High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a rapid, cost-effective alternative for bacterial species identification. Identifying 60 blind-coded nonfermenting bacteria samples, this international study (using eight laboratories) achieved 98.75% interlaboratory reproducibility. Only 6 of the 480 samples were misidentified due to interchanges (4 samples) or contamination (1 sample) or not identified because of insufficient signal intensity (1 sample).

Ligand-induced conformational changes in the human retinoic acid receptor detected using monoclonal antibodies.

The mechanism by which the naturally occurring ligand for a nuclear hormone receptor regulates transcription remains largely unknown. One approach combines the specificity of monoclonal antibodies, which recognize a three-dimensional epitope, with ligand binding. Using purified retinoic acid receptor gamma D and E domains, a panel of six unique monoclonal antibodies were isolated and characterized using solid-state receptor binding and retinoic acid receptor (RAR)-RXR heterodimer supershift formation. Three antibodies are specific for RARgamma (mAbI, mAbII, and mAbV) and four recognize a three-dimensional epitope (mAbI, mAbIV, mAbV, and mAbVI). Three antibodies (mAbIII, mAbV, and mAbVI) dissociate from the receptor in electrophoretic mobility shift assays upon the addition of retinoic acid. In particular, the binding characteristics of mAbIII, whose epitope was mapped to a region identified as an omega-loop (amino acids 207-222), suggest a model for ligand binding to the receptor. In this model, ligand binding causes a positioning of helix 12 into a favorable conformation for interaction with the transcriptional machinery. The Omega-loop then closes in order to stabilize this "active" position. The results reported here also suggest that a region of the hinge or D domain of the receptor (amino acids 156-188), an area that can play a role in protein-protein interactions, may also be important in ligand-induced functional changes.

Identification of TonB homologs in the family Enterobacteriaceae and evidence for conservation of TonB-dependent energy transduction complexes.

The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli requires the TonB-dependent energy transduction system. A set of murine monoclonal antibodies (MAbs) was generated against an E. coli TrpC-TonB fusion protein to facilitate structure and function studies. In the present study, the epitopes recognized by these MAbs were mapped, and their distribution in gram-negative organisms was examined. Cross-species reactivity patterns obtained against TonB homologs of known sequence were used to refine epitope mapping, with some epitopes ultimately confirmed by inhibition experiments using synthetic polypeptides. Epitopes recognized by this set of MAbs were conserved in TonB homologs for 9 of 12 species in the family Enterobacteriaceae (including E. coli), including previously unidentified TonB homologs in Shigella, Citrobacter, Proteus, and Kluyvera species. These homologs were also detected by a polyclonal alpha-TrpC-TonB serum that additionally recognized the known Yersinia enterocolitica TonB homolog and a putative TonB homolog in Edwardsiella tarda. These antibody preparations failed to detect the known TonB homologs of either Pseudomonas putida or Haemophilus influenzae but did identify potential TonB homologs in several other nonenteric gram-negative species. In vivo chemical cross-linking experiments demonstrated that in addition to TonB, auxiliary components of the TonB-dependent energy transduction system are broadly conserved in members of the family Enterobacteriaceae, suggesting that the TonB system represents a common system for high-affinity active transport across the gram-negative outer membrane.

Energy transduction between membranes. TonB, a cytoplasmic membrane protein, can be chemically cross-linked in vivo to the outer membrane receptor FepA.

TonB, a cytoplasmic membrane protein, couples cytoplasmic membrane protonmotive force to active transport across the outer membrane of Escherichia coli. In vivo cross-linking studies were initiated to analyze TonB interactions with other cell envelope proteins. Four TonB-specific cross-linked complexes were detected with apparent molecular masses of 195, 77, 59, and 43.5 kDa. The 195-kDa complex was shown to contain both TonB and FepA, the outer membrane receptor for the siderophore enterochelin. The 195-kDa complex is absent in strains missing either TonB or FepA and can be detected by either TonB-specific or FepA-specific monoclonal antibodies. This is the first direct in vivo evidence that TonB can span the periplasmic space to interact physically with outer membrane receptors. Consistent with that observation, the outer membrane protease OmpT was shown to play a role in TonB turnover, both in the presence and absence of ExbB results in the rapid degradation of TonB. The absence of OmpT could be used to stabilize TonB in an exbB::Tn10 strain such that steady state levels of TonB protein are identical to a wild-type strain. Under those conditions, the absence of ExbB results in greatly reduced TonB activity, indicating that ExbB plays a direct role in energy transduction and probably secondarily protects TonB protein from proteolysis. The 59-kDa complex was absent in an exbB::Tn10 strain, suggesting either that ExbB is in the complex with TonB or that ExbB is required to form the 59-kDa complex. A tolQ nonsense mutation had no effect on the cross-linking profile observed, confirming that its participation in TonB-dependent phenomena is minor and most likely the result of evolutionary cross-talk.

