Inadequacies of micronutrient intake in normal weight and overweight young adults aged 18-25 years: a cross-sectional study.

OBJECTIVES: This study aims to assess adequacy in micronutrient intake in comparison with reference nutrient intakes (RNI) and to identify differences in intakes between normal weight and overweight individuals. STUDY DESIGN: A sample of 542 university students (18-25 years), normal weight (N = 369) and overweight (N = 173), were included in a cross-sectional study. METHODS: A three-day diet diary was used to assess energy and nutrient intake. Body mass index (BMI) and waist circumference were measured. RESULTS: Mean dietary vitamin D intake was lower than RNI in both men (4.44 µg) and women (5.04 µg). Mean intakes of calcium (597.44 mg), iron (8.62 mg) and folate (171.29 mg) were also lower than recommendations in women. Weight status (normal weight versus overweight) was significantly associated with micronutrient intake, and a trend towards a decrease in vitamin and mineral intake with increasing weight was noted. CONCLUSIONS: Results suggest the need to increase the intake of some micronutrients to meet the RNI, to ensure optimal health. This study provides a helpful tool to reinforce recommendations and potential health promotion and intervention strategies in university settings and could influence manufacturers involved in new food product development targeted to this young population.

The role of Clostridium difficile in the paediatric and neonatal gut - a narrative review.

Clostridium difficile is an important nosocomial pathogen in adults. Its significance in children is less well defined, but cases of C. difficile infection (CDI) appear to be increasingly prevalent in paediatric patients. This review aims to summarize reported Clostridium difficile carriage rates across children of different age groups, appraise the relationship between CDI and factors such as method of delivery, type of infant feed, antibiotic use, and co-morbidities, and review factors affecting the gut microbiome in children and the host immune response to C. difficile. Searches of PubMed and Google Scholar using the terms 'Clostridium difficile neonates' and 'Clostridium difficile children' were completed, and reference lists of retrieved publications screened for further papers. In total, 88 papers containing relevant data were included. There was large inter-study variation in reported C. difficile carriage rates. There was an association between CDI and recent antibiotic use, and co-morbidities such as immunosuppression and inflammatory bowel disease. C. difficile was also found in stools of children with diarrhoea attributed to other pathogens (e.g. rotavirus). The role of C. difficile in the paediatric gut remains unclear; is it an innocent bystander in diarrhoeal disease caused by other organisms, or a pathogen causing subclinical to severe symptoms? Further investigation of the development of serological and local host response to C. difficile carriage may shed new light on disease mechanisms. Work is underway on defining a framework for diagnosis and management of paediatric CDI.

Inducible liver-specific knockdown of protein tyrosine phosphatase 1B improves glucose and lipid homeostasis in adult mice.

AIMS/HYPOTHESIS: Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin signalling. Hepatic PTP1B deficiency, using the Alb-Cre promoter to drive Ptp1b deletion from birth in mice, improves glucose homeostasis, insulin sensitivity and lipid metabolism. The aim of this study was to investigate the therapeutic potential of decreasing liver PTP1B levels in obese and insulin-resistant adult mice. METHODS: Inducible Ptp1b liver-specific knockout mice were generated using SA-Cre-ER(T2) mice crossed with Ptp1b floxed (Ptp1b(fl/fl)) mice. Mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity and insulin resistance. Tamoxifen was administered in the HFD to induce liver-specific deletion of Ptp1b (SA-Ptp1b(-/-) mice). Body weight, glucose homeostasis, lipid homeostasis, serum adipokines, insulin signalling and endoplasmic reticulum (ER) stress were examined. RESULTS: Despite no significant change in body weight relative to HFD-fed Ptp1b(fl/fl) control mice, HFD-fed SA-Ptp1b(-/-) mice exhibited a reversal of glucose intolerance as determined by improved glucose and pyruvate tolerance tests, decreased fed and fasting blood glucose and insulin levels, lower HOMA of insulin resistance, circulating leptin, serum and liver triacylglycerols, serum NEFA and decreased HFD-induced ER stress. This was associated with decreased glycogen synthase, eukaryotic translation initiation factor-2α kinase 3, eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase 2 phosphorylation, and decreased expression of Pepck. CONCLUSIONS/INTERPRETATION: Inducible liver-specific PTP1B knockdown reverses glucose intolerance and improves lipid homeostasis in HFD-fed obese and insulin-resistant adult mice. This suggests that knockdown of liver PTP1B in individuals who are already obese/insulin resistant may have relatively rapid, beneficial therapeutic effects.

