Cloning of vascular adhesion protein 1 reveals a novel multifunctional adhesion molecule.

Vascular adhesion protein 1 (VAP-1) is a human endothelial sialoglycoprotein whose cell surface expression is induced under inflammatory conditions. It has been shown previously to participate in lymphocyte recirculation by mediating the binding of lymphocytes to peripheral lymph node vascular endothelial cells in an L-selectin-independent fashion. We report here that the VAP-1 cDNA encodes a type II transmembrane protein of 84.6 kD with a single transmembrane domain located at the NH2-terminal end of the molecule and six potential N-glycosylation sites in the extracellular domain. In vivo, the protein exists predominantly as a homodimer of 170-180 kD. Ax endothelial cells transfected with a VAP-1 cDNA express VAP-1 on their cell surface and bind lymphocytes, and the binding can be partially inhibited with anti-VAP-1 mAbs. VAP-1 has no similarity to any currently known adhesion molecules, but has significant identity to the copper-containing amine oxidase family and has a monoamine oxidase activity. We propose that VAP-1 is a novel type of adhesion molecule with dual function. With the appropriate glycosylation and in the correct inflammatory setting, its expression on the lumenal endothelial cell surface allows it to mediate lymphocyte adhesion and to function as an adhesion receptor involved in lymphocyte recirculation. Its primary function in other locations where it is expressed, such as smooth muscle, may depend on its inherent monoamine oxidase activity.

CD73 is involved in lymphocyte binding to the endothelium: characterization of lymphocyte-vascular adhesion protein 2 identifies it as CD73.

We have recently described a monoclonal antibody (mAb) 4G4 recognizing a 70-kD molecule constitutively expressed on human endothelial cells and on subpopulations of lymphocytes. We showed that this molecule, which we named lymphocyte-vascular adhesion protein 2 (L-VAP-2), mediates lymphocyte adhesion to cultured endothelial cells. Protein sequencing of tryptic peptides from immunoaffinity-purified L-VAP-2 revealed sequence identity between L-VAP-2 and CD73 (ecto-5'-nucleotidase, E.C.3.1.3.5), and COS cells transfected with a CD73 cDNA were positively stained with the mAb 4G4, which recognizes L-VAP-2. mAb 4G4 was also able to partially inhibit the ecto-5'-nucleotidase activity of peripheral blood lymphocytes. Moreover, cross-precipitation studies performed with mAb 4G4 and a CD73 workshop mAb 1E9 showed that these two antibodies recognize the same molecule. Since the tissue distribution and biochemical characteristics of the two molecules are also similar, we conclude that L-VAP-2 and CD73 are the same glycoprotein. Adhesion experiments showed significantly increased binding of freshly isolated lymphocytes to COS cells transfected with a CD73 cDNA, as compared to mock-transfected COS cells, and binding of lymphocytes to CD73-expressing COS cells was inhibited by the presence of mAb 4G4 in the adhesion assay. CD73 is a glycosyl phosphatidylinositol-linked molecule previously shown to have a cosignalling role in T lymphocyte proliferation. Our data suggest that it also has a function in mediating lymphocyte adhesion to the endothelium.

Waldenstrom macroglobulinemia involving the superior rectus muscle.

PURPOSE: We present the first reported case of Waldenstrom macroglobulinemia in the right superior rectus causing diplopia. OBSERVATIONS: A 72-year-old man with a 6-month history of untreated asymptomatic Waldenstrom macroglobulinemia presented with 2 years of diagonal binocular diplopia that was previously thought to be due to ocular myasthenia gravis. Examination showed mild right proptosis and right hypotropia, and MRI revealed a focal lesion of the right superior rectus muscle. Orbital biopsy was performed, and histopathology showed lymphoplasmacytic infiltration among the skeletal muscle fibers of the rectus muscle. Immunostaining confirmed a B-cell preponderance, along with more extensive staining for IgM than IgG, and in situ hybridization confirmed lambda restriction. These findings corresponded with those of his previous bone marrow biopsy, confirming Waldenstrom macroglobulinemia as the etiology for the extraocular muscle mass. CONCLUSIONS AND IMPORTANCE: Lymphoma of an extraocular muscle is a rare manifestation of orbital lymphoma, and the tumors are usually mucosa-associated lymphoid tissue (MALT) lymphomas (i.e. extranodal marginal zone lymphomas). There are 4 previous reports of lymphoplasmacytic lymphoma of an extraocular muscle; however this is the first reported case of such a lesion in a patient with concurrent Waldenstrom macroglobulinemia at the time of diagnosis.

