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The nitric oxide/cGMP system in the antennal lobe of Apis mellifera is implicated in integrative processing of chemosensory stimuli.

The high concentration and the localization of nitric oxide synthase in the olfactory system of both vertebrates and invertebrates suggest that the diffusible messenger nitric oxide plays a central role in the processing of chemosensory information. This paper describes the nitric oxide releasing system in the antenna and the antennal lobes of Apis mellifera using the NADPH diaphorase technique, and analyses the contribution of the nitric oxide system in the neuronal processing of chemosensory signals using a behavioral assay in vivo. In the antenna the strongest NADPH diaphorase staining is found in non-neuronal auxiliary and/or epithelial cells, while the sensory cells and the antennal nerve are stained at a low level. At the major site of chemosensory signal integration, the antennal lobes, the highest nitric oxide synthase activity is located in the glomeruli, which are ideally suited to act as diffusion compartments. We demonstrate that inhibition of nitric oxide synthase in the antennal lobes specifically interferes with neuronal processing of repetitive chemosensory stimuli but does not affect the response to single stimuli, and is independent of parameters such as satiation level, stimulus strength, interstimulus interval and duration of sensory stimuli. Since inhibition of the soluble guanylate cyclase, a major target of nitric oxide, also particularly affects the adaptive component, the physiological effects of nitric oxide appear to be mediated by the action of cGMP. These findings suggest that the nitric oxide/cGMP system in the antennal lobes is a component of the molecular machinery involved in adaptive and/or integrative mechanisms during chemosensory information processing in vivo.

Animals

Odour coding is bilaterally symmetrical in the antennal lobes of honeybees (Apis mellifera).

The primary olfactory neuropil, the antennal lobe (AL) in insects, is organized in glomeruli. Glomerular activity patterns are believed to represent the across-fibre pattern of the olfactory code. These patterns depend on an organized innervation from the afferent receptor cells, and interconnections of local interneurons. It is unclear how the complex organization of the AL is achieved ontogenetically. In this study, we measured the functional activity patterns elicited by stimulation with odours in the right and the left AL of the same honeybee (Apis mellifera) using optical imaging of the calcium-sensitive dye calcium green. We show here that these patterns are bilaterally symmetrical (n=25 bees). This symmetry holds true for all odours tested, irrespective of their role as pheromones or as environmental odours, or whether they were pure substances or complex blends (n=13 odours). Therefore, we exclude that activity dependent mechanisms local to one AL determine the functional glomerular activity. This identity is genetically predetermined. Alternatively, if activity dependent processes are involved, bilateral connections would have to shape symmetry, or, temporal constraints could lead to identical patterns on both sides due to their common history of odour exposure.

Acyclic Monoterpenes

Modification of potassium movement through the retina of the drone (Apis mellifera male) by glial uptake.

Intracellular recordings were made in photoreceptors and glial cells (outer pigment cells) of the superfused cut head of the honey-bee drone (Apis mellifera male). When the [K+] in the superfusate was abruptly increased from 3.2 mM to 17.9 mM both photoreceptors and glial cells depolarized. The time course of the depolarization of the photoreceptors was slower with increasing depth from the surface. Half time of depolarization was plotted against depth: this graph was compatible with the arrival of K+ being exclusively by diffusion through the extracellular clefts. However, as we then showed, this interpretation is inadequate. The time course of depolarization of the glial cells was almost the same at all depths. This indicates that they are electrically coupled. Consequently, current-mediated K+ flux (spatial buffering) through glial cells will contribute to the transport of K+ through the tissue: K+ ions enter the glial syncytium in the region of high external potassium concentration, [K+]0, and an equivalent quantity of K+ ions leave in regions of low [K+]0. Intracellular K+ activity (aiK) was measured with double-barrelled K+-sensitive micro-electrodes in slices of retina superfused on both faces. When [K+] in the superfusate was increased from 7.5 mM to 17.9 mM an increase in aiK was observed in glial cells at all depths in the slice (initial rate 1.7 mM min-1, S.E. of the mean = 0.2 mM min-1), but there was little increase in the photoreceptors (0.3 +/- 0.2 mM min-1). The increase in aiK in glial cells near the centre of the slice could not have been caused by spatial buffering; it presumably resulted from net uptake. We conclude that when [K+] is increased at the surface of this tissue, the build up of K+ in the extracellular clefts depends on extracellular diffusion, spatial buffering and net uptake. The latter two processes, which have opposing effects, involve about 10 times as much K+ as the first. This is in rough agreement with less direct experiments on mammalian brain (Gardner-Medwin, 1977, 1983b).

