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Refined NMR structure of alpha-sarcin by 15N-1H residual dipolar couplings.

(15)N-(1)H residual dipolar couplings (RDC) have been used as additional restraints to refine the solution structure of the ribotoxin alpha-sarcin. The RDC values were obtained by partial alignment of alpha-sarcin in the binary mixture of n-dodecyl hexa(ethylene glycol)/hexanol. A total of 131 RDCs were measured and 106 were introduced in the final steps of the calculation protocol following the main calculation based on nuclear Overhauser enhancements and torsion angle restraints. A homogeneous family of 81 conformers was obtained. The resulting average pairwise root-mean-square deviation corresponding to the superposition of the 20 best structures is 0.69+/-0.12 A for the backbone and 1.29+/-0.14 A for all heavy atoms. The new structural features derived from the refined structure, compared with the non-refined structure of alpha-sarcin, consist of new hydrogen bonds and a better definition of the backbone conformation. In particular, the loop segment spanning Gly 60 to Lys 70 shows a single conformation, corresponding to the most populated family of conformers observed in the unrefined structure. The information derived from the analysis of the refined structure and the comparison with the homologous protein restrictocin could help in establishing further structure-function relationships concerning alpha-sarcin which can be reasonably extrapolated to other members of the ribotoxin family.

Algorithms↗

Mutation analysis of the HFE gene associated with hereditary hemochromatosis in a Venezuelan sample.

The frequency of the C282Y, H63D and S65C alleles of the HFE gene was determined in a sample of the Venezuelan population. Two new sets of primers were tested for amplifying the regions mapping these mutations, and genotyping was performed by restriction fragment length polymorphism (RFLP). DNA sequencing was used to validate the RFLP analysis. Serum ferritin levels were also determined. Two hundred and fourteen individuals were tested, extracting DNA from whole blood cells (n=177) or from serum (n=37). The frequency of heterozygous subjects was 3.7, 18.2 and 1.7% for the C282Y, H63D and S65C mutations, respectively, and the allele frequencies were 0.019+/-0.01 for C282Y, 0.119+/-0.016 for H63D and 0.009+/-0.005 for S65C. The results suggest that the admixture of native populations with subjects of South European origin might have had an important role in the diffusion of HFE alleles in Venezuela. C282Y homozygous subjects were not found in this study. No HFE genotype studied here was associated with a significant elevation of serum ferritin concentrations, except for C282Y/H63D compound heterozygote found in one asymptomatic male. This finding supports the theory that the H63D mutation could be involved in alterations of iron parameters when inherited together with C282Y. Our results indicate that C282Y homozygotes will be rarely detected. Performance of HFE mutation analysis in individuals with high iron determinations would be recommended.

Adolescent↗

Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer.

The high-level pigment-producing Monascus strain IBCC1 was characterized by random amplification of polymorphic DNA as M. purpureus. This technique allowed us to distinguish between M. purpureus and M. ruber strains. Transformation of Monascus species has not been previously reported. Protoplast formation and regeneration from M. purpureus IBCC1 was optimized by modification of growth media, lytic enzyme mixture, osmotic stabilizer and regeneration media. Of the Monascus transformants, 60% were found to be mitotically stable and retained the plasmid inserted in the chromosome after repeated sporulation cycles. Additionally, an Agrobacterium-mediated DNA transfer system was developed. The transformants obtained by Agrobacterium-mediated DNA transfer remained fully stable (98%) after four sporulation rounds and showed bands of hybridization corresponding to integration of the plasmid in different sites of the genome. The green fluorescent protein marker was well expressed in the M. purpureus transformants. The development of transformation systems is a basic tool for advanced genetic manipulation of the natural pigment producers, M. purpureus and M. ruber.

DNA, Bacterial↗

Unknown internal carotid artery atherosclerotic stenoses detected with biphasic multidetector computed tomography for head and neck cancer.

The possibility of detecting unknown internal carotid artery stenoses in patients undergoing biphasic multidetector computed tomography (MDCT) for current or previous head and neck cancer was investigated in 52 patients who underwent four-row MDCT (4 mm x 1.25 mm collimation, pitch 3, kVp 120, mAs 140; 90-100 ml of non-ionic contrast agent at 2 ml/s, early and late phases). Vessel stenoses were classed as severe (70-99%), moderate (30-69%), or mild (<30%). Internal carotid arteries (ICAs) with vessel stenosis was found in 37 patients (age 67.0+/-9.8 years, median 69 years) and 67 ICAs. Four patients (age 65.5+/-9.7 years) had severe stenosis, all of them associated with contralateral moderate stenosis. Seventeen patients (age 70.35+/-9.78 years) had moderate stenosis, bilateral (n=7), associated with contralateral mild stenosis (n=8), unilateral (n=2). Sixteen patients (age 64.8+/-9.8 years) had mild stenosis, bilateral (n=10) or unilateral (n=6). Of the four patients with unknown MDCT-detected severe stenosis, three had a change of therapy: one endarterectomy before tumor surgery, one combined vascular and tumor surgery, and one patient with a previously treated hypopharyngeal cancer had carotid stenting. In conclusion, of 52 patients, four (7.7 %) had unknown severe ICA stenoses, three of them with relevant impact on therapy. ICAs should be carefully evaluated for atherosclerotic disease using biphasic MDCT for head and neck cancer.

