Direction determination in the minus-end-directed kinesin motor ncd

Motor proteins of the kinesin superfamily transport intracellular cargo along microtubules. Although different kinesin proteins share 30-50% amino-acid identity in their motor catalytic cores, some move to the plus end of microtubules whereas others travel in the opposite direction. Crystal structures of the catalytic cores of conventional kinesin (a plus-end-directed motor involved in organelle transport) and ncd (a minus-end-directed motor involved in chromosome segregation) are nearly identical; therefore, the structural basis for their opposite directions of movement is unknown. Here we show that the ncd 'neck' made up of 13 class-specific residues next to the superfamily-conserved catalytic core, is essential for minus-end-directed motility, as mutagenesis of these neck residues reverses the direction of ncd motion. By solving the 2.5 A structure of a functional ncd dimer, we show that the ncd neck (a coiled-coil) differs from the corresponding region in the kinesin neck (an interrupted beta-strand), although both necks interact with similar elements in the catalytic cores. The distinct neck architectures also confer different symmetries to the ncd and kinesin dimers and position these motors with appropriate directional bias on the microtubule.     

Neither dopamine nor dobutamine corrects mesenteric blood flow depression caused by positive end-expiratory pressure in a rat model of acute lung injury

OBJECTIVE: To determine if either dopamine or dobutamine would counteract the deleterious effect that positive end-expiratory pressure (PEEP) has on cardiac output and mesenteric blood flow in a rat model of acute lung injury. DESIGN: Prospective, randomized, controlled trial in a clinically relevant model of acute lung injury. SETTING: Microcirculation research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: The animals were anesthetized with pentobarbital (30 mg/kg) by intraperitoneal injection. They underwent tracheostomy, jugular and femoral vein cannulation, femoral artery cannulation, carotid artery thermistor placement, and bowel preparation for in vivo video microscopy. Acute lung injury was created by administering 0.1 N hydrochloric acid (1 mL/kg) via the tracheostomy. Dopamine or dobutamine (2.5 or 12.5 microg/kg/min), followed by two intravenous fluid boluses, was administered to rats ventilated with 5, 10, 15, and 20 cm H2O of PEEP. MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure, thermodilution cardiac output, mesenteric arteriolar diameter, and red blood cell velocity were measured and mesenteric blood flow was calculated. Cardiac output was depressed in rats exposed to 20 cm H2O of PEEP by 32+/-2%. The corresponding values for cardiac output depression at 20 cm H2O of PEEP in rats receiving 2.5 and 12.5 microg/kg/min of dopamine and 2.5 and 12.5 microg/kg/min of dobutamine were 31+/-1%, 21+/-1%, 29+/-0%, and 24+/-2%, respectively. Mesenteric blood flow was depressed in rats ventilated with 20 cm H2O of PEEP by 74+/-3%, while the corresponding values in rats exposed to 20 cm H2O of PEEP and receiving 2.5 or 12.5 microg/kg/min of dopamine or 2.5 or 12.5 microg/kg/min of dobutamine were 86+/-3%, 77+/-3%, 73+/-3%, and 66+/-3%, respectively. Fluid boluses did not correct the deficits in cardiac output or mesenteric blood flow caused by the combination of acute lung injury and PEEP. CONCLUSIONS: The higher doses of dopamine and dobutamine partially, but insignificantly, corrected the cardiac output depression caused by PEEP in a model of acute lung injury. Neither dose of dopamine nor dobutamine was able to improve PEEP-induced mesenteric blood flow depression.     

Cardiac autonomic dysfunction in survivors of acute lymphoblastic leukemia in childhood

Lower levels of baseline ventilatory function have consistently been associated with increased risk of cardiovascular mortality in prospective studies, but the underlying mechanisms are not known. Increased risk of coronary heart disease is associated with higher serum insulin levels. This report examines the relationship between ventilatory function and indirect measures of insulin resistance. Cross-sectional data from 922 nondiabetic participants in the Normative Aging Study were analysed using multiple linear regression models with adjustment for potential confounders. Forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) were examined in relation to indicators of insulin resistance, i.e. fasting insulin and the fasting insulin resistance index (FIRI). Diabetics were excluded because impaired insulin secretion interferes with the validity of these as measures of insulin resistance. Fasting insulin and FIRI were negatively correlated with FVC and FEV1 (all p< 0.001). These associations persisted after adjusting for potential confounders including age, height, body mass index, waist to hip circumference ratio, physical activity, alcohol intake and smoking in separate multiple linear regression models, for both insulin (all p< or =0.0008) and FIRI (all p< or =0.0001). Negative cross-sectional associations between ventilatory function and indirect measures of insulin resistance were found in nondiabetic males. Insulin resistance may contribute to the previously unexplained association between ventilatory function impairment and cardiovascular mortality. Mechanisms underlying the relationship between insulin resistance and decreased ventilatory function remain to be elucidated.     

Strategies for positioning fluorescent probes and crosslinkers on formyl peptide ligands

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1-3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.     

