Strategies for physician education in therapeutic drug monitoring

The upstream region of the gene coding for Clostridium botulinum type B (BoNT/B) neurotoxin was cloned and sequenced. There were two open reading frames, which were identified as a nontoxic-nonhemagglutinin component (ntnh/B) and a 22 kDa adjacent open reading frame (orf22/B). Deduced primary structure of ntnh/B showed that it was composed of 1,197 amino acid residues. Pairwise comparisons of the ntnh/B component with other botulinum toxin types showed high degree of homology to ntnh/A (82% identity). Northern blot analysis revealed that toxin gene could be transcribed alone or co-transcribed with the ntnh gene. The orf22/B gene encoding for 178 amino acids (M.W. 21.6 kDa) was located between the 33 kDa hemagglutinin gene and the ntnh gene. Orf22/B also showed high degree of homology to orf22/A (98.9% identity). These results suggested that the upstream region of the BoNT/B gene (containing the ntnh/B and orf22/B genes) might be evolutionarily closely related to the counterparts of the BoNT/A.     

Evaluation of adsorption preconcentration/capillary zone electrophoresis/nanoelectrospray mass spectrometry for peptide and glycoprotein analyses

The use of an on-line adsorption preconcentrator coupled with capillary zone electrophoresis/nanoelectrospray mass spectrometry (PC/CZE/nESMS) is described for the analysis of peptides and protein digests. The investigation was focused on the production of disposable preconcentrators made of large particle size (40 microns irregular packing), thereby eliminating the use of a retaining frit without loss of performance. These preconcentration devices were made of commercially available components which can be easily interfaced to current CZE/nESMS systems. Practical issues such as the composition of the stationary phase, the elution volume and sample breakthrough and carry-over were evaluated in order to optimize the analytical performance of this technique. Under optimized elution conditions, the PC/CZE/nESMS technique provided separation efficiencies in excess of 100,000 theoretical plates for a sample loading of 8 microliters. Sample carry-over was minimized by proper reconditioning of the preconcentrator prior to the CZE separation. Alternatively, the sample carry-over resulting from small elution volumes could be used advantageously to provide multiple analyses from a single injection of sample. The application of this technique is demonstrated for the analysis of proteolytic peptides from a Bauhinia purpurea lectin at a concentration level of 30 nM. Further structural information was obtained using on-line tandem mass spectrometry to elucidate the structure of N-linked glycans and the amino acid sequences of the glycopeptides.     

Protein identification by capillary zone electrophoresis/microelectrospray ionization-tandem mass spectrometry at the subfemtomole level

A method for the identification of proteins by their amino acid sequence at the low-femtomole to subfemtomole sensitivity level is described. It is based on an integrated system consisting of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization triple- quadrupole tandem mass spectrometer (ESI-MS/MS) via a microspray interface. The method consists of proteolytic fragmentation of a protein, peptide separation by CZE, analysis of separated peptides by ESI-MS/MS, and identification of the protein by correlation of the collision-induced dissociation (CID) patterns of selected peptides with the CID patterns predicted from all the isobaric peptides in a sequence database. Using standard peptides applied to a 20-microns-i.d. capillary, we demonstrate an ESI-MS limit of detection of less than 300 amol and CID spectra suitable for searching sequence databases obtained with 600 amol of sample applied to the capillary. Successful protein identification by the method was demonstrated by applying 50 and 38 fmol of a tryptic digest of the proteins beta-lactoglobulin and bovine serum albumin, respectively, to the system.     

Isolation, cloning, and expression of a 70-kilodalton plasminogen binding protein of Borrelia burgdorferi

Surface receptors for plasminogen are expressed by many gram-positive and gram-negative bacteria and may play a role in the dissemination of organisms by binding plasminogen, which upon conversion to plasmin can digest extracellular matrix proteins. Two plasminogen binding proteins have been identified for Borrelia burgdorferi, outer surface protein A and a 70-kDa protein (BPBP). We purified BPBP by plasminogen affinity chromatography and obtained its amino acid sequence by Edman degradation of a tryptic digest. The gene coding for BPBP was isolated from a lambda-ZAP II genomic library with probes developed from sequenced portions of the protein. This gene was expressed in Escherichia coli; the recombinant product was seen by antibody raised against native BPBP and also bound 125I-labeled plasminogen. The experimentally derived amino acid sequences corresponded to the predicted sequence encoded by the BPBP gene. The deduced amino acid sequence for BPBP revealed significant similarity to p30, a 30-kDa protein of B. burgdorferi (54% identity and 65% similarity), to a 60-kDa protein in Borrelia coriaceae (66% identity and 80% similarity), to oligopeptide binding protein A of E. coli (34% identity and 57% similarity), and, more generally, to the periplasmic oligopeptide binding family of proteins.     

