Identification of a BRCA1-associated kinase with potential biological relevance

A biochemical approach was used to identify proteins which interact with human BRCA1. Through this work, a kinase activity which co-purifies with BRCA1 has been identified. This kinase activity, which phosphorylates BRCA1 in vitro, was originally identified in Sf9 insect cells but is also present in cells of human origin including breast and ovarian carcinoma cell lines. The BRCA1 kinase activity in vitro is associated with a fragment of BRCA1 encompassing amino acids 329-435. This peptide is also phosphorylated in various human cell lines. A computer-assisted sequence analysis revealed that this peptide was a potential substrate for phosphorylation by PKA, PKC, or CKII. However, phosphorylation by these kinases could not be demonstrated in vitro indicating the presence of another kinase activity. Phosphorylation in vitro requires a minimal domain of BRCA1 encompassing amino acids 379-408. Notably, deletion of this minimal domain abolishes growth suppression by BRCA1 indicating that this domain, as well as phosphorylation within this domain, may be important for BRCA1 function.     

Endocrine effects of IGF-I on normal and transformed breast epithelial cells: potential relevance to strategies for breast cancer treatment and prevention

We studied the origin, length, external diameter, disposition, branching patterns and the perforators of short central artery in the circle of Willis in eighty fresh, unfixed cerebral hemispheres (40 brains). We also examined the relationship of the short central artery, the recurrent artery of Heubner, and M1 perforators to the anterior perforated substance, caudate and putamen. The short central artery arises at 5.2 +/- 3.36 mm from the origin of the anterior cerebral artery, hidden and overlapped by the internal carotid artery bifurcation. The recurrent artery of Heubner arises distally to the origin of the short central artery. Four main anatomical variations were found: 1. Presence of short central artery and recurrent artery of Heubner (37.5%). 2. Isolated presence of the short central artery (27.5%). 3. Isolated presence of the recurrent artery of Heubner (21.25%). 4. Absence of short central artery and recurrent artery of Heubner (13.75%). Two different branching patterns were observed: 1. 2-4 straight perforators from the short central artery, perforators from the recurrent artery of Heubner and lenticulostriate from the most medial portion of the M1, in 42 of 80 hemispheres (52.5%). 2. 8-10 perforators from only short central artery in 10 of 80 hemispheres (12.5%) with absent or hypoplastic recurrent artery of Heubner and lenticulostriates from M1 and M2.     

Mg(2+)-mediated binding of 6-substituted quinolones to DNA: relevance to biological activity

The interaction of a number of novel 6-substituted quinolone derivatives with DNA in the presence/absence of magnesium ions has been investigated by fluorometric techniques. The drug-single-stranded nucleic acid interaction is invariantly mediated by the metal ion. In all cases optimal complex formation is found at physiological Mg2+ concentration. From titrations at different [Mg2+] the binding constant for the ternary drug-DNA-Mg2+ complex (KT) has been evaluated. Interestingly, a good relationship is found between KT and gyrase poisoning activity of the test quinolones (IC50), which confirms that DNA-affinity of the quinolone, modulated by Mg2+, plays an important role in poisoning the cleavable gyrase-DNA complex and, consequently, in eliciting antibacterial activity in this family of drugs. The results obtained with different 6-substituted compounds supports the idea that position 6 of the drug, besides playing a pharmacokinetic role, is involved in recognition of the enzyme pocket. Our data do not support a mechanism of action based upon quinolone intercalation into B-DNA.     

Infections due to group A streptococcus: new concepts and potential treatment strategies

Important new information has been gained on the pathogenesis and treatment of life-threatening invasive infections caused by group A streptococci (GAS), i.e. the streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF). Both STSS and NF lead to superantigen reactions with activation of up to 10% of the CD4+ lymphocytes and release of large amounts of cytokines; mainly tumour necrosing factor beta, interferon gamma, interleukin 1 and interleukin 6. Streptococcal products known to trigger the superantigen reactions are the pyrogenic exotoxins, spe A, spe B and spe C and the M-proteins. Therapeutically clindamycin has been shown to reduce mortality in animal experiments in comparison to penicillin treatment. A possible mechanism is the effect of clindamycin on protein synthesis which might decrease the production of superantigens. In man, the use of intravenous immunoglobulin has been shown to significantly reduce mortality in STSS and NF. The most probable mechanism is neutralisation of superantigens by antibodies in the immunoglobulin preparations used.     

