Molecular basis of the catalytic differences among DT-diaphorase of human, rat, and mouse

DT-diaphorase (EC 1.6.99.2), also referred to as NAD(P)H:(quinone-acceptor) oxidoreductase, is involved in the reductive activation process of several cytotoxic antitumor quinones and nitrobenzenes. It has been observed in our and other laboratories that the rat enzyme is significantly more effective in activating these drugs than the human and mouse enzymes. These results indicate that the available cytotoxic drugs are better substrates for the rat enzyme and are not the most ideal prodrugs for activation by DT-diaphorase in human tumors. In this study, using site-directed mutagenesis to replace residues in the rat enzyme with the human sequences and residues in the human enzyme with the rat sequences, we have found that residue 104 (Tyr in the rat enzyme and Gln in the human and mouse enzymes) is an important residue responsible for the catalytic differences between the rat and the human (and mouse) enzymes. With an exchange of a single amino acid, the rat mutant Y104Q behaved like the wild-type human enzyme, and the human mutant Q104Y behaved like the wild-type rat enzyme in their ability to reductively activate the cytotoxic drug CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The study also confirms the conclusion of the x-ray structural analysis of rat enzyme that residue 130 (Thr in the rat enzyme and Ala in the human and mouse enzymes) is positioned near the binding region of the nicotinamide portion of NAD(P)H. This structural information is very important for designing suitable drugs and approaches for human cancer chemotherapy mediated by DT-diaphorase.     

Neuropeptide Y release in the paraventricular nucleus is decreased during transient hyperphagia induced by microinjection of colchicine into the ventromedial nucleus of rats

The adenosine A2a receptors (A2aR) play an important role in the purinergic mediated neuromodulation. The presence of A2aR in the brain is well established. In contrast, little is known about their expression in the periphery. The purpose of this study was to investigate the expression of A2aR gene in the autonomic (otic, sphenopalatine, ciliary, cervical superior ganglia and carotid body) and in the dorsal root ganglia of normal rat. Hybridization histochemistry with S35-labelled radioactive oligonucleotide probes was used. An expression of A2aR gene was found in the large neuronal cells of the rat dorsal root ganglia. The satellite cells showed no expression of A2aR gene. In the superior cervical ganglion, isolated ganglion cells expressed A2aR. In the carotid body clusters of cells with a strong A2aR gene expression were found. In contrast, the ciliary and otic ganglia did not expressed A2aR gene, and only few small sized A2aR expressing cells were demonstrated in the sphenopalatine ganglion. The discrete distribution of A2aR gene expression in the peripheral nervous system speaks for a role of this receptor in the purinergic modulation of sensory information as well as in the sympathetic nervous system.     

Adjunctive use of inhaled nitric oxide during implantation of a left ventricular assist device

Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B' (delta) subunit, was phosphorylated at serine residues of B' in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 microM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B', respectively. The Km value of A-kinase for CAB' was 0.17 +/- 0.01 microM in the presence of OA. The major in vitro phosphorylation sites of B' were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B' did not dissociate B' from CA, and stimulated the molecular activity of CAB' toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a.     

Reduction ventriculoplasty for the cardiomyopathic heart: a case report

On the basis of long clinical experience principles are substantiated and schemes are suggested of therapy of childhood disseminated sclerosis (DS), these being as follows: 1) pulse therapy with high dose intravenous corticosteroids, a switch-over to oral intake followed by synacten-depo during the phase of exacerbation; 2) long-term maintenance therapy during transition to the phase of remission. The above algorithm allowed the patients to be helped out of the exacerbation. Analysis of 3-to-11 yr catamnesis permitted assessing the efficacy and outcomes of the proposed therapy in children. Of the 21 cases, thirteen showed stabilization of the process as evidenced by continuing remission of one to 3 years in duration. Eight children showed short-time remission, with neurological deficit slowly progressing, resulting in invalidism in 7 patients. In summary, the proposed therapeutic regimen for DS exacerbation in children permits achieving quick regression of the neurologic symptomatology, returning progression of neurological deficit, which observation was recordable in two thirds of the patients. In 1/2 of children DS runs an aggressive course that does not respond even to intensive therapy; in these, long-term remission was not achievable, this resulting in progression of the neurologic symptomatology and, in the long run, disability under protracted course of the condition.     