Cyclin dependent kinase regulation.

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and their activities are consequently tightly regulated. Recent developments in the field of CDK regulation have included the discovery and characterization of CDK inhibitors. These developments have had an impact on our understanding of how other signalling pathways may be linked to the cell cycle machinery.

Induction of cell cycle regulatory proteins in anti-immunoglobulin-stimulated mature B lymphocytes.

Progression through the cell cycle is a tightly controlled process that integrates signals generated at the plasma membrane with the proteins that form the cell cycle machinery. The current study chronicles the induction of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors in low density primary mouse B lymphocytes after anti-immunoglobulin plus interleukin 4 (IgM + IL-4) stimulation. In this system, > 85% of cells remain in the G0/G1 phase of cell cycle for an initial 24-h period, followed by entry of up to 50% of the cells into S phase, commencing around 30 h and peaking at 48 h. Extensive time course analyses of these anti-IgM + IL-4-stimulated B cells revealed that the G1-associated D-type cyclins D2 and D3 were induced by 3 h after stimulation, and that cyclins E, A, and B were subsequently induced sequentially, beginning at mid-G1, G1/S transition, and S phase, respectively. The G1-associated cyclin D1 was not expressed at any stage of the anti-Ig + IL-4-induced B cell cycle. cdk2, cdk4, and cdk6 were induced during G1, whereas cell division cycle-2 (cdc2) was induced concomitantly with S phase. Irrespective of their expression, the kinases cdk2 and cdc2 were only active from S phase onwards, suggesting that productive cyclin/kinase complex formation did not occur until that time. Cell cycle inhibitors p21 and p19 were induced by anti-Ig + IL-4, peaking in expression at mid-G1 and S phase, respectively. Stimulation of low density B cells with anti-Ig + IL-4 caused rapid down regulation of the p27 inhibitor, however this protein was reexpressed at 54-96 h after stimulation. In contrast, B cells stimulated with anti-CD40, a stimulus which induces long-term B cell proliferation, permanently down regulated p27. These findings are consistent with the concept that p27 reexpression contributes to the G1 arrest that follows antigen receptor crosslinking. Low density B cells cultured in the viability-enhancing cytokine IL-4 alone also showed induction of D2 and D3 cyclin expression. However, the D2 expression was transient, and the D3 expression was substantially lower than that observed in B cells induced to proliferate by anti-Ig + IL-4. This partial induction of D2 and D3 expression may explain IL-4's ability to promote B cell entry into G1 but not S phase of cell cycle, and furthermore, its ability to truncate G1 progression when B cells are subsequently stimulated with anti-Ig.

Transgene expression of bcl-xL permits anti-immunoglobulin (Ig)-induced proliferation in xid B cells.

Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice (xid). While B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here induction of the complete complement of cell cycle regulatory molecules, though the level of induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-xL. Ectopic expression of Bcl-xL in xid B cells permitted anti-Ig induced cell cycle progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly regulate Bcl-xL.

Association of human cyclin E with a periodic G1-S phase protein kinase.

G1 cyclins control the G1 to S phase transition in the budding yeast, Saccharomyces cerevisiae. Cyclin E was discovered in the course of a screen for human complementary DNAs that rescue a deficiency of G1 cyclin function in budding yeast. The amounts of both the cyclin E protein and an associated protein kinase activity fluctuated periodically through the human cell cycle; both were maximal in late G1 and early S phases. Cyclin E-associated kinase activity was correlated with the appearance of complexes containing cyclin E and the cyclin-dependent kinase Cdk2. Thus, the cyclin E-Cdk2 complex may constitute a human G1-S phase-specific regulatory protein kinase.