Doppler echocardiographic measurement of pulmonary artery pressure from ductal Doppler velocities in the newborn.

The ductal flow velocities in 37 newborns (group 1: persistent pulmonary hypertension [n = 16], transient tachypnea [n = 3], other [n = 2]; group 2: respiratory distress syndrome [n = 16]) were prospectively evaluated by Doppler ultrasound for the purpose of deriving systolic pulmonary artery pressures. Maximal tricuspid regurgitant Doppler velocity in 21 of these patients was used to validate the pulmonary artery pressures derived from ductal flow velocities. There was a significant linear correlation between tricuspid regurgitant Doppler velocity and pulmonary artery systolic pressure derived from ductal Doppler velocities in patients with unidirectional (pure left to right or pure right to left) ductal shunting (p less than 0.001, r = 0.95, SEE 8) and in those with bidirectional shunting (p less than 0.001, r = 0.95, SEE 4.5). Systolic pulmonary artery pressure in group 1 (67 +/- 13 mm Hg) was significantly higher than that in group 2 (39 +/- 10 mm Hg) (p less than 0.001). In those with bidirectional shunting, duration of right to left shunting less than 60% of systole was found when pulmonary artery pressure was systemic or less, whereas duration greater than or equal to 60% was associated with suprasystemic pulmonary artery pressures. Serial changes in pulmonary artery systolic pressure, reflected by changes in ductal Doppler velocities, correlated with clinical status in persistent pulmonary hypertension of the newborn. Persistently suprasystemic pulmonary artery pressure was associated with death in five group 1 patients. It is concluded that ductal Doppler velocities can be reliably utilized to monitor the course of pulmonary artery systolic pressures in newborns.

A mouse molecular mimic of human vascular adhesion protein-1.

Human vascular adhesion protein-1 (VAP-1) is an endothelial sialoglycoprotein which exists in forms of Mr 90000 and 170000 and mediates lymphocyte binding to vessels under shear. VAP-1 is functionally defined by an inhibitory mouse mAb 1B2. A large-scale immunoaffinity purification of VAP-1 from human tonsil lysates was performed to determine the protein sequence for VAP-1 cDNA cloning. A dominant protein of molecular weight 90000 was obtained which yielded an N-terminal sequence of 20 amino acids which bore no significant identity to any protein sequence in the data banks. A mouse mAb (5B11) against a synthetic peptide from this sequence was raised and found to stain tissues in an identical manner to mAb 1B2, to inhibit lymphocyte adhesion to endothelial cells and to recognize VAP-1. Later, the N-terminal sequence obtained from the 1B2 immunoprecipitations was found to be identical to a mouse cyclophilin C associated protein (mCyCAP) subsequently published by others. We show here by several criteria at the protein and DNA level that VAP-1 is distinct from mCyCAP. Moreover, we elucidate the mechanism which results in binding of mCyCAP to mAb 1B2 during antibody synthesis in hybridoma cells and the sequelae of co-precipitation of mCyCAP during the immunoaffinity chromatography. Binding of mCyCAP to a mouse mAb has not been described before and suggests a new function for this molecule in immunoglobulin synthesis and/or secretion. Moreover, these data indicate that the N-terminal peptide of mCyCAP is a molecular mimic of a functionally important epitope of VAP-1.

Tartrate-resistant acid phosphatase from human bone: purification and development of an immunoassay.