Adult↗

[Primary chylopericardium--stepwise diagnosis and therapy of a differential diagnostically important illness].

Primary chylopericardium is a rare disease with a highly variable clinical course. We report on a 24-year old female with chylopericardium detected during a pulmonary infection. Despite successful treatment of the infectious disease, the chylopericardium persisted and led to cardiac tamponade. From this case, as well as from the literature, it is intriguing to postulate an inflammatory injury of preexisting anomalous lymphatic vessels leading to onset or aggravation of primary chylopericardium. The clinical hallmark of chylopericardium is a milky white, but odorless pericardial fluid at pericardiocentesis. For cases where conservative treatment and pericardiocentesis fail, we newly introduced the method of pericardio-peritoneal shunting by a pericardial window. With postoperative reaccumulation of pericardial fluid, total parenteral nutrition followed by medium chain triglyceride diet was successfully reinitiated. This combined surgical and conservative approach was performed for the first time and may have helped to avoid the more aggressive treatment of thoracic duct ligation and resection. During 2 years of follow-up the patient was asymptomatic and had no recurrence of pericardial effusion.

Adult↗

[Bradycardia despite hyperthyroidism].

Hyperthyroidism is usually associated with tachycardia, hypothyroidism with bradycardia. After observing clinically inapparent hyperthyroidism in patients requiring pacemaker implantation, we studied the occurrence of hyperthyroidism in patients receiving a first permanent pacemaker. Of 237 patients (age 71.4 +/- 8.9 years; 54.9% females), 16 (6.75%) had subclinical (TSH < 0.1 mE/l and fT3 < or = 9.0 pmol/l) and 4 (1.69%) overt hyperthyroidism (TSH < 0.1 mE/l and fT3 > 9.0 pmol/l). Prevalence of hyperthyroidism was similar to that in the general population. Compared to euthyroid patients, in the patients with subclinical or overt hyperthyroidism there were significantly more females (n = 16) than males (n = 4; p = 0.018). Hyperthyroid patients were older (75.0 +/- 9.6 vs. 70.7 +/- 8.9 years; p = 0.015). At follow-up, all patients had a relevant proportion of pacemacer-induced beats. Clinical signs of hyperthyroidism or cardiac symptoms were not different between groups. In conclusion, bradycardia does not exclude the presence of hyperthyroidism. Temporary pacing is recommended in thyreotoxicosis with bradycardia. In contrast, primary implantation of a permanent pacemaker appears to be adequate in patients with bradycardia, cardiovascular disease and an additional diagnosis of hyperthyroidism.

Age Factors↗

Pathocycles: Ustilago maydis as a model to study the relationships between cell cycle and virulence in pathogenic fungi.

Activation of virulence in pathogenic fungi often involves differentiation processes that need the reset of the cell cycle and induction of a new morphogenetic program. Therefore, the fungal capability to modify its cell cycle constitutes an important determinant in carrying out a successful infection. The dimorphic fungus Ustilago maydis is the causative agent of corn smut disease and has lately become a highly attractive model in addressing fundamental questions about development in pathogenic fungi. The different morphological and genetic changes of U. maydis cells during the pathogenic process advocate an accurate control of the cell cycle in these transitions. This is why this model pathogen deserves attention as a powerful tool in analyzing the relationships between cell cycle, morphogenesis, and pathogenicity. The aim of this review is to summarize recent advances in the unveiling of cell cycle regulation in U. maydis. We also discuss the connection between cell cycle and virulence and how cell cycle control is an important downstream target in the fungus-plant interaction.

Cell Cycle↗

Bile duct injury during laparoscopic cholecystectomy: myth or reality of the learning curve?

