A novel image cytometric method for quantitation of immunohistochemical staining of cytoplasmic antigens

A novel method was developed to evaluate the acrosomal status of mammalian spermatozoa. The method is based on the ability of the lectin Pisum sativum agglutinin (PSA) to bind specifically to glycoproteins of the acrosomal matrix released during the acrosome reaction. The amount of released acrosomal content is proportional to the fraction of spermatozoa that underwent acrosome reaction. The released glycoproteins present in the supernatant separated from the cells were detected via an ELISA-like assay. The authors suggest that one of these glycoproteins might be the acrosin as identified by anti-acrosin antibodies, using Western blot analysis. The new method (demonstrated here with ram and bull spermatozoa) correlates well with the results obtained by conventional methods. Its advantages are simplicity, objectiveness, rapidity, and low cost. In addition, many samples can be processed in parallel. The method can be used in experimental as well as clinical applications.     

Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies

Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.     

Alpha 2-adrenergic receptors regulate generation of cyclic AMP in the pineal gland, but not in cerebral cortex of chick

A beta-adrenoceptor agonist isoprenaline potently stimulated cyclic AMP formation in chick cerebral cortical slices. L-Noradrenaline (10-1000 microM) also increased cortical nucleotide synthesis, the effect being antagonized by beta-adrenoceptor blocker propranolol, and not affected by alpha 1- and alpha 2-adrenoceptor blockers, prazosin and yohimbine, respectively. Clonidine, a selective alpha 2-agonist, had no effect on cerebral cyclic AMP production stimulated by both isoprenaline and forskolin. However, clonidine (0.001-10 microM) concentration-dependently suppressed forskolin-driven cyclic AMP synthesis in intact chick pineal glands. In living chicks clonidine suppressed the nocturnal activity of cyclic AMP-dependent serotonin N-acetyltransferase, a rate-limiting enzyme in melatonin biosynthesis, the effect being prevented by yohimbine. The data suggest that the cyclic AMP generating system of the pineal gland, but not that of cerebral cortex in chick, is negatively regulated by alpha 2-adrenergic receptor-mediated signal.     

Biochemical disease-free survival following 125I prostate implantation

OBJECTIVES: The purpose of our study was to validate the incentive spirometry (IS) as a simple mean to follow pulmonary function at the bedside after lung surgery. MATERIALS AND METHODS: We studied prospectively 19 patients (16 men, 3 women; mean +/- SE age, 60 +/- 2.8 years) undergoing lobectomy for lung cancer. All the patients had an obstructive pattern with FEV1/FVC below 75%. Lung volumes, including functional residual capacity (FRC) and residual volume (RV), measured using spirometry and the helium dilution technique, and IS were measured preoperatively and postoperatively at days 1, 2, 3, and 8, and at 2 months. RESULTS: Our results showed that in the postoperative period after lung resection, IS performance was well correlated (R) during the first 8 postoperative days with vital capacity (VC) (R between 0.667 and 0.870) mainly due to the excellent correlation with the inspiratory reserve volume (IRV, R between 0.680 and 0.895) but was poorly correlated with expiratory reserve volume (R below 0.340), RV (R below 0.180), and FRC (R below 0.470). CONCLUSIONS: IS can be used as a simple mean to follow lung function, especially VC and IRV, in the postoperative period in spontaneously breathing patients. IS is noninvasive and can be performed repeatedly at the bedside in the intensive care setting.     

A model of peptide-induced lupus autoimmune B cell epitope spreading is strain specific and is not H-2 restricted in mice

Anti-Sm is a common and specific autoantibody found in systemic lupus erythematosus. The peptide PPPGMRPP from Sm B/B' is an early target of the autoimmune response in some anti-Sm-positive human patients. After immunization with this peptide on a MAP backbone, rabbits develop anti-Sm autoantibodies with B cell epitope spreading of the autoimmune response as well as other features of lupus autoimmunity. Various strains of inbred mice have been immunized with peptide PPPGMRPP or PSQQVMTP (nonantigenic region of Sm B/B') in Freund's adjuvant or with no peptide. All peptide-immunized mouse strains eventually develop high titers of specific anti-peptide of immunization Abs. Mice immunized with Freund's adjuvant alone have no measurable Ab binding to the PPPGMRPP peptide. With time, nearly half the mouse strains tested develop Abs that react with additional regions of Sm B/B' and Sm D. All the regions bound by mouse serum are major epitopes of the human systemic lupus erythematosus anti-Sm response. These same strains also develop significant anti-Sm and anti-nuclear ribonucleoprotein titers. In addition, some of these strains demonstrate positive anti-nuclear Abs and anti-dsDNA Abs. Experiments with congenic H-2 mice demonstrate that the H-2 region does not play a role in spreading the immune response from the peptide of immunization to other epitopes of the spliceosome. These results present a new murine model of B cell epitope spreading and lupus autoimmunity induced by peptide immunization that is strain specific and not apparently dependent upon the loci at H-2.     

Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences

Traditional molecular and biochemical methods, such as schizodeme analysis, karyotyping, DNA fingerprinting, and enzyme electrophoretic profiles, have shown a large variability among Trypanosoma cruzi isolates. In contrast to those results, polymerase chain reaction (PCR) amplification of sequences from the 24S alpha ribosomal RNA gene and from the mini-exon gene nontranscribed spacer indicated a dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 86 T. cruzi field stocks (68 isolated from humans with defined presentations of Chagas' disease and 18 from triatomines) derived from four Brazilian geographic areas were typed by the PCR assay based on the DNA sequences of the mini-exon and 24S alpha rRNA genes. These stocks were ordered into the two major T. cruzi lineages. Lineage 1 was associated mainly with human isolates and lineage 2 with the sylvatic cycle of the parasite.