Impact of polyunsaturated vegetable oils on adiponectin levels, glycaemia and blood lipids in individuals with type 2 diabetes: a randomised, double-blind intervention study.

BACKGROUND: Low adiponectin levels are discussed as risk factor for cardiovascular events. This is of special importance in individuals with type 2 diabetes (T2DM) because they are at higher risk for cardiovascular diseases. The present study aimed to investigate the effect of two plant oils rich in polyunsaturated fatty acids (PUFA), with different content of omega-3 fatty acids, on adiponectin levels, glucose and lipid metabolism in T2DM individuals treated either with insulin or oral anti-diabetics (OAD). METHODS: Ninety-two subjects with T2DM [34 treated with insulin (T2DM-Ins) and 58 treated with OAD (T2DM-OAD)] participated in this randomised, double-blind, parallel intervention study. Individuals received either 9 g of nut oil (n-3:n-6 ratio: 1.3 : 6.1) or mixed oil (n-3:n-6 ratio: 0.6 : 5.7) per day for 10 weeks. The fatty acid profile, tocopherol, adiponectin levels and parameters regarding glucose and lipid metabolism were assessed at baseline, during and after the intervention. RESULTS: Compliance was confirmed by significant increases in γ-tocopherol and PUFA in both oil groups. An increase in adiponectin levels in T2DM-Ins participants (+6.84% in nut oil and +4.47% in mixed oil group after 10 weeks compared to baseline) was observed, albeit not significantly different from T2DM-OAD individuals (P = 0.051). Lipid and glucose metabolism were not affected by the intervention. CONCLUSIONS: The present study provides evidence that a small and easy change in dietary behaviour towards better fat quality moderately increases adiponectin levels in T2DM-Ins subjects, independently of the administered plant oil.

Effects of unconjugated bilirubin on chromosomal damage in individuals with Gilbert's syndrome measured with the micronucleus cytome assay.

Circulating unconjugated bilirubin (UCB) has been reported to protect against lung and colorectal cancer. The present study aimed to explore, for the first time, whether mildly elevated circulating UCB, as found in Gilbert`s syndrome (GS), is associated with changes of DNA damage. A random 76 individuals, matched for age and gender, were recruited from the general population and allocated into the GS group (UCB ≥ 17.1 µM; n = 38) or control group (UCB <17.1 µM; n = 38). Chromosomal and cytological changes were determined in lymphocytes and buccal cells using the cytokinesis-block micronucleus cytome assay (CBMN) and buccal micronucleus cytome assay (BMcyt). No significant differences were found between GS subjects and the control group in the CBMN and BMcyt determined endpoints. Subsequently, when age dependency of effects were analysed, lower formation of buccal micronucleated cells (by 73.3%) and buccal nuclear buds (by 70.9%) in the GS subgroup ≥ 30 years were found, compared to the GS subgroup <30 years. These findings suggest DNA protection in epithelial tissue of older individuals with GS.

Insights into erythroid differentiation obtained from studies on avian erythroblastosis virus.

Analysis of the oncogenes v-erbB and v-erbA and their normal proto-oncogene counterparts has revealed several novel aspects of erythroid differentiation. A new erythroid progenitor capable of extended self-renewal has been described, tyrosine kinase receptors and steroid hormone receptors have been found to cooperate in controlling self-renewal, and dramatic alterations in the cell cycle have been found to accompany induction of terminal differentiation.

Translation control: bridging the gap between genomics and proteomics?

mRNA profiling enables the expression levels of thousands of transcripts in a cell to be monitored simultaneously. Nevertheless, analyses in yeast and mammalian cells have demonstrated that mRNA levels alone are unreliable indicators of the corresponding protein abundances. This discrepancy between mRNA and protein levels argues for the relevance of additional control mechanisms besides transcription. As translational control is a major mechanism regulating gene expression, the use of translated mRNA in profiling experiments might depict the proteome more closely than does the use of total mRNA. This would combine the technical potential of genomics with the physiological relevance of proteomics.

Erythroid progenitor renewal versus differentiation: genetic evidence for cell autonomous, essential functions of EpoR, Stat5 and the GR.

The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.

Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis.

Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes v-ErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal differentiation under appropriate conditions, were established from fetal livers of p53-/- mice. Expression and activation of the EGF-receptor (HER-1/c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal differentiation. Upon inhibition of ErbB function, differentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5- and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms.

Increased levels of dihydrofolate reductase mRNA can be measured in normal, growth-stimulated mouse fibroblasts.

