Accumulation in fetal muscle and localization to the neuromuscular junction of cAMP-dependent protein kinase A regulatory and catalytic subunits RI alpha and C alpha.

Using probes specific for cAMP-dependent protein kinase, we have analyzed by in situ hybridization the patterns of expression of regulatory and catalytic subunits in mouse embryos and in adult muscle. RI alpha transcripts are distributed in muscle fibers exactly as acetylcholinesterase, showing that this RNA is localized at the neuromuscular junction. The transcript levels increase upon denervation of the muscle, but the RNA remains localized, indicating a regulation pattern similar to that of the epsilon subunit of nicotinic acetylcholine receptor. RI alpha transcripts have accumulated in the muscle by day 12 of mouse embryogenesis, and localization is established by day 14, at about the time of formation of junctions. This localization is maintained throughout development and in the adult. Immunocytochemical analysis has demonstrated that RI alpha protein is also localized. In addition, RI alpha recruits C alpha protein to the junction, providing at this site the potential for local responsiveness to cAMP. PKA could be implicated in the establishment and/or maintenance of the unique pattern of gene expression occurring at the junction, or in the modulation of synaptic activity via protein phosphorylation. Embryonic skeletal muscle shows a high level of C alpha transcripts and protein throughout the fiber; the transcripts are already present by day 12 of embryogenesis, and their elevated level is maintained only through fetal life. In the adult, the C alpha hybridization signal of muscle is weak and homogeneous.

The expert witness in psychology and psychiatry.

The involvement of psychologists and psychiatrists within the legal arena continues to grow rapidly but remains highly controversial. Extensive research on clinical judgement provides a scientific basis for clarifying the growing disputes about the values of such professional activities. Studies show that professionals often fail to reach reliable or valid conclusions and that the accuracy of their judgements does not necessarily surpass that of laypersons, thus raising substantial doubt that psychologists or psychiatrists meet legal standards for expertise. Factors that underlie the research findings and implications for courtroom testimony are discussed.

Clinical versus actuarial judgment.

Professionals are frequently consulted to diagnose and predict human behavior; optimal treatment and planning often hinge on the consultant's judgmental accuracy. The consultant may rely on one of two contrasting approaches to decision-making--the clinical and actuarial methods. Research comparing these two approaches shows the actuarial method to be superior. Factors underlying the greater accuracy of actuarial methods, sources of resistance to the scientific findings, and the benefits of increased reliance on actuarial approaches are discussed.

Image modulation in corona discharge photography.

Photographic images obtained by the Kirlian technique are principally a record of corona activity during an exposure interval. Most of the variations in the images of the corona of a living subject who is in contact with the photographic film can be accounted for by the presence of moisture on or within the subject's surface. During exposure, moisture is transferred from the subject to the emulsion surface of the photographic film and causes an alteration of the electric charge pattern on the film, hence the electric field at the surface of the subject. As a result, large variations in the density of corona images, corona streamer trajectories, and image coloration can be brought about. The radial extent of corona images--that is, the range of corona streamers--is an inverse function of the resistance in the circuit formed by the high-voltage supply, the subject, and the film-electrode configuration. This is because the voltage at which corona is initiated is dependent on the rate of rise of the voltage impressed between the subject and the electrode, and the rate of rise is governed by the applied voltage waveform and the voltage drop across the resistance. The range of streamers is proportional to the corona onset voltage. However, we have not seen any influence of large changes in skin resistance on streamer range. Presumably, this is due to the shunting effect of skin capacitance. In general, the photographic response to moisture suggests that corona discharge photography may be useful in the detection and quantification of moisture in animate and inanimate specimens through the orderly modulation of the image due to various levels of moisture.

Polymorphisms in the methylenetetrahydrofolate reductase gene are determinant for vascular complications after liver transplantation.