Peptides related to the carboxyl terminus of human platelet factor IV with antibacterial activity.

A peptide (C13) corresponding to the last 13 amino acids of the carboxyl terminus of human platelet factor IV was found to be antibacterial. Amino acid substitutions predicted to disrupt either the amphipathic or alpha-helical nature of C13 rendered the peptide inactive. Antibacterial activity was demonstrated in normal human serum on bacteria which had been previously exposed to low levels of cefepime, a beta-lactam antibiotic. Peptide analogues were examined for more potent antibacterial activity in an antibacterial assay that employed normal human serum and low levels of cefepime. A peptide analogue (C18G) with 80-fold more antibacterial activity than C13 was identified. Studies in C8-deficient sera confirmed an essential role of human serum complement for optimal antibacterial activity. Additional studies showed low levels of cefepime, although not essential, enhanced the antibacterial activity of C18G. Animal protection experiments demonstrated that either peptide C18G or an analogue with all D amino acids (C18X) significantly increased the survival of neutropenic mice when coadministered with a low level of cefepime. This work has resulted in the identification of a new group of antibacterial peptides.

Beta-lactam antibiotics potentiate magainin 2 antimicrobial activity in vitro and in vivo.

The ability of magainin 2 to augment antibiotic therapy was examined. Susceptibility to magainin 2 was determined on Escherichia coli incubated in the presence and absence of sublethal concentrations of antibiotics both in vitro and in vivo. Experiments in buffer and normal human serum revealed that E. coli exposed to sublethal amounts of cefepime, a beta-lactam antibiotic, was significantly more susceptible to the antimicrobial activity of magainin 2. Bacteria incubated with subinhibitory concentrations of other beta-lactam type antibiotics, but not amikacin (an aminoglycoside) or ciprofloxacin (a quinolone), were also more susceptible to magainin 2 in normal human serum. Bacteria were less susceptible to magainin 2 when they were examined in heat-inactivated serum. Complement was shown to be required for magainin 2 activity in serum by using C8-deficient sera. The combination of magainin 2 and cefepime was shown to be more antimicrobial in normal human serum for a variety of bacterial strains. Magainin 2 was completely inactive as a therapeutic agent when it was administered alone (2 mg per mouse) but significantly increased the survival of mice when it was administered with a low level of cefepime.

Platelet function, size and yield in whole blood and in platelet-rich plasma prepared using differing centrifugation force and time in domestic and food-producing animals.

The effects of centrifugation force and time upon platelets function, mean platelet volume and platelet yield were compared with whole blood platelet counts and size in citrated blood samples from the bovine, canine, caprine, equine, feline, ovine and porcine species. The results were similar, for a given species, irregardless of sample volume. Bovine, caprine, feline and ovine platelet yields and mean platelet volumes were maximal when platelet-rich plasma was prepared using longer centrifugation times and lower gravitational forces. Canine, equine and porcine platelet yields and mean platelet volumes were maximal when platelet-rich plasma was prepared using shorter centrifugation times and higher gravitational forces. Platelet aggregation to adenosine diphosphate or arachidonic acid was not effected by the method of platelet-rich plasma preparation in bovine, caprine, feline, ovine or porcine platelets. Equine platelet aggregation was maximal when platelet-rich plasma was prepared using longer centrifugation times and lower gravitational forces. Canine platelet aggregation, particularly arachidonic acid-induced aggregation, was maximal when platelet-rich plasma was prepared using short centrifugation times and higher gravitational forces. It appeared that the effects of centrifugation parameters upon platelet yield depended upon the relative difference between platelet and red blood cell volumes.

Serotonin accumulation in granules of storage pool-deficient platelets of Chediak-Higashi cattle.

Platelets from cattle with the Chediak-Higashi (CH) syndrome are virtually devoid of dense granules, serotonin (5-HT), and stored ATP and ADP. The present study determined how the handling of 5-HT in normal cattle platelets differed from that in CH cattle platelets. Normal and CH platelets accumulated 5-[14C]HT to the same extent. After normal and CH platelets were incubated with 5-HT for 12 h most 5-HT is still intact, indicating that it was protected from metabolism. Part of the newly acquired 5-HT in normal and CH platelets was in a pool that was rapidly released by 5 U/ml of thrombin, suggesting that 5-HT was, in part, within granules. Subcellular fractionation studies showed that, whereas most of the newly acquired 5-HT in normal platelets was located in the dense granule fractions, about one fourth was found in the lighter granule fraction that was enriched in alpha-granules. The dense granule fraction was virtually absent in CH platelets, and most of the granule 5-HT was associated with the lighter granule fraction. The mixed granule fraction from CH platelets accumulated 5-HT but the uptake was about 10% of that from normal platelets. Unlike normal granules the uptake of 5-HT by CH granules was only slightly inhibited by reserpine but was reversed by NH4Cl and nigericin treatment.