Identification of an overlapping binding domain on Cdc20 for Mad2 and anaphase-promoting complex: model for spindle checkpoint regulation.

Activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis in mammalian cells. Cdc20, which specifically recognizes APC substrates involved in the metaphase-to-anaphase transition, plays a pivotal role in APC activation through direct interaction with the APC. The activation of the APC by Cdc20 is prevented by the interaction of Cdc20 with Mad2 when the spindle checkpoint is activated. Using deletion mutagenesis and peptide mapping, we have identified the sequences in Cdc20 that target it to Mad2 and the APC, respectively. These sequences are distinct but overlapping, providing a possible structural explanation for the internal modulation of the APC-Cdc20 complex by Mad2. In the course of these studies, a truncation mutant of Cdc20 (1-153) that constitutively binds Mad2 but fails to bind the APC was identified. Overexpression of this mutant induces the formation of multinucleated cells and increases their susceptibility to undergoing apoptosis when treated with microtubule-inhibiting drugs. Our experiments demonstrate that disruption of the Mad2-Cdc20 interaction perturbs the mitotic checkpoint, leading to premature activation of the APC, sensitizing the cells to the cytotoxic effects of microtubule-inhibiting drugs.

Sequences within the conserved cyclin box of human cyclin A are sufficient for binding to and activation of cdc2 kinase.

Cyclins are pivotal in the coordinate regulation of the cell cycle. By physical association, they are able to activate at least one of the cyclin-dependent kinases, cdc2. How this association between the catalytic moiety and cyclins leads to subsequent activation of the kinase remains unclear. In this report, we describe experiments to investigate this event at a physical level. Our approach was to map the regions required on the cyclin A molecule for interaction with cdc2. We have mapped the contact regions to two small noncontiguous stretches of amino acids, residues 189 to 241 and 275 to 320, both located within the conserved cyclin box domain of the protein. We have further shown that this region not only represents a contact site for cdc2 but apparently represents an intact functional domain with respect to cdc2 activation. This region alone is sufficient to stimulate maturation when injected into immature Xenopus laevis oocytes. This observation implies that events leading to the activation of cdc2 kinase can be mediated through small regions of the cyclin molecule that are located in the cyclin box. These regions contain some of the most highly conserved residues found between all the cyclin members so far identified. This suggests that the cyclin family members may have conserved a similar mechanism to bind and activate cyclin-dependent kinases.

Cyclin D-CDK subunit arrangement is dependent on the availability of competing INK4 and p21 class inhibitors.

The D-type cyclins and their major kinase partners CDK4 and CDK6 regulate G0-G1-S progression by contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Assembly of active cyclin D-CDK complexes in response to mitogenic signals is negatively regulated by INK4 family members. Here we show that although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only p16(INK4a) can form stable, binary complexes with both CDK4 and CDK6 in proliferating cells. The other INK4 family members form stable complexes with CDK6 but associate only transiently with CDK4. Conversely, CDK4 stably associates with both p21(CIP1) and p27(KIP1) in cyclin-containing complexes, suggesting that CDK4 is in equilibrium between INK4 and p21(CIP1)- or p27(KIP1)-bound states. In agreement with this hypothesis, overexpression of p21(CIP1) in 293 cells, where CDK4 is bound to p16(INK4a), stimulates the formation of ternary cyclin D-CDK4-p21(CIP1) complexes. These data suggest that members of the p21 family of proteins promote the association of D-type cyclins with CDKs by counteracting the effects of INK4 molecules.

Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.

The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.

Cyclin E associates with components of the pre-mRNA splicing machinery in mammalian cells.

Cyclin E-cdk2 is a critical regulator of cell cycle progression from G1 into S phase in mammalian cells. Despite this important function little is known about the downstream targets of this cyclin-kinase complex. Here we have identified components of the pre-mRNA processing machinery as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitated a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B' and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. We demonstrate that SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and that its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.

