Selective inhibition of factor Xa in the prothrombinase complex by the carboxyl-terminal domain of antistasin

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2-6-fold more potently and 2-3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.     

Open trial of nefazodone for combat-related posttraumatic stress disorder

BACKGROUND: Because of its ability to block 5-HT2 receptors postsynaptically and inhibit 5-HT reuptake presynaptically and/or its enhancement of sleep quality, nefazodone may be useful for symptom management in posttraumatic stress disorder (PTSD) patients. METHOD: Ten patients with combat-related DSM-IV posttraumatic stress disorder (PTSD) entered an open-label 12-week trial of nefazodone with a 4-week follow-up, beginning with 100 mg/day and increasing as necessary to achieve a maximal response or until reaching a maximum dosage of 600 mg/day. RESULTS: Nefazodone was well tolerated, and no significant changes in sexual function were reported. Based on Clinical Global Impressions-Improvement scores, all 10 patients were rated as much improved. All PTSD symptoms (except self-reported PTSD reexperiencing symptoms), sleep, and clinician-rated depression significantly improved at week 12. At follow-up, significant changes were maintained, and self-reported PTSD reexperiencing symptoms had also significantly improved. Effect sizes for all changed symptoms were moderate to large at week 12 and at follow-up. Self-reported and clinician-rated anger significantly improved. Self-reported depression failed to improve. Improvement in social and occupational functioning was minimal. CONCLUSION: These preliminary data suggest that nefazodone may be effective in reducing the 3 primary PTSD symptom clusters and may be particularly helpful in improving sleep and decreasing anger.     

Gamma-globin gene promoter elements required for interaction with globin enhancers

The solution structure of ganglioside G(M1) carbohydrate moiety at the surface of a 102-kDa lipid-modified-G(M1) micelle is investigated by high-resolution 1H-NMR in H2O. The micellar surface can be considered a cluster-like lateral distribution of the gangliosides, each single monomer being anchored in a carbohydrate-enriched model membrane matrix. 1H NOESY measurements at short mixing times reveal a rigid trisaccharide core -beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal- and a more flexible beta-Gal-(1-3)-beta-GalNAc- terminal glycosidic bond. In the lipid-modified G(M1) ganglioside micellar system, there is no evidence that intermolecular side-by-side carbohydrate interactions modulate, or alter in any way, the head-group spatial arrangement. Possible intermonomer interactions at the level of the branched trisaccharide portion were further investigated on mixed micelles of natural N-glycolyl- and N-acetylneuraminic acid containing G(M1) in D2O, taking advantage of the different NMR features of N-glycolyl- and N-acetylneuraminic acids, which allow discrimination between sialic acid ring proton signals. Measurements of the water/ganglioside-OH proton chemical exchange rates suggest hydroxyl group involvement at position 8 of sialic acid in strong intramolecular interaction processes.     

Inhibition of human squamous cell carcinoma growth in vivo by epidermal growth factor receptor antisense RNA transcribed from the U6 promoter

BACKGROUND: Squamous cell carcinomas of the head and neck (SCCHN), unlike normal mucosal squamous epithelial cells, overexpress epidermal growth factor receptor (EGFR) messenger RNA and protein. EGFR protein is required to sustain the proliferation of SCCHN cells in vitro. To determine whether EGFR expression contributes to tumor growth, we investigated the effect of suppressing EGFR expression in tumor xenografts through in situ expression of antisense oligonucleotides. METHODS: Intratumoral cationic liposome-mediated gene transfer was used to deliver plasmids capable of expressing sense or antisense EGFR sequences into human head and neck tumors, which were grown as subcutaneous xenografts in nude mice. The oligonucleotides were expressed under the control of the U6 RNA promoter. RESULTS: Direct inoculation of the EGFR antisense (but not the corresponding sense) plasmid construct into established SCCHN xenografts resulted in inhibition of tumor growth, suppression of EGFR protein expression, and an increased rate of apoptosis (programmed cell death). Sustained antitumor effects were observed for up to 2 weeks after the treatments were discontinued. CONCLUSION: These results suggest that interference with EGFR expression, using an antisense-based gene therapy approach, may be an effective means of treating EGFR-overexpressing tumors, including SCCHN.     