Levels of mRNA for the enzyme dihydrofolate reductase (EC 1.5.1.3) were determined in growth-stimulated 3T6 cells which contained wild-type dosage of the gene coding for this enzyme. As in the case of methotrexate-resistant cells having highly amplified levels of genes for dihydrofolate reductase, an increase in dihydrofolate reductase mRNA by a factor of 2-4 can be determined when cells enter the S phase. This increase is inhibited by sodium butyrate (which inhibits growth-stimulated 3T6 cells in mid G1 phase) but not by hydroxyurea (which inhibits in early S phase). We conclude that with the available methods it is possible to study the regulation of S phase-specific enzymes after growth stimulation at the level of the mRNA, even if gene amplification is not possible or desirable.

Mouse thymidine kinase stability in vivo and after in vitro translation.

Using a combination of centrifugal elutriation and recultivation of synchronised cell populations we could show that murine thymidine kinase (TK) is rapidly degraded during mitosis in polyoma virus-transformed mouse fibroblasts, in parallel to the time-course for loss of cyclin A. Transformation is no prerequisite for the instability phenotype since artificial overexpression of TK under the control of a constitutive promoter in normal mouse fibroblasts also resulted in rapid turnover of TK during mitosis. The decay of TK protein could be partially mimicked in vitro with enzymatically active protein translated in a rabbit reticulocyte lysate: full length polypeptide was lost slightly more rapidly in the presence of G2/M cytosolic extracts than with G1/S preparations. In addition, an enzymatically active C-terminal truncation of 37 amino acids at Gln-196 was completely stable under the conditions tested, confining the instability domain between residues 196 to 233. These experiments also indicated the border for intact TK since translation products up to Tyr-189 or less were completely inactive. This was also confirmed by a mutant TK protein from mouse F9tk- teratocarcinoma cells which harboured a similar deletion.

Post-transcriptional repression of thymidine kinase expression during cell cycle and growth stimulation.

In vertebrates, endogenous thymidine kinase (TK) gene expression is strictly growth-dependent. Here we report that in continuously cycling Ltk-mouse fibroblasts, stably transfected with a vector expressing human TK cDNA from a constitutive promoter, enzyme activity rises 8-fold at the G1/S phase transition and declines again in G2. The mechanism did not involve changes in protein stability. When hTK was put under the control of a hormone-inducible promoter, production of high mRNA levels following addition of dexamethasone did not result in any enzyme activity in resting NIH-3T3tk- cells. After growth stimulation with serum, TK activity rose together with the onset of DNA synthesis only in the simultaneous presence of the hormone.

Reliability of mRNA profiling: verification for samples with different complexities.

Normalization of mRNA profiling data remains an open issue, which turns critical when comparing divergent samples or mRNA populations with different complexities. To address this question, we generated samples with different RNA amounts and complexities by subcellular fractionation of cytoplasmic RNA into the mutually exclusive ribosome-free and polysome-bound RNA pools. For each of the 563 mRNAs analyzed, the hybridization signal corresponding to the cytoplasmic sample equals the sum of signals from the ribosome-free plus the polysome-bound targets (cytoplasmic mRNA = ribosome-free mRNA + polysome-bound mRNA). This intuitive equation was fulfilled only after data normalization following "spiking" of the samples with an exogenous RNA. This is the first demonstration that spiking allows one to correct not only for differences in reaction efficiencies but also to reflect the variations in amount and complexity between the initial mRNA populations.

Thymidine kinase is expressed differently in transformed versus normal-cells - a novel test for malignancy.

Using a cytofluorometric assay, we studied the time course of thymidine kinase activity during the cell-cycle in logarithmically growing cells. Two different patterns were found. Many cells, including all strains of normal diploid fibroblasts, exhibited a transient stimulation of enzyme activity during early S-phase. On the contrary, virally transformed cells or lines derived from tumors also increased enzyme activity during G1/S, but kept this level until mitosis. In the latter case, the absolute enzyme activity was significantly higher during all phases of the cell-cycle than in normal cells. Beside its importance for the understanding of the transformed state, this phenomenon has obvious benefits for tumor diagnosis.

Dynamics of cell cycle regulators: artifact-free analysis by recultivation of cells synchronized by centrifugal elutriation.