Gastric mucous neck cell and intestinal goblet cell phenotypes in gastric adenocarcinoma

AIM: To investigate the phenotype of cells comprising diffuse and intestinal-type gastric cancers using monoclonal antibodies to two antigens. One antigen (designated D10) is characteristic of gastric mucous neck cells, cardiac glands, pyloric glands, and Brunner's glands. The second antigen (designated 17NM) is specific to the mucous vacuole of intestinal goblet cells. METHODS: Thirty two gastrectomy specimens with adenocarcinoma were studied. Serial paraffin sections were stained immunohistochemically for D10 and 17NM and histochemically for acid and neutral mucins. The cancers were classified histologically as of either diffuse or intestinal type according to Lauren. RESULTS: Of 15 diffuse-type gastric carcinomas, 11 showed the majority of cancer cells staining for D10 while four were typical signet ring cell cancers staining predominantly for 17NM; five tumours displayed both phenotypes with the two phenotypes segregated in different areas of the tumours. In contrast, of 16 intestinal-type cancers, six expressed 17NM, three D10, five neither antigen, and two expressed both antigens. One indeterminate-type cancer expressed both antigens. The staining of individual cells for D10 and 17NM was mutually exclusive in both diffuse and intestinal types. In contrast to the diffuse cancers, intestinal-type cancers typically expressed either antigen only in occasional small groups of cells and individual cells. CONCLUSIONS: In disease, the gastric stem cell can assume the capacity of the duodenal stem cell for divergent differentiation into either intestinal goblet cells (for example, as in intestinal metaplasia) or Brunner's gland cells (for example, as in pyloric gland/Brunner's gland metaplasia). With neoplastic transformation, this potential for divergent differentiation is maintained and gives rise to diffuse-type cancers that display either the D10 phenotype, the 17NM phenotype, or the clonal expression of both phenotypes. In the more cell cohesive (intestinal-type) tumours, differentiation for antigen expression is poorly developed and more frequently directed towards the intestinal goblet cell phenotype.     

Spinal cord coordination of hindlimb movements in the turtle: intralimb temporal relationships during scratching and swimming

Hindlimb interlimb coordination was examined in turtles during symmetrical "same-form" behaviors in which both hindlimbs utilized the same movement strategy ("form") and during asymmetric "mixed-form" behaviors in which the form exhibited by one hindlimb differed from that of its contralateral partner. In spinal turtles, three forms of scratching were examined: rostral, pocket, and caudal. Bilateral symmetrical same-form scratching was studied for each of the forms. Asymmetric mixed-form scratching (rostral scratching of a hindlimb and pocket scratching of the other hindlimb) was also examined. In intact turtles, two forms of swimming were examined: forward swimming and back-paddling. The symmetrical behavior of bilateral forward same-form swimming and the asymmetric behavior of turning mixed-form swimming (forward swimming of 1 hindlimb and back-paddling of the other hindlimb) were studied. For all behaviors examined, most episodes displayed absolute or 1:1 coordination; in this type of coordination, during each movement cycle that began and ended with the onset of ipsilateral hip flexion, there was a single onset of contralateral hip flexion. For most of these episodes there was out-of-phase coordination between hip movements; the onset of contralateral hip flexion occurred near the onset of ipsilateral hip extension midway through the ipsilateral movement cycle. Bilateral caudal/caudal same-form scratching displayed out-of-phase 1:1 coordination during some episodes and in-phase 1:1 coordination during other episodes. During in-phase coordination, the onset of contralateral hip flexion occurred near the onset of ipsilateral hip flexion close to the start of the ipsilateral movement cycle. In a few cases of bilateral same-form scratching there were episodes of relative or 2:1 coordination; in this type of coordination, during each movement cycle of the slowly moving limb that began and ended with ipsilateral hip flexion, there were two distinct occurrences of the onset of contralateral hip flexion. The observation that out-of-phase movements of the hip occurred during symmetrical as well as asymmetric behaviors is consistent with the hypothesis that timing signals related to hip movement play a major role in interlimb phase control. The neural mechanisms responsible for interlimb phase control are not well understood in vertebrates. The present demonstration of bilateral scratching in spinal turtles suggests that this preparation may be suitable for additional experiments to examine mechanisms of vertebrate interlimb phase control.     