Animals

Use of the API rapid NFT system for identifying nonfermentative and fermentative marine bacteria.

Thirty-five American Type Culture Collection type strains of marine bacteria were used to evaluate the Rapid NFT system (API Analab Products, Plainview, N.Y.) for use in identifying heterotrophic marine bacteria. The 21 biochemical and assimilation tests on the Rapid NFT test strips were treated according to the manufacturer's protocol, which included use of AUX medium (provided with the Rapid NFT system) for preparing assimilation tests, and by substituting phenol red broth base (BBL Microbiology Systems, Cockeysville, Md.) with and without an oil overlay for the AUX medium. A seven-digit numerical profile was obtained for each NFT test strip from each of the three procedures and matched to its corresponding number in the Rapid NFT identification codebook. Also, all biochemical and assimilation test results were analyzed with SASTAXAN and SAS/GRAPH programs (SAS Institute, Inc., Cary, N.C.); similarity matrices were computed for all 35 strains. For comparison purposes, bacterial strains were grouped at a similarity level of 70%. The results indicated a low efficacy of identification for all three procedures. In addition, similarity matrix analysis showed more cohesive grouping based on results of phenol red broth base-treated strains than for the AUX medium provided by the manufacturer. However, none of the three treatments provided exclusive grouping of type strains at the genus level. Thus, the reliability of the data obtained from the NFT system and modifications thereof should be evaluated carefully when environmental isolates are characterized.

Bacteria

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.

Bacteriological Techniques

Rapid identification of Enterobacteriaceae with the micro-ID system versus API 20E and conventional media.

The Micro-ID system for rapid (4 h) identification of Enterobacteriaceae was evaluated by testing 433 enteric bacilli and 9 other gram-negative bacilli. Each isolate was identified with conventional tubed media and was also tested in the Micro-ID and API 20E systems. The overall accuracy of both systems was 97%. Micro-ID tests for the Voges-Proskauer reaction, indole and H2S production, and ornithine and lysine decarboxylase all demonstrated a 97 to 99% correlation with conventional methods. Only 86% of the Micro-ID urease tests agreed with Christenson urea agar. Two inoculum densities were tested in Micro-ID panels, with 157 stock cultures. Over 90% of the tests were unaffected by changes in inoculum density. Tests with four control strains suggested that the Micro-ID system was more reproducible when a light inoculum was used. The Micro-ID system was found to be a very convenient method for rapid, accurate, and precise identification of the Enterobacteriaceae.

Bacteriological Techniques

Rapid identification of Prototheca species by the API 20C system.

The conventional auxanographic method of testing for the assimilation of carbohydrates and alcohols by the various species of Prototheca requires at least 2 weeks of incubation at 25 to 30 degrees C before definitive results are obtained. Even though Prototheca spp., in culture as well as in fixed tissues, can be identified more rapidly by fluorescent-antibody techniques in which species-specific reagents are used, such diagnostic facilities and reagents are not available in most diagnostic laboratories. The API 20C clinical yeast identification system, a commercially available ready-to-use micromethod, was found to permit the definitive identification of P. stagnora, P. wickerhamii, and P. zopfii within 4 days.

Carbohydrate Metabolism

API ZYM system for identification of Bacteroides spp., Capnocytophaga spp., and spirochetes of oral origin.

A total of 80 oral strains of Bacteroides gingivalis, B. asaccharolyticus, B. melaninogenicus subsp. intermedius, B. melaninogenicus subsp. melaninogenicus, Capnocytophaga, Treponema denticola, and T. vincentii were characterized with the API ZYM system for 19 enzyme activities. Comparison of anaerobic and aerobic incubation with nine reference strains of these organisms showed no important differences. The key differential tests for black-pigmented Bacteroides strains and treponemes of oral origin were trypsin, alpha-glucosidase, and N-acetyl-beta-glucosaminidase. All Capnocytophaga strains produced distinctive aminopeptidase activities but varied in their glycosidic capabilities. The presence of a trypsin-like activity in B. gingivalis, T. denticola, and a group of Capnocytophaga strains may contribute to tissue destruction in periodontal disease.

Acetylglucosaminidase

Enzymatic characterization of Aeromonas hydrophila complex by the API ZYM system.

