Comparison of single cell sensitivities to acetone, 1-octen-3-ol and 3-methylphenol in the riverine tsetse species Glossina fuscipes fuscipes and G. palpalis palpalis.

Action potentials from individual cells were recorded from antennae (funiculi) of living tsetse flies, Glossina p. palpalis and Glossina f. fuscipes using a "surface-contact" recording technique. Stimuli were vapours of 1-octen-3-ol, acetone and 3-methylphenol. Of the 101 and 128 olfactory cells tested for their sensitivity to odour stimuli in G. p. palpalis and G. f. fuscipes, respectively, the majority (83 and 77%) were activated by more than one chemical. The numbers of these "generalist" cells were 20 and 15% higher in females than in males. Response intensity increased with increasing odour dose. Temporal patterns of excitation were phasic-tonic and showed cells with relatively rapid cessation of spike activity after the end of stimulation and cells which continued firing for several seconds or even minutes after stimulation. Inhibition by odours only occurred in a minority of cells and was dose-dependent. For each of the three substances the excitatory response was significantly higher in G. f. fuscipes than in G. p. palpalis, whereas no significant differences between inhibitory responses were found. In G. f. fuscipes each stimulus evoked equal excitatory responses. In G. p. palpalis, however, acetone induced significantly higher responses than 1-octen-3-ol and 3-methylphenol. Response intensities to each of the three chemicals did not differ between male and female G. p. palpalis, whereas in G. f. fuscipes 1-octen-3-ol evoked significantly higher responses in males. Possible mechanisms of receptor cell odour coding and behavioural effects of the various cell type activities are discussed.

Diagnosis and treatment of (disease-related) in-hospital malnutrition: the performance of medical and nursing staff.

BACKGROUND & AIMS: Malnutrition continues to be an important problem in health care which is still under recognized and underrated in developed countries. This study aims to describe current practice in diagnosing and treating malnutrition by medical doctors, medical students and nurses prior, during and after hospitalisation. METHODS: Prospective analysis of current practice in assessing nutritional status and prescribing treatment by medical and nursing staff in a cohort of hospitalised patients from the general medical wards of the VU University Medical Center, Amsterdam. Comparison of objective identification of malnutrition by an independent observer with subjective identification by the medical and nursing staff. Quantification of diagnosing, treating and communicating malnutrition before, during and following hospital stay by medical doctors, medical students and nurses by evaluating the written information in medical and nursing charts, and referral and discharge letters. RESULTS: Three hundred and ninety-five women and men, aged 19-96 years, were included from June to September 2005. The prevalence of malnutrition was 31.9%. Nutritional information was not mentioned in written referrals. Medical doctors performed nutritional assessment in 15.3%, medical students in 52.8%, and nurses in 29.9% of their patients. Medical doctors were the most capable of differentiating between malnourished and well-nourished patients as a basis for undertaking nutritional assessment, although this was still inadequate. Little nutritional intervention was applied during hospital stay. Information on nutritional status was lacking in most discharge letters. Nutritional follow-up was appointed in 1.2%. CONCLUSIONS: Nutritional assessment and intervention were not sufficiently applied by any professional at any stage of the pre-, actual and post-hospitalisation period.

Background odour induces adaptation and sensitization of olfactory receptors in the antennae of houseflies.

The presence of background odour was found to have a small but significant effect on the sensitivity of the antennal olfactory system of houseflies, Musca domestica Linnaeus (Diptera: Muscidae), to new pulses of odour. We show that cross-adaptation and cross-sensitization between a background odour of (+/-)-1-octen-3-ol and pulses of (+/-)-1-octen-3-ol, 2-pentanone and R-(+)-limonene can occur, confirming that olfactory receptor cells are sensitive to different odours. Background odour can increase the responses to low concentration odour pulses and decrease the responses to higher concentration odour pulses. It is suggested that background odour has a larger effect on olfactory receptor cells that respond with a tonic increase of spike frequency, giving information about the level of odour concentration, i.e. the 'static' environment. Cells that respond in a phasic way only provide information on the dynamics of the olfactory environment.

Electrophysiological characterization of olfactory cell types in the antennae and palps of the housefly.

