Dual effects of allergen-mPEG conjugates. Induction of immunological suppression and inactivation of sensitized mast cells.
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Allergens↗
Biomedical subjects
G Lang
Publications and source records attributed to G Lang.
Structure/function relationships of mitochondrial monoamine oxidase A and B chimeric forms.
Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH2-terminus, which contains an ADP-binding consensus sequence, little is known about their structural features or the sequences involved in the binding of substrates. In the present paper, we have studied the structure/function relationships of MAOs by constructing 18 different chimeric forms of MAO, engineered by moving progressively the junction between the NH2-terminus of one MAO form with the COOH-terminus of its isoenzyme. After transient expression in HEK-293 cells, the properties of these chimeric enzymes were investigated using both selective and nonselective substrates and inhibitors. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimeras with increasing length of the NH2-terminus of MAO-A (up to residue 256) showed a marked decrease in affinity towards the MAO-B substrate phenylethylamine and the inhibitor N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide . HCl (lazabemide) when compared to wild-type MAO-B. No major changes were observed in the kcat values of these chimeras. From the data obtained, two sequences, i.e. 62-103 and 146-220, appeared of particular importance in constituting the binding site of MAO-B. On the other hand, the catalytic properties and specificity of MAO-A appeared to be relatively insensitive to substitution of both the NH2- (up to position 112) and COOH-termini (from residue 395) of MAO-A with the corresponding MAO-B sequences. However, further modification of the central 283-residue sequence of MAO-A did not appear compatible with enzymic activity. None of the engineered chimeras showed a shift in specificity from one isoform to the other.
Base Sequence↗
Composite and plain tubular synthetic graft conduits in right ventricle-pulmonary artery position: fate in growing lambs.
Our goal was to identify the most appropriate material for right ventricle-pulmonary artery conduits in growing animals. We used 100 lambs that were 3 to 4 weeks old (mean weight 11.7 kg). Follow-up was up to 24 months. Group I received plain tubular conduits: (1) Dacron knitted fabric, (2) collagen-coated knitted fabric, (3) Milliknit and Microknit material, (4) woven Dacron fabric, (5) three-dimensional Dacron fabric (crossweave 500 and 800), or (6) polytetrafluoroethylene. Group II received either a (1) woven Dacron fabric conduit with a built-in tissue valve or (2) polytetrafluoroethylene graft with a built-in St. Jude Medical valve. We did angiograms and catheterizations every 3 to 6 months and killed the lambs at 6, 12, 18, or 24 months. Tubular Dacron fabric woven or knitted grafts, regardless of matrix, pore size, thickness, or coating, caused formation of a thick acellular pseudointima buildup, which led to progressive obstruction starting as early as 3 months. Polytetrafluoroethylene grafts in groups I and II showed the formation of thin inner and outer capsules (0.5 mm) and none developed obstruction despite wall calcification. Conduits of woven Dacron fabric with a built-in tissue valve degenerated rapidly, leading to calcification thrombosis and obstruction within 3 months; no lamb survived 12 months. Polytetrafluoroethylene conduits with a St. Jude Medical valve in lambs receiving anticoagulants remained free of obstruction and continued to function well. It appears that synthetic conduits of polytetrafluoroethylene perform well in either of the situations here tested and may be the best choice at present.
Animals↗
Cloning and characterization of a soluble kynurenine aminotransferase from rat brain: identity with kidney cysteine conjugate beta-lyase.
In this study, we describe the cloning and characterization of a soluble form of kynurenine aminotransferase (KAT, EC 2.6.1.7) present in rat brain. Soluble KAT was purified from rat kidney and the amino acid sequences of four tryptic peptides determined. These peptides were found to belong to the amino acid sequence reported for rat kidney soluble cysteine conjugate beta-lyase, indicating that rat kidney KAT and beta-lyase represent the same molecular entity. Oligonucleotide probes derived from the beta-lyase cDNA were then used as primers for PCR of reverse-transcribed rat brain poly(A)+ RNA. After subcloning of the resulting PCR fragment and sequencing of the isolated rat brain clone, its oligonucleotide sequence was found to be identical to that reported for the beta-lyase cDNA. Further evidence that the isolated rat brain clone encoded for KAT was obtained by transfecting HEK-293 cells with a construct containing the coding sequence for the enzyme. The transfected cells exhibited KAT activity and, in the presence of 2 mM pyruvate and 2-oxoglutarate, the Km values for L-kynurenine were 1.2 mM and 86.3 microM, respectively. Northern blot analysis of rat kidney, liver, and brain RNA revealed a single species of KAT/beta-lyase mRNA of approximately 2.1 kb.
