Two new mutations and three novel polymorphisms in the<Emphasis Type="Italic"> RB1</Emphasis> gene in Ecuadorian patients

RB1 is the gene responsible for retinoblastoma, the most common malignant intraocular tumor of infancy and early childhood. There are no reports about this gene in Ecuadorian populations, and only a few studies have been published in Latin America about this subject. There is a spectrum of more than 370 mutations described in the RB1 gene mutation database (http://www.d-lohmann.de/Rb/mutations.html), and alterations have been found in 25 of the 27 exons. During the exon-by-exon analysis of 31 tumor and blood samples from Ecuadorian patients, we found two new mutations and three novel polymorphisms. One of the polymorphisms is located in intron 26 where no alterations of the gene have been described previously. The polymorphisms were found in all of the patients tumor samples, but not in normal population, suggesting there might be a relationship between these polymorphisms and the development of retinoblastoma in the Ecuadorian population.The nucleotide sequence data reported are available in the GenBank database under the accession numbers: AY243567, AY260472, AY260473, AY273783     

Altered gating of HERG potassium channels by cobalt and lanthanum

Activation of the rapid, delayed rectifier K current (IKr) is important for normal repolarization of cardiac action potentials, especially in mammalian ventricular muscle. The study of this current has been greatly aided by the discovery that the human ether-a-go-go-related gene (HERG) encodes the pore-forming alpha subunits of these channels. As for other voltage-activated K+ channels, divalent and trivalent cations affect the gating of HERG channels by screening negative membrane surface charges or by direct interaction with the channel gating mechanism. Previous studies have reported that IKr of myocytes, and HERG channels heterologously expressed in Xenopus oocytes, are reduced by external Co2+ and La3+. We have reinvestigated the "blocking" effect of Co2+ and La3+ on HERG channels expressed in Xenopus oocytes. At concentrations previously reported to block IKr or HERG current (IHERG), Co2+ (10 mM) and La3+ (10 microM) had only small effects on the magnitude of fully activated IHERG. The apparent block results from altered kinetics and voltage dependence of gating, similar to the effects of Ca2+ on HERG channels. Under control conditions, the half-points for voltage-dependent activation and inactivation of HERG were -35+/-2.1 and -76.3+/-1.7 mV, respectively. Co2+ and La3+ accelerated the rate of deactivation, decreased the rate of current activation, and shifted the half-point of the HERG channel activation curve by +53 and +65 mV, respectively. Co2+ shifted the voltage dependence of inactivation by + 14 mV, whereas La3+ had no effect. Co2+ also slowed the onset of IHERG inactivation and accelerated the rate of recovery from inactivation. These results indicate that reduction of IHERG by Co2+ (10 mM) and La3+ (10 microM) during depolarizing pulses is caused by a positive shift in the voltage dependence of activation, and does not result from pore block.     

Recognition surfaces of MHC class I

Summary: Recent crystallographic results have provided dose to atomic resolution views of the recognition events mediated by MHC class I molecules. The specificity-conferring interaction of MHC class I/peptide with a T-cell antigen receptor (TCR) appears dependent on certain key interactions with the MHC scaffold. These interactions, in particular those of the TCR Va domain, define a standard orientation for TCR binding. Previous studies on biologically significant variations in the TCR recognition surface presented by a series of MHC/variant peptide complexes can be reassessed in the light of this TCR-building mode. The interaction of CDS with MHC class I resembles that between antibody and antigen in the use of loops from the CD8 structure. The interaction is of very low affinity and buries equivalent surface area to that between the TCR and MHC class I but while the TCR/MHC interface shows poor surface shape complementarity the match in the conservative interaction between MHC and CDS is precise.     

Laboratory detection of Haemophilus influenzae with decreased susceptibility to nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin due to GyrA and ParC mutations

The detection of clinical isolates with decreased fluoroquinolone susceptibilities and a resistance mechanism is of epidemiological and clinical interest. We studied the susceptibilities of 62 clinical isolates and 2 American Type Culture Collection reference strains of Haemophilus influenzae to ciprofloxacin, levofloxacin, moxifloxacin, and nalidixic acid by the microdilution and disk diffusion methods. The ciprofloxacin MICs for 34 of the isolates were >/=0.12 micro g/ml (range, 0.12 to 32 micro g/ml), and the ciprofloxacin MICs for 28 matched control isolates were /=0.5 micro g/ml and the vast majority of those for which nalidixic acid MICs were >/=32 micro g/ml exhibited amino acid changes in GyrA and ParC. Nalidixic acid and the other three fluoroquinolones studied could be used to screen H. influenzae isolates for the detection of decreased susceptibilities to quinolones due to the acquisition of two amino acid changes in the QRDRs of GyrA and ParC (sensitivity, >95%; specificity, >80%).     

Specific IgE determination to Ani s 1, a major allergen from Anisakis simplex, is a useful tool for diagnosis.

