Acute and chronic cold exposure differentially affect cardiac control, but not cardiorespiratory function, in resting Atlantic salmon (Salmo salar).

No studies have examined the effects of cold temperatures (∼0-1 °C) on in vivo cardiac function and control, and metabolism, in salmonids. Thus, we examined: 1) how acclimation to 8 °C vs. acclimation (>3 weeks) or acute exposure (8-1 °C at 1 °C h-1) to 1 °C influenced cardiorespiratory parameters in resting Atlantic salmon; and 2) if/how the control of cardiac function was affected. Oxygen consumption ( M ˙ O 2 ) and cardiac function [i.e., heart rate (f H) and cardiac output ( Q ˙ ) ] were 50% lower in the acutely cooled and 1oC-acclimated salmon as compared to 8 °C fish, whereas stroke volume (VS) was unchanged. Intrinsic f H was not affected by whether the fish were acutely exposed or acclimated to 1 °C (values ∼51, 24 and 21 beats min-1 in 8 and 1 °C-acclimated fish, and 8-1 °C fish, respectively), and in all groups f H was primarily under adrenergic control/tone (cholinergic tone 13-18%; adrenergic tone 37-70%). However, ß-adrenergic blockade resulted in a 50% increase in VS in the 1oC-acclimated group, and this was surprising as circulating catecholamine levels were ∼1-3 nM in all groups. Overall, the data suggest that this species has a limited capacity to acclimate to temperatures approaching 0 °C. However, we cannot exclude the possibility that cardiac and metabolic responses are evoked when salmon are cooled to ∼ 0-1 °C, and that this prevented further declines in these parameters (i.e., they 'reset' quickly). Our data also provide further evidence that VS is temperature insensitive, and strongly suggest that changes in adrenoreceptor mediated control of venous pressure/capacitance occur when salmon are acclimated to 1 °C.

Detection of condensed-phase explosives via laser-induced vaporization, photodissociation, and resonant excitation.

We investigate the remote detection of explosives via a technique that vaporizes and photodissociates the condensed-phase material and detects the resulting vibrationally excited NO fragments via laser-induced fluorescence. The technique utilizes a single 7 ns pulse of a tunable laser near 236.2 nm to perform these multiple processes. The resulting blue-shifted fluorescence (226 nm) is detected using a photomultiplier and narrowband filter that strongly block the scatter of the pump laser off the solid media while passing the shorter wavelength photons. Various nitro-bearing compounds, including 2,6-dinitrotoluene (DNT), 2,4,6-trinitrotoluene (TNT), pentaerythritol tetranitrate (PETN), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) were detected with a signal-to-noise of 25 dB. The effects of laser fluence, wavelength, and sample morphology were examined.

The immunogenicity of a conformationally restricted peptide mimetic of meningococcal lipooligosaccharide.

Life-threatening meningitis and septicaemia caused by Neisseria meningitidis are a public health priority, and their prevention by vaccination is a major objective. Meningococcal capsular polysaccharide-based vaccines are effective against the major invasive serogroups, except for serogroup B, the capsule of which mimics human polysaccharides and is poorly immunogenic. An alternative vaccine candidate that has the potential to offer cross-protection against antigenically diverse meningococci is the lipooligosaccharide (LOS). The structurally constrained peptide mimetic, C22, of a bactericidal antibody epitope within LOS was previously shown to elicit cross-reactive antibodies to meningococcal LOS when complexed to NeutrAvidintrade mark as a carrier protein. The immunogenicity of this antigen in H-2(d) (BALB/c) and H-2(k) (C3H/HeN) haplotype mice was further investigated. Anti-LOS immunoglobulin G (IgG) antibody titres increased with the vaccine dose and correlated with the anti-C22 peptide antibody titres in both haplotypes. Antigen-stimulated Th1/Th2 cytokine secretion by splenocytes and antibody isotypes indicated a Th2-type immune response with IgG1 antibodies and a low titre of IgG2b. There was no serum bactericidal activity observed against the meningococcus.