Fixed temporization and bone-augmented ridge stabilization with transitional implants

Utilization of dental implants in full-mouth restorations is now a well-accepted treatment modality, with numerous modifications and implant systems documented in the literature. The efficacy of the treatment procedure generally requires an extended postplacement healing period prior to loading the implant fixture with the stress of mastication. Until recently, clinicians have not been able to address patient comfort requirements during the healing period. The teaching objective of this article is to present and evaluate a transitional implant system used to provide function during the healing phase. The system consists of thin titanium transitional implants and a three-component overdenture that is intended to absorb the pressure during function and protect the augmented implant site and the definitive implant fixtures from the stress of immediate loading. Treatment objectives for the transitional and definitive implants are made during the initial treatment planning. Three cases are presented to document and illustrate the clinical procedure.     

Selective disruption of lymphotoxin ligands reveals a novel set of mucosal lymph nodes and unique effects on lymph node cellular organization

Lymphotoxin (LT) provides a critical signal for the genesis of lymph nodes (LN) in mice. Here we show that mice treated in utero with LT beta-R-Ig, which binds to the membrane LT alpha 1 beta 2 heterotrimer, lacked most LN, yet retained a set of mucosal surface draining LN. Since mice genetically deficient in LT alpha lack all LN, including the mucosal set, we hypothesize that a novel LT alpha-dependent pathway controls their genesis. This novel set of mucosal LN cannot be discriminated on the basis of addressin expression. The discovery of LN in mice treated with LT beta-R-Ig fusion protein in utero allowed us to compare the roles of membrane LT alpha beta or soluble LT alpha/tumor necrosis factor (TNF) in the development of cellular organization in LN and spleen. Our results indicate that both membrane LT alpha beta and soluble LT alpha/TNF mediate T-B cell segregation and the organization of B cell follicles in spleen and LN. Interestingly, while antagonism of membrane LT alpha beta or soluble LT alpha/TNF prevented germinal center (GC) formation in spleen, antagonism of soluble LT alpha/TNF had no effect on LN formation. The data suggest that multiple LT/TNF ligands control B cell follicle organization in the spleen and LN of adult mice, and that the requirements for LT/TNF ligands in GC formation are distinct in the different lymphoid organs.     

Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy

Induction and maintenance of cytotoxic T lymphocyte (CTL) activity specific for a primary endogenous tumor was investigated in vivo. The simian virus 40 T antigen (Tag) expressed under the control of the rat insulin promoter (RIP) induced pancreatic beta-cell tumors producing insulin, causing progressive hypoglycemia. As an endogenous tumor antigen, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) was introduced also under the control of the RIP. No significant spontaneous CTL activation against GP was observed. However, LCMV infection induced an antitumor CTL response which efficiently reduced the tumor mass, resulting in temporarily normalized blood glucose levels and prolonged survival of double transgenic RIP(GP x Tag2) mice (137 +/- 18 d) as opposed to control RIP-Tag2 mice (88 +/- 8 d). Surprisingly, the tumor-specific CTL response was not sustained despite the facts that the tumor cells continued to express MHC class I and LCMV-GP-specific CTLs were present and not tolerized. Subsequent adoptive transfer of virus activated spleen cells into RIP(GP x Tag2) mice further prolonged survival (168 +/- 11 d), demonstrating continued expression of the LCMV-GP tumor antigen and MHC class I. The data show that the tumor did not spontaneously induce or maintain an activated CTL response, revealing a profound lack of immunogenicity in vivo. Therefore, repetitive immunizations are necessary for prolonged antitumor immunotherapy. In addition, the data suggest that the risk for induction of chronic autoimmune diseases is limited, which may encourage immunotherapy against antigens selectively but not exclusively expressed by the tumor.     

Complementary nursing--an acute care case management model. Part I--development and implementation

Complementary nursing was developed in response to the need for maintaining high quality care while controlling health care costs. The complementary nurse provides comprehensive management of complex patients through an entire episode of an acute illness, to transition them back to a prehospital state through interdisciplinary discharge planning. In this article, the authors describe the process used in developing and implementing this new integrated role of acute care case management. The article contains role responsibilities, communication tools, and lessons learned from experience.