Mutations within the selectivity filter of the NMDA receptor-channel influence voltage dependent block by 5-hydroxytryptamine

Background and purpose: Voltage-dependent block by Mg2+ is a cardinal feature of NMDA receptors which acts as a coincidence detector to prevent the receptor from over-activation. Inhibition of NMDA receptor currents by 5-hydroxytryptamine (5-HT) indicated that 5-HT, similar to Mg2+, binds within the membrane electric field. In the present study, we assessed whether point mutations of critical asparagine residues located within the selectivity filter of NR1 and NR2A subunits of NMDA receptor-channel affect voltage-dependent block by 5-HT. Experimental approach: The mode of action of 5-HT and Mg2+ on wild-type and mutated NMDA receptor-channels expressed in Xenopus oocytes was investigated using the two-electrode voltage clamp recording technique. Key results: The mutation within the NR1 subunit NR1(N0S or N0Q) strongly reduced the voltage dependent block by 5-HT and increased the IC50. The corresponding mutations within the NR2 subunits NR2A(N0Q or N + 1Q) reduced the block by 5-HT to a lesser extent. This is in contrast to the block produced by external Mg2+ where a substitution at the NR2A(N0) and NR2A(N + 1) sites but not at the NR1(N0) site significantly reduced Mg2+ block. Conclusion and implications: The block of NMDA receptor-channels by 5-HT depends on the NR1-subunit asparagine residue and to a lesser extent on the NR2A-subunit asparagine residues. These data suggest that the interaction of 5-HT with functionally important residues in a narrow constriction of the pore of the NMDA receptor-channel provides a significant barrier to ionic fluxes through the open channel due to energetic factors governed by chemical properties of the binding site and the electric field.

Modulation of channel activity and gadolinium block of MscL by static magnetic fields

The magnetic field of the Earth has for long been known to influence the behaviour and orientation of a variety of living organisms. Experimental studies of the magnetic sense have, however, been impaired by the lack of a plausible cellular and/or molecular mechanism providing meaningful explanation for detection of magnetic fields by these organisms. Recently, mechanosensitive (MS) ion channels have been implied to play a role in magnetoreception. In this study we have investigated the effect of static magnetic fields (SMFs) of moderate intensity on the activity and gadolinium block of MscL, the bacterial MS channel of large conductance, which has served as a model channel to study the basic physical principles of mechanosensory transduction in living cells. In addition to showing that direct application of the magnetic field decreased the activity of the MscL channel, our study demonstrates for the first time that SMFs can reverse the effect of gadolinium, a well-known blocker of MS channels. The results of our study are consistent with a notion that (1) the effects of SMFs on the MscL channels may result from changes in physical properties of the lipid bilayer due to diamagnetic anisotropy of phospholipid molecules and consequently (2) cooperative superdiamagnetism of phospholipid molecules under influence of SMFs could cause displacement of Gd3+ stop ions from the membrane bilayer and thus remove the MscL channel block.

The effect of caudal block on functional residual capacity and ventilation homogeneity in healthy children

Caudal block results in a motor blockade that can reduce abdominal wall tension. This could interact with the balance between chest wall and lung recoil pressure and tension of the diaphragm, which determines the static resting volume of the lung. On this rationale, we hypothesised that caudal block causes an increase in functional residual capacity and ventilation distribution in anaesthetised children. Fifty-two healthy children (15-30 kg, 3-8 years of age) undergoing elective surgery with general anaesthesia and caudal block were studied and randomly allocated to two groups: caudal block or control. Following induction of anaesthesia, the first measurement was obtained in the supine position (baseline). All children were then turned to the left lateral position and patients in the caudal block group received a caudal block with bupivacaine. No intervention took place in the control group. After 15 nun in the supine position, the second assessment was performed. Functional residual capacity and parameters of ventilation distribution were calculated by a blinded reviewer. Functional residual capacity was similar at baseline in both groups. In the caudal block group, the capacity increased significantly (p < 0.0001) following caudal block, while in the control group, it remained unchanged. In both groups, parameters of ventilation distribution were consistent with the changes in functional residual capacity. Caudal block resulted in a significant increase in functional residual capacity and improvement in ventilation homogeneity in comparison with the control group. This indicates that caudal block might have a beneficial effect on gas exchange in anaesthetised, spontaneously breathing preschool-aged children with healthy lungs.

