Functional analysis of the p57 KIP2 gene mutation in Beckwith-Wiedemann syndrome

p57 ^KIP2 is a potent tight-binding inhibitor of several G₁ cyclin/cyclin-dependent kinase (Cdk) complexes, and is a negative regulator of cell proliferation. The gene encoding p57 ^KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome (BWS). Previously we demonstrated that p57 ^KIP2 is imprinted and only the maternal allele is expressed in both mice and humans. We also showed mutations found in p57 ^KIP2 in patients with BWS that were transmitted from the patients’ carrier mothers, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. In the study reported here, we performed functional analysis of the two mutated p57 ^KIP2 genes. We showed that the nonsense mutation found in the Cdk inhibitory domain in a BWS patient rendered the protein inactive with consequent complete loss of its role as a cell cycle inhibitor and of its nuclear localization. We also showed that the mutation in the QT domain, although completely retaining its cell cycle regulatory activity, lacked nuclear localization and was thus prevented from performing its role as an active cell cycle inhibitor. Consequently, no active p57 ^KIP2 would have existed, which might have caused the disorders in BWS patients. Received: 7 November 1998 / Accepted: 19 December 1998     

Identification and Validation of Quantitative Trait Loci for Agronomic Traits in Advanced Backcross Breeding Lines Derived from Oryza rufipogon × Oryza sativa Cultivar MR219

A backcross breeding strategy was used to identify quantitative trait loci (QTLs) associated with 14 traits in a BC₂F₂ population derived from a cross between MR219, an indica rice cultivar and an accession of Oryza rufipogon (IRGC 105491). A total of 261 lines were genotyped with 96 microsatellite markers and evaluated for plant morphology, yield components and growth period. The genetic linkage map generated for this population with an average interval size of 16.2?cM, spanning 1,553.4?cM (Kosambi) of the rice genome. Thirty-eight QTLs were identified with composite interval mapping (CIM), whereas simple interval mapping (SIM) resulted in 47 QTLs (LOD >3.0). The O. rufipogon allele was favourable for 59% of QTLs detected through CIM. Of 261 BC₂F₂ families, 26 advanced backcross breeding lines (BC₂F₅) were used for QTL validation. These lines were selected on the basis of the yield traits potentiality in BC₂F₃ and BC₂F₄ generations. The field trial was conducted at three different locations in Malaysia using randomized complete block design with three replications. Trait based marker analysis was done for QTL determination. Twenty-five QTLs were detected in BC₂F₅ generation whereas 29 QTLs were detected in BC₂F₂ generation of the same population. Two QTLs (qPL-1 and qSPL-7) were not considered for validation due to their low R ² values and two QTLs (qPSS-3-2 and qGW-3-2) were not detected in the BC₂F₅ population. Fifteen QTLs showed the beneficial effect to enhance the trait value of the breeding lines. QTL validation aided to select the promising lines for further utilization.     

Serogroup,Virulence, and Genetic Traits of Vibrio parahaemolyticus in the Estuarine Ecosystem of Bangladesh

Forty-two strains of Vibrio parahaemolyticus were isolated from Bay of Bengal estuaries and, with two clinical strains, analyzed for virulence, phenotypic, and molecular traits. Serological analysis indicated O8, O3, O1, and K21 to be the major O and K serogroups, respectively, and O8:K21, O1:KUT, and O3:KUT to be predominant. The K antigen(s) was untypeable, and pandemic serogroup O3:K6 was not detected. The presence of genes toxR and tlh were confirmed by PCR in all but two strains, which also lacked toxR. A total of 18 (41%) strains possessed the virulence gene encoding thermostable direct hemolysin (TDH), and one had the TDH-related hemolysin (trh) gene, but not tdh. Ten (23%) strains exhibited Kanagawa phenomenon that surrogates virulence, of which six, including the two clinical strains, possessed tdh. Of the 18 tdh-positive strains, 17 (94%), including the two clinical strains, had the seromarker O8:K21, one was O9:KUT, and the single trh-positive strain was O1:KUT. None had the group-specific or ORF8 pandemic marker gene. DNA fingerprinting employing pulsed-field gel electrophoresis (PFGE) of SfiI-digested DNA and cluster analysis showed divergence among the strains. Dendrograms constructed using PFGE (SfiI) images from a soft database, including those of pandemic and nonpandemic strains of diverse geographic origin, however, showed that local strains formed a cluster, i.e., “clonal cluster,” as did pandemic strains of diverse origin. The demonstrated prevalence of tdh-positive and diarrheagenic serogroup O8:K21 strains in coastal villages of Bangladesh indicates a significant human health risk for inhabitants.Vibrio parahaemolyticus, a halophilic bacterium, is a causative agent of seafood-related gastroenteritis worldwide (5, 13, 41) and one of the major causes of seafood-associated gastroenteritis in the United States, Asia, Europe, and countries where sporadic cases and outbreaks occur regularly (12, 13). The bacterium is prevalent in brackish and marine waters (43). Historically first identified as the causative agent of a gastroenteritis outbreak in Japan in 1950 (14), V. parahaemolyticus is now recognized as one of the most important food-borne pathogens in Asia, causing approximately half of food poisoning outbreaks in Taiwan, Japan, Vietnam, and Southeast Asian countries.The gene encoding the thermostable direct hemolysin (TDH)—manifested as beta-hemolysis when V. parahaemolyticus is plated onto Wagatsuma blood agar (43), i.e., the Kanagawa phenomenon (KP)—has been shown to be present in more than 90% of clinical strains and less than 1% of environmental strains (31, 39). Some strains also possess the gene trh, encoding the TDH-related hemolysin (TRH), or both tdh and trh (18, 43). Another gene, the thermolabile hemolysin gene (tlh), was reported to be present in V. parahaemolyticus (36) and subsequently in all V. parahaemolyticus strains tested (38).V. parahaemolyticus gastroenteritis is a multiserogroup affliction, with at least 13 O serogroups and 71 K serotypes detected (19, 42). In 1996, serogroup O3:K6 was first reported from diarrhea patients in Kolkata, India (32), and subsequently worldwide, as an increasing incidence of gastroenteritis caused by the serogroup O3:K6 was reported in many countries (41). Rapid spreading of serogroup O3:K6 infections in Asia (27, 32), and subsequently in the United States (12), Africa (3), Europe (25), and Latin America (15), indicated its potential as a pandemic pathogen (34, 43). In addition, V. parahaemolyticus serogroup O3:K6 possesses the group-specific (GS) gene sequence in the toxRS operon and ORF8, of the 10 known open reading frames (ORFs) of the O3:K6-specific filamentous phage f237. The GS gene and ORF8 provide genetic markers distinguishing O3:K6 from other serogroups (27, 29). Recent studies have shown O4:K68, O1:K25, O1:K26, O1:K untypeable (O1:KUT), and O3:K46 serogroups to share genetic markers specific for the pandemic serogroup O3:K6 (7, 10, 27, 34, 41). The non-O3:K6 serogroups with pandemic traits are increasingly found worldwide, and therefore, their pandemic potential cannot be ruled out.In Bangladesh, strains of different serogroups having genetic markers for the serogroup O3:K6 of V. parahaemolyticus were reported to have been isolated from hospitalized gastroenteritis patients in Dhaka (7). A systematic surveillance of the coastal areas bordering the Bay of Bengal where diarrheal disease is endemic (1) has not been done. This study, the first of its kind, was undertaken to investigate virulence potential, as well as phenotypic and genotypic traits of V. parahaemolyticus strains occurring in the estuarine ecosystem of Bangladesh.     