Amino Acid Sequence↗
BLT-esterase in infectious mononucleosis.
Peripheral blood lymphocytes of three patients suffering from infectious mononucleosis due to Epstein-Barr virus (EBV) infection were analysed for BLT-esterase expression in peripheral blood lymphocytes by a well established cytochemical staining method. During the acute phase of disease with presence of clinical symptoms a very high level of up to 90% BLT-esterase-expressing lymphocytes were detected. The increased percentage of lymphocytes expressing BLT-esterase coincided with the time of greatest symptoms and the peak elevation of hepatocellular enzymes. The still moderately elevated level only gradually decreased to normal during the further recovery period of 2 months during which the patients described episodes of weakness. Peripheral blood lymphocyte phenotype analysis revealed a marked CD8 lymphocytosis, a CD4/CD8 ratio of about 0.2, low number of CD19+ B cells, and a high level of DR+ CD3+ lymphocytes. Reduction of BLT esterase expression during the recovery period coincided with reduction of CD8+ DR+ lymphocytes. By a combination of BLT-esterase staining with immunocytochemical phenotype analysis, 95% of CD8+ lymphocytes were found to be BLT-esterase-positive. BLT-esterase might be involved in the immunodefence against EBV in infectious mononucleosis by inducing apoptosis in EBV-transformed B cells.
Adolescent↗
Retinoic acid inhibits both the basal activity and phorbol ester-mediated activation of the HIV long terminal repeat promoter.
OBJECTIVE: To establish whether the steroid hormone retinoic acid (RA) was able to modulate the activity of the HIV-1 promoter. DESIGN: The effect of RA and nuclear factor (NF)-kappa B on HIV-1 promoter activity was investigated using HIV-1 long terminal repeat (LTR)-based reporter constructs. METHODS: The activity of wild type and mutant LTR sequences was compared in a variety of stably and transiently transfected human cell lines. RESULTS: It was shown that RA treatment inhibits both the basal activity of the HIV-1 LTR and its stimulation by phorbol ester treatment. This inhibition is observed in both HeLa cells (in the presence of exogenously supplied RA receptors) and the naturally RA-responsive U937 monocyte cell line. RA can also inhibit the stimulation of the HIV-1 LTR by overexpression of NF-kappa B subunits, while linkage of the NF-kappa B sites in the HIV promoter to a heterologous promoter results in its inhibition by RA. CONCLUSIONS: The data presented clearly demonstrate a negative effect of RA on HIV-1 LTR activity that may contribute to its effect on viral replication.
Base Sequence↗
Endoscopic management of the symptomatic caliceal diverticular calculus.
Caliceal diverticular calculi are most often asymptomatic and of little clinical significance. In certain cases they may be associated with flank pain, pyuria and chronic urinary tract infections. Treatment has evolved from open surgical techniques to a purely endoscopic approach. Percutaneous techniques are frequently used to address the diverticular stone burden and to dilate the diverticular neck, improving drainage. Small volume caliceal diverticular calculi and those in the anterior portion of the collecting system represent a greater technical challenge to the endoscopist. We describe a purely retrograde endoscopic technique for treating small stone burdens trapped in caliceal diverticula. Flexible ureteroscopy combined with 3F dilating balloons passed through the endoscopic working channel facilitated treatment. This retrograde technique was combined with a simultaneous primary percutaneous puncture into the diverticulum to treat larger stone burdens and calculi within long-necked diverticula. This combination facilitated prompt, through and through access of a percutaneously placed guide wire, increasing the overall efficiency of treatment. In conclusion, a retrograde endoscopic technique using the actively deflectable, flexible ureteroscope can successfully treat certain caliceal diverticular calculi. By combining this technique with a simultaneous percutaneous puncture, caliceal diverticular calculi throughout the collecting system may be cleared expeditiously.
Diverticulum↗
Flexible ureteroscopically assisted percutaneous renal access.