Studies on the molecular properties of cell cycle regulators in animal cells require cell preparations highly enriched in particular cell cycle phases. Centrifugal elutriation is frequently used to synchronize cells because this technique was thought to cause only minimal distortions in protein expression or metabolic functions. However, in primary chicken erythroblasts, we consistently observed artefacts in mitotic cyclin mRNA expression and p70 S6 kinase activity, which were clearly caused by the elutriation procedure. Therefore, we modified the standard protocol by reseeding various elutriated fractions into preconditioned medium, a process termed recultivation, and harvesting after an appropriate amount of time. This avoided the pleiotropic effects caused by stress and lack of growth factor supply during elutriation. Using this recultivation procedure, highly synchronous progression starting from any given cell cycle phase could be achieved for a variety of cell types, including primary, factor-dependent cells of hematopoietic origin. Mitotic cyclin expression and S6 kinase activity was found to be normal again in recultivated cultures, as opposed to elutriated ones. Finally, monitoring of mitosis-specific cyclin A degradation in recultivated G2 phase cells showed that recultivation provided an excellent tool to follow cells through M phase into G1 without the requirement for a chemical cell cycle block.

Post-transcriptional control via iron-responsive elements: the impact of aberrations in hereditary disease.

Tight regulation of iron metabolism is crucial to avoid formation of deleterious radicals and is mainly executed at the post-transcriptional level. The regulatory loops are exerted by trans-acting iron regulatory proteins (IRPs) and cis-acting stem-loop motifs, termed iron-responsive elements (IREs), located in the untranslated regions (UTRs) of target mRNAs. Iron scarcity induces binding of IRPs to a single IRE in the 5'-UTR of ferritin, eALAS, aconitase and SDHb mRNAs, which specifically suppresses translation initiation. Simultaneous interaction of IRPs with multiple IREs in the 3'-UTR of transferrin receptor (TfR) mRNA selectively causes its stabilization. The pattern is reverted under iron overload: IRP-mRNA binding affinity is reduced, which results in efficient protein synthesis of target transcripts harboring IREs in the 5'-UTR and rapid degradation of TfR mRNA. Although multiple evidences support this model, several studies reported massive alterations in the regulation of iron homeostasis under specific physiological conditions, raising the possibility for additional regulatory events. Intensive analysis of the palindromic IRE consensus sequence revealed the critical elements for the formation of a functional structure and demonstrated the consequences of IRE mutations in IRP binding. Recent investigations indicated the involvement of naturally occurring IRE mutations of the ferritin L subunit in the hyperferritinemia-cataract syndrome, a hereditary disorder. This review summarizes the apparent links between iron-dependent post-transcriptional control and its abnormalities, governed by the properties of a single mRNA stem-loop structure.

[Re-integration of children into the biological (partial) family]. / Die Re-Integration von Kindern in die leibliche (Teil-) Familie.

The article deals with some aspects of a study about reintegration of children from Austrian SOS Children's Villages into their original families. The knowledge that children actually not always return to their original families is of outstanding importance. In the meantime, very often the family has substantially changed because family members joined or left the family. Therefore the term 'reintegration into the original family' reflects the situation insufficiently. Ignoring these changes, many times lead to difficulties within family life.

A stem-loop in the 3' untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm.

Expression of the human transferrin receptor (hTR) and its mRNA is strongly induced by iron deprivation. By measuring transcription elongation rates, levels of hTR-specific nuclear RNA, and mRNA half-lives, we found this regulation to occur posttranscriptionally in the cytoplasm. Analysis of hTR cDNA mutants with deletions in the 3' untranslated region revealed the existence of two distinct domains, both of which are essential for regulation in mouse L cells. The regulated phenotype correlates with the presence of a stem-loop structure predicted by a computer algorithm. Expression of point and deletion mutants affecting the stem-loop confirmed the requirement for this secondary structure in regulation. The 3' untranslated region of hTR cDNA was sufficient to confer iron-dependent regulation on another gene.

Relationship between background activity and subclinical seizure pattern.

Open- and closed-eyed EEG records were made in frontal, temporal and occipital regions, in order to examine the time relationships between various of their characteristics, in connection with the appearance of subclinical seizure patterns. 1. In the range of alpha frequency, both with open and closed eyes, the standard deviation of frequency over all brain areas decreased both before and after subclinical seizure patterns as compared to periods without pattern, and the number of temporal waves was reduced. Frontally, the mean amplitude was increased only in open-eyed records. The time relationship of the appearance of alpha maxima differed over all regions, but was closest in frontal records in both open- and closed-eyed records, and independent of the pathological pattern. In closed-eyed frontal records, a changeover in the time lead between the hemispheres could be observed, with one side leading during the period preceding the pattern, and the other after the pattern. 2. In the range of theta frequency, occipital open-eyed records showed slowing of the frequency both before and after the subclinical seizure pattern, with an increase in the standard deviation. In closed-eyed records made in the period preceding the pattern, an increase of the mean amplitude was observed frontally and occipitally, as well as an increase of the standard deviation of the amplitude in all regions. Slowing of the theta frequency was encountered in the frontal regions. Differences in the periods preceding and following subclinical seizures were to be seen only in the theta range.