In the absence of Rauber's sickle material,no blood islands are formed in the avian blastoderm

Using the quail-chick chimera technique, we followed the fate of Rauber's sickle cells in older whole blastoderms (cultured for approximately 2 days): after removal of the autochthonous Rauber's sickle from an unincubated chicken blastoderm, a quail Rauber's sickle was grafted isotopically and isochronically in its place. In transverse sections through these chimeras, the grafted quail Rauber's sickle cells were seen to have transformed into a broad row or ridge of quail junctional endoblast cells extending at the inner border of the area containing blood islands. After unilateral removal of the junctional endoblast from an intermediate streak chicken blastoderm (Stage 3; Hamburger and Hamilton [1951] J Morphol 88:49-92), we observed during further in vitro culture that at the operated side, in the area previously occupied by this junctional endoblast, blood islands no longer developed. If after such a unilateral removal of the chicken junctional endoblast quail junctional endoblast was apposed in its place, then blood islands reappeared in the operated area. The intimate contact between the apposed quail junctional endoblast and the recently formed blood islands, derived from peripherally migrating mesoderm, was very obvious on sections through such chimeras. We further demonstrate that Rauber's sickle vs. junctional endoblast is indispensable for the anlage of blood islands in avian blastoderms. Indeed, in the absence of Rauber's sickle material no blood islands develop (even when mesoderm is present after ingression of the upper layer via a primitive streak) in the isolated central region of the area centralis of unincubated chicken blastoderms after culture in vitro. Also, no junctional endoblast and no sickle canal appear in these explants. By contrast, if a Rauber's sickle fragment is placed on such an isolated central blastoderm region, then blood islands develop. These blood islands start to develop from peripherally migrating mesoderm in the neighborhood of the Rauber's sickle-derived junctional endoblast.     

DNA base excision repair of uracil residues in reconstituted nucleosome core particles

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.     

The role of medial temporal lobe structures in implicit learning: an event-related FMRI study

The medial temporal lobe (MTL) has been associated with declarative learning of flexible relational rules and the basal ganglia with implicit learning of stimulus-response mappings. It remains an open question of whether MTL or basal ganglia are involved when learning flexible relational contingencies without awareness. We studied learning of an explicit stimulus-response association with fMRI. Embedded in this explicit task was a hidden structure that was learnt implicitly. Implicit learning of the sequential regularities of the "hidden rule" activated the ventral perirhinal cortex, within the MTL, whereas learning the fixed stimulus-response associations activated the basal ganglia, indicating that the function of the MTL and the basal ganglia depends on the learned material and not necessarily on the participants' awareness.     

Methylpurine DNA glycosylase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA glycosylase (MPG) function. Such enzymes have previously been characterized from bacteria and eukarya, but not from archaea. We identified activity for the release of methylated bases from DNA in cell-free extracts of Archaeoglobus fulgidus, an archaeon growing optimally at 83 degrees C. An open reading frame homologous to the alkA gene of Escherichia coli was overexpressed and identified as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA. The purified AfalkA protein differs from E. coli AlkA by excising alkylated bases only, from DNA, in the following order of efficiency: 3-methyladenine (m(3)A) > 3-methylguanine approximately 7-methyladenine > 7-methylguanine. Although the rate of enzymatic release of m(3)A is highest in the temperature range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a temperature that does not support growth of A. fulgidus. At temperatures above 75 degrees C, nonenzymatic release of methylpurines predominates. The results suggest that the biological function of AfalkA is to excise m(3)A from DNA at suboptimal and maybe even mesophilic temperatures. This hypothesis is further supported by the observation that the afalkA gene function suppresses the alkylation sensitivity of the E. coli tag alkA double mutant. The amino acid sequence similarity and evolutionary relationship of AfalkA with other MPG enzymes from the three domains of life are described and discussed.     

Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.     

Effects of emotion-oriented care on elderly people with cognitive impairment and behavioral problems in residential homes

This study investigates the effects of emotion-oriented care on the behavior of elderly people with cognitive impairment and behavioral problems. This approach is mainly based on the validation approach, but uses also insights from other approaches like reminiscence and sensory stimulation. Sixteen homes for the aged with structured day care units were randomly allocated to an intervention or control group. 151 Residents with cognitive impairment and behavioral problems were included in the study. The eight intervention homes received a training program with regard to emotion-oriented care. In the eight control homes usual care was continued. Measurements were performed at baseline and after three, six and twelve months of follow-up (assessment by caregivers and relatives). The primary outcome measure was the change in behavior of the residents. The results of multilevel analyses (overall, subgroup and per protocol) showed no statistically significant, nor clinically relevant effects in favor of the intervention group on the behavioral outcome measures. A possible reason for this is that the intervention did not result in significant contrast between the study groups. There is insufficient scientific evidence yet to justify the implementation of emotion-oriented care in residential homes, for residents with moderate to severe cognitive impairment and behavioral problems, on a large scale. Additional studies are needed in which special attention should be given to the implementation process.     