OBJECTIVE: The aim of this study was to evaluate the role of the C677T-MTHFR (methylenetetrahydrofolate reductase)-polymorphism (CC, CT and TT) for vascular complications in liver transplant recipients. DESIGN: Retrospective study. SETTING: Hepatology-Transplantation-Unit, Johann Wolfgang Goethe-University, Frankfurt am Main. SUBJECTS: 48 liver transplant recipients were included, no dropouts. METHODS: MTHFR polymorphism was detected by PCR amplification and digestion with Hinfl restriction enzyme. Vascular complications after liver transplantation were detected from the patients' records. The total serum homocysteine (HCY) was analyzed with high-pressure liquid chromatography. RESULTS: In the wild-type group (CC), the HCY levels were slightly high (14.0+/-1 micro M). Among the patients with the CT polymorphism, the HCY values were elevated (22.5+/-3 micro M). In the homozygous TT group, there was a significant increase (31.2+/-6 micro M, P<0.01) of the HCY values. The percentage of vascular complications was higher in the heterozygous CT (47%) and homozygous TT (62.5%) group compared with wild-type CC (21%). Patients with a homozygous TT genotype of the MTHFR polymorphism with a vascular complication had a highly significant elevated HCY level compared to the other genotype groups, both with and without any vascular complications (P<0.001). Recipients with an elevated HCY and the TT polymorphism have a higher probability of developing a vascular complication after transplantation (odds ratio: 4.3 and 11.0; 95% confidence interval: 1.15, 12.25 and 1.41, 85.24). CONCLUSIONS: The C677T polymorphism in the MTHFR gene and subsequent elevation of the total serum HCY is significantly associated with an increased incidence of vascular complications in liver transplant recipients.

The folic acid metabolite L-5-methyltetrahydrofolate effectively reduces total serum homocysteine level in orthotopic liver transplant recipients: a double-blind placebo-controlled study.

OBJECTIVE: Hyperhomocysteinemia is a described risk factor of cardiovascular diseases. The aim of this study was the treatment of hyperhomocysteinemia in liver transplant recipients with L-5-methyltetrahydrofolate (L-5-MTHF; 1 mg) vs folic acid (1 mg) vs placebo in a double-blind placebo-controlled study and to compare the relative responsiveness of these patients to L-5-MTHF and folic acid. SUBJECTS/METHODS: Patients were recruited from Hepatology-Transplantation-Unit at Johann Wolfgang Goethe-University, Frankfurt. Sixty patients were included in this study and 12 patients dropped out for different reasons. The patients were treated over 8 weeks with supplemental L-5-MTHF or folic acid or placebo. Serum homocysteine (HCY) was analyzed with high-performance liquid chromatography (HPLC) beside routine lab tests. RESULTS: We observed only a significant decrease of total serum HCY in the L-5-MTHF group during the study period (at week 0: 15+/-7.7 microM; after 8 weeks treatment: 9.41+/-2.6 microM, P<0.001). There was no significant decrease of total serum HCY neither in the folic acid group nor in the placebo group. CONCLUSION: The effects of L-5-MTHF are significantly more potent than folic acid itself. Therefore, lowering serum HCY in liver transplant recipients is effective with L-5-MTHF.

Climate deteriorations and Neanderthal demise in interior Iberia.

Time and circumstances for the disappearance of Neanderthals and its relationship with the advent of Modern Humans are not yet sufficiently resolved, especially in case of the Iberian Peninsula. Reconstructing palaeoenvironmental conditions during the last glacial period is crucial to clarifying whether climate deteriorations or competition and contacts with Modern Humans played the pivotal role in driving Neanderthals to extinction. A high-resolution loess record from the Upper Tagus Basin in central Spain demonstrates that the Neanderthal abandonment of inner Iberian territories 42 kyr ago coincided with the evolvement of hostile environmental conditions, while archaeological evidence testifies that this desertion took place regardless of modern humans' activities. According to stratigraphic findings and stable isotope analyses, this period corresponded to the driest environmental conditions of the last glacial apart from an even drier period linked to Heinrich Stadial 3. Our results show that during Marine Isotope Stages (MIS) 4 and 2 climate deteriorations in interior Iberia temporally coincided with northern hemisphere cold periods (Heinrich stadials). Solely during the middle MIS 3, in a period surrounding 42 kyr ago, this relation seems not straightforward, which may demonstrate the complexity of terrestrial climate conditions during glacial periods.