Comparative study of platelet dense granule constituents.

Cat, cattle, dog, horse, human, mink, pig, and rabbit platelets were separated from plasma by gel filtration. The gel-filtered platelets (GFP) were treated with thrombin to induce maximal granule secretion and the potential dense granule constituents ATP, ADP, serotonin (5-HT), Ca2+, and Mg2+ were measured in GFP and in the control and thrombin-treated platelets and in the respective supernatants. The amount of Ca2+, Mg2+, 5-HT, ATP, and ADP within the nonreleasable pool for all species varied between 3.1 and 10.0 mumol/10(11) platelets for Ca2+ and Mg2+ was less than 1.5 mumol/10(11) platelets for ADP and 5-HT and was between 2.0 and 5.0 mumol/10(11) platelets for ATP. Marked differences were observed in the releasable fraction. Human platelets were characterized by the largest releasable Ca2+ pool (greater than 10 mumol/10(11) platelets), the smallest secretable 5-HT and Mg2+ pool (less than 0.5 mumol/10(11) platelets), and the lowest ATP-to-ADP ratio (greater than 1.0). Pig platelets had the highest amount of releasable Mg2+ (approximately 8.0 mumol/10(11) platelets). Rabbits platelets released the most 5-HT (greater than 3.0 mumol/10(11)) and had the highest ATP/ADP (greater than 5.0). The releasable pool of Ca2+, Mg2+, ATP, and ADP in the remaining species varied in mumol/10(11) platelets from approximately 1.5-4.0, approximately 1.0-3.0, 0.5-3.5, and approximately 0.5-1.5, respectively.

Evaluation of the platelet storage pool deficiency in the feline counterpart of the Chediak-Higashi syndrome.

Cats with the Chediak-Higashi (CH) syndrome have abnormal hemostasis with prolonged bleeding times and normal coagulation times. Platelet aggregation induced by serotonin, ADP, and collagen was impaired. Platelets from normal and CH cats were incubated with 14C-adenine and then gel-filtered. Gel-filtered platelets (GFP) from CH cats contained 63% of the ATP, 38% of the ADP, 100% of the Ca2+, and 75% of the Mg25 of normal platelets. Serotonin could not be detected in CH platelets. Acid hydrolase and total platelet protein of CH platelets was similar to normal platelets. Gel-filtered platelets were treated with thrombin to induce maximal secretion. Secretion of ATP, Ca2+, and Mg2+ was 1.9%, 12.4%, and 16% respectively of normal platelets. ADP secretion by CH platelets was not detectable. The ATP/ADP ratio in the 14C-labeled metabolic pool of normal platelets was similar to that of total measured nucleotide pool of CH platelets. These findings suggest that in feline CH platelets, as in platelets from CH mink and cattle, there is storage pool deficiency that is virtually complete, and the virtual absence of ADP and 5HT may in part account for the abnormal hemostasis. Aggregation of platelets from CH cats was impaired, but these platelets did aggregate to arachidonate, serotonin-induced biphasic aggregation, and the aggregation response to ADP and collagen varied according to the amount of serotonin-induced TxB2 formed. These findings support a major role for arachidonate in platelet activation.

Characterization of platelets from normal mink and mink with the Chediak-Higashi syndrome.

Bleeding times of mink with the Chediak-Higashi (CH) syndrome was markedly prolonged. Platelet counts were normal but there was an impaired platelet aggregation response to collagen. The metabolic adenine nucleotide pool of platelets from normal and CH mink was labeled with 14C-adenine and the platelets were gel-filtered. Gel-filtered platelets (GFP) from CH mink contained only 37.9% of the adenosine triphosphate (ATP) and 9.6% of the adenosine diphosphate (ADP) found in normal platelets and the ATP/ADP ratio was similar to the 14C-ATP/14C-ADP ratio. Platelet content of Ca2+, Mg2+, and in particular 5-hydroxytryptamine was decreased. When GFP were incubated with thrombin to induce maximal secretion, only negligible amounts of ATP and ADP were released. The specific activity of the extracellular nucleotides approximated that within the platelet. These findings suggest that the stored nucleotide pool in CH platelets is virtually absent and that the abnormalities in platelet function may be due, in part, to the essential absence of secretable ADP and serotonin. The release of Ca2+ and Mg2+ by CH platelets was 56% and 27.8% of normal, respectively.