Cyclin E associates with BAF155 and BRG1, components of the mammalian SWI-SNF complex, and alters the ability of BRG1 to induce growth arrest.

SWI-SNF complexes have been implicated in transcriptional regulation by chromatin remodeling. We have identified an interaction between two components of the mammalian SWI-SNF complex and cyclin E, an essential cell cycle regulatory protein required for G1/S transition. BRG1 and BAF155, mammalian homologs of yeast SWI2 and SWI3, respectively, are found in cyclin E complexes and are phosphorylated by cyclin E-associated kinase activity. In this report, we show that overexpression of BRG1 causes growth arrest and induction of senescence-associated beta-galactosidase activity, which can be overcome by cyclin E. Our results suggest that cyclin E may modulate the activity of the SWI-SNF apparatus to maintain the chromatin in a transcriptionally permissive state.

A human RNA polymerase II complex containing factors that modify chromatin structure.

We have isolated a human RNA polymerase II complex that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8, the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA polymerase II.

A novel growth- and cell cycle-regulated protein, ASK, activates human Cdc7-related kinase and is essential for G1/S transition in mammalian cells.

A novel human protein, ASK (activator of S phase kinase), was identified on the basis of its ability to bind to human Cdc7-related kinase (huCdc7). ASK forms an active kinase complex with huCdc7 that is capable of phosphorylating MCM2 protein. ASK appears to be the major activator of huCdc7, since immunodepletion of ASK protein from the extract is accompanied by the loss of huCdc7-dependent kinase activity. Expression of ASK is regulated by growth factor stimulation, and levels oscillate through the cell cycle, reaching a peak during S phase. Concomitantly, the huCdc7-dependent kinase activity significantly increases when cells are in S phase. Furthermore, we have demonstrated that ASK serves an essential function for entry into S phase by showing that microinjection of ASK-specific antibodies into mammalian cells inhibited DNA replication. Our data show that ASK is a novel cyclin-like regulatory subunit of the huCdc7 kinase complex and that it plays a pivotal role in G1/S transition in mammalian cells.

Cyclin E, a potential prognostic marker for breast cancer.

A fundamental cause of cancer is changed properties of genetic material, which may deregulate normal development of the tissue or provide selective growth advantage to the tumor cell. This deregulation of cell proliferation results from altered production of a handful of proteins that play key roles in progression through the eukaryotic cell cycle. Some of these proteins include tumor suppressor genes or oncogenes. However, no one general change or alteration of a critical gene has yet been found in all cancers. Using surgical material obtained from patients with various malignancies, we show that breast cancers and other solid tumors, as well as malignant lymphocytes from patients with lymphatic leukemia, show severe quantitative and qualitative alterations in cyclin E protein production independent of the S-phase fraction of the samples. Hence, these alterations represent a true difference between normal versus tumor tissue. In addition, in breast cancer, the alterations in cyclin E expression become progressively worse with increasing stage and grade of the tumor, suggesting its potential use as a new prognostic marker.

BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines.

Human BRG1 is a component of the evolutionarily conserved SWI-SNF chromatin remodeling complex. BRG1 has been implicated in growth control through its interaction with the tumor suppressor pRb and may consequently serve as a negative regulator of proliferation. Postulating that BRG1 may itself be a tumor suppressor gene, we screened a panel of tumor cell lines to determine whether the gene is targeted for mutation. We report that the COOH-terminal region of BRG1 is homozygously deleted in two carcinoma cell lines, prostate TSU-Pr1 and lung A-427. In addition, biallelic inactivations of BRG1 were observed in four other cell lines derived from carcinomas of the breast, lung, pancreas, and prostate; their mutations in BRG1 included three frameshift lesions and one nonsense lesion. Point mutations were also discovered in a number of other cell lines, however in most cases any effect of these mutations on BRG1 function remains to be established. A variety of different mutations within BRG1, in several cell lines, suggest that BRG1 may be targeted for disruption in human tumors. Significantly, reintroduction of BRG1 into cells lacking BRG1 expression was sufficient to reverse their transformed phenotype inducing growth arrest and a flattened morphology. These data strongly support the model that BRG1 may function as a tumor suppressor and strengthen the hypothesis that the regulation of gene expression through chromatin remodeling is critical for cancer progression. It will be important to confirm these observations in primary tumors.