Density-dependent regulation of cell growth by contactinhibin and the contactinhibin receptor.

BACKGROUND: The number of cells within mammalian tissues is maintained by growth-stimulating and growth-inhibiting mechanisms, with inhibitory signals being superimposed over growth stimuli. This is reflected, in the culture of normal adherent cells, by the phenomenon of density-dependent inhibition of growth: cells cease proliferation after becoming a confluent monolayer. We have shown previously that a plasma membrane glycoprotein, contactinhibin, is a major effector of negative growth regulation. Although transformed cells express contactinhibin in a functionally active form, they are not growth-inhibited, suggesting that the defects that lead to their aberrant growth are located 'downstream' of contactinhibin. RESULTS: Here, we provide evidence that a 92 kD plasma membrane protein, which we call CiR, binds specifically to contactinhibin and acts as a receptor mediating the contact-dependent inhibition of growth of cultured human fibroblasts. When polyclonal antibodies against CiR were introduced into cells using liposomes, confluent cells were released from density-dependent growth control. By contrast, cross-linking CiR that is localized to the plasma membrane, using anti-CiR antibodies, led to growth inhibition, suggesting that CiR is a signalling molecule and implicating CiR oligomerization in signal generation. This conclusion is supported by the finding that binding of contactinhibin by CiR is strongly dependent on the local concentration of both molecules and has a sharp threshold. When CiR was isolated by immuno-precipitation under conditions favouring phosphorylation, it was hyperphosphorylated on serine and threonine residues and had reduced contactinhibin-binding capacity; the binding capacity of CiR was restored after treatment with potato acid phosphatase. Fibroblasts transformed with simian virus 40 had reduced CiR expression, higher CiR phosphorylation levels, and a strongly reduced capacity of CiR to bind to contactinhibin. Phosphatase treatment of the CiR isolated from transformed cells only partially restored its contactinhibin-binding capacity. CONCLUSIONS: Homeostasis is the net result of a highly balanced network of growth-stimulating and growth-inhibitory signals. We have shown that density-dependent inhibition of growth in vitro is mediated by the interaction of contactinhibin with a 92 kD plasma membrane glycoprotein, CiR, the contactinhibin-binding capacity of which is regulated by phosphorylation.

Hepatocyte nuclear factor 1alpha gene inactivation impairs chromatin remodeling and demethylation of the phenylalanine hydroxylase gene.

Hepatocyte nuclear factor 1 alpha (HNF1alpha) is a homeoprotein that is expressed in the liver, kidney, pancreas, and digestive tract. Its inactivation in mouse resulted in decreased transcription of known target genes such as albumin and alpha1-antitrypsin. In contrast, the phenylalanine hydroxylase (PAH) gene was totally silent and unresponsive to normal inducers like glucocorticoids and cyclic AMP in the liver. DNase I and micrococcal nuclease digestion of liver nuclei showed that HNF1alpha inactivation had drastic effects on the chromatin structure of the PAH regulatory regions. Three DNase I-hypersensitive sites (HSSI, HSSII, and HSSIII), typical of the actively transcribed PAH gene, were undetectable in liver from HNF1alpha-deficient animals. Both HSSII and HSSIII elements harbor HNF1 sites, but only the latter has detectable enhancer activity in transient-transfection assays. In addition, the PAH promoter in livers of HNF1alpha-deficient animals was methylated. These results suggest that HNF1alpha could activate transcription through two mechanisms. One implies participation in the recruitment of the general transcription machinery to the promoter, and the second involves the remodeling of chromatin structure and demethylation that would allow transcription factors to interact with their cognate cis-acting elements.

The activity of the highly inducible mouse phenylalanine hydroxylase gene promoter is dependent upon a tissue-specific, hormone-inducible enhancer.