Metabolic mapping of the rat brain after subanesthetic doses of ketamine: potential relevance to schizophrenia

Several lines of evidence suggest the molecular and functional entity of muscarinic M1 receptors in mammalian heart. We have reported that acetylcholine (ACh) reduces the maximum upstroke velocity of action potential (Vmax) through activation of muscarinic M1 receptors, which is followed by a muscarinic M2 receptor-mediated increase. The present study sought to determine whether activation of beta-adrenergic receptors modulates the muscarinic M1 and M2 receptor-mediated effects on Vmax in isolated mouse right atria. Intracellular recordings of spontaneous action potential were done using the conventional glass microelectrode technique. Isoproterenol (3 nM) completely antagonized ACh (5 microM)-induced reduction in Vmax. The antagonism was accompanied by a subsequent increase in Vmax. Propranolol (0.3 microM) abolished the effects of isoproterenol on ACh-induced changes in Vmax. Isoproterenol antagonized McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride) (300 microM, a muscarinic M1 receptor agonist)-induced reduction in Vmax. Oxotremorine (0.03 microM), a muscarinic M2 receptor agonist, did not affect Vmax by itself, but significantly increased it in the presence of 3 nM isoproterenol. The effects of isoproterenol were mimicked by cholera toxin (100 nM, 1 hr), a Gs-protein activator, and forskolin (10 nM), a direct activator of adenylyl cyclase. H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide++ +, 1 microM), a selective protein kinase (PK)-A inhibitor, abolished the antagonism by isoproterenol of ACh-induced reduction in Vmax. The present results suggest that activation of the beta-adrenergic-Gs-adenylyl cyclase system antagonizes ACh-induced reduction (muscarinic M1-mediated) and potentiates the subsequent increase (muscarinic M2 receptor-mediated) in Vmax. The beta-adrenergic antagonism of ACh-induced reduction in Vmax may involve cross-talk between PK-A and PK-C signaling pathways.     

A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell adhesion in brain

90Yttrium-labeled monoclonal antibodies (mAbs) are likely to be important to radioimmunotherapy (RAIT) of a variety of cancers. The goal of this study was to select and evaluate a form of [90Y]mAb suitable for RAIT and determine conditions for high-yield, reproducible radiolabelings. 90Y-Labelings, at 2-40 mCi levels, of cdr-grafted versions of anti-B-cell lymphoma (hLL2) and anti-CEA (hIMMU-14) mAbs were optimized to >90% incorporations using the macrocyclic chelator DOTA as the metal carrier. In in vitro challenge assays, the stability of mAbs labeled with [90Y]DOTA was better than that of the corresponding [90Y]benzyl-DTPA conjugates. The retention of [90Y]DOTA-hLL2 on Raji tumor cells in vitro was similar to that of the same mAb labeled with [90Y]benzyl-DTPA and was about twice as much as with [125I]hLL2, indicating residualization of metalated mAb. Both [90Y]hLL2 conjugates, prepared using DOTA and Bz-DTPA, had similar maximum tolerated doses of 125 muCi in BALB/c mice and showed no discernible chelator-induced immune responses. Animal biodistribution studies in nude mice bearing Ramos human B-cell lymphoma xenografts revealed similar tumor and tissue uptake over a 10 day period, with the exception of bone uptake which was up to 50% lower for [88Y]DOTA-hLL2 compared to [88Y]Bz-DTPA-hLL2 at time points beyond 24 h. With [90Y]DOTA-hLL2 fragments, in vivo animal tumor dosimetries were inferior to those for the IgG, and kidney uptake was relatively high even with D-lysine administration. The ability of [111In]DOTA-hLL2 to accurately predict [90Y]DOTA-hLL2 biodistribution was established. These preclinical findings demonstrate that [90Y]DOTA-(CDR-grafted) mAbs are suitable for examination in clinical RAIT.     

Induction of endothelial cell adhesion molecules by serum and immunoglobulins G from a patient with vasculitis and monoclonal gammapathy: potential relevance to vasculitis

Interactions between endothelial cell adhesion molecules and their beta2 integrin adhesive receptors on leukocytes are thought to play a role in the pathogenesis of inflammatory diseases and probably vasculitis. We describe a case in whom leukocytoclastic vasculitis was associated to a monoclonal immunoglobulin G2 kappa (IgG2K). During the vasculitic crisis, the patient's serum and the isolated IgG from this serum induced the expression of E-selectin, VCAM-1 and ICAM-1 at the HUVEC surface, but not tissue factor activity, whereas normal, control serum and patient serum at remission were without any effect. A close relationship between the vasculitis and the serum level of the monoclonal IgG was observed. We suggest that the monoclonal IgG might induce the vasculitis by increasing the expression of E-selectin, VCAM-1 and ICAM-1 which facilitate the interaction of leukocytes with vascular endothelium.     