Tartrate-resistant acid phosphatase (TRAP) was purified 20,000-fold to apparent homogeneity from human bone. The purified enzyme consisted of one 32 kd subunit, which was cleaved by beta-mercaptoethanol into two subunits of 15 kd and 20 kd, as shown by sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The purified enzyme was identified by N-terminal amino acid sequencing, and it was shown to be homologous with previously purified TRAPs from other sources. We developed a polyclonal antiserum against the purified enzyme in mice. In immunohistochemistry, the antiserum recognized osteoclasts from human bone and alveolar macrophages from human lung tissue, but no cells from human spleen tissue. It also stained osteoclasts from rat bone cells cultured on bovine bone slices. Purified TRAP could be inhibited by vanadate and molybdate, but not by tartrate, and it was activated 2-fold by beta-mercaptoethanol. The glycoprotein structure of human bone TRAP was analyzed, and it was shown to contain only high-mannose type carbohydrates. We used the polyclonal antibody to develop a competitive fluorescence immunoassay for measuring serum TRAP concentrations. According to the assay, children have higher serum TRAP concentrations than adults, and postmenopausal women have higher concentrations than premenopausal women. Postmenopausal women also have higher serum TRAP concentrations than postmenopausal women on estrogen replacement therapy.

Demonstration of the predominant urine osteocalcin fragments detectable by two-site immunoassays.

We have isolated and characterized human osteocalcin (OC) fragments from pubertal urine. The fragments were isolated by immunoaffinity chromatography based on monoclonal antibody 6F9 and further purified by reverse phase chromatography. The major isolated forms, which were detectable with two-site immunofluorometric assays for serum OC, span residues 6-30 and 7-30 as determined by mass spectrometry and N-terminal amino acid sequencing. Full-length OC was not detectable in the supernatant fraction of urine but could be extracted with guanidinium hydrochloride from the sediment of urine samples. Urine samples from subjects with different menopausal status were measured by two different two-site assays. Urine OC (uOC) concentrations were 12- to 16-fold higher in the pubertal group than in the adult group. Also, the uOC concentration in a postmenopausal group was significantly higher than in a premenopausal group. The difference was 125% and 75% (values for p < 0.0001), respectively, when measured with the two assays. uOC concentrations in postmenopausal subjects on hormone replacement therapy were indistinguishable from the premenopausal subjects. The fact that uOC can be measured by a noncompetetive two-site assay design offers improved analytical sensitivity. Urine as the sample matrix is also especially interesting because the predominant markers of bone resorption, collagen type I peptides or cross-links, are performed on urine samples. Our results from the technical validation of two-site assays for uOC and from applying these to human pubertal and pre- and postmenopausal samples calls for more extensive clinical validation.

A dual-label immunofluorometric assay for human osteocalcin.

Circulating human osteocalcin (hOC) has been shown to be comprised of two main forms: the intact 1-49 form and the proteolytic N-terminal midfragment (N-mid) spanning amino acid residues 1-43 or 1-44. We used three monoclonal antibodies (MAbs) raised against hOC and bovine osteocalcin in developing a dual-label assay for the simultaneous measurement of the proportions of the intact and N-mid forms in serum samples. The assay is based on time-resolved fluorescence utilizing differently labeled trace MAbs. Biotinylated MAb 2H9 is used as a capture antibody for both the intact hOC and the N-mid. Tracer MAb 6F9 labeled with a Europium (III)-chelate binds to the intact the N-mid and the intact hOC, whereas tracer MAb 3G8 labeled with a Terbium (III)-chelate binds to the intact hOC only. The simultaneous binding of the antibodies was tested by comparing full-length hOC purified from human bone and hOC shortened from the C terminus by four amino acid residues with carboxypeptidase Y. Serum hOC measurements with the dual-label assay were in agreement with the corresponding single-label assays (r = 0.96 for intact + N-mid assay and r = 0.81 for intact assays, n = 91). The lower correlation between the intact assays was attributable to proteolytic susceptibility of the intact form due to one additional freezing and thawing cycle in carrying out the dual-label assay. As measured with the dual-label assay, the levels (mean +/- SD) of serum intact + N-mid OC were 6.2 +/- 2.1 ng/ml in the premenopausal group (n = 44), 13.9 +/- 4.9 ng/ml in the postmenopausal group without hormone replacement therapy (HRT; n = 13), and 7.5 +/- 3.4 ng/ml in the postmenopausal group with HRT (n = 13). The levels of intact hOC in the same groups were 4.8 +/- 1.4 ng/ml, 9.8 +/- 2.9 ng/ml, and 5.3 +/- 2.1 ng/ml, respectively. Whether the main forms of OC or their relative proportions in serum can be used for predicting bone diseases or for monitoring the progression and management of diseases awaits further investigations.