Expression of the phenylalanine hydroxylase gene in livers and kidneys of rodents is activated at birth and is induced by glucocorticoids and cyclic AMP in the liver. Regulatory elements in a 10-kb fragment upstream of the mouse gene have been characterized. The promoter lacks TAATA and CCAAT consensus sequences and shows only extremely weak activity in transitory expression assays with phenylalanine hydroxylase-producing hepatoma cells. No key elements for regulation of promoter activity are localized within 2 kb of upstream sequences. However, a liver-specific DNase I-hypersensitive site at kb -3.5 comprises a tissue-specific and hormone-inducible enhancer. This enhancer contains multiple protein binding sites, including sites for ubiquitous factors (NF1 and AP1), the glucocorticoid receptor, and the hepatocyte-enriched transcription factors hepatocyte nuclear factor 1 (HNF1) and C/EBP. Mutation revealed that the last two sites are critical not only for basal activity but also for obtaining a maximal hormone response. Efficient transcription from the highly inducible promoter shows absolute dependence upon the enhancer at kb - 3.5, which in turn requires HNF1 and C/EBP as well as hormones. The regulatory region of the mouse phenylalanine hydroxylase gene differs totally from that of humans, even though the genes of both species are expressed essentially in the liver. Furthermore, the phenylalanine hydroxylase gene of mice shows an expression pattern very similar to those of the rodent tyrosine aminotransferase and phosphoenolpyruvate carboxykinase genes, yet each shows a different organization of its regulatory region.

p16INK4 mediates contact-inhibition of growth.

Growth of non-transformed cells in vitro is regulated by density-dependent mechanisms via cell-cell contacts, leading to arrest in late G1-phase at confluency (contact-inhibition of growth). In the present study it is shown that this results from p16INK4-mediated dissociation of the complex cdk4-cyclin D1, which is responsible for the inactivation of the gate keeper of G1-S transition, the retinoblastoma protein pRb. As a consequence of the inactivation of cdk4, downstream the activation of cdk2 and hyperphosphorylation and thus inactivation of pRb was impaired. Direct evidence for the central role of p16INK4 in growth control comes from the observation that a competitive inhibitor of p16INK4 repressed contact inhibition of growth. These findings provide an explanation for the high incidence of mutation or loss of INK4 in human tumours.

Involvement of protein kinase Cdelta in contact-dependent inhibition of growth in human and murine fibroblasts.

There is evidence that protein kinase C delta (PKCdelta) is a tumor suppressor, although its physiological role has not been elucidated so far. Since important anti-proliferative signals are mediated by cell-cell contacts we studied whether PKCdelta is involved in contact-dependent inhibition of growth in human (FH109) and murine (NIH3T3) fibroblasts. Cell-cell contacts were imitated by the addition of glutardialdehyde-fixed cells to sparsely seeded fibroblasts. Downregulation of the PKC isoforms alpha, delta, epsilon, and mu after prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) resulted in a significant release from contact-inhibition in FH109 cells. Bryostatin 1 selectively prevented TPA-induced PKCdelta-downregulation and reversed TPA-induced release from contact-inhibition arguing for a role of PKCdelta in contact-inhibition. In accordance, the PKCdelta specific inhibitor Rottlerin (1 microM) totally abolished contact-inhibition. Interestingly, immunofluorescence revealed a rapid translocation of PKCdelta to the nucleus when cultures reached confluence with a peak in early-mid G1 phase. Nuclear translocation of PKCdelta in response to cell-cell contacts could also be demonstrated after subcellular fractionation by Western blotting and by measuring PKCdelta-activity after immunoprecipitation. Transient transfection of NIH3T3 cells with a dominant negative mutant of PKCdelta induced a transformed phenotype. We conclude that PKCdelta is involved in contact-dependent inhibition of growth.

RNA polymerase II of Drosophila. Relation of its 140,000 Mr subunit to the beta subunit of Escherichia coli RNA polymerase.