The prospects for immunotherapy in smoking cessation

Context Smoking is a major preventable cause of death and disability that is maintained by dependence on nicotine. Smoking cessation reduces mortality and morbidity. Although existing pharmacological aids to smoking cessation and relapse prevention (nicotine replacement therapy and bupropion) improve on unassisted quitting and behavioural methods, they are only modestly effective. More effective pharmacological methods are required that improve compliance, reduce side-effects, and can be used in combination with existing cessation methods. Starting point A nicotine vaccine is a promising immunotherapeutic approach to smoking cessation and relapse prevention. Such a vaccine would induce the immune system to form specific antibodies to nicotine to prevent it from crossing the blood-brain barrier to act on receptor sites in the central nervous system. Recent studies in rats provide proof of principle by showing that nicotine-specific antibodies can prevent the reinstatement of nicotine self-administration (N Lindblom et al, Respiration 2002; 69: 254–60) and block dopamine release in the shell of the nucleus accumbens (Sde Villiers et al, Respiration 2002; 69: 247–53). A phase 1 trial of a human cocaine vaccine has also recently been successfully completed (T Kosten et al, Vaccine 2002; 20: 1196–204). A safe and effective human nicotine vaccine would potentially have fewer side-effects and better compliance than existing smoking-cessation pharmacotherapies. It could also be used in combination with some of them (eg, bupropion). Where next? The most promising clinical application of a human nicotine vaccine is likely to be in relapse prevention in abstinent smokers. A vaccine may also have a role in preparing smokers to quit. Clinical trials of safety and efficacy in human smokers and ex-smokers are warranted. If a nicotine vaccine proves to be safe and effective, the health-care system will need to ensure that it is registered for clinical use and that the poorer members of the community (among whom smoking prevalence is now highest in developed countries) have access to the vaccine. The community will need to be appropriately informed about the role of a nicotine vaccine to ensure that it is not prematurely used for preventive purposes in children and adolescents.

A randomized and controlled trial of a participative ergonomics intervention to reduce injuries associated with manual tasks: physical risk and legislative

A participative ergonomics approach to reducing injuries associated with manual tasks is widely promoted; however only limited evidence from uncontrolled trials has been available to support the efficacy of such an approach. This paper reports on a randomized and controlled trial of PErforM, a participative ergonomics intervention designed to reduce the risks of injury associated with manual tasks. One hundred and seventeen small to medium sized food, construction, and health workplaces were audited by government inspectors using a manual tasks risk assessment tool (ManTRA). Forty-eight volunteer workplaces were then randomly assigned to Experimental and Control groups with the Experimental group receiving the PErforM program. Inspectors audited the workplaces again, 9 months following the intervention. The results showed a significant decrease in estimates of manual task risk and suggested better legal compliance in the Experimental group.