Different responses of tobacco antioxidant enzymes to light and chilling stress

The effect of elevated light treatment (25 degrees C, PPFD 360 mumol m-2 sec-1) or chilling temperatures combined with elevated light (5 degrees C, PPFD 360 mumol m-2 sec-1) on the activity of six antioxidant enzymes, guaiacol peroxidases, and glutathione peroxidase (GPx, EC 1.11.1.9) protein accumulation were studied in tobacco Nicotiana tabacum cv. Petit Havana SR1. Both treatments caused no photooxidative damage, but chilling caused a transient wilting. The light treatment increased the activities of ascorbate peroxidase (APx, EC 1.11.1.11) and guaiacol peroxidases while catalase (EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were unchanged. In contrast, chilling treatment did not increase any of the antioxidant enzyme activities, but decreased catalase and to a lesser extent DHAR activities. Glutathione peroxidase protein levels increased sporadically under light treatment and constantly under chilling. Both chilling and light stress caused induction of glutathione synthesis and accumulation of oxidised glutathione, although the predominant part of the glutathione pool remained in the reduced form. Antioxidant enzymes from the chilling treated plants were measured at both 25 degrees C and 5 degrees C. Measurements at 5 degrees C revealed a 3-fold reduction in catalase activity, compared with that measured at 25 degrees C, indicating that the overall reduction in catalase after four days of chilling was approximately 10-fold. The overall reduction in activity for the other antioxidant enzymes after four days of chilling was 2-fold for GR and APx, 1.5-fold for MDHAR, 3.5-fold for DHAR. The activity of SOD was the same at 25 and 5 degrees C. These results indicate that catalase and DHAR are most strongly affected by the chilling treatment and may be the rate-limiting factor of the antioxidant system at low temperatures.     

Two pregnancies after preimplantation genetic diagnosis for osteogenesis imperfecta type I and type IV

Osteogenesis imperfecta (OI) is an autosomal dominant genetic disorder characterized by the presence of brittle bones and decreased bone mass (osteopenia), as a result of mutations in the genes that encode the chains of type I collagen, the major protein of bone. The clinical features of the disease range from death in the perinatal period to normal life span with minimal increase in fractures. The present report describes two polymerase chain reaction (PCR)-based assays allowing preimplantation genetic diagnosis (PGD) on the one hand for OI type I, the mildest form, and on the other hand for OI type IV, which is intermediate in severity between OI type I and OI type III. In the couple referred for PGD for OI type I, the female partner carried a 1-bp deletion in exon 43 of the COL1A1 gene, resulting in a premature stop codon in exon 46. The synthesis of too little type I procollagen results from such a non-functional or COL1A1 null allele. In the other couple, referred for PGD for OI type IV, the male partner carried a G to A substitution in exon 19 of the COL1A2 gene, which results in an abnormal gene product due to an alphaGly247 (GGT) to Ser (AGT) substitution (G247S). Both mutations result in the loss of a specific restriction enzyme recognition site and can therefore be detected by PCR amplification followed by restriction fragment analysis. PCR amplification of genomic DNA of the parents-to-be with one of the two primers fluorescently labelled, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified and restricted fragments, allowed a distinction between the healthy and affected genotypes. PCR on single Epstein-Barr-virus (EBV)-transformed lymphoblasts resulted in acceptable amplification efficiencies (87% and 85% for OI type I and OI type IV respectively) and the allele drop-out (ADO) rate was assessed at 11.5% and 11.1% for OI type I and OI type IV respectively. With research blastomeres, 100% amplification rates were obtained and no contamination was observed in the blank controls, which validated the tests for clinical application. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal genotype of the non-affected parent. For OI type I, two frozen-thawed ICSI-PGD cycles and two fresh ICSI-PGD cycles were carried out for the same couple. The transfer of two unaffected embryos in the last cycle resulted in a twin pregnancy. A twin pregnancy was also achieved in one clinical ICSI-PGD cycle for OI type IV.     

The J Domain of Simian Virus 40 Large T Antigen Is Required To Functionally Inactivate RB Family Proteins

Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G₁ arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.