Epitope mapping of nine monoclonal antibodies against osteocalcin: combinations into two-site assays affect both assay specificity and sample stability.

Nine monoclonal antibodies (Mabs) were raised against human recombinant osteocalcin fusion protein (rGST-hOC) or bovine osteocalcin (bOC) and selected to develop two-site immunoassays for human osteocalcin (hOC). The detection system was based on the time-resolved measurement of the fluorescence of europium chelates conjugated to the tracer Mabs. Based on the ability of the Mabs to recognize different forms of hOC (carboxypeptidase Y-digested, alkylated hOC, thermally decarboxylated hOC, recombinant forms of hOC, and tryptic peptides derived from hOC) and the information obtained from combinations of the Mabs in two-site assays, an epitope map was created. The epitope map was useful in understanding the behavior of the two-site combinations of the Mabs with serum samples. The two-site combinations could be divided into subgroups detecting either full-length hOC or full length+large NH2-terminal fragment as stimulated by the carboxypeptidase Y-digested form of hOC (it lacks four COOH-terminal residues), which with intact specific assays showed cross-reactivities ranging from 7 to 14% when compared with full-length hOC. In addition, differences were observed in the ability of the combinations to detect thermally decarboxylated hOC (lacks gamma-carboxylation at residues 17, 21, and 24) with cross-reactivities ranging from 8 to 85% when compared to gamma-carboxylated hOC. The analysis of human serum samples showed considerable differences in the concentration and stability of serum OC. This was attributed as the varying ability of the Mabs to detect different proteolytic fragments derived from hOC and/or differences in the degree of gamma-carboxylation of hOC. The in vitro generation of the large NH2-terminal fragment during incubation of the serum samples at room temperature (RT) and during prolonged storage at -20 degrees C in an undercooled state was detectable as loss of immunoreactivity (ranging from -42 +/- 17 to -50 +/- 15% in 16 h at RT, n = 3) with Mab combinations detecting only full-length hOC. Two-site combinations detecting full-length+large NH2-terminal fragment showed no loss of immunoreactivity after incubation of the serum samples at RT for 16 h. With all assays, an increase of serum OC ranging from 16 to 38% was found in postmenopausal samples (n = 24) when compared with premenopausal samples (n = 17), but the degree of statistical significance varied from not significant to p < 0.01.

Two-site immunoassays for osteoclastic tartrate-resistant acid phosphatase based on characterization of six monoclonal antibodies.

Tartrate-resistant acid phosphatase (TRAP), an enzyme expressed in bone-resorbing osteoclasts, is secreted into the circulation during bone resorption. We used six monoclonal antibodies (MAbs) to optimize direct two-site fluoroimmunoassays for determining serum TRAP concentrations. Four of the MABs, 1F1, 2H1, 4E6, and 5C1, were raised against recombinant human TRAP, and the other two, O1A and J1B, against human bone TRAP. 2H1, J1B, and O1A appeared to be highly specific for TRAP. 1F1 and 4E6 were poor in recognizing bone TRAP and were not useful in the assay. 5C1, while having a good affinity for the bone enzyme, was not specific. Serum TRAP is relatively stable, because 7 days of storage of serum samples at 4 degreesC and -20 degreesC or five thawing-freezing cycles, did not change the TRAP concentration detected using the two-site assays. All studied assays detected an increase in serum TRAP concentrations of postmenopausal women compared with premenopausal women, the difference being highest with MAB pairs 2H1-5C1 and O1A-J1B. These results suggest that serum TRAP may be a useful bone resorption marker, and the MAB pairs 2H1-5C1 and O1A-J1B may be useful in determining the bone resorption rate.

N-terminal mutations in the anti-estradiol Fab 57-2 modify its hapten binding properties.

