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Pyridoxine 5'-phosphate oxidase (PNP Ox) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate. The 53-kDa homodimeric enzyme contains a noncovalently bound flavin mononucleotide (FMN) on each monomer. Three crystal forms of Escherichia coli PNP Ox complexed with FMN have been obtained at room temperature. The first crystal form belongs to trigonal space group P3(1)21 or P3(2)21 with unit cell dimensions a = b = 64.67A, c = 125.64A, and has one molecule of the complex (PNP Ox-FMN) per asymmetric unit. These crystals grow very slowly to their maximum size in about 2 to 4 months and diffract to about 2.3 A. The second crystal form belongs to tetragonal space group P4(1) or P4(3) with unit cell dimensions a = b = 54.92A, c = 167.65A, and has two molecules of the complex per asymmetric unit. The crystals reach their maximum size in about 5 weeks and diffract to 2.8 A. A third crystal form with a rod-like morphology grows faster and slightly larger than the other two forms, but diffracts poorly and could not be characterized by X-ray analysis. The search for heavy-atom derivatives for the first two crystal forms to solve the structure is in progress.

Clear guidelines exist for treating spondylolisthetic deformity and instability. How the surgeon handles adjacent-level degenerative disease is not as well established. Because magnetic resonance imaging now provides us with far more information on the "health" of radiographically normal intervertebral discs, the treatment of dehydrated or degenerated discs adjacent to a fusion is becoming more problematic. In this discussion, two experts discuss their approach to symptomatic lumbosacral spondolisthesis accompanied by adjacent-level disc degeneration. Drs. Herkowitz and Abraham believe strongly that the adjacent segment should be left alone, whereas Dr. Albert recommends extending the fusion in many instances.

Objective: To determine the temporal and spatial relationship between platelet-derived growth factor beta (PDGFbeta) receptors, PDGF-AB/BB, and activated pericytes across the Raynaud's phenomenon (RP) and systemic sclerosis (SSc; scleroderma) disease spectrum., Methods: Monoclonal antibodies against PDGFbeta receptors, PDGF-AB/BB, and high molecular weight-melanoma-associated antigen (HMW-MAA), a marker for activated pericytes, were used to immunohistochemically analyze serial sections of skin biopsy tissue from patients with RP and from scleroderma patients. To delineate cell-specific PDGFbeta receptor expression, double immunofluorescence-stained sections were analyzed using computer-aided image analysis and confocal microscopy., Results: PDGFbeta receptor-expressing cells and HMW-MAA-expressing pericytes were found in biopsy samples from autoimmune RP patients and in both early fibrotic and early nonfibrotic scleroderma skin, but not in normal or primary RP or late-stage scleroderma skin. PDGF-AB/BB was expressed within the epidermis, at the epidermal/dermal junction, and by dermal macrophages. Analysis of juxtaposed serial sections revealed an increased frequency of receptor expression in microvessels from autoimmune RP and early scleroderma skin (P < 0.01). Double-labeling studies using confocal microscopy showed that, in vivo, PDGFbeta receptors were predominantly expressed by microvascular pericytes from both autoimmune RP and early scleroderma skin., Conclusion: PDGFbeta receptors are expressed by activated microvascular pericytes in patients with autoimmune RP and in early SSc patients, but not in those with primary RP or late-stage scleroderma. These findings suggest that features of autoimmune RP are distinct from those of primary RP, and that microvascular pericytes may be an important link between chronic microvascular damage and fibrosis.

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)-acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 microg/ml and the urine method was linear from 2 to 40 microg/ml. The method had a detection limit of 1 microg/ml for vanillin and vanillic acid using 5 microl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.

Pre-clinical evaluation has demonstrated that 2-[4-(((3,5-dimethylanilino)carbonyl)methyl)phenoxy]-2-methylpropi onic acid (RSR13) acts as an allosteric effector of haemoglobin (Hb). RSR13 binding to Hb results in decreased haemoglobin-oxygen (Hb-O2) affinity, improved tumour oxygenation, and enhanced radiation-induced cell killing in several experimental tumour systems. In the present work, ex vivo clonogenic survival analyses are applied in two murine tumour systems to characterize the relationship between the magnitude of decrease in Hb-O2 affinity and radiosensitization, the influence of inspired pO2 upon this effect, and the efficacy of combining RSR13 and radiation during a course of repeated radiation exposures. For FSaII tumours in C3H mice breathing air, 100 mg kg(-1) RSR13 administered intraperitoneally produced an enhancement ratio (ER) of 1.3, but there was marked desensitization at a RSR13 dose of 300 mg kg(-1) (ER 0.6). The most likely reason for the increased radioresistance was insufficient oxygen loading of Hb in the pulmonary circulation due to reduced haemoglobin-oxygen affinity because carbogen breathing combined with 300 mg kg(-1) RSR13 reversed the effect and produced an ER of 1.8. In SCCVII tumours in C3H mice irradiated with eight fractions of 2.5 Gy over 4 days, the surviving fraction was reduced to 58-67% of control values when RSR13 was combined with radiation on days 1 and 2, days 3 and 4, or days 1-4. These results confirm that combining RSR13 and irradiation within a fractionated course of clinically relevant low-dose exposures provides significant radiosensitization. Additional preclinical experimentation is needed to define better the optimum dose-scheduling conditions for clinical applications.

Theoretically, if the arterial partial oxygen pressure (PaO2) does not change, a right shift in the oxygen equilibrium curve (OEC) of hemoglobin should reduce arterial oxygen saturation. In this study we investigate whether a right shift in the OEC of hemoglobin decreases transcutaneous oxygen saturation (Tc-SO2) following the administration of an allosteric effector, 2-[4-(((3, 5-dichloroanilino)-carbonyl) methyl) phenoxy]-2-methylpropionic acid (RSR-4). The effect of RSR-4 on hemoglobin oxygen affinity was studied in four New Zealand white male rabbits. Following intraperitoneal administration of RSR-4, Tc-SO2 decreased in a dose-dependent manner. P50 (partial oxygen pressure at 50% hemoglobin oxygen saturation) in whole blood increased as the concentration of RSR-4 increased. Tc-SO2 decreased as whole-blood affinity (1/P50) decreased. There was no positive correlation between Tc-SO2 and PaO2. We concluded that a decrease in hemoglobin oxygen affinity following RSR-4 administration reduced arterial oxygen saturation. This decrease in the presence of an allosteric effector such as RSR-4 in vivo can be detected and monitored as a reduction in Tc-SO2.