We have determined the nucleotide sequence of the gene coding for the 140,000 Mr subunit of the DNA-dependent RNA polymerase II from Drosophila melanogaster. This analysis revealed features that are typical for a household-function gene. The codon usage is relaxed and there is no apparent TATA box upstream from the transcription start site. A comparison of the deduced amino acid sequence with that of the Escherichia coli RNA polymerase beta subunit shows a total of nine regions of homology. These regions are also conserved in chloroplast DNA of tobacco. This supports the notion that the two large subunits of the eukaryotic RNA polymerases are the structural and functional equivalents of E. coli beta' and beta subunits.

13C-methacetin breath test as liver function test in patients with chronic hepatitis C virus infection.

BACKGROUND: The 13C-methacetin breath test enables the quantitative evaluation of the cytochrome P450-dependent liver function. AIM: To find out whether this breath test is sensitive in noncirrhotic patients also with chronic hepatitis C in early stages of fibrosis. METHODS: Sixty-one healthy controls and 81 patients with chronic hepatitis C underwent a 13C-methacetin breath test. In all patients, a liver biopsy was performed. The liver histology was classified according to the histology activity index-Knodell score. RESULTS: Delta over baseline values of the patients at 15 min significantly differed from controls (19.2 +/- 9.2 per thousand vs. 24.1 +/- 5.7 per thousand; P < 0.003). The cumulative recovery after 30 min in patients was 11.4 +/- 4.8% and in healthy controls 13.8 +/- 2.8% (P < 0.002). However, patients with early fibrosis (histology activity index IVB) did not differ in delta over baseline values of the patients at 15 min (23.2 +/- 7.9 per thousand vs. 22.6 +/- 7.2 per thousand; P = 0.61) or cumulative recovery (13.6 +/- 3.7% vs. 13.2 +/- 3.8%; P = 0.45) from patients with more advanced fibrosis (histology activity index IVC). Patients with clinically nonsymptomatic cirrhosis (histology activity index IVD; Child A) metabolized 13C-methacetin to a significantly lesser extent (delta over baseline values of the patients at 15 min: 8.3 +/- 4.9 per thousand; P < 0.005 and cumulative recovery after 30 min: 5.6 +/- 3.2%; P < 0.003). The 13C-methacetin breath test identified cirrhotic patients with 95.0% sensitivity and 96.7% specificity. CONCLUSION: The non-invasive 13C-methacetin breath test reliably distinguishes between early cirrhotic (Child A) and noncirrhotic patients, but fails to detect early stages of fibrosis in patients with chronic hepatitis C.

Does the liver ever age? Results of liver transplantation with donors above 80 years of age.

INTRODUCTION: Facing an increasing shortage of donor organs, donor criteria become more extended and so-called marginal organs are accepted for transplantation. For liver donation donor age above 70 years is accepted as a risk factor concerning primary dysfunction or nonfunction. Therefore, the aim of this study was to compare the early outcome of grafts older versus younger than 80 years of age. PATIENTS AND METHOD: Between August 2002 and February 2004, 40 adult liver transplants were performed using triple immunosuppression with tacrolimus, MMF, and low-dose corticosteroids. Recipients with HCC received low-dose rapamycin after postoperative day 14. The outcome of grafts from donors under 80 years of age (n=35) was compared with those from donors 80 years old or more (n=5). For statistical analysis Mann-Whitney-U-Test and Fisher's Exact Test were used with P < .05 considered statistically significant. RESULTS: The average donor age of our population was 54.4 +/- 17.3 years with five donors older than 80 years (80-83 years). These donors all had additional risk factors. The recipients of the latter grafts suffered from HCC and liver cirrhosis Child A (n=2) or from viral hepatitis (n=3). One recipient had advanced cirrhosis with severe complications. The outcomes of both groups were comparable concerning intraoperative and postoperative courses. All recipients of old liver grafts left the hospital with stable graft function. CONCLUSION: Liver grafts over 80 years can be transplanted with good results, especially if given to recipients with malignancy and otherwise stable liver function.

Conserved as well as divergent regulatory elements account for expression of the human and rodent phenylalanine hydroxylase genes.