Separation and characterization of humic acids and metal humates by electrophoretic methods

A modified buffer for the separation of humic acids (HA) by capillary zone electrophoresis (CZE) was studied. The addition of hydroxycarboxylic acids to borate buffer makes a more efficient separation of HA possible. Two sharp peaks are obtained. Preparative isolation of these two fractions can be reached by applying the conditions used in CZE to free-flow electrophoresis (FFE). The fractions obtained were characterized by repetition of the CZE procedure. Also metal complexes of HA were investigated by electrophoretic methods. In the presence of metal cations, HA form stable complexes of different electrophoretic mobility. These complexes can be separated by free-flow isotachophoresis (FF-ITP). The different fractions are analyzed by CZE, ultraviolet-visible and atomic absorption spectrometry. Significant changes in both the ITP-pattern of the FFE and the 'humic hump' in CZE demonstrate the influence of complexation on the macromolecules.     

MR imaging of soft-tissue changes after percutaneous transluminal angioplasty and stent placement

A cDNA library enriched for human fetal-specific liver genes was constructed by suppressive subtractive hybridization. EST fls223 generated from this library was found to represent a novel putative serine/threonine (Ser/Thr) kinase. A full-length clone isolated for this gene encodes a protein of 396 amino acids. The amino acid sequence has 40% identity over 305 amino acids with the B1R Ser/Thr protein kinase of vaccinia virus. This gene has therefore been named VRK1 (vaccinia virus B1R kinase related kinase). VRK1 was also found to have sequence identity (62.0% over 481 nucleotides) to a database EST. A full-length clone for this EST was isolated and sequenced. Conceptual translation predicts a protein of 508 amino acids that, like VRK1, has similarity to B1R kinase (38.7% identity over 300 amino acids). This gene has been named VRK2. Comparison of VRK1 with VRK2 indicates that they encode structurally related putative Ser/Thr protein kinases. Northern analysis shows that expression of both genes is widespread and elevated in highly proliferative cells, such as testis, thymus, and fetal liver. B1R kinase is reported to be essential for DNA replication of vaccinia virus. The similarity of VRK1 and VRK2 to B1R indicates that these genes may have similar functions.     

Identification of metallothionein isoforms with capillary zone electrophoresis using a polyacrylamide-coated tube

Botulinum C2 toxin is composed of two nonlinked protein components, component-I (light chain) and component-II (heavy chain). It is produced by Clostridium botulinum types C and D, and is thought to play a lethal pathogenic role. These biological activities of C2 toxin may be due to the ADP-ribosylation of non-muscle actin by component-I of the toxin. We were able to isolate two overlapping gene fragments encoding component-I from the chromosomal DNA of Clostridium botulinum type C strain (C)-203U28, and determine the complete nucleotide sequence of component-I gene. The gene for component-I, bc21, consists of one open reading frame (ORF) encoding 431 amino acid residues (1293 nucleotides) without signaling peptide sequence. The molecular mass calculated from the deduced amino acid sequence was 49400.37 Da. Mono-ADP-ribosyltransferase activity was demonstrated in the lysate from E. coli transformed by the recombinant plasmid, pGEM-C2 encompassing whole component-I gene with its own promoter.     

Synovial sarcoma of the pericardium

Differences between the type B neurotoxin gene sequence of Clostridium botulinum type A(B) and Cl. botulinum type B, including a six nucleotide deletion, were recently proposed as a cause of the lack of expression of this gene in the type A toxigenic strains. A polymerase chain reaction (PCR) based on two sets of primers was designed to investigate the absence of the 6-nucleotide sequence in the apparently unexpressed type B toxin gene of 42 strains of Cl. botulinum type A(B). Thirty-five strains were shown to exhibit a deletion in their type B toxin gene; two strains did not have the deletion and actually produced small amounts of type B toxin when tested by the mouse bioassay. This two-step PCR might be useful for the rapid determination of the presence of the six nucleotide deletion and consequently, whether the type B toxin is likely to be produced.     

Isolation, crystallization, and primary amino acid sequence of human platelet factor 4

Human platelet factor 4 was purified by a method employing affinity chromatography on heparin/agarose. The amino acid sequence of the protein was determined by automatic Edman degradations and carboxypeptidase Y digestion. There are 70 amino acids in the protein with 5 of the 8 negatively charged residues clustered near the NH2 terminus and 10 of the 13 positively charged residues in clusters of 3 and 4 elsewhere in the protein. Small crystals have been obtained from ammonium sulfate solutions which give a promising preliminary x-ray diffraction pattern.     

cDNA cloning of nuclear localization signal binding protein NBP60, a rat homologue of lamin B receptor, and identification of binding sites of human lamin B receptor for nuclear localization signals and chromatin