Recombinant antibodies often contain N-terminal mutations arising from the use of degenerate cloning primer sets and/or the introduction of restriction sites in the framework 1 regions. We studied the effects of such mutations in a recombinant anti-estradiol Fab fragment derived from the hybridoma cell line 57-2. The 5' ends of the heavy and light chain genes were originally modified to introduce the restriction sites XhoI and SacI, respectively, for cloning purposes. However, the affinity and specificity of the recombinant Fab were lowered compared to the proteolytic Fab' fragment of the parental hybridoma IgG. Replacing the mutated sites with authentic amino acid coding sequences restored the binding properties as well as increased the bacterial production levels fivefold and 10-fold at 30 and 37 degrees C, respectively. Local changes in the antigen binding site were probed by determining the affinity constants (Kd) for estradiol and four related steroids. It was found that the mutated heavy chain amino terminus specifically increased the Kd for testosterone whereas the mutated light chain amino terminus decreased the Kd for all of the steroids to the same extent; the heavy and light chain effects were additive. Analysis of a newly determined crystal structure of the authentic Fab 57-2 in complex with estradiol suggests that mutations in the residue 2 in V(H), and 2 and 4 in the V(L) domain were those responsible for the observed effects. Their general roles as structure-determining residues for the CDR3 loops imply that similar effects can occur with other recombinant antibodies as well.

Outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP) are released in experimental Gram-negative sepsis.

We previously showed that Escherichia coli bacteria incubated in normal human serum release complexes that contain three conserved Gram-negative bacterial outer membrane proteins (OMPs) and LPS. We have identified the OMPs as outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP). These OMPs are conserved among enteric Gram-negative bacteria and are bound by IgG in antisera raised to heat-killed rough bacteria such as E. coli J5 (J5 IgG). The present experiments were performed to further analyze the release of these OMPs in a rat wound infection model of sepsis. Plasma was collected from thermally injured rats with E. coli O18 sepsis and filtered. LPS was affinity-purified from plasma filtrates using monoclonal antibody specific for the O-polysaccharide side chain of E. coli O18 LPS. Plasma filtrates were also incubated with J5 IgG conjugated to magnetic beads. Affinity-purified samples were analyzed for the OMPs by immunoblotting. OmpA, PAL, and MLP were released into septic rat blood in complexes with LPS. PAL was consistently present in samples affinity-purified using J5 IgG. The results indicate that OmpA, PAL, and MLP are released and circulate in experimental Gram-negative sepsis and suggest that a proportion of released OMPs are tightly associated with LPS.

Thermal gradients in microtitration plates. Effects on enzyme-linked immunoassay.

Temperature studies of microtitration plates demonstrate that the use of a common bacteriology incubator for heating the plates can cause a phase log of over 30 min for the fluid in the wells to reach 37 degrees C from ambient temperature, and that a temperature gradient of as much as 1.6 degree C can exist between the peripheral and center wells. This gradient is a cause of the "rim" of edge effect noted in enzyme immunoassay using microtitration plates. The problem is corrected by the use of a specially designed forced air microtitration plate incubator.

Aspartate aminotransferase from an alkalophilic Bacillus contains an additional 20-amino acid extension at its functionally important N-terminus.

Aspartate aminotransferase (AspAT), responsible for a minor part of the total AspAT enzymic activity in alkalophilic Bacillus circulans, was purified, its N-terminal amino acid sequence was determined, and its gene was cloned as two separate fragments. DNA sequencing showed an open reading frame of 432 amino acids (M(r) 47,439) exhibiting moderately low homology with AspATs from other sources. Sequence alignment of the enzyme with chicken mitochondrial, chicken cytoplasmic and Escherichia coli AspATs was performed with the MULTALIN program and further optimized assuming that the three-dimensional structures of the proteins were conserved. The primary structure of the studied AspAT diverged markedly from the others in the catalytically important small domain and in a segment of 31 amino acids in the large domain. The functional N-terminal arm was about two times longer than those of AspATs from other sources. According to the molecular model, the unique regions of B. circulans AspAT are all located together, forming a continuous network of contacts. Additional contacts formed by the elongated N-terminal arm may result in some limitation of domain movements in the alkalophilic enzyme in comparison to in other known AspATs.

Antiendotoxin strategies.