Does nitric oxide contribute to the basal vasodilation of pregnancy in conscious rabbits?

Pregnancy produces marked systemic vasodilation, but the mechanism is unknown. Experiments were performed in conscious rabbits to test the hypotheses that increased nitric oxide (NO) production contributes to the increased vascular conductance, but that the contribution varies among vascular beds. Rabbits were instrumented with aortic and vena caval catheters and ultrasonic flow probes implanted around the ascending aorta, superior mesenteric artery, terminal aorta, and/or a femoral artery. Hemodynamic responses to intravenous injection of N(omega)-nitro-L-arginine (L-NA; 20 mg/kg or increasing doses of 2, 5, 10, 15, and 20 mg/kg) were determined in rabbits first before pregnancy (NP) and then at the end of gestation (P). L-NA produced similar increases in arterial pressure between groups, but the following responses were larger (P < 0.05) when the rabbits were pregnant: 1) decreases in total peripheral conductance [-3.7 +/- 0.3 (NP), -5.0 +/- 0.5 (P) ml x min(-1) x mmHg(-1)], 2) decreases in mesenteric conductance [-0.47 +/- 0.05 (NP), -0.63 +/- 0.07 (P) ml x min(-1) x mmHg(-1)], 3) decreases in terminal aortic conductance [-0.43 +/- 0.05 (NP), -0.95 +/- 0.19 ml x min(-1) x mmHg(-1) (P)], and 4) decreases in heart rate [-41 +/- 4 (NP), -62 +/- 5 beats/min (P)]. Nevertheless, total peripheral and terminal aortic conductances remained elevated in the pregnant rabbits (P < 0.05) after L-NA. Furthermore, decreases in cardiac output and femoral conductance were not different between the reproductive states. We conclude that the contribution of NO to vascular tone increases during pregnancy, but only in some vascular beds. Moreover, the data support a role for NO in the pregnancy-induced increase in basal heart rate. Finally, unknown factors in addition to NO must also underlie the basal vasodilation observed during pregnancy.

Stabilized plasmid-lipid particles for systemic gene therapy.

The structure of 'stabilized plasmid-lipid particles' (SPLP) and their properties as systemic gene therapy vectors has been investigated. We show that SPLP can be visualized employing cryo-electron microscopy to be homogeneous particles of diameter 72 +/- 5 nm consisting of a lipid bilayer surrounding a core of plasmid DNA. It is also shown that SPLP exhibit long circulation lifetimes (circulation half-life >6 h) following intravenous (i.v.) injection in a murine tumor model resulting in accumulation of up to 3% of the total injected dose and concomitant reporter gene expression at a distal (hind flank) tumor site. In contrast, i v. injection of naked plasmid DNA or plasmid DNA-cationic liposome complexes did not result in significant plasmid delivery to the tumor site or gene expression at that site. Furthermore, it is shown that high doses of SPLP corresponding to 175 microg plasmid per mouse are nontoxic as assayed by monitoring serum enzyme levels, whereas i.v. injection of complexes give rise to significant toxicity at dose levels above 20 microg plasmid per mouse. It is concluded that SPLP exhibit properties consistent with potential utility as a nontoxic systemic gene therapy vector.

Stabilized plasmid-lipid particles for regional gene therapy: formulation and transfection properties.