We previously purified and characterized a nuclear localization signal (NLS) binding protein, NBP60, in rat liver nuclear envelopes. In this study, we cloned and sequenced the cDNA of rat NBP60, and predicted an amino acid sequence comprising 620 amino acids. The sequence revealed that NBP60 is a rat homologue of lamin B receptor (LBR), and is 79 and 63% identical in amino acids to human and chicken LBR, respectively. Using three fusion proteins containing different parts of the amino-terminal domain of human LBR, it was shown that the stretch comprising amino acids 1 to 89, which contains a Ser-Arg rich region (RS region), binds to nucleoplasmin and that the binding was inhibited by a common NLS-peptide. These results suggested that the amino-terminal domain of LBR contains an NLS-binding site. Furthermore, it was shown that the stretch comprising amino acids 1 to 53, which does not contain the RS region or the predicted DNA-binding site, binds to Xenopus laevis sperm chromatin.     

Covalent structure of botulinum neurotoxin type E: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains

Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an approximately 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992); Biochem. Biophys. Res. Commun. 183, 107-113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein--at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)--is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419-421 (Gly-Ile-Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1-Lys 418 constitute the approximately 50-kDa light chain, Lys 422-Lys 1251 constitute the approximately 100-kDa heavy chain; Cys 411-Cys 425 and Cys 1196-Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The approximately 150-kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without 14C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence.     

Characterization and biological properties of a new staphylococcal exotoxin

Staphylococcus aureus strain D4508 is a toxic shock syndrome toxin 1-negative clinical isolate from a nonmenstrual case of toxic shock syndrome (TSS). In the present study, we have purified and characterized a new exotoxin from the extracellular products of this strain. This toxin was found to have a molecular mass of 25.14 kD by mass spectrometry and an isoelectric point of 5.65 by isoelectric focusing. We have also cloned and sequenced its corresponding genomic determinant. The DNA sequence encoding the mature protein was found to be 654 base pairs and is predicted to encode a polypeptide of 218 amino acids. The deduced protein contains an NH2-terminal sequence identical to that of the native protein. The calculated molecular weight (25.21 kD) of the recombinant mature protein is also consistent with that of the native molecules. When injected intravenously into rabbits, both the native and recombinant toxins induce an acute TSS-like illness characterized by high fever, hypotension, diarrhea, shock, and in some cases death, with classical histological findings of TSS. Furthermore, the activity of the toxin is specifically enhanced by low quantities of endotoxins. The toxicity can be blocked by rabbit immunoglobulin G antibody specific for the toxin. Western blotting and DNA sequencing data confirm that the protein is a unique staphylococcal exotoxin, yet shares significant sequence homology with known staphylococcal enterotoxins, especially the SEA, SED, and SEE toxins. We conclude therefore that this 25-kD protein belongs to the staphylococcal enterotoxin gene family that is capable of inducing a TSS-like illness in rabbits.     

Truncated SNAP-25 (1-197), like botulinum neurotoxin A, can inhibit insulin secretion from HIT-T15 insulinoma cells

We and others have previously shown that insulin-secreting cells of the pancreas express high levels of SNAP-25 (synaptosomal-associated protein of 25 kDa), a 206-amino acid t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) implicated in synaptic vesicle exocytosis. In the present study, we show that SNAP-25 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A (BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins and caused significant reductions in K+ and glucose-evoked secretion of insulin. To determine whether the inhibition of release was due to the depletion of functional SNAP-25 or the accumulation of proteolytic by-products, we transfected cells with SNAP-25 proteins from which the C-terminal nine amino acids had been deleted to mimic the effects of the toxin. This modified SNAP-25 (amino acids 1-197) remained bound to the plasma membrane but was as effective as the toxin at inhibiting insulin secretion. Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K(+)-mediated plasma membrane depolarization. Electron microscopy revealed that cells transfected with either BoNT/A or truncated SNAP-25 contained significantly higher numbers of insulin granules, many of which clustered close to the plasma membrane. Together, these results demonstrate that functional SNAP-25 proteins are required for insulin secretion and suggest that the inhibitory action of BoNT/A toxin on insulin secretion is in part caused by the production of the plasma membrane-bound cleavage product, which itself interferes with insulin granule docking and fusion.     