OBJECTIVE: To combine retrograde flexible ureteroscopic techniques with a simultaneous percutaneous puncture to gain precise antegrade renal access in selected patients. METHODS: Patients with minimally dilated collecting systems and complex stone burdens (including caliceal diverticular calculi) underwent adjunctive flexible ureteroscopy in an attempt to expedite percutaneous renal access. This combined retrograde and antegrade approach was also used in treating obese patients and those in whom prior attempts at percutaneous renal access had failed. The prone split-leg position and flexible retrograde ureteroscopy were employed. The exact calyx for percutaneous puncture was selected under direct vision using an actively deflectable, flexible ureteroscope. Access to narrow infundibula and caliceal diverticula was facilitated by employing injectable guidewires as well as small-diameter balloon dilators passed through the working channel of the flexible endoscope. A fluroscopically guided percutaneous puncture was then performed. The tip of the intrusive needle was visualized both fluoroscopically and endoscopically. An antegrade guidewire was advanced through a ureteroscopically positioned snare and withdrawn out the urethra. With this through-and-through guidewire access, standard percutaneous tract dilation and nephroscopic lithotripsy were performed expeditiously. RESULTS: Seven patients with renal disease or body habitus that made precise percutaneous access difficult underwent adjunctive retrograde flexible ureteroscopy. One patient had a large perinephric hematoma from previous attempts at nephrostomy placement. The other six patients presented with: tightly branched staghorn calculi (three patients) and lateral/anterior caliceal diverticular calculi (three patients). Four patients were morbidly obese (240-320 lb), which also complicated antegrade access. Percutaneous renal access was obtained in < 30 min in all cases. CONCLUSION: A precise percutaneous puncture into a complex collecting system and establishing a through-and-through safety guidewire can be facilitated with simultaneous retrograde flexible ureteroscopic techniques.
Catheterization↗
Mass spectrometric analysis of human soluble catechol O-methyltransferase expressed in Escherichia coli. Identification of a product of ribosomal frameshifting and of reactive cysteines involved in S-adenosyl-L-methionine binding.
Technological advances in the field of mass spectrometry (MS) are providing powerful analytical means for the investigation of proteins and peptides. In the present work we have used pneumatically assisted electrospray (ion-spray) MS for the biochemical characterization of recombinant human catechol O-methyltransferase (rhCOMT). hCOMT could be expressed in Escherichia coli in large quantities but in two forms of different size, both enzymically active. Electrospray MS analysis showed that the smaller rhCOMT protein had a molecular mass of 24352 +/- 2 Da, corresponding to the calculated value for native hCOMT (without the initiating methionine), whereas that mass of the larger protein was of 25775 +/- 4 Da. To investigate the molecular differences between the two proteins, they were digested with trypsin and the peptides produced analysed by electrospray MS. Neither protein apparently contained disulfide bridges and the observed molecular masses of the tryptic peptides corresponded to the calculated values. It was possible to determine, however, that the larger protein contained an extended C-terminus with the correct sequence GPGSEAGP plus an additional stretch, EDLR. This C-terminal extension resulted from ribosomal frameshift at the codon of the last proline (CCC, rare codon in prokaryotes). In fact, rightward frameshifting would produce a hCOMT form with an additional stretch of 11 amino acid (EDLRSHHHHHH) and the calculated molecular mass of this protein (25773.5 Da) is in good agreement with our experimental result. The differential reactivity of the cysteine residues of the correct rhCOMT enzyme, in the presence and in the absence of S-adenosyl-L-methionine (AdoMet) and MgCl2, was also studied. 5-Iodoacetamido fluorescein (5-IAF) was used as thiol-modifying reagent. Under the conditions used, 5-IAF rapidly inactivated rhCOMT but the presence of AdoMet and MgCl2 partially protected it from inactivation. The 5-IAF-labeled tryptic peptides were separated by HPLC and then submitted to electrospray MS and tandem MS. Several cysteine residues appeared to be readily available to chemical modification by 5-IAF. Incorporation of 5-IAF occurred to a larger extent into Cys32, Cys68, Cys94 and Cys172. AdoMet and MgCl2 markedly reduced the label incorporation into Cys68 and Cys94, therefore suggesting that these residues belong to a region at or near the binding site of AdoMet.
Amino Acid Sequence↗
Molecular cloning and functional expression of human 3-hydroxyanthranilic-acid dioxygenase.