BACKGROUND: Bile duct injury (BDI) is a severe complication of laparoscopic cholecystectomy (LC) that is probably related to the effects of the learning curve. The aim of this prospective, institutional, and longitudinal study is to compare the incidence of BDI during LC in relation to the progressive experience of surgeons. METHODS: A total of 784 LC were examined during a 6-year period. They were divided into the following three consecutive groups: group A (1993-94), group B (1995-96), and group C (1997-98). Incidence and type of BDI, experience of the surgeon, intra- or postoperative diagnosis, treatment performed to repair the injury, and early and late morbidity and mortality were evaluated. RESULTS: The overall incidence of BDI was 1.4%. There were three cases of transection of the common bile duct, four partial lesions of the bile duct, and four cystic leakages. The number of BDI was maintained over the three different time periods; there were no statistical differences in the proportion of injuries among groups. Most BDI were incurred by experienced surgeons. In all, 36% of BDI were recognized intraoperatively. Hepaticojejunostomy, direct suture over a T-tube, and closure of the cystic stump were done to repair BDI. There was no additional morbidity or mortality in the patients with BDI. CONCLUSIONS: No relation was found between the experience of the surgeon and the number of BDI over the different periods of time. Therefore, BDI during LC cannot be attributed solely to the learning curve.

Adult↗

Distal RTA with nerve deafness: clinical spectrum and mutational analysis in five children.

Distal renal tubular acidosis (RTA) with nerve deafness is caused by mutations in the ATP6V1B1 gene causing defective function of the H+ -ATPase proton pump. We report five acidotic children (four males) from four unrelated families: blood pH 7.21-7.33, serum bicarbonate 10.8-14.7 mEq/l, minimum urinary pH 6.5-7.1 and fractional excretion of bicarbonate in the presence of normal bicarbonatemia 1.1-5.7%. Growth retardation and nephrocalcinosis, but not hypercalciuria, were common presenting manifestations. Hearing was normally preserved in one of the patients whose sister was severely deaf. One child was homozygous for a known mutation in exon 1: C>T (R31X). Three children were homozygous for a splicing mutation, intron 6 + 1G>A. The other patient was a compound heterozygote, having this mutation and a previously unreported mutation in exon 10: G>A (E330K). Our report shows that hearing loss is not always present in the syndrome of distal renal tubular acidosis with nerve deafness and the absence of hypercalciuria at diagnosis and describes a new mutation responsible for the disease in the ATP6V1B1 gene.

Acidosis, Renal Tubular↗

[The phantom and stump phenomena interview (PSPI)].

RESEARCH QUESTION: A structured German-language interview was developed for the assessment of painful and non-painful phantom and stump phenomena after amputation. The aim was a thorough assessment of the quality, quantity and time course of these phenomena, which is of scientific as well as therapeutic relevance. METHODS: Each phenomenon was assessed using visual analogue scales as well as qualitative descriptors adapted from the McGill Pain Inventory and from literature reports. The factor structure and psychometric properties of the interview were evaluated in a sample of 139 upper and lower limb amputees. Test-retest coefficients were obtained in a subset of 20 amputees. RESULTS: As expected, all pain-related scales showed a two-dimensional internal structure with the factors "affective pain" and "sensory pain". For the non-painful phantom sensations, three factors "general/kinesthetic phantom sensations", "phantom movements" and "paresthesias" were obtained, while for non-painful stump sensations only one general factor emerged. The internal consistency was high with respect to the pain-related scales and was still satisfying for the scales that cover nonpainful phantom and stump phenomena. All scales have sufficient validity. Test-retest coefficients suggest a satisfactory stability of all scales that assess present phenomena, while the stability of the retrospective scales is markedly lower and in some cases insufficient. CONCLUSIONS: The phantom and stump phenomena interview is a highly reliable and valid instrument to assess present perceptual phenomena after amputation. Only the included retrospective scales apparently show low stability scores over time. This raises the more general question of the validity of retrospective pain reports.

Adult↗

[Cortical reorganization and pain. Empirical findings and therapeutic implication using the example of phantom pain].

The neuroscientific research of the past years has shown that extensive plastic change occurs in the adult human brain. The functional reorganization of the somatosensory and motor maps related to phantom limb pain is described. Subsequent to deafferentation amputees with phantom limb pain show a shift of neighbouring representation zones into the deafferented zone of the somatosensory and motor cortex that can be detected with noninvasive neuroimaging methods. This reorganization of the sensory and motor maps is not found in painfree amputees and persons with congenital loss of a limb. We discuss a model of the development of phantom limb pain that incorporates both peripheral and central factors and assigns an important role to chronic pain before the amputation. The modulation of plasticity and phantom limb pain by anesthesiological interventions is described and the results of preemptive analgesia for the prevention of phantom limb pain are discussed. Opioid treatment and behaviorally relevant stimulation are effective means to eliminate phantom limb pain and cortical somatosensory pain memories.

