrophic activity was monitored for the hydrocoral Mi. alicornis, which also experienced higher bleaching levels compared to Mu. hartii. Overall, we showed that the levels of ONOO- in coral tissue, combined to the heterotrophic capacity, are two good proxies explaining the intensity of coral bleaching.

CDKN2B methylation status and isolated chromosome 7 abnormalities predict responses to treatment with 5-azacytidine.

5-Azacytidine, a DNA methyl transferase inhibitor, is effective in patients with myelodysplastic syndromes (MDS). Whether responses to 5-Azacytidine are achieved by demethylation of key genes or by cytotoxicity is unclear. Of 34 patients with MDS or acute myeloid leukaemia (AML) treated with 5-Azacytidine, 7 achieved complete remissions (CR) (21%) and 6 achieved haematological improvement. All six had less than 5% bone marrow (BM) blasts at the time of haematological improvements (HI) (2 had pre-existing refractory anaemia (RA), 4 had refractory anaemia with excess blasts (RAEB)). A further patient with RAEB had blast reduction to less than 5% without HI. Five of the seven (71%) complete responders had chromosome 7 abnormalities. BM CR predicted longer overall survival (OS) (median 23 versus 9 months, P=0.015). Bisulphite genomic sequencing (BGS) of the CDKN2B (p15(INK4b)) promoter showed low level, heterogeneous pretreatment methylation (mean 12.2%) in 14/17 (82%) patients analysed. Lower baseline methylation occurred in responders (9.8% versus 16.2% in non-responders P=0.07). No response was seen in patients with >24% methylation, in whom p15(INK4b) mRNA was not expressed. 5-Azacytidine reduced CDKN2B methylation by mean 6.8% in 8/17 (47%) patients, but this did not correlate with response. At 75 mg/m(2), cell death (reduced BM cellularity (P=0.001) and increased apoptosis (P=0.02)) rather than demethylation of CDKN2B correlates with response. Patients with >24% methylation may benefit from alternative dosing or combination strategies.

A large patient study confirming that facioscapulohumeral muscular dystrophy (FSHD) disease expression is almost exclusively associated with an FSHD locus located on a 4qA-defined 4qter subtelomere.

Facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant disorder, represents the third most common human muscular dystrophy. The FSHD disease locus, at chromosome 4q35, is associated with large contractions of the polymorphic repeat sequence array D4Z4. In addition to FSHD disease association with large D4Z4 deletions, a biased interaction with a specific 4qter subtelomeric sequence has been described in patients. Two distinct 4qter subtelomeres, defined as types 4qA and 4qB, have been identified and shown to be equally prevalent in the Caucasian population. In almost all 4q35-linked patients with FSHD, however, disease expression only occurs when large D4Z4 deletions are located on 4qA-defined 4qter subtelomeres. Conversely, large D4Z4 repeat contractions situated on 4qB-defined subtelomeres either are not disease-causing or exhibit a greatly reduced disease penetrance. This study was initiated to confirm this direct FSHD disease association data by measuring the frequency of type 4qA-defined and 4qB-defined subtelomeric sequences in a large cohort of 164 unrelated patients with FSHD from Turkey and the UK, all known to have large D4Z4 deletions. An almost complete association was found between large D4Z4 repeat array deletions located on 4qA-defined 4qter subtelomeres and disease expression in our large FSHD patient cohort. The observed failure of probes 4qA and 4qB to hybridise to two patient-derived DNA samples confirms the presence of an additional rare type of 4qter subtelomeric sequence in humans.

The B-cell and neuronal forms of the octamer-binding protein Oct-2 differ in DNA-binding specificity and functional activity.

B lymphocytes contain an octamer-binding transcription factor, Oct-2, that is absent in most other cell types and plays a critical role in the B-cell-specific transcription of the immunoglobulin genes. A neuronal form of this protein has also been detected in brain and neuronal cell lines by using a DNA mobility shift assay, and an Oct-2 mRNA is observed in these cells by Northern (RNA) blotting and in situ hybridization. We show that the neuronal form of Oct-2 differs from that found in B cells with respect to both DNA-binding specificity and functional activity. In particular, whereas the B-cell protein activates octamer-containing promoters, the neuronal protein inhibits octamer-mediated gene expression. The possible role of the neuronal form of Oct-2 in the regulation of neuronal gene expression and its relationship to B-cell Oct-2 are discussed.

Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1).

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27(KIP1) through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27(KIP1) promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27(KIP1) appears to be critical in the regulation of cell survival since mere ectopic expression of p27(KIP1) was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27(KIP1) null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27(KIP1) transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

Fanconi anaemia complementation group B presenting as X linked VACTERL with hydrocephalus syndrome.