Inactivation and stabilization of urokinase fibrinolytic activity in vitro

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene were characterized in eight Babesia canis isolates of differing geographic origin, vector specificity, and pathogenicity to dogs. The genotypes determined by sequencing segregated into three clearly separated groups close to or near the species level and correspond to the previously proposed subspecies B. canis canis, B. canis vogeli, and B. canis rossi. The three genotypes can be distinguished by Sau96I digestion of the polymerase chain reaction (PCR)-amplified rDNA target.     

Casein kinase I: spatial organization and positioning of a multifunctional protein kinase family

The casein kinase I family of serine/threonine protein kinases is highly conserved from yeast to humans. Until only recently, both the function and regulation of these enzymes remained poorly uncharacterised in that they appeared to be constitutively active and were capable of phosphorylating an untold number of other proteins. While relatively little was known regarding the exact function of the higher eukaryotic isoforms, the casein kinase I (CKI) isoforms from yeast have been genetically linked to vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. All five S. cerevisiae isoforms are known to be associated with discrete cellular compartments and this localization has been shown to be absolutely essential for their respective functions. New evidence now suggests that the CKI isoforms in more complex systems also exhibit non-homogeneous subcellular distributions that may prove vital to defining the function and regulation of these enzymes. In particular, CKIalpha, the most-characterized vertebrate isoform, is associated with cytosolic vesicles, the mitotic spindle and structures within the nucleus. Functions associated with these localizations coincide with those previously reported in yeast, suggesting a conservation of function. Other reports have indicated that each of the remaining CKI isoforms have the capacity to make associations with components of several signal transduction pathways, thereby channeling CKI function toward specific regulatory events. This review will examine what is now known about the higher eukaryotic CKI family members from the perspective localization as a means of gaining a better understanding of the function and regulation of these kinases.     

Importance of heart failure as a cause of death. Changing contribution to overall mortality and coronary heart disease mortality in Scotland 1979-1992

AIMS: As heart failure is a syndrome arising from another condition, such as coronary heart disease, it is rarely officially coded as the underlying cause of death regardless of the cause recorded by the physician at the time of certification. We sought to assess the true contribution of heart failure to overall mortality and coronary heart disease mortality and to examine how this contribution has changed over time. METHODS AND RESULTS: We carried out a retrospective analysis of all death certificates in Scotland between 1979 and 1992 for which heart failure was coded as the underlying or a contributory cause of death. From a total of 833622 deaths in Scotland between 1979 and 1992, heart failure was coded as the underlying cause in only 1.5% (13695), but as a contributory cause in a further 14.3% (126073). In 1979, 28.5% of male and 40.4% of female deaths attributed to coronary heart disease (coded as the underlying cause of death) also had a coding for heart failure. In 1992 these percentages had risen significantly to 34.1% and 44.8%, respectively (both P<0.001). Mortality rates for heart failure as the underlying or contributory cause of death, standardized by age and sex, fell significantly over the period studied in all ages and in both sexes: by 31% in men and 41% in women <65 years and 15.8% in men and 5.1% in women > or =65 years, respectively (P<0.01 for all changes). CONCLUSIONS: Death from heart failure is substantially underestimated by official statistics. Furthermore, one third or more of deaths currently attributed to coronary heart disease may be related to heart failure and this proportion appears to be increasing. While the absolute numbers of deaths caused by heart failure remains constant, this study is the first to show that standardized mortality rates are declining.     