A set of odours was presented to the housefly Musca domestica and the electrophysiological responses of single olfactory receptor cells in the antennae and palps were recorded. The olfactory cells in the antennae of the housefly showed a large variability of response profiles, but multidimensional cluster analysis suggested a moderate clustering in olfactory response types. Receptor cells with similar or with different odour response profiles can reside in one and the same sensillum. No fixed spatial distribution of olfactory response types over the antennal of palpal surface was found. The odours of 1-octen-3-ol, amyl acetate, 3-methylphenol, 2-pentanone and R(+)limonene elicited the largest responses in antennal cells. Most odours elicited responses in cells of only a few of the clusters, but 1-octen-3-ol was detected by cells of almost all clusters of the antenna. Surprisingly, rather low responses were found to acetic acid, skatole, indole and muscalure, odours that are known to attract flies. Response profiles of palpal cells differed considerably from those of antennal cells. Palpal cells mostly responded to 3-methylphenol and 2-pentanone. In the palps, the clusters of cells responding to 3-methylphenol and 2-pentanone are clearly separated from the other olfactory cells.

CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies.

The aim of this study was to find a cell culture model of the intestinal epithelium for use in studies of CYP3A4-mediated first-pass metabolism of drugs and also for studies of the interplay between CYP3A4 metabolism and P-glycoprotein efflux. For this purpose, the expression of CYP3A4, CYP3A5, and MDR1 mRNA was studied in three cell lines of the normal human intestinal epithelium and three transformed cell lines of colonic (Caco-2) origin. Surprisingly, only transformed cell lines were induced by 1alpha,25-dihydroxy vitamin D3 (D3) to express high amounts of CYP3A4. In contrast to the original findings for this model, the monolayer integrity was maintained during D3 treatment. Levels of CYP3A mRNA expression in Caco-2 and TC7 cells differed dramatically. The highest levels of CYP3A4 and lowest levels of CYP3A5 mRNA expression were observed in D3 treated Caco-2 cells of high passage numbers, resulting in a CYP3A4/3A5 expression ratio greater than fourfold higher than that seen in TC7 cells. Functional studies, using the CYP3A probe testosterone, showed that CYP3A activity was completely absent only in uninduced Caco-2 cells. After D3 induction, high levels of the metabolite were produced in both cell lines (149.4 +/- 12.3 and 86.5 +/- 3.8 pmol 6beta-OH testosterone/min/mg cellular protein from 75 microM testosterone in Caco-2 and TC7 cells, respectively). The maximum velocity (Vmax) and the apparent Michaelis constant (Km) for the 6beta-hydroxylation of testosterone by CYP3A4 in intact Caco-2 monolayers were similar to those obtained from human intestinal microsomes. Only minor changes in P-glycoprotein activity were observed when the metabolically stable P-glycoprotein substrate celiprolol was used. In conclusion, these results show that the features of the generally available Caco-2 cell line from American Type Culture Collection make it suitable for studies of CYP3A4-mediated first-pass metabolism and also for studies of the interplay between CYP3A4 and drug efflux mechanisms.

Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase.

P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport. Analysis of the structure and function of P-type ATPases has been limited by the lack of active recombinant ATPases in quantities suitable for crystallographic studies aiming at solving their three-dimensional structure. We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2, equipped with a His(6)-tag, in the yeast Saccharomyces cerevisiae. The H+-ATPase could be purified both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation of the H+-ATPase/14-3-3 protein complex. Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-ATPase polypeptide. The purified H(+)-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles. Electron cryo-microscopy of the crystals yielded a projection map at approximately 8 A resolution, the p22(1)2(1) symmetry of which suggests a dimeric protein complex. Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2.

Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43.

On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.

Characterization and functional analysis of two common human cytochrome P450 1B1 variants.