BACKGROUND: The VACTERL with hydrocephalus (VACTERL-H) phenotype is recognised to be a severe manifestation of autosomal recessive Fanconi anaemia. Several families have been described in which the VACTERL-H phenotype segregates as an X linked syndrome. The mutations which cause X linked VACTERL-H syndrome are not known. OBJECTIVE: To determine if mutations in FANCB, which are known to cause Fanconi anaemia complementation group B, are a cause of X linked VACTERL-H syndrome. METHODS: A three generation pedigree with X linked VACTERL-H syndrome was investigated. X inactivation was tested in carrier females, and fibroblasts from an affected male fetus were analysed for increased sensitivity to diepoxybutane. FANCB coding exons and flanking splice sites were screened for mutations by direct sequencing of polymerase chain reaction (PCR) fragments amplified from genomic DNA. cDNA from affected fetal fibroblasts was analysed by PCR and direct sequencing using specific exonic primers. RESULTS: A FANCB mutation which results in a premature stop codon by causing skipping of exon 7 was identified. Chromosomes from the affected fetus showed increased sensitivity to diepoxybutane, and carrier women were found to have 100% skewed X inactivation in blood. CONCLUSIONS: Mutations in FANCB are a cause of X linked VACTERL-H syndrome. The data presented are of relevance to the genetic counselling of families with isolated male cases of VACTERL-H and Fanconi anaemia.

Molecular investigation of a dicentric 13;17 chromosome found in a 21-week gestation fetus with multiple congenital abnormalities.

We report a 21-week gestation fetus terminated because of multiple congenital abnormalities seen on ultrasound scan, including ventriculomegaly, possible clefting of the hard palate, cervical hemivertebrae, micrognathia, abnormal heart, horseshoe kidney and a 2-vessel umbilical cord. On cytogenetic examination, the fetus was found to have a male karyotype with 45 chromosomes with a dicentric chromosome, which appeared to consist of the long arms of chromosomes 13 and 17. Molecular genetic investigations and fluorescence in situ hybridization (FISH) unexpectedly showed that the derivative chromosome contained two interstitial blocks of chromosome 17 short arm sequences, totalling approximately 7 Mb, between the two centromeres. This effectively made the fetus monosomic for approximately 15 Mb of 17p without the concurrent trisomy for another chromosome normally seen following malsegregation of reciprocal translocations. It also illustrates the complexity involved in the formation of some structurally abnormal chromosomes, which can only be resolved by detailed molecular investigations.

The complex nature of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes.

OBJECTIVE: To describe the systematic analysis of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes, characterise the structural chromosome rearrangements, map the translocation breakpoints, and report detectable genomic imbalances. METHODS: DNA microarrays were used with a resolution of 1 Mb for the detailed genome-wide analysis of the patients. Array CGH was used to screen for genomic imbalance and array painting to map chromosome breakpoints rapidly. These two methods facilitate rapid analysis of translocation breakpoints and screening for cryptic chromosome imbalance. Breakpoints of rearrangements were further refined (to the level of spanning clones) using fluorescence in situ hybridisation where appropriate. RESULTS: Unexpected additional complexity or genome imbalance was found in six of 10 patients studied. The patients could be grouped according to the general nature of the karyotype rearrangement as follows: (A) three cases with complex multiple rearrangements including deletions, inversions, and insertions at or near one or both breakpoints; (B) three cases in which, while the translocations appeared to be balanced, microarray analysis identified previously unrecognised imbalance on chromosomes unrelated to the translocation; (C) four cases in which the translocation breakpoints appeared simple and balanced at the resolution used. CONCLUSIONS: This high level of unexpected rearrangement complexity, if generally confirmed in the study of further patients, will have an impact on current diagnostic investigations of this type and provides an argument for the more widespread adoption of microarray analysis or other high resolution genome-wide screens for chromosome imbalance and rearrangement.

The retinoblastoma protein is partially phosphorylated during early G1 in cycling cells but not in G1 cells arrested with alpha-interferon.

The retinoblastoma protein (pRB) is thought to act as a tumour suppressor which is inactivated by phosphorylation. In quiescent (G0) cells pRB exists in a hypophosphorylated form (pRB110), but proliferating cells in G1 contain a significant proportion of phosphorylated pRB (pRB112-114). Studies of synchronized or elutriated cells have suggested that the phosphorylated forms of pRB disappear as cells pass from G2/M to G0/G1 and that pRB is phosphorylated again to pRB114 at the G1/S border. In this study we used two-parameter flow cytometry and cell sorting to isolate cycling cells in early and late G1 (G1A and G1B), and we show that partially phosphorylated pRB is present in cycling human lymphoid cells even in G1A. These G1A cells contain intermediate forms of pRB which become further phosphorylated to pRB112-114 as cells pass into G1B. Therefore pRB is at least partially phosphorylated from early G1 onwards. Cell cycle arrest by alpha-interferon (alpha-IFN) results in an accumulation of cells in both G1A and G1B, and these cells contain mainly pRB110. Since pRB110 is thought to prevent cell proliferation, the cytostatic effect of alpha-IFN may therefore occur by preventing the initial phosphorylation of pRB during or prior to G1A.

The phosphorylation state of the retinoblastoma (RB) protein in G0/G1 is dependent on growth status.