Previous work (Wheeler et al, Gene Therapy 1999; 6: 271-281) has shown that plasmid DNA can be entrapped in 'stabilized plasmid-lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). These SPLP exhibit low transfection potencies in vitro, due in part to the long residence time of the PEG-CerC20 on the SPLP surface. In this work we employed SPLP stabilized by PEG attached to ceramide containing an octanoyl acyl group (PEG-CerC8), which is able to quickly exchange out of the SPLP, to develop systems that give rise to optimized in vitro and in vivo (regional) transfection. A particular objective was to achieve cationic lipid contents that give rise to maximum transfection levels. It is shown that by performing the dialysis procedure in the presence of increasing concentrations of citrate, SPLP containing up to 30 mol% of the cationic lipid dioleoydimethylammonium chloride (DODAC) could be generated. The SPLP produced could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution (62 +/- 8 nm, as determined by freeze-fracture electron microscopy) and a high plasmid-to-lipid ratio of 65 microg/micromol (corresponding to one plasmid per particle) regardless of the DODAC content. It was found that isolated SPLP containing 20-24 mol% DODAC resulted in optimum transfection of COS-7 and HepG2 cells in vitro, with luciferase expression levels comparable to those achieved for plasmid DNA-cationic lipid complexes. In vivo studies employing an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP also demonstrated maximum luciferase expression for DODAC contents of 20-24 mol% and significantly improved gene expression in tumor tissue as compared with complexes. We conclude that SPLP stabilized by PEG-CerC8 and containing 20-24 mol% cationic lipid are attractive alternatives to plasmid DNA-cationic lipid complexes for regional gene therapy applications.

Multilayers of cells growing on a permeable support. An in vitro tumour model.

A system for growing three-dimensional cell culture has been developed which exhibits many features of solid tumours. This system comprises cells growing as a thick mat on a semipermeable membrane suspended in stirred media. SiHa cells grown as these multilayered cell cultures (MCCs) have produced cultures up to ca. 20 cell diameters in thickness. The MCCs, like solid tumours growing in vivo. develop diffusion-dependent necrosis and hypoxia and the cell packing acts as a barrier to the diffusion of drugs. These cultures can, therefore, be used to study aspects of cancer biology and drug transport that are difficult to study using other techniques.

The effect of thalidomide on experimental tumors and metastases.

Thalidomide has recently been shown to antagonize basic fibroblast growth factor-induced angiogenesis in the rat corneal micropocket assay. We have investigated the effect of thalidomide on growth, radiosensitivity and metastasis in murine SCCVII and Lewis Lung tumors. We found that daily thalidomide administration (0.77 mmol/kg/day, i.p.) does not alter primary tumor growth of SCCVII or Lewis Lung tumors. However, thalidomide administration does reduce radiosensitivity of the Lewis Lung tumor, and increases its sensitivity to combined treatment with radiation and the bioreductive cytotoxin tirapazamine. These findings suggest that thalidomide is elevating tumor hypoxia in the Lewis Lung tumor, presumably via an anti-angiogenic mechanism. We also found that thalidomide administration reduces the incidence of lung metastases from primary Lewis Lung tumors. Thalidomide may therefore have utility in the management of solid tumors, especially when combined with drugs that are selectively toxic to cells at reduced oxygen tension (e.g. bioreductive cytotoxins).

An empirical study of the influence of demographic variable on the choice criteria for assisted living facilities.

Despite the growth and prevalence of assisted-living facilities, empirical marketing research for these facilities is scarce. The objectives of this study were to determine the relative importance of three sets of evaluation criteria in the initial selection of an assisted-living facility, and determine whether the relative importance of these three sets of criteria differed by gender, marital status, level of household, age, or income of the consumer. Survey responses from 279 households indicates that primary service criteria are relatively more important than facilities amenities or organized social activities in the initial selection of an assisted-living facility. The relative importance of the choice criteria differed markedly by gender of the consumer, but marital status, level of household income, and age of the consumer did not have as great an impact on consumers' choice criteria.

Patient expectations of dental services. Image affects expectations, and expectations affect perceived service quality.

The authors construct a theoretical model of the antecedents of expectations for dental services by analyzing survey responses from 240 dental patients. The patients' image of the dentist, tangible cues, situational factors, and patient satisfaction with prior service encounters have the greatest influence on expectations of service, whereas marketing variables, such as price and advertising, appear to have no effect.

Pharmacological but not physiological modulation of cortical acetylcholine release by cholinergic mechanisms in the nucleus basalis magnocellularis.