Cytochrome P450 1B1 (CYP1B1) is a human extrahepatic P450 that activates procarinogens, metabolizes 17beta-estradiol, and may well have a role in the pathogenesis of some forms of cancer. Besides rare deleterious mutations reported for the CYP1B1 gene, six single-nucleotide polymorphisms have been reported, of which four cause amino acid exchanges. We have expressed two of the common CYP1B1 alleles in yeast cells and mammalian COS-1 cells in order to functionally characterize the alleles with respect to kinetic properties and protein stability. The CYP1B1.2 variant contains the two linked amino acid substitutions R48G and A119S compared to CYP1B1.1. The kinetic parameters of two structurally unrelated CYP1B1 substrates for the two variants were examined. No kinetic differences were seen of 17beta-estradiol hydroxylation activities between the two CYP1B1 variants and an only minor increase in the apparent Km for ethoxyresorufin was observed for CYP1B1.2. It therefore appears that they have very similar catalytic properties and the substitutions do not appear to alter CYP1B1 catalytic function. The two CYP1B1 variants were similarly stable when expressed in mammalian COS-1 cells, indicating that the substitutions have no effect on protein folding or stability. The combined results indicate that these two CYP1B1 variants show very similar properties with respect to catalytic activities and protein stability and do not alter CYP1B1 function.

Arginines 97 and 108 in CYP2C9 are important determinants of the catalytic function.

Human cytochrome P450 2C9 (CYP2C9) is one of the major drug metabolising enzymes which exhibits a broad substrate specificity. The B-C loop is located in the active-site but has been difficult to model, owing to its diverse and flexible structure. To elucidate the function of the B-C loop we used homology modelling based on the Cyp102 structure in combination with functional studies of mutants using diclofenac as a model substrate for CYP2C9. The study shows the importance of the conserved arginine in position 97 and the arginine in position 108 for the catalytic function. The R97A mutant had a 13-fold higher K(m) value while the V(max) was in the same order as the wild type. The R108 mutant had a 100-fold lower activity with diclofenac compared to the wild-type enzyme. The other six mutants (S95A, F100A, L102A, E104A, R105A, and N107A) had kinetic parameters similar to the CYP2C9 wild-type. Our homology model based on the CYP102 structure as template indicates that R97, L102, and R105 are directed into the active site, whereas R108 is not. The change in catalytic function when arginine 97 was replaced with alanine and the orientation of this amino acid in our homology model indicates its importance for substrate interaction.

Olfactory responses to attractants and repellents in tsetse.

The aims of this study were to investigate how antennal olfactory cells of tsetse (Diptera: Glossinidae) code odour quality and how they are able to discriminate between attractive and repellent odours. For Glossina pallidipes Austen, a survey is presented of the cells' responses to attractive (1-octen-3-ol, acetone, 3-methylphenol, carbon dioxide) and repellent stimuli (2-methoxyphenol, acetophenone, lactic acid, naphthalene). In addition, the responses of these cells to binary mixtures and the dose-response curves of 1-octen-3-ol, 3-methylphenol, 2-methoxyphenol and acetophenone are presented. A minority of the cells responded to one attractant or repellent only, whereas the vast majority were excited by more than one of the attractive and/or repellent stimuli. It is proposed that the peripheral olfactory cells of tsetse discriminate between different compounds via an across-fibre pattern coding, in which the cells that specifically code for attractants or repellents may play a substantial role in composing a unique excitation pattern that informs the central nervous system about the specificity of odours.

Heterologous expression and kinetic characterization of human cytochromes P-450: validation of a pharmaceutical tool for drug metabolism research.

Drug metabolism studies in the early phases of drug discovery and development will improve the selection of new chemical entities that will be successful in clinical trials. To meet the expanding demands for these studies on the numerous chemicals generated through combinatorial chemistry, we have heterologously expressed nine human drug-metabolizing cytochromes P-450 (CYPs) in Saccharomyces cerevisiae. The enzymes were characterized using known marker substrates CYP1A1/1A2 (ethoxyresorufin), 2C8 (paclitaxel), 2C9 (diclofenac), 2C19 (S-mephenytoin), 2D6 (bufuralol), 2E1 (chlorzoxazone), and 3A4/3A5 (testosterone). All of the CYPs showed the expected substrate specificity except for chlorzoxazone hydroxylation, which, in addition to CYP2E1 and 1A2, was also catalyzed by CYP1A1 with a high turnover. The apparent Michaelis-Menten parameters obtained for each CYP were within the ranges of those reported in the literature using human liver microsomes and/or recombinant CYPs. The K(m) for CYP2E1-catalyzed chlorzoxazone hydroxylation was, however, much higher (177 microM) than that obtained using liver microsomes (40 microM). CYP-selective inhibitors, alpha-naphthoflavone (CYP1A1/1A2), quercetin (2C8), sulfaphenazole (2C9), quinidine (2D6), and ketoconazole (3A4/3A5) showed significant isoform-selective inhibitory effects. We have shown that ticlopidine is a potent inhibitor of CYP2C19 (IC(50) = 4. 5 microM) and CYP2D6 (IC(50) = 3.5 microM) activities. We have therefore successfully set-up and validated an "in-house" heterologous system for the production of human recombinant CYPs for use in metabolism research.