The product of the retinoblastoma gene (RB) is a nuclear phosphoprotein which is thought to regulate the proliferation of cells. Its phosphorylation state changes with passage through the cell cycle and it has been proposed that RB protein in its hypo-phosphorylated form prevents cells proliferating. We have investigated the phosphorylation state of the RB protein in an actively-dividing human B-lymphoblastoid cell line and after cell cycle arrest caused by alpha-Interferon (alpha-IFN). We show that the phosphorylation state of the RB protein in cells with 2N DNA content depends on whether the cells are actively cycling. Our data is compatible with the proposal that dephosphorylation of the RB protein allows cells to enter a quiescent state. This study sheds light on the molecular mechanisms which may mediate the cytostatic effects of alpha-IFN.

BCR signals target p27(Kip1) and cyclin D2 via the PI3-K signalling pathway to mediate cell cycle arrest and apoptosis of WEHI 231 B cells.

Cross-linking of the B cell antigen receptor (BCR) on immature WEHI 231 B cells results in G1 cell cycle arrest and apoptosis. Here we investigated the molecular mechanisms that are necessary and sufficient for these changes to occur. We show that BCR stimulation of WEHI 231 cells results in down-regulation of cyclin D2 and up-regulation of p27(Kip1), which are associated with pocket protein hypophosphorylation and E2F inactivation. Ectopic expression of p27(Kip1) by TAT-fusion protein or retroviral transduction is sufficient to cause G1 cell cycle arrest, followed by apoptosis. In contrast, over-expression of cyclin D2 overcomes the cell cycle arrest and apoptosis induced by anti-IgM, indicating that down-regulation of cyclin D2 is necessary for the cell cycle arrest and apoptosis activated by BCR stimulation. Thus, cyclin D2 and p27(Kip1) have opposing roles in these pathways and our data also suggest that cyclin D2 functions upstream of p27(Kip1) and the pRB pathway and therefore plays an essential part in integrating the signals from BCR with the cell cycle machinery. We next investigated which signal transduction pathways triggered by the BCR regulate cell proliferation and apoptosis via cyclin D2 and p27(Kip1). Inhibition of PI3-K signalling by LY294002 down-regulated cyclin D2 and up-regulated p27(Kip1) expression at both protein and RNA levels, mimicking the effects of BCR-stimulation. Furthermore, ectopic expression of a constitutively active form of AKT blocked the cell cycle arrest and apoptosis triggered by anti-IgM and also abrogated down-regulation of cyclin D2 and up-regulation of p27(Kip1) expression induced by BCR-engagement. These results indicate that BCR activation targets p27(Kip1) and cyclin D2 to mediate cell cycle arrest and apoptosis and that down-regulation of PI3-K/AKT activity post BCR stimulation is necessary for these to occur.

The synthesis of p58cyclin A and the phosphorylation of p34cdc2 are inhibited in human lymphoid cells arrested in G1 by alpha-interferon.

Daudi cells arrest in G1 in the presence of alpha-interferon. Such cells have little p58cyclin A, probably due to inhibition of p58cyclin A synthesis. The phosphorylation-associated migration shift of p34cdc2 is not seen in alpha-interferon-arrested cells. Cells arrested in late G1 by aphidicolin have abundant p58cyclin A and phosphorylated p34cdc2. Cell sorting showed that p58cyclin A increases in proliferating cells in late G1 and coincides with phosphorylation of p34cdc2.

Cloning and characterization of cDNAs encoding the epsilon-subunit of eukaryotic initiation factor-2B from rabbit and human.

A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the epsilon-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two V8 fragments of purified nonrecombinant eIF-2B epsilon in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2B epsilon. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2B epsilon mRNA were obtained by 5' RACE. A human eIF-2B epsilon cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in lambda gt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2B epsilon polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single approximately 2.8 kilobase mRNA species which is ubiquitously expressed.

Variable expression of p16 protein in patients with acute myeloid leukemia without gross rearrangements at the DNA level.

The p16 protein is an inhibitor of cyclin-dependent kinases (cdk) 4 and 6. Both cdk4 and cdk6 phosphorylate the retinoblastoma protein (pRb) and are thought to be required for cell proliferation. Mutations and homozygous deletions in the p16 gene have been found in a number of cell lines but the incidence of abnormalities in primary tumours is controversial. We have studied the p16 gene in 76 cases of acute myeloid leukemia (AML) and 18 hematologically normal controls by quantitative Southern blotting. No deletions or rearrangements were detected. Twenty-five cases also showed no abnormal band patterns by RT-PCR-SSCP analysis of the coding sequence. We analyzed 60 AML samples for p16 protein expression by Western blot analysis. A reduced level of p16 was observed in six cases, however, none of them showed methylation of the 5'-CpG island. Over-expression of p16 protein was detected in six cases. Studies in cell lines have suggested a feedback loop between p16 and pRb with a futile overproduction of p16 protein in cells containing abnormal pRb. In accordance with these findings, four of the AML cases with high levels of p16 had abnormal pRb (two alteration in band size, two absent). From our data we suggest that gross abnormalities in the p16 gene are rare in AML, but that p16 levels are variable and high levels are associated with pRb abnormalities in a subset of cases.