Increased cerebral levels of the endogenous excitotoxin quinolinic acid (QUIN) have been speculatively linked to neuronal damage following neurological and inflammatory disorders. 3-Hydroxyanthranilic-acid dioxygenase (3-HAO; 3-hydroxyanthranilate 3,4-dioxygenase, EC 1.13.11.6) is the enzyme that catalyzes the synthesis of QUIN from 3-hydroxyanthranilic acid, and evidence suggests that it could play a role in disorders associated with altered tissue levels of QUIN. In this report, we describe the isolation of a full-length cDNA clone encoding human 3-HAO (h3-HAO). Degenerate oligonucleotides were designed from the amino acid sequences of tryptic peptides of rat liver 3-HAO, and they were used as primers for reverse transcription-polymerase chain reaction of rat liver RNA. The resulting rat cDNA product was used to screen a human hepatoma cell line (HepG2) cDNA library and to isolate a human 3-HAO cDNA clone. This clone was found to have an insert of 1276 nucleotides. The deduced primary structure of h3-HAO is composed of 286 amino acid residues with a predicted molecular mass of approximately 32.6 kDa. The human sequence exhibits high similarity (94%) to the rat partial amino acid sequence deduced from the rat reverse transcription-polymerase chain reaction fragment. Insertion of the h3-HAO coding sequence into a eukaryotic expression vector yielded relatively high amounts of the active enzyme in human embryonic kidney HEK-293 cells. The Km value of 3-HANA for recombinant h3-HAO (approximately 2 microM) was in good agreement with that reported for the native enzyme. Immunoblot analysis of recombinant h3-HAO revealed a polypeptide with an apparent molecular mass of 32 kDa, as predicted from the deduced amino acid sequence. RNA blot analysis of human liver and HepG2 cells revealed one major species of h3-HAO mRNA of approximately 1.3 kilobases.
3-Hydroxyanthranilate 3,4-Dioxygenase↗
Practical solution to the Monte Carlo sign problem: Realistic calculations of 54Fe.
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Journal Article↗
Hypertonic hydroxyethyl starch restores hepatic microvascular perfusion in hemorrhagic shock.
The influence of small-volume resuscitation (hypertonic saline-10% hydroxyethyl starch, HS/HES) on liver microcirculation (intravital fluorescence microscopy) was studied in a nonheparinized hemorrhagic shock model [mean arterial pressure (MAP) 40 mmHg for 1 h] in rats. Resuscitation was performed with Ringer lactate (RL, 4-fold shed volume/20 min; n = 7), 10% hydroxyethyl starch 200/0.6 (HES, shed volume/5 min; n = 6), or 7.2% NaCl-10% hydroxyethyl starch 200/0.6 (HS/HES, 10% shed volume/2 min; n = 7). One hour after resuscitation, MAP increased in all groups, but it did not return to preshock values (P < 0.05). HES (16 +/- 2% nonperfused sinusoids) and HS/HES (14 +/- 2% nonperfused sinusoids), but not RL (24 +/- 2% nonperfused sinusoids), reduced (P < 0.05) shock-induced sinusoidal perfusion failure (28 +/- 3%) with restoration of leukocyte velocity in sinusoids (S) and postsinusoidal venules (V). Shock-induced stasis/adherence of leukocytes was further increased (P < 0.05) after resuscitation with RL (S, 38 +/- 6%; V, 55 +/- 20%) and HES (S, 31 +/- 8%; V, 23 +/- 14%). In contrast, resuscitation with HS/HES prevented increased leukocyte stasis in sinusoids (-4 +/- 4%) as well as adherence to endothelial lining of postsinusoidal venules (-5 +/- 10%). We conclude that replacement of only 10% of actual blood loss by means of small-volume resuscitation (HS/HES) can restore hepatic microvascular perfusion and prevent reperfusion-induced leukocyte stasis/adherence.
Animals↗
Photoageing of hair fiber and photoprotection.
