Improved DOP-PCR-based representational whole-genome amplification using quantitative real-time PCR.

In many cases, only a minute amount of partially degraded genomic DNA can be extracted from archived clinical samples. Diverse whole-genome amplification methods are applied to provide sufficient amount of DNA for comparative genome hybridization, single-nucleotide polymorphism, and microsatellite analyses. In these applications, the reliability of the amplification techniques is particularly important. In PCR-based approaches, the plateau effect can seriously alter the original relative copy number of certain chromosomal regions. To eliminate this distorting effect, we improved the standard degenerate oligonucleotide-primed PCR (DOP-PCR) technique by following the amplification status with quantitative real-time PCR (QRT-PCR). With real-time detection of the products, we could eliminate DNA overamplification. Probes were prepared from 10 different tumor samples: primary and metastatic melanoma tissues, epidermoid and bronchioloalveolar lung carcinomas, 2 renal cell carcinomas, 2 colorectal carcinomas, and a Conn and Cushing adenoma. Probes were generated by using nonamplified and amplified genomic DNA with DOP-PCR and DOP-PCR combined with QRT-PCR. To demonstrate the reliability of the QRT-PCR based amplification protocol, altogether 152 relative copy number changes of 44 regions were determined. There was 85.6% concordance in copy number alterations between the QRT-PCR protocol and the nonamplified samples, whereas this value was only 63.8% for the traditional DOP-PCR. Our results demonstrate that our protocol preserves the original copy number of different chromosomal regions in amplified genomic DNA than standard DOP-PCR techniques more accurately.     

On the pharmacological phenocopying of memory mutations inDrosophila: Alkylxanthines accelerate memory decay

Theophylline and 3-isobutyl-1-methylxanthine, two cyclic nucleotide phosphodiesterase inhibitors, when fed to wild-typeDrosophila adults, cause the rapid decay of learning index after training in a shock-odor learning paradigm. The drugs practically do not affect the olfactory acuity of flies, hence they influence the learning/memory process itself. The time courses of memory decay resemble those of the memory mutantsrutabaga andamnesiac and, to a lesser extent,dunce ² anddunce ^M11. Theophylline further deteriorates the learning performance ofdunce ^M11. Biochemical characterization of the inhibition of the two major phosphodiesterase isoenzymes inDrosophila by theophylline predicts only a slight inhibition of these enzymesin vivo, in accordance with the unchanged level of cAMP in wild-type fly heads during drug feeding. 8-Phenyltheophylline, an adenosine receptor antagonist in mammals, slightly retards memory decay in the wild-type. It is suggested that alkylxanthines induce memory decay inDrosophila by interfering with cAMP dynamics at more than one point of its metabolism.This work was supported by Grants OTKA and OKKFT Tt to P.F.     

Negative regulatory effect of histamine in DNFB-induced contact hypersensitivity

Histamine plays an important role in the regulation of various immunological functions. To evaluate the role of histamine in contact hypersensitivity, contact dermatitis was induced with dinitrofluorobenzene (DNFB) in histidine decarboxylase knockout (HDC-/-) histamine-deficient and wild-type mice. The DNFB-induced increase of the ear thickness was significantly higher in HDC-/- mice than in wild-type mice. Using flow cytometry, significantly lower percentages of CD4+ Th and CD8+ Tc cells, and significantly higher percentages of CD45R+ B cells were observed in the regional lymph nodes in HDC-/- mice than in wild-type mice. In the ear specimens of both groups, the majority of the infiltrating cells were neutrophils and macrophages at 24 and 48 h after challenge. Using immunohistochemistry, we observed significantly more CD45+ leukocytes in HDC-/- mice than in wild-type mice. The expression of Th1 (IL-2, IFN-gamma, TNF-alpha) and Th2 (IL-4) mRNAs was examined by quantitative real time RT-PCR in the ear samples. The levels of Th1 cytokine mRNAs both at 24 and 48 h after challenge and IL-4 mRNA at 48 h showed a significantly higher increase in HDC-/- mice than in wild-type mice. These results suggest that histamine plays a negative immunoregulatory role in DNFB-induced contact hypersensitivity.     

Theory of mind and motion perception in schizophrenia

This study investigated the relationship between theory of mind (ToM) deficits and visual perception in patients with schizophrenia (N=52; 17 remitted and unmedicated) compared with healthy controls (N=30). ToM was assessed with the Eyes Test, which asked participants to choose which of 4 words best described the mental state of a person whose eyes were depicted in a photograph. Visual perception was evaluated with form and motion coherence threshold measurements. Results revealed that patients with schizophrenia (both remitted and nonremitted) showed deficits on the Eyes Test and the motion coherence task. ToM dysfunctions were associated with higher motion coherence thresholds and more severe negative symptoms. This suggests that ToM deficits are related to motion perception dysfunctions, which indicates a possible role of motion-sensitive areas in the pathophysiology of schizophrenia.     