We have uncovered a fundamental difference in the regulation of the rodent and the human phenylalanine hydroxylase (PAH) genes: expression of human PAH is independent of glucocorticoids and/or cAMP in contrast to the mouse gene which is not only highly inducible but dependent upon hormones for expression. Nevertheless, the two genes do exhibit similarities: DNaseI hypersensitive sites are identically located in the regulatory regions, and the sequences around these sites are partially conserved and associated with regulatory elements sharing similar function. In transient transfections, the human proximal promoter is tissue-specific and presents significant activity compared to the extremely low and ubiquitous activity of the mouse promoter. DNA fragments corresponding to the two upstream hypersensitive sites of both genes have enhancer activity that depends upon the liver-enriched transcription factor binding sites for hepatocyte nuclear factor (HNF) 1 and/or CCAAT/enhancer binding protein (C/EBP). While expression of the rodent gene relies upon two modules in the HSIII enhancer, one activated by HNF1 and C/EBP and the other required for the hormone response, the human equivalent has conserved only the liver-specific transcription factor binding module. Even though the more proximal enhancer is not necessary for full reporter gene activity in transient transfection assays in Pah-expressing hepatoma cells, this enhancer could be required in both species for activation during development.

The empiricist and his new clothes: DSM-III in perspective.

The authors examine and critique the methodological underpinnings and programmatic goals of DSM-III's underlying doctrine--strict empiricism. The methodological program of DSM-III emphasizes description and the reduction of inference and theory. Regardless of appearances to the contrary, these goals have not been approached, cannot be attained, and should not be pursued, at least in their extreme form. They are throwbacks to earlier and now revised views of science. These goals give the highest priority to things that either should not receive it or that are secondary outgrowths of other accomplishments, and they have the potential to discourage the conceptual and theoretical developments that are the prime movers of scientific progress, according to the authors.

Alternative 5'-exons of the mouse cAMP-dependent protein kinase subunit RIalpha gene are conserved and expressed in both a ubiquitous and tissue-restricted fashion.

The activity of cAMP-dependent protein kinase is controlled by its regulatory subunits. Mouse RIalpha regulatory subunit expression is initiated from five different non-coding 5'-regions (exons 1a, 1b, 1c, 1d and 1e). This organization appears to be conserved among species. All mouse tissues accumulate exon 1a and 1b transcripts and most contain more 1b than 1a, except brain, heart and oesophagus. Exon 1d and 1e transcripts are found in several tissues, while exon 1c is testis-specific. All five transcripts are in RIalpha-rich tissues: gonads and adrenal glands.

Moderately elevated blood lead levels: effects on neuropsychologic functioning in children.

Investigations of moderately elevated lead levels and children's cognitive functioning have yielded conflicting results, although studies showing no effects used measures of limited sensitivity and breadth. In this study, a comprehensive neuropsychologic battery was used to determine whether deficits would be revealed. An experimental group of 15 children with a past history of moderately elevated lead levels, but subsequently without increased lead levels for at least a year, were compared with a control group matched by residential area, socioeconomic status, parental IQ, age, and gender. The experimental groups' performance was lower on the battery overall and on measures of motor skill, memory, language, advanced spatial functions, and concentration. The results suggest that exposure to moderately elevated lead levels exerts significant and nontransient effects on cognitive functions.

Effects of the label "schizophrenia" on causal attributions of violence.

We investigated the relation between the label of "schizophrenia" and causal attributions of violence. Undergraduates read 1 of 10 scenarios in which two variables were manipulated: a psychiatric label and environmental stress. The scenario described an employee who acted violently toward his boss. Subjects made causal attributions for the employee's behavior by completing an adapted version of the Causal Dimension Scale II. Subjects also completed a questionnaire designed to explore several issues concerning the effects of the schizophrenia label on perceptions of behavior. Contrary to the primary hypothesis, the schizophrenia label did not lead subjects to make significantly more personality causal attributions for violent behavior. With increasing environmental stress, subjects did make significantly fewer personality attributions. A follow-up study using practicing clinicians as subjects yielded similar findings. The results of these studies are discussed in light of perceived stereotypes of persons with schizophrenia and conceptual issues in attribution research.