A review of the use of the basic cation saturation ratio and the "ideal" soil

The use of 'balanced' Ca, Mg, and K ratios, as prescribed by the basic cation saturation ratio (BCSR) concept, is still used by some private soil-testing laboratories for the interpretation of soil analytical data. This review aims to examine the suitability of the BCSR concept as a method for the interpretation of soil analytical data. According to the BCSR concept, maximum plant growth will be achieved only when the soil’s exchangeable Ca, Mg, and K concentrations are approximately 65 % Ca, 10 % Mg, and 5 % K (termed the ‘ideal soil’). This ‘ideal soil’ was originally proposed by Firman Bear and co-workers in New Jersey (USA) during the 1940s as a method of reducing luxury K uptake by alfalfa (Medicago sativa L.). At about the same time, William Albrecht, working in Missouri (USA), concluded through his own investigations that plants require a soil with a high Ca saturation for optimal growth. Whilst it now appears that several of Albrecht’s experiments were fundamentally flawed, the BCSR (‘balanced soil’) concept has been widely promoted, suggesting that the prescribed cationic ratios provide optimum chemical, physical, and biological soil properties. Our examination of data from numerous studies (particularly those of Albrecht and Bear, themselves) would suggest that, within the ranges commonly found in soils, the chemical, physical, and biological fertility of a soil is generally not influenced by the ratios of Ca, Mg, and K. The data do not support the claims of the BCSR, and continued promotion of the BCSR will result in the inefficient use of resources in agriculture and horticulture.

hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA

Segregation of mRNAs in the cytoplasm of polar cells has been demonstrated for proteins involved in Xenopus and Drosophila oogenesis, and for some proteins in somatic cells. It is assumed that vectorial transport of the messages is generally responsible for this localization. The mRNA encoding the basic protein of central nervous system myelin is selectively transported to the distal ends of the processes of oligodendrocytes, where it is anchored to the myelin membrane and translated. This transport is dependent on a 21-nucleotide cis-acting segment of the 3'-untranslated region (RTS). Proteins that bind to this cis-acting segment have now been isolated from extracts of rat brain. A group of six 35-42-kDa proteins bind to a 35-base oligoribonucleotide incorporating the RTS, but not to several oligoribonucleotides with the same composition but randomized sequences, thus establishing specificity for the base sequence in the RTS. The most abundant of these proteins has been identified, by Edman sequencing of tryptic peptides and mass spectroscopy, as heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a 36-kDa member of a family of proteins that are primarily, but not solely, intranuclear. This protein was most abundant in samples from rat brain and testis, with lower amounts in other tissues. It was separated from the other polypeptides by using reverse-phase HPLC and shown to retain preferential association with the RTS. In cultured oligodendrocytes, hnRNP A2 was demonstrated by confocal microscopy to be distributed throughout the nucleus, cell soma, and processes.

Type 1 and Type 2 cytokines: from basic science to fungal infections

At the present time, it is clear that Th1 responses afford protection against the fungi; however, the development, maintenance and function of the protective immune responses are complex mechanisms and are influenced by multiple factors. The route of infection has been shown to affect initial cytokine production and, consequently, the induction of protective Th1 responses. The ability of different isolates of the same fungal agent to induce and sustain a protective response has also been emphasized. Protective immune responses have been shown to vary in genetically different mouse strains after infection. In addition, these protective responses, such as cellular influx and cytokine production, also vary within the same animal depending on the tissue infected. The functional dominance of certain cytokines over others in influencing development and maintenance of protective responses has been discussed. Certain cytokines may act differently in hosts lacking important components of their innate or immune repertoire. It is evident from these presentations that a more comprehensive understanding of the protective mechanisms against different fungal agents is emerging. However, there is still much to learn before cytokine modulatory therapy can be used effectively without risk in the human host.

TFEC is a macrophage-restricted member of the microphthalmia-TFE subfamily of basic helix-loop-helix leucine zipper transcription factors

The murine homologue of the TFEC was cloned as part of an analysis of the expression of the microphthalmia-TFE (MiT) subfamily of transcription factors in macrophages. TFEC, which most likely acts as a transcriptional repressor in heterodimers with other MiT family members, was identified in cells of the mononuclear phagocyte lineage, coexpressed,vith all other known MiT subfamily members (Mitf, TFE3, TFEB), Northern blot analysis of several different cell lineages indicated that the expression of murine TFEC (mTFEC) was restricted to macrophages. A 600-bp fragment of the TATA-less putative proximal promoter of TFEC shares features with many known macrophage-specific promoters and preferentially directs luciferase expression in the RAW264.7 macrophage cell line in transient transfection assays. Five of six putative Ets motifs identified in the TFEC promoter bind the macrophage-restricted transcription factor PU,I under in vitro conditions and in transfected 3T3 fibroblasts; the minimal luciferase activity of the TFEC promoter could be induced by coexpression of PU.1 or the related transcription factor Ets-2. The functional importance of the tissue-restricted expression of TFEC and a possible role in macrophage-specific gene regulation require further investigation, but are likely to be linked to the role of the other MiT family members in this lineage.