Examination by transmission electron microscopy of hair exposed to sunlight has revealed important damage. Findings indicate an alteration of various cell components. Damage occurs in the cuticle and leads to its loss. Separation of macrofibrils and destruction of melanin pigments result in cortex damage. Some of the chemical and physical changes which occur in hair exposed to sunlight were studied: formation of carbonyl groups, cystine destruction, modification of the proteins obtained by reduction of the disulfide bonds, losses in mechanical strength and discoloration are discussed. Effects of UV and visible radiations were studied. All of them cause modification in hair properties. Experiments with artificial light sources were carried out to reproduce these alterations. The role of water during exposure was studied. In particular, discoloration of brown hair is largely affected by it. We showed that Xenon lamps were useful to follow the photooxidation of hair and simulate natural alterations. The properties of some protective materials were examined.
Hair↗
In vivo and in vitro primed lymphocytes. Correlation of cytochemically detected BLT-specific lymphoid serine protease with cytotoxic activity.
We describe the validation of a cytochemical method to detect a cytolytic cell-specific lymphoid serine protease which can be upregulated during viral infection and allogeneic stimulation. The cytolytic cell specificity was ascertained by demonstrating a high correlation between BLT substrate-specific serine protease (SP) activity and cytotoxicity of in vivo and in vitro stimulated lymphocytes. The presence of SP in peripheral blood lymphocytes was compared with their capacity to kill K562 targets in a lectin-dependent cytotoxicity assay. The correlation coefficient was 0.92 and 0.93 at E:T ratios 10:1 and 20:1 respectively. In allogeneic mixed lymphocyte cultures an increase of SP activity in effector lymphocytes was paralleled by an augmentation of cytotoxic capacity towards stimulator target cells. SP+ granules showed intracellular polarization to the effector/target cell interface during conjugate formation. These results together with previous studies suggest that this method provides a sensitive assay which predicts the cytolytic potential present in a lymphocyte population.
CD4-Positive T-Lymphocytes↗
Characterisation of wild-type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line.
Monoamine oxidase (MAO)-A and MAO-B are FAD-containing mitochondrial enzymes which catabolize biogenic and xenobiotic amines. The N-terminal regions of both forms of MAO contain an ADP-binding consensus sequence found in several dinucleotide-dependent enzymes, but otherwise show remarkable sequence differences. In order to investigate whether the N-terminal region of MAOs participates in the different catalytic properties and inhibitor specificities exhibited by MAO-A and MAO-B, we constructed chimeric A/B forms and expressed them in a human embryonic kidney cell line (293 cells). The MAO-A chimeric form containing the N-terminus (36 amino acids) of MAO-B and the B chimera having the first 45 amino acid sequence of MAO-A were both catalytically active. Compared to the respective wild-type form, they did not show any significant difference in their catalytic properties (Km, kcat) towards the substrates tested or in their sensitivity towards inhibitors. This indicates that the N-terminal region of the two isoenzymes is not involved in the different specificities of MAO-A and MAO-B. Substitution of Cys-397 of MAO-B, i.e. the residue covalently anchoring FAD, with an Ala or a His residue resulted in the total loss of enzymatic activity, suggesting that the covalent coupling of FAD to MAO occurs specifically at the-SH group of cysteine.
Cells, Cultured↗
Inhibition by ascorbic acid of NMDA-evoked acetylcholine release in rabbit caudate nucleus.
The interaction of ascorbic acid and of dehydroascorbic acid with acetylcholine (ACh) release in rabbit caudate nucleus was investigated. The presence of ascorbic acid in the superfusion medium decreased the release of ACh evoked by N-methyl-D-aspartate (NMDA), but not by electrical stimulation. The pH of the buffer was always maintained at 7.4. Inhibition occurred even at 570 mumol/l ascorbic acid, a concentration which is widely employed in transmitter release experiments. In vivo this concentration may be reached extracellularly in brain tissue. Both ascorbic acid and dehydroascorbic acid inhibited the NMDA-evoked ACh release to the same degree in a non-competitive manner. The nearly identical action of ascorbic acid and dehydroascorbic acid makes a mode of action by lipid peroxidation or by redox phenomena unlikely. The mechanism of action underlying the described effects is unknown.
2-Amino-5-phosphonovalerate↗
Melorheostosis--a very rare entity in neurosurgery.
Melorheostosis is of minor importance in neurosurgery. A case of this disease is reported, which required neurosurgical activity and the literature is reviewed.
Adolescent↗
[Medium-term results of the uncemented threaded SCL prosthesis. International experience apropos of 5,341 cases since 1982. Groupe International d'Etude].
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Alloys↗