Perivascular infiltrates of inflammatory cells are a hallmark of lesional skin in scleroderma. We have explored the potential for scleroderma fibroblasts to modulate mononuclear leucocyte migration across endothelial cell monolayers in tissue culture, and to regulate expression of endothelial cell adhesion molecules. Fibroblasts were grown from skin biopsies of eight patients with active diffuse cutaneous scleroderma and from four healthy controls. Co-culture and conditioned medium transfer experiments examined the effect of soluble fibroblast products on mononuclear leucocyte (U937) cell migration across endothelial cell (1E-7) monolayers grown on tissue culture inserts. Co-culture of scleroderma, but not control fibroblasts, promoted transendothelial migration of U937 cells. Scleroderma fibroblast-conditioned medium had qualitatively similar effects and equivalent results were obtained using Jurkat-6 (T lymphocyte) cells, and with peripheral blood mononuclear cells from a patient with diffuse cutaneous scleroderma. Promotion of leucocyte migration does not appear to result from increased endothelial adhesion molecule expression, since fibroblast-conditioned medium did not up-regulate endothelial cell expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) or E-selectin. Moreover, leucocyte migration across cytokine-activated endothelial cell layers in co-culture with fibroblasts was less than across resting cells, although the selective effect of scleroderma fibroblast co-culture persisted. Recombinant monocyte chemoattractant protein-1 (MCP-1) or IL-8 increased passage of mononuclear leucocytes across endothelial cell monolayers, whilst anti-MCP-1, but not anti-IL-8 antibodies, significantly reduced the effect of fibroblast conditioned medium. These data suggest that systemic sclerosis (SSc) fibroblasts promote leucocyte migration across endothelial cell monolayers in tissue culture via an MCP-1-dependent mechanism. These findings may be relevant to the perivascular mononuclear leucocyte infiltrates characteristic of early SSc lesions.

Over the last 10 years, the annual number of spinal procedures performed in the United States has more than doubled. In 1996, there were roughly 29,000 thoracic or dorsal fusion procedures, which made up almost 13% of all spine fusions performed. Scoliosis was the most common condition necessitating posterior thoracic fusion. The first half of this article focuses on the indications for thoracic and lumbar fusions; whereas, the second half of this article discusses the trends in use of thoracic and lumbar spine fusions.

Anterior cervical decompression and arthrodesis has evolved over the last 40 years and has become the preferred procedure for managing many cervical spine disorders. The first half of this article discusses the indications for cervical fusion in the management of traumatic, degenerative, neoplastic, infectious, and congenital conditions of the cervical spine. The second half of this article discusses the recent trends in use of cervical spine fusions that demonstrate the increasing frequency of this procedure in the United States over the last 10 years.

Patients with the nondeletion genotype of hemoglobinopathy H (HbH or beta4) disease have higher proportions of HbH and more severe tissue hypoxia than patients with the deletion genotype. Because these patients' red blood cells (RBCs) contain mainly two Hb species, HbH and HbA, the high proportion of HbA can be exploited by lowering its oxygen affinity; this would probably increase oxygen delivery to the RBCs and improve the patients' clinical phenotype. Allosteric effectors that induce a low-affinity Hb may be useful in this regard. We investigated the effect of a bezafibrate derivative, RSR-4, on the oxygen affinity of RBCs and purified hemolysates containing HbA and HbH. This allosteric effector crosses RBC membranes and binds reversibly to the alpha-chains of deoxy-Hb, decreasing hemoglobin oxygen affinity. The blood used was obtained from a patient with HbH disease (alphaTSaudi homozygote) whose HbH level was 33.5% as measured by high-performance liquid chromatography. Oxygen binding studies were performed in RBCs and purified hemolysates. RBCs incubated in the presence of 500 microM RSR-4 (2-[[[(3,5-dichloroanilino)-carbonyl]methyl]phenoxy]-2-methylpropi onic acid) in standard conditions (pH 7.4, 0.14 M NaCl, 37 degrees C) displayed an increase in their P50 value from 14.5 to 35.2 mm Hg. Oxygen binding studies in purified stripped hemolysates (pH 7.2, 0.1 M NaCl, 25 degrees C) showed that addition of both 500 microM RSR-4 and 1 mM of 2,3 diphosphoglycerate (DPG) led to an 11-fold decrease in oxygen affinity, whereas the addition of the natural effector DPG or RSR-4 alone produced a 2.7- and 5.7-fold decrease, respectively. In both cases, the oxygen equilibrium curves (OECs) were biphasic due to the presence of the noncooperative, high-oxygen-affinity HbH (beta4) component. After addition of RSR-4, the lower part of the OEC (corresponding to HbH) was not shifted compared with the upper part (corresponding to HbA). These results were confirmed by kinetic studies of CO recombination. Both experiments demonstrated that RSR-4 does not affect beta4 Hb. Our findings provide an experimental model for lowering the oxygen affinity of HbA in HbH-containing cells and suggest that the oxygen delivery capability of the latter would be thereby improved.