Enzymatic characterization of 48 Aeromonas hydrophila complex isolates from various sources was determined with the API ZYM system (Analytab Products, Plainview, N.Y.). All isolates lacked valine and cystine aminopeptidases, chymotrypsin, alpha-mannosidase, alpha-fucosidase, alpha-galactosidase, and beta-glucuronidase but possessed caprylate esterase-lipase, leucine aminopeptidase, acid phosphatase, phosphoamidase, and N-acetyl-beta-glucosidase. Variability was found in the presence of alkaline phosphatase, butyrate esterase, myristate lipase, trypsin, beta-galactosidase, alpha-glucosidase, and beta-glucosidase. No significant differences were evident among the enzymatic profiles of isolates from various sources.

Aeromonas

Identification of coagulase-negative staphylococci with the API STAPH-IDENT system.

A group of 300 clinically derived isolates of coagulase-negative staphylococci were tested in parallel with the API STAPH-IDENT system (Analytab Products) and 14 conventional biochemical tests contained in Kloos and Schleifer's simplified scheme for identification of human Staphylococcus species. STAPH-IDENT is a miniaturized biochemical test strip that incorporates four synthetic chromogenic substrates, urea, arginine, and four carbohydrates and that requires only a 5-h test period. Use of the STAPH-IDENT system alone allowed correct or partly correct classification of 67% (201 of 300) of the study isolates. However, if a supplemental test was performed (most often novobiocin susceptibility), correct classification of an additional 25.7% (77) was possible, for a total of 92.7% of isolates identified to the species level. Species correctly identified included 94% (116 of 123) of Staphylococcus epidermidis isolates, 98% (63 of 64) of S. saprophyticus, 71% (34 of 48) of S. hominis, 100% (22) of S. simulans, 100% (18) of S. haemolyticus, 100% (17) of S. warneri, and 100% (8) of S. capitis. Fourteen percent (42 of 300) of profile codes encountered in this study were not included in the STAPH-IDENT profile register, but were included in Analytab Products' expanded computer data base.

Bacteriological Techniques

Clinical comparison of the AutoMicrobic system gram-positive identification card, API Staph-Ident, and conventional methods in the identification of coagulase-negative Staphylococcus spp.

In an effort to rapidly identify coagulase-negative staphylococci (CNS), a clinical comparison was conducted with the AutoMicrobic system Gram-Positive Identification Card (GPI) (Vitek Systems, Inc.), the API Staph-Ident (Analytab Products), and the conventional methods of W. E. Kloos and K. H. Schleifer (W. E. Kloos and K. H. Schleifer, J. Clin. Microbiol. 1:82-88, 1975). CNS isolates tested included 157 from blood and 33 from urine in pure culture at greater than 10(5) CFU/ml. S. epidermidis accounted for 79.6 and 60.6% of the isolates from blood and urine, respectively. S. saprophyticus was the next most frequent urine isolate (27.4%). Other CNS species were isolated from blood and urine specimens with frequencies of less than 5%. Overall, the GPI correctly identified 158 (83.2%) of the 190 CNS, whereas the Staph-Ident identified 124 (65.3%) without further testing. This resulted in the GPI and Staph-Ident correctly identifying 95.9 and 74.5% of the S. epidermidis and 100 and 33% of the S. saprophyticus, respectively. The GPI misidentified 8 (47%) of the S. hominis and S. warneri isolates as S. saprophyticus, indicating the need for novobiocin testing. These data suggest that the GPI is a more definitive method for the rapid identification of S. epidermidis than the Staph-Ident and that both systems require additional testing to identify S. saprophyticus.

Bacteriological Techniques

Use of the API NeIdent system for identification of pathogenic Neisseria spp. and Branhamella catarrhalis.

The API NeIdent system (Analytab Products, Plainview, N.Y.) was evaluated for identifying Neisseria spp. and Branhamella catarrhalis commonly isolated from clinical specimens. The system identified 90% of 303 Neisseria gonorrhoeae isolates, 71% of 113 Neisseria meningitidis isolates, and 63% of 16 Neisseria lactamica isolates but failed to identify any of 22 B. catarrhalis isolates. Testing of gonococcal strains of various auxotypes revealed no relationship between nutritional requirements and NeIdent profile numbers. With the Neisseria species, interpretation of the cinnamaldehyde-coupled beta-naphthylamine reactions was difficult and resulted in profile numbers not listed in the Profile Register. Positive resazurin-glucose reactions resulted in unlisted numbers for all B. catarrhalis strains. Inconsistent results were also obtained when 62 N. gonorrhoeae isolates were tested more than once on the strip. In all cases, profile variability and failure to identify these organisms were related to the beta-naphthylamide substrate tests. Expansion of the data base and modification of the substrate formulations or their interpretive criteria may increase the reliability of the NeIdent system for identifying Neisseria spp. and B. catarrhalis.