Olfactory sensitivities of mosquitoes with different host preferences (Anopheles gambiae s.s., An. arabiensis, An. quadriannulatus, An. m. atroparvus) to synthetic host odours.

Responses of antennal olfactory cells associated with sensilla trichodea were recorded in females of four Anopheles species (Diptera, Culicidae) with different host preferences: the anthropophilic An. gambiae s.s., the opportunistic An. arabiensis, and the zoophilic An. quadriannulatus and An. maculipennis atroparvus. Stimuli were vapours of synthetic host-odours: ethanoic, propanoic, butanoic, 3-methyl propanoic, 4-methyl butanoic acid, 1-octen-3-ol, and 3- and 4-methyl phenol. On stimulation with fatty acids and phenols either excitation or inhibition of spike activity was found, whereas responses to 1-octen-3-ol were invariably excitatory. The odour spectra of the cells could include activating as well as inhibiting substances. Differences in host preferences may be reflected in the numbers of olfactory cells responding to different odours and/or in the sensitivities of these cells. In An. gambiae more cells were excited by fatty acids than in An. arabiensis and An. m. atroparvus, whereas inhibition occurred more often in the latter two species. In addition, the fatty acid-excited cells in An. gambiae were more sensitive to these substances than in An. m. atroparvus and An. quadriannulatus. On the contrary, in the latter two species cells were more responsive to 1-octen-3-ol. In An. arabiensis, responses of stimulus-excited cells were intermediate between those in the anthropophilic and zoophilic species.

Neural coding in antennal olfactory cells of tsetse flies (Glossina spp.).

Spike trains from individual antennal olfactory cells of tsetse flies (Glossina spp.) obtained during steady-state conditions (spontaneous as well as during stimulation with 1-octen-3-ol) and dynamic stimulation with repetitive pulses of 1-octen-3-ol were investigated by studying the spike frequency and the temporal structure of the trains. In general, stimulation changes the intensity of the spike activity but leaves the underlying stochastic structure unaffected. This structure turns out to be a renewal process. The only independently varying parameter in this process is the mean interspike interval length, suggesting that olfactory cells of tsetse flies may transmit information via a frequency coding. In spike records with high firing rates, however, the stationary records had significant negative first-order serial correlation coefficients and were non-renewal. Some cells in this study were capable of precisely encoding the onset of the odour pulses at frequencies up to at least 3 Hz. Cells with a rapid return to pre-stimulus activity at the end of stimulation responded more adequately to pulsed stimuli than cells with a long increased spike frequency. While short-firing cells process information via a frequency code, long-firing cells responded with two distinctive phases: a phasic, non-renewal response and a tonic, renewal response which may function as a memory of previous stimulations.

Olfactory sensitivity in tsetse flies: a daily rhythm.