Census of the Viral Metagenome within an Activated Sludge Microbial Assemblage

The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.Bacteriophages play an active role in the ecology of natural environments, influencing prokaryotic population dynamics (5, 15) and mediating lateral gene transfer between diverse bacterial species, for example. Activated sludge (AS) microbial assemblages in wastewater treatment plants have been shown to harbor great numbers of viruses with a wide range of genome sizes (7, 9, 10, 16). Historically, the focus of wastewater viral studies has been on specific host-virus interactions, the application of phages as tools in microbial source tracking, or the use of phages to improve the efficiency of the wastewater treatment process (e.g., foam and pathogen control) (2, 4, 8, 12, 17). Despite the interest in the wastewater viral community, a census of the activated sludge total viral community has not, to our knowledge, been investigated using both culture-based and metagenomic approaches.     

Circulating Mucosal Associated Invariant T Cells Are Activated in Vibrio cholerae O1 Infection and Associated with Lipopolysaccharide Antibody Responses

Background

Mucosal Associated Invariant T (MAIT) cells are innate-like T cells found in abundance in the intestinal mucosa, and are thought to play a role in bridging the innate-adaptive interface.

Methods

We measured MAIT cell frequencies and antibody responses in blood from patients presenting with culture-confirmed severe cholera to a hospital in Dhaka, Bangladesh at days 2, 7, 30, and 90 of illness.

Results

We found that MAIT (CD3⁺CD4⁻CD161^hiVα7.2⁺) cells were maximally activated at day 7 after onset of cholera. In adult patients, MAIT frequencies did not change over time, whereas in child patients, MAITs were significantly decreased at day 7, and this decrease persisted to day 90. Fold changes in MAIT frequency correlated with increases in LPS IgA and IgG, but not LPS IgM nor antibody responses to cholera toxin B subunit.

Conclusions

In the acute phase of cholera, MAIT cells are activated, depleted from the periphery, and as part of the innate response against V. cholerae infection, are possibly involved in mechanisms underlying class switching of antibody responses to T cell-independent antigens.     

Novel benzimidazole phosphonates as potential inhibitors of protein prenylation

Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.     

Preimplantational embryo development and incidence of blastomere apoptosis in bovine somatic cell nuclear transfer embryos reconstructed with long-term cultured donor cells

This study was performed to investigate whether types and/or age of donor cells affect preimplantational embryo development and the incidence of apoptosis in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine fetal or adult ear fibroblasts were isolated, cultured in vitro and categorized into fresh or long-term cultured cells in terms of population doublings (PD): in fetal fibroblasts, <16 being considered fresh and >50 being long-term cultured; in adult ear fibroblasts, <16 being considered fresh and >30 being long-term cultured. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199, enucleated and reconstructed by SCNT. The reconstructed oocytes were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39 degrees C in a humidified atmosphere of 5% CO(2) air for 7 days. The early development of SCNT embryos was monitored under a microscope and the quality of blastocysts was assessed by differential counting of inner cell mass (ICM) and trophectoderm (TE) cells and by apoptosis detection in blastomeres using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. As results, types and/or age of donor cells did not affect the rate of blastocyst formation and the number of ICM and TE cells. However, a significant increase in apoptotic blastomeres was observed in SCNT embryos reconstructed with long-term cultured fetal or adult ear fibroblasts compared to those in SCNT embryos derived from fresh fetal or adult ear fibroblasts. In conclusion, these results indicated that the long-term culture of donor cells caused increased the incidence of apoptosis in bovine SCNT embryos but did not affect the developmental competence and the cell number of blastocysts.