Endotoxin is a potent stimulator of the inflammatory response and is believed to initiate the pathology in Gram-negative sepsis. Agents are being developed that bind and neutralize or block the effects of endotoxin, with the goal of improving outcome in the treatment of sepsis. Strategies discussed in this article include anti-LPS antibodies, LPS binding proteins and lipoproteins, polymyxin B conjugates, lipid A analogues, and extracorporeal techniques for endotoxin removal.

Alteration of the hormonal bioactivity of parathyroid hormone-related protein (PTHrP) as a result of limited proteolysis by prostate-specific antigen.

OBJECTIVES: To discover whether the proteolytic activity of prostate-specific antigen (PSA) affects the structure and function of parathyroid hormone-related protein (PTHrP), as both are abundant components of human seminal plasma. METHODS: The ability of PTHrP to act as a substrate was studied by incubating a synthetic polypeptide, consisting of 34 amino acid residues of the amino-terminal domain of PTHrP, with purified PSA. The incubate was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-pressure liquid chromatography separation, amino-terminal peptide sequencing, and mass spectrometry. The physiologic effect of the proteolytic activity of PSA on PTHrP was studied by measuring any alteration in PTHrP (1-34)-induced elevation of cyclic adenosine monophosphate (cAMP) production by UMR-106 rat osteosarcoma cells in culture. All cell culture experiments were performed with PSA and PTHrP (1-34) at physiologic concentrations. RESULTS. Our data show that PSA proteolytically cleaves PTHrP (1-34) after either residue 22 or 23, generating three peptide fragments. Both cleavages occur carboxy terminally of a phenylalanine residue. The cAMP production in rat osteosarcoma cells, induced by the amino-terminal portion of PTHrP (1-34), as a result of its structural similarity with parathyroid hormone (PTH), was abated by PSA in a dose- and time-dependent fashion. In contrast, heat-inactivated PSA had no effect on cAMP production. CONCLUSIONS: Our study demonstrates that PTHrP is a substrate for PSA. The cleavage of the amino-terminal portion of PTHrP completely disrupts its ability to interact with the PTH/PTHrP receptor and thus inhibits its PTH-like activity. The proteolytic processing of PTHrP by PSA may play an important role in the post-translational/post-secretional regulation of prostatic PTHrP activities, which are believed to include regulation of prostate growth and differentiation.

RT-PCR from eosinophil-depleted leukocytes without RNA extraction: cell selection using streptavidin PCR tubes.

OBJECTIVES: The major RNase activity of leukocytes has been attributed to eosinophil-derived neurotoxin EDN. Depletion of eosinophils enables RT-PCR from 10(5) leukocytes without RNA extraction. In this study we introduced streptavidin-coated PCR tube strips for the selection of eosinophil-free leukocytes for RT-PCR analysis. DESIGN AND METHODS: Polypropylene 0.2 ml PCR tube strips were coated with streptavidin and biotinylated antibodies against cell surface antigens were attached to the tubes. CD7-positive T-lymphocytes, CD19-positive B-lymphocytes and CD16-positive cells (mainly neutrophils and monocytes) were positively selected by incubating of 1-2 x 10(5) leukocytes in the antibody-coated PCR tubes for 30 min at 23 degrees C. RESULTS: The mean amount of cells bound into a tube was 31,500 (CV25%) T-cells and 8,600 (CV61%) B-cells from 12 blood samples, and 23,600 (CV22%) CD16+ cells from 17 samples. The influence of selected cell lysate on the RT-PCR analysis of Philadelphia chromosome (bcr/abl translocation) from 100 K562 cells was small: 78% (CV28%) of the leukocyte-free signal was obtained in the presence of CD16+ cells or 89% (CV15%) and 99% (CV11%) and in the presence of T-cells and B-cells, respectively. CONCLUSIONS: These results suggest that through the introduction of eosinophil-free cell population into RT-PCR a reproducible method with reasonable leukocyte yield and avoiding RNA extraction was developed.

Construction of an Escherichia coli export-affinity vector for expression and purification of foreign proteins by fusion to cyclomaltodextrin glucanotransferase.