Both CD4(+) and CD8(+) lymphocytes reduce the severity of tissue lesions in murine systemic candidiasis, and CD4(+) cells also demonstrate strain-specific immunopathological effects

The role of T lymphocytes in host responses to sublethal systemic infection with Candida albicans was evaluated by mAb depletion of CD4(+) and CD8(+) cells from BALB/c and CBA/CaH mice, which develop mild and severe tissue damage, respectively. Depletion of CD4(+) lymphocytes from BALB/c mice markedly increased tissue damage, but did not alter the course of infection. In CBA/CaH mice, depletion of CD4+ cells abrogated tissue destruction in both brain and kidney at day 4 after infection, and significantly decreased fungal colonization in the brain. However, the severity of tissue lesions increased relative to controls from day 8 onwards. A small increase in tissue damage was evident in both mouse strains after depletion of CD8(+) cells. There were no major differences between days 4 end 8 after infection in cDNA cytokine profiles of CD4(+) lymphocytes from either BALB/c or CBA/CaH mice. After passive transfer into infected syngeneic recipients, spleen cells from infected CBA/CaH mice markedly increased tissue damage when compared to controls, and also caused a significant increase in fungal colonization in the brain. A similar transfer in BALB/c mice increased the number of inflammatory cells in and around the lesions, but had no effect on the fungal burden in brain and kidney. The data demonstrate that both CD4(+) and CD8(+) lymphocytes contribute to the reduction of tissue damage after systemic infection with C. albicans, and that the development and expression of CD4(+) lymphocyte effector function is influenced by the genetic background of the mouse.

The line structure of balanced ternary designs with block size three, index three and rho(2) = 1, 2

In this paper necessary and sufficient conditions for a vector to be the fine structure of a balanced ternary design with block size 3, index 3 and rho(2) = 1 and 2 are determined with one unresolved case.

Magnetic resonance imaging (MRI) and diseases of the liver and biliary tract. Part 1. Basic principles, MRI in the assessment of diffuse and focal hepatic disease

Magnetic resonance imaging (MRI) relies on the physical properties of unpaired protons in tissues to generate images. Unpaired protons behave like tiny bar magnets and will align themselves in a magnetic field. Radiofrequency pulses will excite these aligned protons to higher energy states. As they return to their original state, they will release this energy as radio waves. The frequency of the radio waves depends on the local magnetic field and by varying this over a subject, it is possible to build the images we are familiar with. In general, MRI has not been sufficiently sensitive or specific in the assessment of diffuse liver disease for clinical use. However, because of the specific characteristics of fat and iron, it may be useful in the assessment of hepatic steatosis and iron overload. Magnetic resonance imaging is useful in the assessment of focal liver disease, particularly in conjunction with contrast agents. Haemangiomas have a characteristic bright appearance on T-2 weighted images because of the slow flowing blood in dilated sinusoids. Focal nodular hyperplasia (FNH) has a homogenous appearance, and enhances early in the arterial phase after gadolinium injection, while the central scar typically enhances late. Hepatic adenomas have a more heterogenous appearance and also enhance in the arterial phase, but less briskly than FNH. Hepatocellular carcinoma is similar to an adenoma, but typically occurs in a cirrhotic liver and has earlier washout of contrast. The appearance of metastases depends on the underlying primary malignancy. Overall, MRI appears more sensitive and specific than computed tomography with contrast for the detection and evaluation of malignant lesions. (C) 2000 Blackwell Science Asia Pty Ltd.