Cervical epidural steroid injection with intrinsic spinal cord damage. Two case reports

STUDY DESIGN: Intrinsic cervical spinal cord damage represents the serious and permanent complications that can occur if cervical epidural steroid injections are administered while the patient is sedated. Two case reports are presented. OBJECTIVES: To draw attention to the dangerous consequences that can arise from sedating a patient before administering a cervical epidural steroid injection. SUMMARY OF BACKGROUND DATA: Reported complications of cervical epidural steroid injections have been minor and infrequent. No reports of intrinsic cervical cord damage could be found in a comprehensive English language literature search. METHODS: Two case reports of permanent intrinsic cervical cord damage in patients who had been administered cervical epidural steroid injections while under intravenous sedation are presented. Magnetic resonance imaging was performed before and after the administration of cervical epidural steroid injections. Each patient had herniated nucleus pulposus before they received cervical epidural steroid injections and intrinsic cord damage on postinjection magnetic resonance images. RESULTS: Both patients developed increased pain and neurologic symptoms within 24 hours of injection. To date, these symptoms appear to be permanent. However, Patient 1 had pain relief in her right arm and shoulder after undergoing a microdiscectomy, but pain was still persistent in her left leg, and she has developed a positive Lhermitte's sign. CONCLUSION: These case reports indicate fluoroscopic guidance will not insure or prevent intrathecal perforation or spinal cord penetration during the administration of cervical epidural steroid injections. In addition, although intravenous sedations during cervical epidural steroid injections have been used numerous times without reported complications, it appears intravenous sedation in these two cases resulted in the inability of the patient to experience the expected pain and paresthesias at the time of spinal cord irritation. Therefore, the authors conclude that the patient should be fully awake during the administration of cervical epidural steroid injections, with only local anesthetic in the skin used for analgesia.     

Desoxyepothilone B is curative against human tumor xenografts that are refractory to paclitaxel

The epothilones are naturally occurring, cytotoxic macrolides that function through a paclitaxel (Taxol)-like mechanism. Although structurally dissimilar, both classes of molecules lead to the arrest of cell division and eventual cell death by stabilizing cellular microtubule assemblies. The epothilones differ in their ability to retain activity against multidrug-resistant (MDR) cell lines and tumors where paclitaxel fails. In the current account, we focus on the relationship between epothilone and paclitaxel in the context of tumors with multiple drug resistance. The epothilone analogue Z-12,13-desoxyepothilone B (dEpoB) is >35,000-fold more potent than paclitaxel in inhibiting cell growth in the MDR DC-3F/ADX cell line. Various formulations, routes, and schedules of i.v. administration of dEpoB have been tested in nude mice. Slow infusion with a Cremophor-ethanol vehicle proved to be the most beneficial in increasing efficacy and decreasing toxicity. Although dEpoB performed similarly to paclitaxel in sensitive tumors xenografts (MX-1 human mammary and HT-29 colon tumor), its effects were clearly superior against MDR tumors. When dEpoB was administered to nude mice bearing our MDR human lymphoblastic T cell leukemia (CCRF-CEM/paclitaxel), dEpoB demonstrated a full curative effect. For human mammary adenocarcinoma MCF-7/Adr cells refractory to paclitaxel, dEpoB reduced the established tumors, markedly suppressed tumor growth, and surpassed other commonly used chemotherapy drugs such as adriamycin, vinblastine, and etoposide in beneficial effects.     

Can energy transfer be an indicator for DNA intercalation?

Contact energy transfer from DNA bases to various ligands, which can be represented by the ratio of the fluorescence intensity Q(lambda)/Q310nm, is measured by conventional fluorometer. 4',6-Diamidino-2-phenylindole and 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole can accept energy from DNA bases and exhibit the ratio Q(lambda)/Q310nm, similar to that of intercalators, although these molecules are known to bind preferentially to the minor groove of the adenine-thymine rich region of DNA. When porphyrin is intercalated in DNA or poly[d(G-C)2], the shape of the ratio Q(lambda)/Q310nm is distinct from that of the ethidium-DNA complex with two maxima at 250 nm and 280 nm. The ratio Q(lambda)/Q310nm of the porphyrin-poly[d(A-T)2] complex, in which porphyrin is known to bind 'outside' of the DNA stem, is similar to that of intercalators. Therefore, energy from excited DNA bases can be transferred not only to an intercalated ligand through direct contact but also to that bound in the minor groove. It follows from this observation that using energy transfer as a criterion for DNA intercalation requires extreme caution.