Active cdk6 complexes are predominantly nuclear and represent only a minority of the cdk6 in T cells.

We have performed biochemical analyses of cdk6 complexes in T cells. By gel filtration chromatography we observed at least three cdk6 containing complexes in the cell, the most abundant eluting at 450 kDa and 50-70 kDa and a minor complex eluting at 170 kDa. Cyclin D was present in the minor 170 kDa complex which co-eluted with the peak of cdk associated in vitro Rb kinase activity. Analysis of proteins that co-immunoprecipitated with cdk6 showed that the 450 kDa complex contained both Hsp90 and CDC37. The 50-70 kDa complex was made up of two moieties, a 66 kDa complex containing cdk6 bound to p19INK4d and monomeric cdk6. The subcellular localisation of the cdk6 complexes was analysed by preparing cytoplasmic and nuclear extracts. The 450 kDa complex was shown to be predominantly cytoplasmic, whereas the 170 kDa cyclin D/cdk6 and the 50-70 kDa complexes were present in both nuclear and cytoplasmic compartments. When these same extracts were assayed for cdk6 associated kinase activity, only the nuclear cdk6 complexes were active. These data suggest that even though there are cdk6/cyclin D complexes detectable in both the cytoplasm and nucleus, only the cdk6 that is in the nucleus is active.

Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases.

Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II is important for basal transcriptional processes in vivo and for cell viability. Several kinases, including certain cyclin-dependent kinases, can phosphorylate this substrate in vitro. It has been proposed that differential CTD phosphorylation by different kinases may regulate distinct transcriptional processes. We have found that two of these kinases, cyclin C/CDK8 and cyclin H/CDK7/p36, can specifically phosphorylate distinct residues in recombinant CTD substrates. This difference in specificity may be largely due to their varying ability to phosphorylate lysine-substituted heptapeptide repeats within the CTD, since they phosphorylate the same residue in CTD consensus heptapeptide repeats. Furthermore, this substrate specificity is reflected in vivo where cyclin C/ CDK8 and cyclin H/CDK7/p36 can differentially phosphorylate an endogenous RNA polymerase II substrate. Several small-molecule kinase inhibitors have different specificities for these related kinases, indicating that these enzymes have diverse active-site conformations. These results suggest that cyclin C/CDK8 and cyclin H/CDK7/p36 are physically distinct enzymes that may have unique roles in transcriptional regulation mediated by their phosphorylation of specific sites on RNA polymerase II.

The cdk2 kinase is required for the G1-to-S transition in mammalian cells.

In the cell cycle of fission and budding yeast, the p34cdc2/CDC28 kinase is required for both the G1-to-S and G2-to-M phase transitions. In vertebrates, the homologous p34cdc2 kinase is required for G2-to-M phase transitions but appears to be dispensable for DNA synthesis. We have investigated the function of a related kinase, p33cdk2, using serum-stimulated quiescent human fibroblasts. While the p33cdk2 protein was expressed at constant levels throughout the cell cycle, p33cdk2 kinase activity was first detected a few hours prior to the onset of DNA synthesis. Microinjection of anti-p33cdk2 antibodies blocked cells from entering S phase. Pre-adsorption of these antibodies with cdk2 protein abrogated their blocking effect suggesting that the G1 arrest caused by these antibodies is cdk2-specific. These results indicate that p33cdk2 is required for the G1-to-S phase transition in mammalian cells. We also show evidence to suggest that the cyclin E/p33cdk2 complex is likely to be required for entry into S phase since the timing of the cyclin E-associated kinase activity was coincident with that of p33cdk2 and preclearing of either component abolished the majority of the histone H1 kinase activity present in the lysates harvested from the late G1.