BACKGROUND: The most serious limitation of the serodiagnosis of parasitoses is the occurrence of cross-reactions. OBJECTIVE: The possible use of Anisakis simplex major allergen Ani s 1 for diagnosis. PATIENTS AND METHODS: Forty-nine non-fish-allergic patients with Anisakis simplex hypersensitivity, 21 patients without allergic episodes suffering intestinal anisakiasis with obstruction of the intestinal lumen, and 10 unrelated sera as a control were included in this study to determine specific immunoglobulin (Ig)E and IgG to Anisakis simplex major allergen Ani s 1 by immunoblotting. RESULTS: Eighty-six percent of patients with Anisakis simplex hypersensitivity showed specific IgE directed to Ani s 1. Identical result was obtained for IgG detection in this group. Among patients with intestinal anisakiasis, 86% showed specific IgE, but only 29% had specific IgG (P < 0.001, two-tailed Fisher exact test). One of the 10 control subjects was positive both for IgE and IgG (P < 0.001). CONCLUSIONS: Determination of specific IgE directed to Anisakis simplex major allergen Ani s 1 is a useful tool for the diagnosis of hypersensitivity and intestinal anisakiasis. Further, measurement of specific IgG directed to Anisakis simplex major allergen Ani s 1 is only valid for Anisakis simplex allergy.     

Different applications of polymerases with and without proofreading activity in single-nucleotide polymorphism analysis

With the completion of the human genome project, single-nucleotide polymorphisms (SNPs) have become the focus of intense study in biomedical research. Polymerase-mediated primer extension has been employed in a variety of SNP assays. However, these SNP assays using polymerase without proofreading function are compromised by their low reliability. Using a newly developed short amplicon harboring restriction enzyme site, EcoR-I, we were able to compare the single-base discrimination abilities of polymerases with and without proofreading function in primer extension in a broad range of annealing temperatures. Thermodynamic analysis demonstrated a striking single-nucleotide discrimination ability of polymerases with proofreading function. Using unmodified 3'-end allele-specific primers, only template-dependent products were generated by polymerase with proofreading activity. This powerful single-base discrimination ability of exo(+) polymerases was further evaluated in primer extension using three types of 3' terminally modified allele-specific primers. As compared with the poor fidelity in primer extension of polymerases lacking 3' exonuclease activity, this study provides convincing evidence that the use of proofreading polymerases in combination with 3'-end modified allele-specific primers can be a powerful new strategy for the development of SNP assays.     

Ultrastructural identification of the splenic follicular dendritic cells in the chicken

Background: There is a need to identify the follicular dendritic cells (FDC) of the chicken spleen at the ultrastructural level during a secondary immune response. Methods: The cells were identified after intravenous priming BSA and boosting with biotinylated BSA conjugated to colloidal gold particles. Monoclonal antibodies raised specifically either to chicken IgG or IgM were used to characterize these immune complex-trapping cells. Results: The FDC had an irregular morphology which varied through time, supporting the existence of two types of FDC in the chicken spleen, one showing filiform cell processes, the other provided with beaded dendrites. When the filiform dendrites were observed, the FDC bound the antigen on their surfaces. These dendrites showed an intrincate convoluted configuration, forming tightly wrapped networks near the cell body. The networks had the same features as those described in mammals as antigen retaining reticulum (ARR). In chickens, the ARR, which represents sites of antigen localization on FDC, reached maximum development on day 5 after the second injection of BSA and had disappeared by day 8. At this time FDC had beaded dendrites. Conclusions: Antigen is retained on FDC in the chicken spleen for long periods of time. © 1995 Wiley-Liss, Inc.     

Interaction between α- and β-adrenoceptors in rat pineal adenosine cyclic 3′,5′-monophosphate phosphodiesterase activation

Summary The adrenergic regulation of the low-Km pineal cAMP phosphodiesterase (PDE) activity was studied in adult female rats. PDE activity showed a transient enhancement (up to 42%) during the process of degeneration of pineal sympathetic nerve terminals that followed superior cervical ganglionectomy (SCGx), thus confirming the neural modulation of the enzyme. Treatment with isoproterenol (0.3–5.0 mg/Kg) increased significantly PDE activity within 2 hours. Phenylephrine induced a significant increase of pineal PDE only at a 10 mg/Kg dose, while at a lower dose (1 mg/Kg) it potenciated the stimulatory effect of isoproterenol. Treatment of pineal organ cultures with 100 M propranolol inhibited norepinephrine (NE)-induced PDE activity while 100 M phentolamine had no significant effect. Propranolol at doses unable to alter thein vitro NE-induced stimulation of pineal PDE activity (1 M), antagonized such NE effect when used in combination with 1 uM phentolamine. At equimolecular concentrations (1 M) the mixed --adrenergic agonist NE was more effective than the -adrenergic agonist isoproterenol to increase pineal PDEin vitro. These resultsSupported by grant no. 6638, Consejo Nacional de Investigaciones Cientificas y Técnicas de la RepÚblica Argentina (CONICET), Argentina.Established Investigator, CONICET.     

Immediate repair of urethroperineal fistulas after abdominoperineal amputation by means of a cutaneo-muscular flap of the gluteus maximus. Apropos of 2 successfully treated cases

Although a rare complication, the development of a urethro-perineal urinary fistula immediately after abdomino-perineal amputation is a difficult problem to solve. The fragility of the membranous urethra immediately in contact with the exenterated pelvic cavity which takes several weeks to fill in makes any attempt at isolated direct suture very hazardous. Secondary repair is also frequently difficult and the "functional prognosis is far from being always favourable". The major handicap resulting from the perpetuation of this type of fistula has led us to propose an attempt at immediate repair as soon as it is diagnosed with filling of the pelvi-perineal cavity by a cutaneo-muscular flap taken from gluteus maximus. In the two cases in which this treatment was performed, the fistula was cured and a good quality functional result was obtained.     

Predictors of health-related quality of life in Parkinson’s disease: the impact of overlap between health-related quality of life and clinical measures

Quality of Life Research - This study aimed to determine predictors of health-related quality of life (HRQoL) in Parkinson's disease (PD) and to explore their predictive value before and after...