Distribution of immunoreactive dynorphin in discrete brain nuclei; comparison with vasopressin

The distribution of the opioid peptide, dynorphin, has been studied in discrete, microdissected hypothalamic nuclei, and compared with the distribution of vasopressin. Both peptides were found in relatively high concentration in the supraoptic and paraventricular nuclei. However, dynorphin-like immunoreactivity (DYN-LI) was much more widely distributed in the hypothalamus than vasopressin-like immunoreactivity, with highest concentrations in the anterior hypothalamic and ventromedial nuclei. DYN-LI was also observed in some extra-hypothalamic structures; concentrations which were comparable to the highest levels in the hypothalamus were found in the tractus diagonalis and the nucleus interstitialis stria terminalis. Among the three brainstem nuclei examined, DYN-LI levels were highest in the sensory nucleus of the trigeminal nerve.     

Behavioral treatment parameters with primary dysmenorrhea

Fourteen women with primary dysmenorrhea were administered four sessions of systematic desensitization (SD) by either a male or a female therapist. The following measures were taken during the flow periods before and after treatment and at a 6-month follow-up: menstrual symptom checklist, medication usage, invalid hours, and menstrual attitudes. At pretreatment, menstrually distressed women had significantly higher scores on all measures compared to a normative group and an explicitly nondistressed group. At posttreatment, treated women's scores on the dependent variables were significantly reduced. All indices were reduced to a nondistressed level at posttreatment and at 6-month follow-up. Type of dysmenorrhea (congestive vs. spasmodic), trait anxiety level, and therapist sex did not predict differential responsiveness to SD. SD did not affect frontalis EMG, peripheral blood flow, or pain threshold. A Retrospective Symptom Scale of menstrual distress was found to be highly reliable, valid, and sensitive.     

Radiometric detection of carbohydrate catabolism by pathogenic Neisseria.

A liquid scintillation procedure for the catabolism of D-[1-14C]glucose and [U-14C]maltose by pathogenic Neisseria was tested. Definitive results were obtained within a 30-min incubation period.     

Specificity of immunoglobulin E in coccidioidomycosis and correlation with disease involvement.

Serum immunoglobulin E (IgE) antibodies were quantitated in 26 patients with active pulmonary coccidioidomycosis, 59 patients with active disseminated disease, 12 patients in clinical remission, and 91 healthy subjects. Significant differences were obtained in IgE serum levels of patients with active disease versus healthy subjects (P less than 0.0001). Patients with pulmonary coccidioidomycosis did not differ in their IgE levels when compared with patients with disseminated disease. However, serum IgE levels were significantly increased in patients with disease involving two or more organ systems when compared with patients with pulmonary disease or extrapulmonary disease involving a single organ system (P less than 0.02). Total serum IgE correlated with anti-Coccidioides IgE (P less than 0.001), but with only six exceptions, patients with anti-Coccidioides IgE also exhibited IgE antibodies to 1 or more of 12 common allergens. The correlation between hyperproduction of IgE and disease severity coupled with the depressed cell-mediated immune status of patients with this disease suggests a defect(s) in the T-lymphocyte population which functions to regulate IgE synthesis.     

SSAHA: a fast search method for large DNA databases

We describe an algorithm, SSAHA (Sequence Search and Alignment by Hashing Algorithm), for performing fast searches on databases containing multiple gigabases of DNA. Sequences in the database are preprocessed by breaking them into consecutive k-tuples of k contiguous bases and then using a hash table to store the position of each occurrence of each k-tuple. Searching for a query sequence in the database is done by obtaining from the hash table the "hits" for each k-tuple in the query sequence and then performing a sort on the results. We discuss the effect of the tuple length k on the search speed, memory usage, and sensitivity of the algorithm and present the results of computational experiments which show that SSAHA can be three to four orders of magnitude faster than BLAST or FASTA, while requiring less memory than suffix tree methods. The SSAHA algorithm is used for high-throughput single nucleotide polymorphism (SNP) detection and very large scale sequence assembly. Also, it provides Web-based sequence search facilities for Ensembl projects.     