Sensitivity Analysis of Respiratory Parameter Estimates in the Constant-Phase Model

The constant-phase model is increasingly used to fit low-frequency respiratory input impedance (Zrs), highlighting the need for a better understanding of the use of the model. Of particular interest is the extent to which Zrs would be affected by changes in parameters of the model, and conversely, how reliable are parameters estimated from model fits to the measured Zrs. We performed sensitivity analysis on respiratory data from 6 adult mice, at functional residual capacity (FRC), total lung capacity (TLC), and during bronchoconstriction, obtained using a 1-25 Hz oscillatory signal. The partial derivatives of Zrs with respect to each parameter were first examined. The limits of the 95% confidence intervals, 2-dimensional pairwise and p-dimensional joint confidence regions were then calculated. It was found that airway resistance was better estimated at FRC, as determined by the confidence region limits, whereas tissue damping and elastance were better estimated at TLC. Airway inertance was poorly estimated at this frequency range, as expected. During methacholine-evoked pulmonary constriction, there was an increase in the uncertainty of airway resistance and tissue damping, but this can be compensated for by using the relative (weighted residuals) in preference over the absolute (unweighted residuals) fitting criterion. These results are consistent with experimental observation and physiological understanding.     

Laparoscopic ileo-cecal resection: case report and review of current issues in laparoscopic colorectal surgery

A successful case of laparoscopic ileocecal resection is presented. The laparoscopic surgical techniques are described. Advantages, disadvantages and controversial issues surrounding laparoscopic or laparoscopically assisted colorectal surgery are discussed. Reviewing the current literature and the authors' collective clinical experience, the conclusion is that laparoscopic or laparoscopically assisted procedures are clear indications for benign colorectal diseases. The authors believe that these approaches can be employed as palliative measures in malignant diseases or as curative procedures in prospective randomised trials.     

Conception without the development of a human being

Conception sometimes results in products that are not capable of developing into an embryo and fetus. This group, designated with the term gestational trophoblastic neoplasia, comprises the benign hydatidiform mole, the invasive mole (chorioadenoma destruens) and the frankly malignant variety, choriocarcinoma. Another type of atypical oocyte activation occurs in parthenogenesis. In the human, two types of tumors, dermoid cysts and teratomas, are believed to result from this process. We elucidate the generation of these abnormal growths and provide explanations as to why they cannot be regarded as human individuals or human beings. We argue that it is not the number of chromosomes that is required for a given form of human life to become a human being but rather the biparental origin of the chromosome set.     

Risk of recurrence in major central nervous system malformations between 1976 and 2005

OBJECTIVE: The goal of the current publication is to review isolated central nervous system malformations (CSMs) using a database in excess of 75 000 cases, with special regard to the risk of recurrence of these malformations alone or in combination. METHODS: In the period between 1 January 1976 and 31 December 2005, among the 75 320 documented cases, consultations were requested due to earlier isolated CSMs in the patients' histories in 3030 cases (4.2%). Processing the data we only considered disorders of genetic origin, and that was why we excluded the cases due to intrauterine infection. Monogenically inherited malformations were also excluded from the analysis. The diagnosis of the malformations was based on the prenatal diagnosis of ultrasonography as well as the findings of the foetopathological examination. RESULTS: In 65% of the cases, the couples sought counselling because of malformation in a previous pregnancy. In these cases, the risk of recurrence was thought to be 5.2%, while in the case of two affected children this figure stood at 21.9%. Analysing the values for the risk of recurrence in 5-year periods, neural tube defects (NTDs) (particularly anencephaly and spina bifida) showed a detectable decrease, which could be attributed to a growing use of folic acid supplementation around the time of conception and during pregnancy. CONCLUSION: There is a clear decrease of risk of recurrence of NTDs, while in the case of the other CSMs in this study, there is no noteworthy chronological change in their risk of recurrence.     

Experiences in the perinatal management of congenital diaphragmatic hernia during the last 15 years in a tertiary referral institute

OBJECTIVE: Review of cases of perinatally diagnosed congenital diaphragmatic hernias with special regard to time of diagnosis, organs herniated into the thorax, associated malformations, and outcomes. METHODS: We analyzed the data of 106 cases between July 1, 1990, and June 30, 2005. The observation period was analyzed in two parts. RESULTS: Prenatal ultrasound was performed in 89.6% (95/106) of the cases. 51.7% (46/89) of the pre- and postnatally verified congenital diaphragmatic hernia cases were diagnosed before the 24th week of gestation. 11% (11/100) of the hernias were identified postnatally. 71% (71/100) of the cases were associated with other malformations. The hernia was on the left side in 86% (86/100) and bilateral in 5% (5/100) of the cases. Between 1990 and 1997, the proportion of right-sided hernias was 4.3% (2/46), while during the second period it was 13% (7/54). 27.5% (14/51) of the newborns survived the perinatal period. The survival rate of the newborns delivered by caesarean section was three times higher than that of infants delivered vaginally. In the latter group, the rate of premature deliveries was considerably higher. The pregnancy was terminated in 45% (40/89) of the cases. 15% (6/40) of the terminated cases were isolated. Intrauterine or intrapartum deaths occurred in 5.6% (5/89) of the prenatally diagnosed cases. Simultaneous thoracic herniation of liver, spleen, or stomach decreased the survival rate. In these cases, the discharge rate was between 6 and 22%. CONCLUSIONS: According to the analysis of 15-year data, the herniated organs, multiple malformations, as well as the mode of delivery influence the survival in a large number of cases. Early diagnosis predicts a large hernia, a higher malformation rate, and a less favourable prognosis.