Improving the delivery of oxygen to the tissues by decreasing the oxygen affinity of haemoglobin has been a major aim of several laboratories over recent years because this may reduce the consequences of anaemia and/or improve tissue oxygenation in cases of decreased blood perfusion. Within the same context, lowering the oxygen affinity may prove valuable in the application of native or recombinant haemoglobin solutions as a blood substitute. The shift of the oxygen equilibrium curve to the right is obtained by various modulators. Among them, the bezafibrate derivatives are considered as a most interesting group. These principles are of the utmost importance in thalassaemia and other haemoglobinopathies where the beneficial effects of the compensatory synthesis of fetal haemoglobin are diminished by the increased oxygen affinity of this pigment. In this paper we present the results of a study initiated to determine whether a potent oxygen affinity modifier, RSR-4, could satisfactorily decrease the oxygen affinity of fetal haemoglobin, thus improving tissue oxygenation. The experiments were carried out on whole blood and on purified haemoglobin solutions and showed that the effector markedly decreased the oxygen affinity of HbF (from 18.7 to 3.73 mmHg in whole blood). At the same time the cooperativity index (n50) and the oxygen saturation levels remained within normal limits under the conditions of the main experiment. These observations have important implications for the potential application of oxygen affinity modifiers in vivo.

Abnormal endothelial cell function is implicated in the development of scleroderma, and in major life-threatening complications of the disease. The nature of the stimulus leading to abnormal endothelial cell function in scleroderma, scleroderma renal crisis, and scleroderma-associated pulmonary hypertension was investigated by measurement of soluble adhesion molecules, shed by activated endothelial cells, in sera from patients with these conditions. In scleroderma renal crisis, mean levels of soluble E-selectin (p < 0.05 limited scleroderma, p < 0.0001 diffuse scleroderma), sVCAM-1 (soluble vascular cell adhesion molecule-1) (p < 0.05 limited scleroderma, p < 0.05 diffuse scleroderma), and sICAM-1 (soluble intercellular adhesion molecule-1) (p < 0.0001 limited scleroderma, p < 0.0001 diffuse scleroderma) were raised, supporting a model of endothelial cell activation in this complication. Evidence for endothelial cell activation in scleroderma-associated pulmonary hypertension was inconsistent, with normal sE-selectin and normal sVCAM-1 in sera from patients with limited scleroderma and pulmonary hypertension. The endothelial cell phenotype in scleroderma-associated pulmonary hypertension may resemble that of unstimulated cells. The pulmonary vascular and renal vascular lesions associated with scleroderma may arise by distinct pathogenic mechanisms.

Among the numerous strategies to design an oxygen carrier, we outline in this work the engineering of a stable homotetrameric hemoglobin, expressed in Escherichia coli. The chimeric globin (Psi) consists of the first 79 residues of human beta globin (corresponding to positions NA1 --> EF3) followed by the final 67 residues of human alpha globin (corresponding to positions EF3 --> HC3). The molecular mass for beta-EF3-alpha (Psi) globin was measured using mass spectrometry to be equal to its theoretical value: 15782 Da. Correct protein folding was assessed by UV/visible and fluorescence spectra. The subunit interaction free energies were estimated by HPLC gel filtration. In the cyanometHb species, the formation of the dimer-tetramer interface is 2 kcal/mol less favorable (Delta G = -7 kcal/mol) than that of Hb A (Delta G = -9 kcal/mol), whereas the dimer-monomer interface is tightly assembled (< -10 kcal/mol) as for the Hb A alpha 1 beta 1 interface. In contrast to Hb A, oxygen binding to Psi Hb is not cooperative. The free energy for binding four oxygen molecules to a Psi homotetramer is slightly increased compared to a Hb A heterotetramer (-28 and -27.5 kcal/4 mol of O2, respectively). The intrinsic O2 affinity of a Psi homodimer is 6-fold higher than that of a homotetramer. The linkage scheme between dimer-tetramer subunit assembly and the noncooperative oxygenation of Psi Hb predicts a stabilization of the tetramer after ligand release. This protein mechanism resembles that of Hb A for which the dimers exhibit a 100-fold higher O2 affinity relative to deoxy tetramers (which are 10(5) times more stable than oxy tetramers). A potent allosteric effector of Hb A, RSR4, binds to Psi Hb tetramers, inducing a decrease of the overall O2 affinity. Since RSR4 interacts specifically with two binding sites of deoxy Hb A, we propose that the chimeric tetramer folding is close to this native structure.

We investigated the potential modulation of cell surface intercellular adhesion molecule-1 (ICAM-1) expression and function by ET-1 in fibroblasts grown from skin biopsies of scleroderma (SSc) patients compared with healthy controls. Surface ICAM-1 expression was quantified by cell-bound ELISA and by FACS analysis. ICAM-1 function was investigated by measuring cell adhesion, and we studied ICAM-1 gene expression using RT-PCR. Fibroblast ET-1 binding sites were measured using 125I-labeled ET-1, and the modulation of ICAM-1 function by ET-1 was determined by measuring the binding of human U937 cells to fibroblasts in the presence of a mixed ETA/B receptor antagonist (bosentan) or a neutralizing anti-ICAM-1 antibody. ICAM-1 expression was significantly higher in SSc fibroblasts compared with normal controls. ET-1 increased ICAM-1 on both normal and SSc fibroblasts to comparable levels. RT-PCR demonstrated that ICAM-1 mRNA was upregulated by ET-1, and results from binding studies showed fibroblasts exposed to ET-1 supported more U937 cells than controls, a process that could be inhibited by bosentan and ICAM-1 neutralizing antibody. Autoradiography showed ET-1 receptors on both normal and SSc fibroblasts. Our findings indicate that SSc fibroblasts express intrinsically elevated levels of surface ICAM-1 and message. ET-1 can induce normal fibroblasts to express some SSc phenotypes and may function as a potent proinflammatory mediator, similar to cytokines, and therefore may also have immunoregulatory functions for immune cells infiltrating and binding to connective tissues.