Adenosine A1 receptor stimulation antagonizes the negative inotropic effects of the PKC activator dioctanoylglycerol

It has been suggested that adenosine cardioprotection occurs via adenosine A1 receptor-mediated activation of protein kinase C (PKC). However, adenosine has well-known vasodilatory effects in the myocardium, whereas PKC is a vasoconstrictor. This study examined whether adenosine A1 receptor activation alters the effects of the PKC activator. 1,2-dioctanoyl-s,n-glycerol (DOG) in isolated perfused rat hearts (left-ventricular developed pressure) and rat ventricular myocytes ([Ca2+]i and cell shortening). Exposure to DOG decreased left-ventricular developed pressure by 30%, an effect that was completely reversible. Pretreatment of isolated hearts with either the PKC inhibitor chelerythrine or the adenosine A1 agonist 2-chloro-N6-cyclo-cyclo-isolated pentlyadenosine (CCPA) attenuated the negative inotropic effects of DOG. In the isolated myocytes, DOG decreased [Ca2+]i and cell shortening by 25 and 28%, respectively, effects that were attenuated by both chelerythrine and CCPA. The CCPA attenuation of the DOG-induced decrease in [Ca2+]i and cell shortening was blocked by pretreating the myocytes with the adenosine A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). These results indicate that in rat ventricular myocardium, adenosine A1 receptor activation attenuates the apparent PKC-dependent negative inotropic effects of DOG via preservation of [Ca2+]i levels.     

Atlantoaxial instability complicating radiation therapy for recurrent nasopharyngeal carcinoma. A case report

STUDY DESIGN: A case report of a patient in whom atlantoaxial instability developed secondary to repeat radiation therapy for recurrent nasopharyngeal carcinoma. OBJECTIVES: To illustrate a dramatic and previously unreported complication of local radiation to the posterior nasopharynx. SUMMARY OF BACKGROUND DATA: Nasopharyngeal carcinoma is an unusual tumor that usually is managed with local, external-beam radiation. It is not thought to involve the cervical spine directly, although local invasion of the skull base is common. METHODS: A review of the medical records and radiographs of the only patient known to develop this complication of radiation used to manage nasopharyngeal carcinoma. RESULTS: Atlantoaxial instability developed in a patient as a result of repeat radiation for a locally recurrent tumor. The instability was associated with intrusion of the anterior arch of C1 into the posterior nasopharynx and was managed successfully with a posterior stabilization using transarticular screws and supplemental wiring. CONCLUSIONS: Patients who have undergone local irradiation for nasopharyngeal carcinoma may be at risk for developing atlantoaxial instability.     

Right hemisphere, white-matter learning disabilities associated with depression in an adolescent and young adult psychiatric population

Four hundred and eighty-four adolescents and young adults at an inpatient psychiatric facility were diagnosed as nonverbal learning disabled, verbal learning disabled, general learning disabled, or normal psychiatric controls. The nonverbal learning disabled group had the highest incidence of depression and was clearly different from the reading disabled group (66.3% vs. 33.3%). Nondisabled subjects were significantly more likely than the other subjects to be diagnosed with adjustment problems. Depressed subjects were significantly younger and more likely to be female. This study supports the contention that right-hemisphere, white-matter, arithmetic-disabled adolescents and young adults in a psychiatric population are at greater risk for depression than are psychiatric patients not showing this pattern.     

Immunohistochemical localisation of alpha1B-adrenergic receptors in the rat iris

This article reports results from a meta-analysis on adult age differences in the negative priming effect (21 studies on identity negative priming and 8 on location negative priming). Both younger and older adults were found to be susceptible to the negative priming effect in identity and location tasks. Effect sizes were homogeneous for both tasks, indicating that the data are adequately described without reference to moderator variables. State trace analysis on identity tasks, in which mean latencies in negative priming conditions were regressed onto mean latencies in baseline conditions, showed (a) that in both age groups the negative priming effect is proportional rather than additive and (b) that the negative priming effect is smaller in older adults as compared with younger adults.     

Protection of mice against SV40 tumours by Pam3Cys, MTP-PE and Pam3Cys conjugated with the SV40 T antigen-derived peptide, K(698)-T(708)

The intraperitoneal injection of Balb/c mice with synthetic analogues of adjuvants S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-cysteine (Pam3Cys) or muramyltripeptide phosphatidylethanolamine (MTP-PE) inhibited the tumourigenic growth of subcutaneously injected VLM cells, a syngeneic simian virus 40 (SV40)-transformed cell line. Furthermore, the Pam3Cys conjugate of K698-T708 (KT), which represents the C-terminal undecapeptide of the SV40 large tumour (T) antigen, was tumour-protective. Also syngeneic spleen cells, preincubated in vitro with this Pam3Cys-KT derivative, which anchores spontaneously at the cell membrane, were, through SV40 tumour mimicry, tumour-protective. The protection was impaired by treatment of the mice with either anti-CD4, anti-CD8 IgG, anti asialo GM1 antiserum or dextrane sulfate, which deplete the CD4+, CD8+ and NK cells or the macrophages, respectively. In summary, SV40 tumour transplantation resistance can be experimentally elicited by a tumour-epitope-specific vaccine. In the absence of an immunogenic epitope protection was obtained by administration of biological response modifiers. Protection is effected by SV40-T-antigen-specific cytotoxic lymphocytes in cooperation with NK cells and macrophages.     

Ischemic heart disease and stroke mortality in African-American, Hispanic, and non-Hispanic white men and women, 1985 to 1991

Electrochemiluminescence (ECL) of 200 ppm 2,3-diaminonaphthalene (2,3-DAN) was studied alone and in conjunction with 100 ppm of 34 different metal and non-metal ions and revealed three relatively intense ECL responses from interactions of 2,3-DAN with Au+, Fe+3 and V+5. ECL responses from Cr+6 or Ru+3 with 2,3-DAN were less intense, but noteworthy, as was the coloured fluorescent product of the non-metal ion Se+4 interaction with 2,3-DAN. Several intense 2,3-DAN-metal ion ECL reactions were studied in greater detail and revealed various titration curves with ionic detection limits in the low ppm range, using a fixed level (200 ppm) of 2,3-DAN.