The diurnal tsetse Glossina morsitans morsitans bites especially in early morning and late afternoon; around midday feeding is at a low. In laboratory apparatus that measures the amount of locomotion under constant conditions over the photophase, the flies display a similar patterning of activity levels. The profile of daily rhythms for G. morsitans reported in the literature includes a number of motor and sensory motor systems that fluctuate cophasically. Lacking is a study on the patterning of the senses' response levels. In this paper we present the first instance of a daily modulation in the sense of smell. We stimulated the antennae with concentration series of host-derived odours and measured the spiking rate of cells at different times during the photophase. The concentration-response curves suggest that the sensitivity of antennal olfactory cells flows in parallel with the other daily rhythms. This was also reflected in electroantennograms (EAGs). The electroantennography was extended to G. fuscipes fuscipes, whose level of spontaneous locomotor activity--instead of following a U-shaped pattern--rises gradually over the photophase. Again, the EAGs appeared to parallel the species' locomotor activity. What we believe happens is that the organism tones down the sensitivity of its odour receptors during periods of anticipated inactivity for reasons of economy.

Search for tsetse attractants: A structure-activity study on 1-octen-3-ol inGlossina fuscipes fuscipes (Diptera: Glossinidae).

Trapping tsetse flies belonging to thepalpalis group still relies totally upon luring by visual cues even though odor-baited trapping is used effectively against themorsitans-group species. Forty-three percent of the antennal olfactory cells ofGlossina f. fuscipes, a member of thepalpalis group, respond to 1-octen-3-ol. For this species we report a structure-activity relationship between 1-octen-3-ol analogs, in which carbon chain length and the configuration of the hydroxyl and π-bond moieties are varied, and biological activity. Although the optimum chain length for all cells sensitive to 1-octen-3-ol is eight and most cells give lower responses when the hydroxyl function is omitted, there is a clear division into two groups. One group is diverse and represents cells that appear indifferent to the presence or position of the π bond; many will respond to such disparate structures as acetone and 3-methylphenol as well as to 1-octen-3-ol. In the other group, the structural requirements for the stimulus are more stringent; the cells appear to be specifically tuned to 1-octen-3-ol. Their thresholds are three orders of magnitude lower than those of the former group. The existence of two clusters points to a functional division in the olfactory sense. We suggest that the latter low-threshold group is involved in host detection from a distance while the former diverse group is involved in host discrimination at close range. Trap harvests with 1-octen-3-ol as a bait may have been disappointing because the appropriate mixture for generating a landing response on the traps is still lacking.

Carbohydrate specificity of the Escherichia coli P-pilus papG protein is mediated by its N-terminal part.

The adherence of pyelonephritic Escherichia coli isolates to mammalian host cells is mediated by the P-pili structures on the bacterial surface. The protein constituting the distal part of the pili structure, papG, interacts with glycan receptors on the host cell. Variation in specificity for different glycoconjugates between the isolates, that may reflect variation in host tropism, has been correlated to three different classes of papG. Truncated variants of the class I, II and III papG adhesins were produced as fusion protein in E. coli and analysed for carbohydrate binding. The results showed that both carbohydrate binding and specificity of the papG adhesin resided in a linear part of the N-terminus of the protein.

Characterization of transcription and processing from plasmids that use polIII and a yeast tRNA gene as promoter to transcribe promoter-deficient downstream DNA.

Transfer RNA (tRNA) with mutations affecting the internal promoters and thereby causing them to be nontranscribable by polIII (exemplified, e.g., by nematode tDNAPro with large insertions between the two internal promoters) could be transcribed by polIII both in vitro (yeast) and in vivo (oocytes) when cloned behind a yeast tRNAArg gene. PolIII initiated RNA synthesis could also proceed into a downstream structural gene normally read by polIII. the resultant yeast-nematode dimeric tRNA linked in front of mRNA sequences was recognized by processing enzymes to give mature tRNA. Thus, a yeast tRNA gene preceded by its 5' flank can function as a promoter for polIII transcription of any DNA, also of DNA coding for genes that otherwise could not have been expressed either in vitro or in vivo.

Reforming Swedish health care in the 1990s: the emerging role of 'public firms'.

A growing number of Swedish county councils have started to develop more flexible methods by which to produce and deliver health services. This paper explores the current status of this reform process both empirically and conceptually. Empirically, it draws upon data obtained by a 1990 questionnaire from all 26 county councils to chart the level of movement across the entire system. Conceptually, it distills from this reform activity a key element that provides an organizational basis for the future, namely the transformation of provider institutions into 'public firms'. The paper concludes that while the precise outcome may be hard to predict, the reform process itself is well underway.