We investigated the effect of endothelin-1 (ET-1) in normal and systemic sclerosis (SSc) dermal fibroblasts. Collagen type I, collagen type III, and MMP-1 levels in culture supernatants were measured by competition ELISA and cellular mRNA expression was examined by Northern blotting. Mitogenic responses to ET-1 were assessed by [3H]TdR incorporation. ET receptor mRNA expression was examined by RT-PCR analysis of fibroblast RNA and with surface binding studies using radiolabeled ET receptor ligands and specific receptor antagonists. ET-1 enhanced release of collagen types I and III by control and SSc fibroblast strains, but the effects were significantly greater for control cells (p < 0.05). This effect appeared to involve both ETA and ETB receptor subtypes. SSc fibroblasts demonstrated lower constitutive MMP-1 production than control fibroblasts (p < 0.01), but ET-1 treatment decreased MMP-1 in normal fibroblasts to levels observed in SSc. Mitogenic response (percent control [3H]TdR incorporation) to ET-1 for SSc fibroblasts was 130 +/- 34, significantly less (p < 0.01) than that for normal fibroblasts strains (290 +/- 25). This response appeared to be predominantly mediated via the ETA receptor subtype. Surface binding studies suggested a significantly lower level of ETA binding sites in SSc compared with normal fibroblasts (p < 0.05). These data suggest that ET-1 induces a fibrogenic phenotype in normal dermal fibroblasts that resembles that seen in fibroblasts grown from lesional SSc skin. Moreover, SSc cells appear to be refractory to these effects, and this reduced responsiveness is associated with an altered ratio of ETA:ETB receptor expression, supporting a role for ET-1 in the fibrotic pathology of SSc.

Nef is a regulatory protein of the human and simian immunodeficiency viruses (HIV and SIV) whose role in infection and the viral life cycle are not fully understood. In T-lymphocytes Nef down-regulates cell-surface CD4, and has been implicated in an increase in infectivity at low primary viral isolate titres. Additionally, the SIV nef gene is necessary for viraemia and AIDS-like pathogenesis in rhesus macaques. We report here in an in vivo murine transgenic model that thymocyte and T-cell-specific nef gene expression results in a marked decrease in thymic cellularity from 16 days post coitus. This reduction in thymocyte cell number is independent of CD4 expression and Nef-induced CD4 down-regulation, but can be restored by expressing a constitutively active p56lckF505 gene. Functional analyses have revealed a severe decrease in thymocyte and T-cell proliferation in response to both T-cell-receptor- and mitogen-mediated stimuli. In addition, a significant proportion of Nef-expressing peripheral T-cells display cell-surface characteristics associated with cellular activation. These results suggest that Nef expression in developing thymocytes can severely reduce the regeneration capacity of the immune system, whereas expression in mature T-cells dramatically decreases their potential to respond to antigen. With the recent recognition of a persistently high viral load in HIV-infected individuals, these findings have important implications for the mechanism of the progressive deterioration of the immune system that leads to AIDS.

Reduced brain tissue oxygenation is frequently seen in severe head injury and after subarachnoid hemorrhage, and this is considered a major cause of secondary ischemic brain injury. In fact, in a previous study, we found a tight correlation between low brain tissue oxygen tension and poor outcome. Therefore, we tested the hypothesis that an allosteric modifier of hemoglobin, which improves oxygen transport to tissue, could reduce the size of an acute infarct in a feline model of human stroke. This compound produces a shift in the hemoglobin dissociation curve to the right and therefore facilitates the unloading of oxygen during low oxygen tension. Seventeen adult cats were studied. Ischemic stroke was induced through a transorbital, permanent, middle cerebral artery occlusion. Seven animals received saline, and 10 received the allosteric Hb modifier RSR-13. Three different endpoints were used to determine the effect of the allosteric modifier. Delta p50 values were measured in the arterial blood; the intra-infarct oxygen tension was measured, and finally, the volume of the infarct was assessed using TTC staining. Mean delta p50 changes varied from 10.4 +/- 9.2 mmHg up to 15.0 +/- 6.8 mmHg. Mean intra-infarct oxygen tension was 27 +/- 6 mmHg for the control group and 33 +/- 7 mmHg for the drug-treated animals. The mean infarct size (measured as percentage of hemisphere volume) in the control group was 32 +/- 9% and for the RSR-13 animals 22 +/- 10% (p < 0.05). A definitive trend towards improvement in brain oxygen tension was seen, such that animals pretreated with RSR-13 showed a higher infarct oxygen tension. Infarct size was significantly reduced in the drug group. Therefore, RSR-13 is potentially beneficial in the treatment of brain ischemia. Since human studies with this compound are already completed, and other compounds which increase oxygen delivery, such as perfluorocarbons, are already being evaluated, it is likely that oxygen delivery enhancement will rapidly become the first 'neuroprotective' modality, employed in patients with severe brain injury, stroke and subarachnoid hemorrhage.

The software program, HINT (Hydropathic INTeractions), which characterizes non-polar-non-polar, polar-polar, and non-polar-polar interactions, has been used to examine subunit interface associations involved in the hemoglobin allosteric transition at a residue and atomic level. HINT differs from many other computational programs in that it is based not on a statistical method or a force-field but employs parameters experimentally determined from solvent transfer experiments. The main focus of this study is to compare HINT scores that are based upon experimentally and thermodynamically derived measurements with experimentally determined thermodynamic results. The HINT analysis yields a good first-order approximation of experimentally measured energies for these interactions as determined by free energies of dimer-tetramer assembly for mutant hemoglobins. The results provide a framework for understanding subunit stabilities based upon individual atom interactions and repulsions. HINT, in agreement with previous analyses, indicates that: (1) the alpha1beta1 and alpha2beta2 subunit contacts are stabilized via several polar and many hydrophobic interactions with few repulsive contact areas in both the T (deoxyhemoglobin) and R (oxyhemoglobin) structures; (2) the alpha1alpha2 subunit contacts are primarily stabilized by polar salt bridge linkages in both T and R states; and (3) the alpha1beta2 and alpha2beta1 contacts have both strong positive and negative interactions in both T and R states with few hydrophobic interactions. The HINT scoring methodology provides a quantitative characterization of the major role of the alpha1beta2 and alpha2beta1 interfaces in the T-->R quaternary transition. HINT also confirms the stronger hydrogen bond formation in mutant Hb Rothschild (Trp 37beta-->Arg) with Asp94alpha1 that gives rise to a low-affinity (deoxy) hemoglobin. HINT shows that the stabilization of the alpha1beta2 interface with mutant Hb Ypsilanti (Asp99alpha-->Tyr) produces a high-affinity (oxy) hemoglobin by reducing hydrophobic-polar contacts in the R state. HINT interaction maps also identified specific sites for mutagenesis at the alpha1beta2 interface that can be explored to shift the allosteric equilibrium in either direction. In addition, the HINT program provides useful diagnostic data for checking the quality of refined crystallographic structures., (Copyright 1997 Academic Press Limited.)