The role of muscarinic transmission in the activation of cholinergic neurons ascending to the neocortex from the nucleus basalis magnocellularis (NBM) was investigated. The release of acetylcholine (ACh) from the neocortex of urethane-anesthetized rats was measured using microdialysis, and a second microdialysis probe was inserted into the NBM to apply drugs to the NBM and to measure ACh release from this area. Cholinergic neurons in the NBM were activated synaptically by stimulating the pedunculopontine tegmentum (PPT). Systemically administered scopolamine greatly increased the PPT stimulation evoked cortical release of ACh when the cortical probe was perfused with the cholinesterase inhibitor neostigmine. PPT stimulation evoked release was also high when the cortical probe was perfused with atropine plus neostigmine, but it was not increased any further by systemic scopolamine or by scopolamine perfused through the NBM probe. When neostigmine was perfused through the NBM probe, PPT stimulation evoked cortical ACh release was halved, but the release was restored when the NBM solution also contained scopolamine. The resting release of ACh within the NBM was increased by local neostigmine, but evoked release in the NBM was large only in the presence of local scopolamine. Both of these increases were blocked by perfusion of the NBM with tetrodotoxin. These results suggest that muscarinic transmission within the NBM does not control the activation of cholinergic neurons under physiological conditions, when the diffusion of ACh is limited by its hydrolysis. However, when ACh is allowed to diffuse to a wider area, it may inhibit the release of an excitatory transmitter, probably glutamate, via presynaptic muscarinic receptors.

Modification of neocortical acetylcholine release and electroencephalogram desynchronization due to brainstem stimulation by drugs applied to the basal forebrain.

Acetylcholine released from the cerebral cortex was collected using microdialysis while stimulating the region of the pedunculopontine tegmentum in urethane-anesthetized rats. Electrical stimulation in the form of short trains of pulses delivered once per minute produced a 350% increase in acetylcholine release and a desynchronization of the electroencephalogram, as measured by relative power in the 20-45 Hz range (low-voltage fast activity). Perfusion of the region of cholinergic neurons believed to be responsible for the cortical release of acetylcholine, the nucleus basalis magnocellularis, was carried out using a second microdialysis probe. Exposure of the nucleus basalis magnocellularis to blockers of neural activity (tetrodotoxin or procaine) or to blockers of synaptic transmission (calcium-free solution plus magnesium or cobalt) produced a substantial decrease in the release of acetylcholine and desynchronization evoked by brainstem stimulation. Exposure of the nucleus basalis magnocellularis to the glutamate antagonist, kynurenate, resulted in a decrease in evoked acetylcholine release and electroencephalogram desynchronization similar in magnitude to that produced by nonspecific blockers, whereas application of muscarinic or nicotinic cholinergic blockers to the nucleus basalis magnocellularis did not reduce acetylcholine release or electroencephalogram desynchronization. Application of tetrodotoxin to the collection site in the cortex abolished the stimulation-evoked acetylcholine release, but not the low baseline release indicating that cholinergic nucleus basalis magnocellularis neurons have a low spontaneous firing rate in urethane-anesthetized animals. The results of this study suggest that the major excitatory input to the cholinergic neurons of the nucleus basalis magnocellularis from the pedunculopontine tegmentum is via glutamatergic and not cholinergic synapses.

Frequency-dependent increase in cortical acetylcholine release evoked by stimulation of the nucleus basalis magnocellularis in the rat.

Acetylcholine was collected from the somatosensory cortex of anesthetized rats, using the microdialysis technique. Electrical stimulation of the nucleus basalis magnocellularis (NBM) with trains of 10 pulses at 100 Hz delivered every second produced a 3-4-fold increase in acetylcholine release. Stimulation with an intratrain frequency of 10, 50, 100 or 200 Hz demonstrated that 100 Hz trains produced the greatest increase, while the other frequencies were about half as effective. The cortical release of acetylcholine in this paradigm supports the hypothesis that the previously demonstrated enhancement by NBM stimulation of cortical sensory inputs is due to cholinergic activation.