Humans↗

Standardized stimuli to assess drug craving and drug memory in addicts.

Due to conditioning processes, originally neutral stimuli become drug-associated cues and can initiate drug craving. Standardized stimuli are required to assess stimulus-induced activation of drug memory and craving in brain imaging and neurophysiology studies. We developed substance-specific visual and olfactory stimuli for alcohol, tobacco, opiate and cannabis abuse and tested them in subjects with the respective addiction and in healthy volunteers. Stimulus-related drug craving differed significantly between the diagnostic groups and indicated that the stimuli are suitable for craving studies.

Adult↗

Alcohol-associated stimuli activate the ventral striatum in abstinent alcoholics.

Alcohol-associated cues may act as conditioned stimuli that activate the brain reward system and motivate alcohol intake in alcoholics. Alcohol-associated visual stimuli were presented during functional magnetic resonance imaging. An activation of the ventral putamen was observed in alcoholics but not in control subjects. Patients with a strong activation of the ventral putamen relapsed during the next three months. This observation supports the hypothesis that alcohol use affects areas involved in brain reward circuits and that their stimulus-induced activation may be associated with an increased risk for relapse.

Adult↗

In vitro and in vivo characterization of MPEP, an allosteric modulator of the metabotropic glutamate receptor subtype 5: review article.

There is a need to identify subtype-specific ligands for mGlu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. To date, most mGlu receptor antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular N-terminal domain. We have characterized novel subtype-selective mGlu(5) receptor antagonists which are structurally unrelated to competitive mGlu receptor ligands. Using a series of chimeric receptors and point mutations we demonstrate that these antagonists act as inverse agonists with a novel allosteric binding site in the seven-transmembrane domain. Recent studies in animal models implicate mGlu(5) receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety.

Animals↗

An expanded view of psychological aspects in head pain: the biopsychosocial model.

Traditionally, headache has been viewed from a limited perspective, both medically and psychologically. The authors propose that a more expanded view of headache that considers each perspective as important, as embodied in the biopsychosocial model, will greatly enhance understanding and be more useful in treatment planning. This model views pain as emanating from a complex interaction of biological, psychological and social variables. This paper describes the key behavioural, affective and cognitive influences and provides pertinent supporting examples from the literature.

Affect↗

Early prediction of anastomotic leak in colorectal cancer surgery by intramucosal pH.

PURPOSE: Adequate oxygenation is necessary for anastomotic healing, and ischemia has been found to be one of the most important factors in anastomotic leakage. This study was designed to assess the value of early postoperative intramucosal pH measurements for the prediction of anastomotic leakage in patients with colorectal anastomosis. METHODS: A prospective study of 90 patients with rectal or sigmoid cancer with primary anastomosis was conducted. In all patients intramucosal pH was determined by using tonometry at the anastomotic and gastric levels during the first 24 and 48 hours postoperatively. Seven other variables also were tested by univariate and multivariate analysis for any association with anastomotic leakage. RESULTS: The rate of clinical anastomotic leakage was 6.6 percent. Multivariate analysis showed that only the intramucosal pH at the anastomosis was an independent factor for the development of anastomotic leakage. The risk of leakage was 22 times higher in patients with an anastomotic intramucosal pH < 7.28 in the first 24 hours after surgery. CONCLUSIONS: Measurement of anastomotic intramucosal pH in the early postoperative period can more accurately predict the risk of anastomotic leakage and benefit those patients who would need additional measures to improve the viability of the anastomosis.

Adult↗

Effect of fish or soybean oil-rich diets on bradykinin, kallikrein, nitric oxide, leptin, corticosterone and macrophages in carrageenan stimulated rats.

We have previously demonstrated that both n-3 and n-6 polyunsaturated fatty acids (PUFA)-rich diets decrease the acute inflammatory response partially explained by the high corticosterone basal levels. The present study aimed to determine the effect of hyperlipidic diets (PUFA n-3 or n-6) on phagocytosis, hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) release by macrophages, bradykinin (BK) and NO release in the paw inflammatory perfusate and Kallikrein (KK), corticosterone and leptin blood levels. Hyperlipidic diets decreased H(2)O(2) release from macrophages stimulated by carrageenan or phorbol-miristate-acetate (PMA), NO release from macrophage stimulated by carrageenan, BK and NO release in the edema perfusate, KK plasma levels and the increase of serum leptin after carrageenan stimulus. These data show that both fish and soybean oil-rich diets promote similar alterations on inflammatory mediators of carrageenan edema and a causal association with the anti-inflammatory effect of these diets.

Animals↗