In addition to their vasoactive action, endothelins are potent peptides in the regulation of both cell proliferation and the turnover of extracellular matrix. Using immunohistochemical, autoradiographic, and molecular analyses, we have studied the localization and expression of endothelin-1 and endothelin A (ETA) and B (ETB) receptors in scleroderma-associated fibrotic lung disease. Increased ET-1 immunoreactivity was found in sclerotic tissue compared with control and was associated with the vasculature, pulmonary interstitium, and bronchial and alveolar epithelium. Microautoradiographic analysis after 125I-labeled ET-1 binding showed a two- to threefold increase in the expression of total ET-1 receptors in scleroderma lung tissue localized to the alveolar epithelium and the pulmonary interstitium which was composed of mainly fibroblastic cells with macrophages and some microvessels. RNAse protection assay revealed significantly reduced ETA receptor and slightly raised ETB message levels in systemic sclerosis lung. Surface expression of functional ET receptors was examined by targeted receptor blocking using mixed and receptor-subtype-selective ligands. A consistent decrease in ETA receptor binding sites was noted primarily within the interstitium and vasculature, in contrast to a slight increase in ETB receptors. Elevated ET-1 and the cell-specific pattern of endothelin receptor expression suggest that the endothelins may represent important mediators that influence the pathology of scleroderma-associated lung disease and other fibrotic conditions.

Objective: To examine whether scleroderma lung fibroblasts show a pattern of aberrant type I collagen (CI) biosynthesis similar to that observed previously in studies of dermal fibroblasts in this disease., Methods: CI secretion and steady-state pro alpha1(I) collagen messenger RNA (mRNA) levels and COL1A2 gene activation were examined in fibroblasts grown from lung biopsy specimens obtained from 16 scleroderma patients with lung fibrosis and from 10 histologically normal lung specimens (controls). The effect of culture in a 3-dimensional (3-D) CI gel matrix culture on CI mRNA levels was also examined., Results: The mean (+/- SEM) collagen secretion in monolayer culture for scleroderma lung fibroblasts was 90.9 +/- 56 ng/ml/10(6) cells, significantly greater (P < 0.05) than controls (40.2 +/- 17.5). Pro alpha1(I) collagen mRNA levels in monolayer cultures were higher in scleroderma (mean +/- SEM collagen:GAPDH ratio 3.7 +/- 0.9) compared with control (1.9 +/- 0.8) lung fibroblasts. Transient expression assays confirmed that genes coding for CI are transcriptionally activated in scleroderma lung fibroblasts compared with control strains. Although all lung fibroblasts induced equivalent contraction of 3-D CI gel matrices, scleroderma strains failed to show a reduction in steady-state pro alpha1(I) collagen mRNA levels in gel culture., Conclusion: We have demonstrated elevated CI biosynthesis and impaired mRNA down-regulation for CI by scleroderma lung fibroblasts. These properties are likely to be highly relevant to the pathogenesis of scleroderma-associated lung fibrosis.

Tissue O2 delivery in excess of metabolic demand may be a factor in the development of high vascular resistance in experimental models of volume-expanded hypertension. This hypothesis was previously tested in rats with an exchange transfusion of red blood cells treated with inositol hexaphosphate or an intravenous infusion of RSR-4, allosteric effectors of hemoglobin. The binding of these drugs with hemoglobin effect a conformational change in the molecule, such that the affinity for O2 is reduced. However, in both preparations, the changes in vascular resistance could have been nonspecific. The present studies used intravenous infusions of RSR-13, which did not share some of the problematic characteristics of RSR-4 and inositol hexaphosphate. Conscious instrumented rats (an electromagnetic flow probe on ascending aorta or an iliac, mesenteric, or renal Doppler flow probe) were studied for 6 h after an RSR-13 infusion of 200 mg/kg in 15 min. This dose significantly increased arterial P50 (PO2 at which hemoglobin is 50% saturated) from 38 +/- 0.8 to 58 +/- 1.4 mmHg at 1 h after the start of the infusion. In the 3rd h cardiac output fell significantly from a control value of 358 +/- 33 to 243 +/- 24 ml.kg-1.min-1 and total peripheral resistance significantly increased from 0.31 +/- 0.03 to 0.43 +/- 0.04 mmHg.ml-1.kg.min. Cardiac output and P50 returned toward control over the next few hours. Neither cardiac output nor total peripheral resistance changed in the group of rats receiving vehicle alone. In a separate group of rats, iliac flow decreased significantly to 60% of control and iliac resistance increased to 160% of control. Iliac flow increased significantly in the group of rats that received vehicle only. Although the mechanism of these changes has not been established, these results suggest that a decreased O2 affinity leads to an increased total peripheral resistance and regional vascular resistance and support the hypothesis that O2 plays a role in the metabolic autoregulation of blood flow.