Bacteriological Techniques

Evaluation of the 24-h API 20A anaerobe system for identification of Clostridium difficile.

Accurate identification of Clostridium difficile is important when antibiotic-associated diarrhea or pseudomembranous colitis is suspected. Presumptive identification of C. difficile was made on the basis of microscopic features and colony characteristics on cycloserine, cefoxitin, fructose, and egg yolk agar medium. We studied the reliability of the 24-h API 20A anaerobe system for definitive identification of C. difficile. This system showed low dependability after the recommended 24 h of incubation by confirming the identity of only 54% of the isolates presumptively identified as C. difficile. There was a marked improvement in the system's capability after 48 h of incubation, when the identity of 95% of the isolates was confirmed.

Anaerobiosis

Comparative evaluation of the API 20S and AutoMicrobic gram-positive identification systems for non-beta-hemolytic streptococci and aerococci.

The API 20S system (Analytab Products, Plainview, N.Y.) and the AutoMicrobic Gram-Positive Identification system (GPI; Vitek Systems, Hazelwood, Mo.) were evaluated for their capacity to identify the non-beta-hemolytic streptococci and aerococci to the species level. The 20S system identified 86% (six of seven strains) of nonhemolytic group B streptococci, whereas 100% of the same group B streptococcal strains were correctly identified by the GPI system. With both systems 99% (134 of 135 strains) of four species of group D enterococcus strains and 92% (24 of 26 strains) of the Aerococcus spp. strains were identified. The 20S system identified 84% (41 of 49 strains) of three species of group D non-enterococcus strains. The GPI system identified 96% of the same group D non-enterococcus strains. The 20S system identified 84% (190 of 226 strains) of 10 species of viridans streptococci; however, supplemental conventional tests were required to identify 49% (110 of the 226 strains) of the viridans strains to the species level. The GPI system identified 79% of the same viridans streptococci without the need for supplemental tests. Both systems identified 84% (161 of 192 strains) of the seven most commonly occurring viridans Streptococcus spp. The 20S system identified 82% (75 of 92 strains) and the GPI system identified 84% (54 of 64 strains) of Streptococcus pneumoniae.

Bacteriological Techniques

Supplementary rapid biochemical test panel for the API 20E bacterial identification system.

The API 20E Analytical Profile Index typically suggests three or four conventional biochemical tests to complete the identification of strains either identified to genus only or that have multiple genera consistent with the profile number. We compiled a simple panel of eight rapid (4-h) tests that can substitute for the supplementary biochemical tests recommended by Analytab Products (Plainview, N.Y.). The rapid test panel (RTP) consisted of adonitol, cellobiose, lactose, raffinose, rhamnose, and xylose utilization, lysine decarboxylase activity, and motility. A total of 114 consecutive clinical isolates that required additional tests to complete the identifications were each tested with the complete RTP, as well as with the recommended conventional biochemicals. All discordant identifications were resolved by using an expanded series of conventional biochemical tests. Overall, 110 (96%) strains were identified to the correct genus, and 109 (95%) strains were identified to the correct species by using the RTP, as compared with 105 (92%) identified to the correct genus and 90 (79%) identified to the correct species with the recommended tests. The identifications based on the two supplementary test systems did not agree for 7 (6.1%) strains. Four discrepancies were resolved in favor of the RTP, and three were resolved in favor of the recommended tests. We were unable to identify five (4.4%) strains with the recommended tests and only one (0.9%) with the RTP. A majority (86%) of the test strains were identified to the species level with the RTP after only 4 h of incubation.

Bacteriological Techniques

Enzymatic characterization of Pseudomonas cepacia by API ZYM profile.

The enzymatic activities of 53 strains of Pseudomonas cepacia were determined by using the API ZYM system. Strong alkaline phosphatase, acid phosphatase, butyrate esterase, caprylate esterase, myristate lipase, leucine arylamidase, and phosphoamidase activities were consistently detected in all strains. Weak activities were observed for valine arylamidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase. No activities could be demonstrated for cystine arylamidase, trypsin, chymotrypsin, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, alpha-glucosidase, alpha-mannosidase, and alpha-fucosidase. Enzymatic activities of pseudomonads may provide useful information about their pathogenesis and information for identification of Pseudomonas species.

Chromogenic Compounds