A novel export-affinity fusion vector employing the gene encoding cyclomaltodextrin glucanotransferase (CGTase; cgt) from Bacillus circulans var. alkalophilus (ATCC 21783) is described. CGTase binds to various sugar polymers, which makes it simple to purify it to near homogeneity in a single step. The CGTase fusion protein vector was constructed by deleting the translational stop codons from the gene encoding CGTase (cgt) by in vitro mutagenesis. As models, genes encoding Escherichia coli alkaline phosphatase (APase; phoA) and Bacillus stearothermophilus (ATCC 12980) alpha-amylase (BStA; amy) were fused to cgt. Overexpression of wild type CGTase and the hybrid proteins under the control of the lac promoter caused a 'leaky phenotype' in E. coli, the outer membrane became permeable, which enabled the adsorption of the fusion proteins directly from the culture medium onto alpha-cyclodextrin (alpha-CD) coupled agarose. The hybrid proteins were eluted from the column with alpha-CD solution under mild conditions at pH 7.5. The CGTase-APase' fusion had a good in vivo stability, whereas the CGTase-BStA' was less stable. In the latter case, according to protein sequencing, the proteolytically sensitive site was on the BStA' side of the fusion. The C-terminus of CGTase was stable against proteolysis as shown by narrow pH range isoelectric focusing. The fused enzymes retained their biological activities.

Expression in E. coli and purification of intracellular proteins by fusion to cyclomaltodextrin glucanotransferase.

A plasmid expression vector was constructed to direct the synthesis of foreign proteins in Escherichia coli as fusions with cyclomaltodextrin glucanotransferase (CGT) with cytoplasmic location (delta ssCGT). The ability of CGT to bind to covalently immobilized cyclodextrins was utilized in purifying fused target proteins. A large proportion of the cytoplasmically synthesized delta ssCGT formed inclusion bodies which adopted the active conformation at considerably high refolding concentration (67 microM delta ssCGT solution). By lowering the cultivation temperature the proportion of the soluble delta ssCGT was slightly increased. Intracellularly expressed delta ssCGT provides a potential affinity handle which forms easily refoldable inclusion bodies increasing the yield and stability, and possibly allows the expression of lethal target proteins. Interestingly, the interaction between one model fusion protein delta ssCGT-CAT (CAT, chloramphenicol acetyltransferase) and the E. coli heat shock protein GroEL was observed.

Venous air embolism, hypotension, and end-tidal nitrogen.

The most sensitive method of venous air embolism (VAE) detection in clinical use is the precordial Doppler detector. Because the Doppler detector may provide false-positive and false-negative data, many clinicians rely on end-tidal gas measurements for verification of VAE in the operating room. End-tidal nitrogen (ETN2) increases soon after experimental VAE are small enough to cause minimal changes in blood pressure; however, decreases in end-tidal CO2 (ETCO2) are more sensitive. A large VAE causes hemodynamic instability, and the effect of low cardiac output on ETN2 has not been evaluated. This study was done to compare the changes in ETN2 and ETCO2 during large bolus and infusion VAE. Five mongrel dogs were anesthetized, intubated, and ventilated (FIO2 1.0, PaCO2 38 +/- 4 (SD) mm Hg). The animals were studied in the supine position; anesthesia and paralysis were maintained with a constant infusion of thiamylal and pancuronium. Maintenance fluids were administered at 5 ml kg-1 h-1. Mean arterial and pulmonary arterial pressures (PAP) and ETN2 and ETCO2 were displayed on a strip chart recorder. The dogs underwent both bolus and infusion VAE in separate experiments 10 to 14 days apart. The air emboli were given in random order by automated syringe over 1 minute (infusion) (1 to 2.5 ml kg-1 min-1) or by hand injection over 5 seconds (bolus) (1 to 2.5 ml kg-1). Changes in precordial Doppler sounds occurred in all animals at all doses. The peak increase in PAP and decrease in ETCO2 were significant after all air doses. ETN2 changes were biphasic. The peak increase was significant after all air doses; the peak decrease was significant in 37 of 40 bolus and infusion VAE episodes, occurred within 1 to 3 minutes, and lasted 20 to 30 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)