The hypothesis that tissue oxygen delivery in excess of metabolic demand results in vasoconstriction and reduced blood flow was tested in the cremaster muscle of anesthetized Sprague-Dawley rats by studying the effects of an intravenous infusion of RSR-13, an allosteric effector of hemoglobin. RSR-13 reduces the affinity of hemoglobin for oxygen, causing a right shift in the oxygen dissociation curve. Thus, oxygen delivery to the tissues was increased without elevations in blood flow or blood pressure. Tissue PO2, arteriolar diameter, and RBC velocity were measured and volume flow was calculated from diameter and RBC velocity in third-order arterioles. In rats receiving RSR-13 at a rate of 200 mg kg-1 in 15 min (n = 18) P50 (the PO2 at which hemoglobin is 50% saturated) increased from 36 +/- 1 to 52 +/- 3 mm Hg, and tissue PO2 increased to a maximum of 146 +/- 12% above its control value. P50 and tissue PO2 did not change in the control group (n = 8) receiving vehicle at a rate equivalent to that in the experimental group. In a separate group of rats receiving RSR-13 (n = 7), P50 increased from 38 +/- 1 to 51 +/- 3 mm Hg, calculated arteriolar flow decreased from 9 +/- 3 to a minimum of 1.4 +/- 1 nl sec-1, and arteriolar diameter decreased from 27 +/- 3 to a minimum of 13 +/- 3 micrograms P50, volume flow, and arteriolar diameter did not change in the control group (n = 10). These results suggest that an increased tissue oxygen delivery, caused by a right shift in the oxygen dissociation curve, may cause an increase in vascular resistance independent of an elevated blood flow.

Examination of the interaction between human immunodeficiency virus (HIV) regulatory gene products and the host immune system is fundamental to understanding the pathogenesis of HIV and could reveal possible targets for therapeutic intervention in the treatment of AIDS. The HIV Tat gene is a potential candidate for this type of strategy. Transgenic mice can be used to investigate the in vivo effects of Tat on the developing and dynamic immune system and on cellular gene expression. Thus, we have generated transgenic mice that harbor the HIV type 1 Tat gene under the transcriptional control of the human CD2 gene regulatory elements. This expression cassette results in high-level, tissue-specific transcription of the transgene within the T-cell compartment. In this report, we demonstrate the effects of Tat on the in vivo immune system. CD2-Tat transgenic mice show no signs of aberrant thymic development and have normal levels of T-cell subsets in the thymus and peripheral lymphoid organs. However, activated T cells from transgenic mice contain increased levels of tumor necrosis factor beta mRNA as well as biologically active tumor necrosis factor protein and express elevated levels of transforming growth factor beta and interleukin-4 receptor mRNA. These increased cytokine levels do not appear to alter mitogen- or antigen-stimulated responses or induce the formation of dermal lesions in ageing mice. Such investigations should provide insight into the combination of host immune factors mediating pathogenesis in HIV infection.

Monoaldehyde allosteric effectors of hemoglobin were designed, using molecular modeling software (GRID), to form a Schiff base adduct with the Val 1 alpha N-terminal nitrogens and interact via a salt bridge with Arg 141 alpha of the opposite subunit. The designed molecules were synthesized if not available. It was envisioned that the molecules, which are aldehyde acids, would produce a high-affinity hemoglobin with potential interest as antisickling agents similar to other aldehyde acids reported earlier. X-ray crystallographic analysis indicated that the aldehyde acids did bind as modeled de novo in symmetry-related pairs to the alpha subunit N-terminal nitrogens. However, oxygen equilibrium curves run on solutions obtained from T- (tense) state hemoglobin crystals of reacted effector molecules produced low-affinity hemoglobins. The shift in the allosteric equilibrium was opposite to that expected. We conclude that the observed shift in allosteric equilibrium was due to the acid group on the monoaldehyde aromatic ring that forms a salt bridge with the guanidinium ion of Arg 141 alpha on the opposite subunit. This added constraint to the T-state structure that ties two subunits across the molecular symmetry axis shifts the equilibrium further toward the T-state. We tested this idea by comparing aldehydes that form Schiff base interactions with the same Val 1 alpha residues but do not interact across the dimer subunit symmetry axis (a new one in this study with no acid group and others that have had determined crystal structures). The latter aldehydes shift the allosteric equilibrium toward the R-state. A hypothesis to predict the direction in shift of the allosteric equilibrium is made and indicates that it is not exclusively where the molecule binds but how it interacts with the protein to stabilize or destabilize the T- (tense) allosteric state.

Four new series of monoaldehyde bisacids and bisaldehyde bisacids with varying chain lengths have been synthesized and evaluated as allosteric effectors of hemoglobin. Molecular modeling, oxygen equilibrium, and crystallographic studies were combined for structure/function studies. Crystallographic analyses of the bisaldehydes reveal that Schiff base interaction occurred exclusively between Val 1 alpha and Lys 99 alpha of the opposite alpha chain even though the two terminal Val 1 alpha nitrogens are ideally spaced to also form cross-links. The reason for the observed mode of binding appears to be the influence of chain direction set by key substitutions on the bisaldehyde molecule. Even longer chain derivatives that could overcome the direction set by the key functional groups bind in the same manner. These studies support the general conclusion that long flexible molecules prefer to bind along cavity walls, like double-sided molecular sticky tape, rather than span large open spaces with few chances for interaction. The cross-linked bisaldehydes bind at the same site when incubated under both allosteric states and exhibit reduced cooperativity with a significant decrease in oxygen affinity. The chain length acts as a molecular ratchet and dictates the degree of allosteric effect observed. The tighter the cross-link, the greater the constraint on the tense- (T-) state and the stronger the allosteric effect that is produced. The monoaldehyde bisacids bind in the same fashion with Schiff base formation at Val 1 alpha while the acid that replaces the second aldehyde moiety forms a salt bridge with Lys 99 alpha of the opposite subunit. This class of molecules has weaker allosteric effector activity as would be expected with replacement of one covalent bond by a salt bridge. The importance of Lys 99 alpha on the allosteric equilibrium is confirmed.

The concept of using affinity and intrinsic activity to analyze drug interactions with receptors has had a long history in pharmacological studies. In the simplest case, the biological response will be proportional to the amount of drug bound, i.e. its affinity. However, the biological response is also mediated by the ability of a drug when bound to exert its maximum effectiveness. This effectiveness is termed the intrinsic activity. Physicochemical processes have been thought to be at the basis of intrinsic activity. Detailed oxygen and solution binding experiments combined with X-ray crystallographic studies on allosteric effectors to hemoglobin demonstrate that these potential drug agents bind at the same site in hemoglobin with similar binding constants yet shift the allosteric equilibrium and the oxygen affinity of the T-structure by different degrees. Therefore some of the effectors with similar binding affinities for the same site exhibit varying degrees of affectiveness, i.e. they possess different intrinsic activities. The intrinsic activity of the effector is defined as the ratio of the oxygen affinity constant to the T-state with drug/oxygen affinity constant to the T-state without drug (KT+drug)/(KT control). The source of the intrinsic activity appears to be the ability of the effectors to interact with key residues such as Lys99 alpha at the binding site. These results suggest a general molecular mechanism for allosteric effector modulation of hemoglobin function that might be of use in other allosteric enzyme systems.