The telomeres of Streptomyces chromosomes contain conserved palindromic sequences with potential to form complex secondary structures

The chromosomes of the gram-positive soil bacteria Streptomyces are linear DNA molecules, usually of about 8Mb, containing a centrally located origin of replication and covalently bound terminal proteins (which are presumably involved in the completion of replication of the telomeres). The ends of the chromosomes contain inverted repeats of variable lengths. The terminal segments of five Streptomyces chromosomes and plasmids were cloned and sequenced. The sequences showed a high degree of conservation in the first 166-168bp. Beyond the terminal homology, the sequences diverged and did not generally cross-hybridize. The homologous regions contained seven palindromes with a few nucleotide differences. Many of these differences occur in complementary pairs, such that the palindromicity is preserved. Energy-optimized modelling predicted that the 3' strand of the terminal palindromes can form extensive hairpin structures that are similar to the 3' ends of autonomous parvovirus genomes. Most of the putative hairpins have a GCGCAGC sequence at the loop, with the potential to form a stable single C-residue loop closed by a sheared G:A pairing. The similarity between the terminal structures of the Streptomyces replicons and the autonomous parvoviral genomes suggests that they may share some structural and/or replication features.     

Architecture of the atrial musculature in and around the triangle of Koch: its potential relevance to atrioventricular nodal reentry

INTRODUCTION: Recent studies suggest that atrial fibers in the approaches to the AV node form part of the dual pathways recognized electrophysiologically in patients with AV nodal reentrant tachycardia (AVNRT). Our aim was to determine, by gross dissection, the arrangement of the superficial musculature in the area of the triangle of Koch in normal hearts and in hearts with documented AVNRT, hoping to ascertain anatomic features that might contribute to the debate. METHODS AND RESULTS: We used blunt dissection to study the architecture of the superficial atrial musculature in 16 autopsied hearts from adults who died of noncardiac disease. A well-defined pattern of architecture of muscle fibers was found in the region of the triangle of Koch, showing marked variations in 7 of the 16 specimens. The relationship of these fibers to the histologically specialized AV node was confirmed by histology in three cases. Two hearts from patients with known AVNRT, treated by ablation in one, were examined further histologically. These sections showed that the site of ablation was well distant from the histologically discrete AV node. CONCLUSION: The variability in the arrangement of the superficial atrial muscle fibers in the area of the triangle of Koch may be one of the factors influencing the route for impulses entering the AV node. Lesions that ablate nodal reentry are within these atrial fibers rather than the histologically specialized AV node.     

Interaction of tacrine and velnacrine with neocortical synaptosomal membranes: relevance to Alzheimer's disease

The acridine-based, potential Alzheimer's disease therapeutic agents, tacrine and velnacrine, were incubated with rat or gerbil neocortical synaptosomal membranes. Electron paramagnetic resonance employing a protein-specific spin label was used to monitor this interaction. Analogous to their effects in erythrocyte membranes [Butterfield and Rangachari (1992) Biochem. Biophys. Res. Commun. 185: 596-603], in the present studies both agents decreased segmental motion of spin labeled synaptosomal membrane proteins, consistent with increased cytoskeletal protein-protein interactions (0.001 < P < 0.005), and tacrine was more potent than velnacrine. These results are discussed with possible relevance to molecular actions of the agents and molecular alterations in Alzheimer's disease.     

Mode of referral to hospital of patients with heart attacks: relevance to home care and special ambulance services

Out of 1250 consecutive patients brought to hospital with heart attacks 956 (76%) were at home when their symptoms began. Of these, 587 (61%) called their general practitioner, and for the remainder an ambulance was summoned by a member of the public. Of the 294 patients who were away from home when the attack occurred 291 were brought to hospital by ambulance. Of these, only 70 (24%) were attended by a general practitioner. Patients for whom ambulances were called by a general practitioner had had their symptoms significantly longer and had significantly lower prehospital and hospital mortalities than those for whom ambulances were summoned by members of the public. Special "cardiac" ambulances appear to be inappropriate for patients who have been seen by a general practitioner, and for this group home care may well be as effective as hospital admission.