New mutations in MID1 provide support for loss of function as the cause of X-linked Opitz syndrome

Opitz syndrome (OS) is a genetically heterogeneous malformation disorder. Patients with OS may present with a variable array of malformations that are indicative of a disturbance of the primary midline developmental field. Mutations in the C-terminal half of MID1, an RBCC (RING, B-box and coiled-coil) protein, have recently been shown to underlie the X-linked form of OS. Here we show that the MID1 gene spans at least 400 kb, almost twice the distance originally reported and has a minimum of six mRNA isoforms as a result of the alternative use of 5' untranslated exons. In addition, our detailed mutational analysis of MID1 in a cohort of 15 patients with OS has resulted in the identification of seven novel mutations, two of which disrupt the N-terminus of the protein. The most severe of these (E115X) is predicted to truncate the protein before the B-box motifs. In a separate patient, a missense change (L626P) was found that also represents the most C-terminal alteration reported to date. As noted with other C-terminal mutations, GFP fusion constructs demonstrated that the L626P mutant formed cytoplasmic clumps in contrast to the microtubular distribution seen with the wild-type sequence. Notably, however, both N-terminal mutants showed no evidence of cytoplasmic aggregation, inferring that this feature is not pathognomonic for X-linked OS. These new data and the finding of linkage to MID1 in the absence of a demonstrable open reading frame mutation in a further family support the conclusion that X-linked OS results from loss of function of MID1.     

Activation of normal murine B cells by Echinococcus granulosus.

Echinococcus granulosus protoscolex (PSC) infection of BALB/c mice led, after 4 days, to raised numbers of cells forming plaques with trinitrophenyl-treated sheep red cells and bromelain-treated mouse red cells. The findings were similar in athymic and euthymic CBA mice. Activation of B cells was accompanied by secretion of immunoglobulin, as indicated by the reverse plaque technique. In addition, co-culture of PSC with the 7OZ/3 pre-B-cell led to the induction of differentiation, resulting in the expression of surface immunoglobulin (Ig). It is concluded that E. granulosus is a polyclonal activator of B cells inducing both transformation and differentiation, and that the effect is thymus-independent.     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.     

Augmentation of oxidant injury to human pulmonary epithelial cells by the Pseudomonas aeruginosa siderophore pyochelin.

Pseudomonas aeruginosa causes acute and chronic infections of the human lung, with resultant tissue injury. We have previously shown that iron bound to pyochelin, a siderophore secreted by the organism to acquire iron, is an efficient catalyst for hydroxyl radical (HO.) formation and augments injury to pulmonary artery endothelial cells resulting from their exposure to superoxide (O2.) and/or H2O2. Sources for O2-. and H2O2 included phorbol myristate acetate (PMA)-stimulated neutrophils and pyocyanin. Pyocyanin, another P. aeruginosa secretory product, undergoes cell-mediated redox, thereby forming O2-. and H2O2. In P. aeruginosa lung infections, damage to airway epithelial cells is probably more extensive than that to endothelial cells. Therefore, we examined whether ferripyochelin also augments oxidant-mediated damage to airway epithelial cells. A549 cells, a human type II alveolar epithelial cell line, was exposed to H2O2, PMA-stimulated neutrophils, or pyocyanin, and injury was determined by release of 51Cr from prelabeled cells. Ferripyochelin significantly increased (> 10-fold) oxidant-mediated cell injury regardless of whether H2O2, neutrophils, or pyocyanin was employed. Apo-pyochelin was not effective, and ferripyochelin was not toxic by itself at the concentrations employed. Spin trapping with alpha-(4-pyrridyl-1-oxide)-N-t-butyl-nitrone-ethanol confirmed the generation of HO., and injury was decreased by a variety of antioxidants, including superoxide dismutase, catalase, and dimethylthiourea. These data are consistent with the hypothesis that the presence of ferripyochelin at sites of P. aeruginosa lung infection could contribute to tissue injury through its ability to promote HO.-mediated damage to airway epithelial cells.