Methods that predict geometries of ligands binding to receptor molecules can facilitate ligand discovery and yield information on the factors governing complementarity. Here, the use of atomic hydrophobicities in evaluating binding modes has been examined with four ligand-receptor complexes of known structure. In each system, hundreds of hypothetical binding orientations were generated with DOCK and evaluated using the HINT (Hydropathic INTeractions) exponential function and atomic hydrophobic constants. In three of the four systems, the experimental binding mode received the best HINT score; in the fourth system, the experimental binding mode scored only slightly lower than a similar, apparently reasonable orientation. The HINT function may be generally useful as a scoring method in molecular docking.

Previous work has shown that the bifunctional alkylating agent chlorambucil induces thermolabile adenine adducts and that the predominant chlorambucil-induced mutations in shuttle vector pZ189 are transversions at AT base pairs. In order to assess the role of thermolabile adducts in generating these transversions, pZ189 was treated with chlorambucil in the presence of distamycin, which specifically blocks formation of thermolabile adenine adducts. Analysis of the mutations resulting from replication of the damaged vector in human 293 cells showed that base substitutions at AT base pairs were specifically suppressed in concert with suppression of thermolabile adducts at specific sites in the supF target gene, strongly supporting a role for these adducts in mutagenesis. Since there is considerable evidence that these adducts are N-3 alkylations, a computer graphics model of such an adduct was constructed. Modeling studies indicated that the adduct could be formed with little distortion of the DNA helix. Analysis of the adduct using the HINT (Hydropathic INTeractions) program was consistent with the proposal that favorable hydrophobic interactions of the phenyl ring of chlorambucil with the wall of the minor groove may promote adenine N-3 alkylation by this drug.

Physical organic structural properties of small molecules and macromolecules such as bond count, branching and proximity between multiple polar fragments contribute significantly to measured hydrophobicity (log P). These structural properties are encoded in the Rekker and Leo methods of calculating log P as structural-dependent factors. Regardless of the size of the atom primitive set, methods predicting log P with only atom primitives can miss subtle structural detail within series of related compounds. The HINT (Hydropathic INTeractions) model for inter- and intramolecular noncovalent interactions calculates atom-based hydrophobic constants, but uses all Leo-type factors in the calculation rather than a large set of atom primitives. Two types of applications of HINT are discussed: evaluation of the binding of an inhibitor (A74704) to HIV-1 protease, where it is shown that modeling of the protonation state (i.e., Asp25, Asp125) in the protein can strongly influence perceived substrate binding; and the use of HINT to calculate a third (hydropathic) field for CoMFA can yield a statistically enhanced and predictive model for molecular design.

The in vivo effects on hemoglobin (Hb)-O2 affinity and tissue PO2 were investigated after intraperitoneal administration of 2-[4-(((dichloroanilino)-carbonyl)methyl)phenoxyl]-2-methyl propionic acid (RSR4; 150 mg/kg) or its 3,5-dimethyl derivative (RSR13; 300 mg/kg) in C3Hf/Sed mice. The Hb-O2 dissociation curve was plotted from tail vein blood samples using an O2 dissociation analyzer before and up to 160 min after compound administration. Twenty to 40 min after injection, the PO2 at 50% saturation of hemoglobin (Hb P50) increased by a mean of 25% (range 18-31%) after RSR4 and 53% (range 36-76%) after RSR13. Tissue PO2 was continuously measured using an O2 microelectrode in thigh muscle before and up to 40 min after RSR4 or RSR13 injection. Twenty to 40 min after administration, tissue PO2 increased by a mean of 78% (range 30-127%) after RSR4 and 66% (range 39-97%) after RSR13 administration in anesthetized mice. No change in tissue PO2 was seen in anesthetized controls.

We tested the effect of certain newly synthesized allosteric modifiers of hemoglobin on the dilation induced by arterial hypoxia, arterial hypotension, and arterial hypercapnia in cerebral arterioles of anesthetized cats equipped with cranial windows for the observation of the cerebral microcirculation. The allosteric modifiers of hemoglobin are isomers of 2-(aryloxy)-2-methylpropionic acid. They shift the oxygen dissociation of hemoglobin to the right, thereby facilitating the local release of oxygen. When these compounds were applied topically by superfusion at a rate of 1 ml/min in a concentration of 0.1 mM, they had no significant effect on baseline arteriolar diameter but reduced significantly the vasodilation from arterial hypoxia and arterial hypotension. They did not influence the vasodilation from arterial hypercapnia. Spectrophotometric measurements of optical densities from pial veins 50-80 microns in diameter indicated that the superfusion with the allosteric compounds reduced hemoglobin oxygen saturation both during room air breathing and during hypoxia. We conclude that the vasodilations from arterial hypoxia and arterial hypotension are mediated by local oxygen-dependent mechanisms. The allosteric modifiers of hemoglobin may be useful as tools in investigating oxygen-dependent mechanisms.

Three new routines (LOCK, KEY and LOCKSMITH) for the program HINT (hydrophobic interactions) are described and demonstrated. The KEY routine uses receptor structure to model the hydropathic profile of the ideal substrate for the receptor. The LOCK routine uses substrate or drug structure to model the hydropathic character of the receptor. LOCKSMITH is an algorithm designed to highlight the significant hydropathic features from a collection of agents. Ten allosteric modifiers of hemoglobin that have been characterized biologically and with X-ray diffraction to determine their protein binding sites/conformations illustrate the KEY and LOCKSMITH routines: The LOCKSMITH composite map correctly identifies the structural features and conformation of the more active modifiers. In addition, many hydropathic features of the "ideal" drug predicted by the KEY map overlap with actual structural features of the most active hemoglobin allosteric modifiers.