Molecular characterization of the clusters of genes encoding the botulinum neurotoxin complex in clostridium botulinum (Clostridium argentinense) type G and nonproteolytic Clostridium botulinum type B

The cluster of genes encoding components of the progenitor botulinum neurotoxin complex has been mapped and cloned in Clostridium botulinum type G strain ATCC 27322. Determination of the nucleotide sequence of the region has revealed open reading frames encoding nontoxic components of the complex, upstream of the gene encoding BoNT/G (botG). The arrangement of these genes differs from that in strains of other antigenic toxin types. Immediately upstream of botG lies a gene encoding a protein of 1198 amino acids, which shows homology with the nontoxic-nonhemagglutinin (NTNH) component of the progenitor complex. Further upstream there are genes encoding proteins with homology to hemagglutinin components (HA-17, HA-70) and a putative positive regulator of gene expression (P-21). Sequence comparison has shown that BoNT/G has highest homology with BoNT/B. The sequence of the BoNT-cluster of genes in non-proteolytic C. botulinum type B strain Eklund 17B has been extended to include the complete NTNH and HA-17, and partial HA-70 gene sequences. Comparison of NTNH/G with other NTNHs reveals that it shows highest homology with NTNH/B consistent with the genealogical affinity shown between BoNT/G and BoNT/B genes.     

Cloning and nucleotide sequence of the alkaline protease gene from Fusarium sp. S-19-5 and expression in Saccharomyces cerevisiae

We have cloned a genomic DNA encoding the alkaline protease (Alp) of Fusarium sp. S-19-5 from a genomic DNA library and sequenced the nucleotides. Complementary DNA encoding Alp was also isolated from the cDNA library after amplifying the gene by PCR using partial sequences of the Alp genomic DNA as primers. The Alp gene has an open reading frame of 1137 nucleotides containing three introns. A TATA box (TAAATA) was observed 112 base pairs upstream from the translation initiation codon in the 5'-non coding region. The Alp protein has a pre region consisting of 14 amino acids and a pro region of 85 amino acids preceding the mature region, which consists of 280 amino acids. The amino acid sequence of Fusarium Alp has 52% homology with that of Aspergillus oryzae and 51% homology with that of Acremonium chrysogenum. The entire cDNA encoding Fusarium Alp was introduced into Saccharomyces cerevisiae, which then secreted enzymatically active Alp into the culture medium.     

Identification of random auditory waveforms. III. Effect of interference

A modified purification method was used to isolate the neurotoxin of proteolytic Clostridium botulinum type B strain Lamanna. The preparation was found to be a mixture of two protein forms. They were of molecular weight 152,000 and could not be separated by ion-exchange chromatography or electrophoresis in polyacrylamide gel. One was a single polypeptide chain, and the other was a dichain molecule (nicked toxin) held together by an interchain disulfide bond(s). Trypsinization increased the toxicity of the toxin preparation and converted the single-chain molecules into dichain forms that were indistinguishable from the endogenously generated nicked toxin. A protease of the type B culture, with substrate specificity similar to that of trypsin, did not change detectably the molecular form of unnicked type E toxin, although toxicity was increased. Higher toxicity was obtained when unnicked type E was trypsinized; the resulting preparation contained only nicked toxin molecules.     

Stress fractures at the base of the second metatarsal in ballet dancers

Buchnera aphidicola is a prokaryotic endosymbiont found in specialized cells of the aphid Schizaphis graminum. Many of the previously cloned B. aphidicola genes are preceded by a poor ribosome-binding site. Ribosomal protein S1 (RpsA) allows the translation of messenger RNAs that lack or have a poor ribosome binding site. We have cloned and sequenced a 4.5-kilobase (kb) B. aphidicola DNA fragment containing four open reading frames corresponding to aroA-rpsA-himD-tpiA. The deduced amino acid sequence of B. aphidicola RpsA was 75% identical to that of the Escherichia coli protein. The major difference was in the number of basic amino acids, which were present in higher numbers in B. aphidicola RpsA. Antiserum to E. coli RpsA was prepared and used to detect B. aphidicola RpsA in cell-free extracts of aphids. During the first 12 days of aphid growth there is a slight decrease in the amount of RpsA per unit of aphid weight. The three additional genes found on the 4.5-kb DNA fragment encoded for proteins involved in aromatic amino acid biosynthesis (aroA), DNA bending (himD), and carbohydrate metabolism (tpiA). The presence of these genes in B. aphidicola is additional evidence of its similarity to free-living bacteria.     

Purification and partial characterization of glyceraldehyde-phosphate dehydrogenase from electric organ of Electrophorus electricus (L.)

The glyceraldehyde-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified to homogeneity from electric organ of Electrophorus electricus (L.) by a hydrophobic chromatography method on deacetylcolchicine-Sepharose. The purification resulted in a 162 fold increase in specific activity of the GAPDH and final yield was approximately 37%. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. The purity of the colchicine-Sepharose isolated material was analysed by isoelectrophocusing and immunoblotting using a heterologous rabbit serum anti-GAPDH. Sequence analysis of the 40-N-terminal amino acids, determined by Edman degradation, revealed its identity to other GAPDHs proteins being the largest number of identical amino acids to lobster (92.5%), rabbit muscle (85%) and human liver (80%) GAPDH.     

Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins

Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.