Two new potent allosteric effectors of hemoglobin, RSR-4 [2-[4-[[(3,5-dichloroanilino)carbonyl]-methyl]phenoxy]-2- methylpropionic acid] and RSR-13 [2-[4-[[(3,5-dimethlanilino)carbonyl]methyl]-phenoxy]-2-methylp rop ionic, are compared to the previously reported compounds L3,5 and L3,4,5 [Lalezari, I., Lalezari, P., Poyart, C., Marden, M., Kister, J., Bohn, B., Fermi, G., & Perutz, M. F. (1990) Biochemistry 29, 1515]. Unlike L3,5 and L3,4,5, RSR-4 and RSR-13 are less impeded by physiological concentrations of serum albumin. RSR-4 has also been shown to be more effective than L3,5 in shifting the allosteric equilibrium of bovine Hb toward the low-affinity T-state. X-ray crystal studies show that both RSR-4 and RSR-13 bind to only one pair of symmetry-related sites in the Hb central water cavity whereas previous studies on L3,5 and L3,4,5 demonstrated a second pair of symmetry-related binding sites near Arg 104 beta. Three major interactions between these allosteric effectors and Hb include the acid group with the guanidinium group of C-terminal Arg 141 alpha, the effector's amide oxygen with the ammonium ion of Lys 99 alpha, and the phi electrons of the halogenated or methylated aromatic ring and Asn 108 beta. No explanation has been found for the difference in number of binding sites observed for RSR-4 and RSR-13 (two sites) compared to L3,5 and L3,4,5 (four sites); also no correlation has been made between the number of binding sites and degree of allosteric shift in the oxygen equilibrium curve.(ABSTRACT TRUNCATED AT 250 WORDS)

X-ray diffraction difference electron density maps at 3 A resolution obtained from di and tetra-ligated T-state hemoglobin (Hb) crystals are reported. Crystals isomorphous with native deoxyhemoglobin were obtained from ammonium sulfate solutions incubated with the synthetic allosteric effector RSR-56. RSR-56 binds at two symmetry-related Hb central water cavity sites and each molecule has major interactions with three different subunit side-chains; one effector with Arg141 alpha 2 HC3, Lys99 alpha 1 G6 and Asn108 beta 1 and the other with the symmetry related residues, Arg141 alpha 1 Lys99 alpha 2 and Asn108 beta 2. Crystals mounted in a nitrogen filled glove box were di-ligated as previously found with polyethyleneglycol Hb crystals. Crystals mounted in air under a layer of mother liquor were bright red and showed all four heme groups ligated. The difference electron density from the di-ligated crystals showed atomic movements to be restricted to the immediate neighborhood of the heme groups and the allosteric effector. By contrast, the tetra-ligated structure showed extended difference electron density near amino acid residues around both alpha and beta heme groups and along the alpha 1/beta 2 interface. Ligation of the beta heme group appears to magnify the difference density around the alpha heme groups. There is no evidence of breakage of the Bohr salt bridge, His146 beta HC3----Asp94 beta FG1, in the crystal. The observed difference electron density maps may help to clarify the way the allosteric mechanism is triggered.

The infiltration of skeletal muscle by leukocytes occurs in a variety of myopathies and frequently accompanies muscle degeneration and regeneration. The latter involves development of new myofibers from precursor myoblasts, and so infiltrating cells may interact with muscle at all stages of differentiation. The authors have investigated the surface expression of ligands for T-cell adhesion during the differentiation of human skeletal muscle in vitro. Myoblasts expressed low levels of ICAM-1 (CD54), which remained constant during muscle cell differentiation and could be induced by cytokines such as gamma-interferon. It is therefore likely that ICAM-1 is involved in the invasive accumulation of lymphocytes during skeletal muscle inflammation. In contrast, LFA-3 (CD58) was expressed at higher levels than ICAM-1 on myoblasts, decreased significantly during myogenesis, and was unaffected by immune mediators. Both ICAM-1 and LFA-3 were able to mediate T cell binding to myoblasts, whereas adhesion to myotubes was independent of the LFA-3 ligand. Although expressed throughout myogenesis, human leukocyte antigen class I and CD44 did not appear to mediate T cell binding. The expression of ligands that facilitate interaction of myogenic cells with lymphocytes may have important implications for myoblast transplantation.

Ethacrynic acid, a loop diuretic, has been shown to inhibit hemoglobin S polymerization. Until now, however, most studies were performed using purified solutions of hemoglobin S. The experiments reported here were designed to examine the effects of ethacrynic acid and its n-butryic acid derivative on the rheological and physiological properties of intact red blood cells. Using net and unidirectional flux measurements, both agents were shown to cause ion and water loss from normal and sickle erythrocytes. Since cell shrinkage adversely influences red cell rheology, it is unlikely that this class of compounds, despite its ability to inhibit hemoglobin S polymerization, will prove useful in the treatment of sickle cell disease.

The crystal state binding of sodium dithionite to deoxyhemoglobin is reported. Dithionite has been used extensively to deoxygenate hemoglobin and myoglobin and there has been considerable interest among users of dithionite about its effect on protein structure and binding site(s). We have determined that dithionite binds to deoxygenated hemoglobin crystals at the interface of two molecules in the crystal lattice. Specific residues involved in hydrogen bonds or salt interactions with dithionite include His116 and His117 of the beta 2 subunit and Lys16 of the alpha 1 subunit of the adjacent hemoglobin molecule. No binding was observed at the symmetry related His116 and 117 beta 1 residues. We have shown that dithionite does not affect the native hemoglobin structure or the binding of several allosteric inhibitors to hemoglobin and can be used to mount T state crystals in the air.

An empirical hydrophobic field-like 3D function has been calculated with the program HINT (hydrophobic interactions) and imported into the SYBYL implementation of CoMFA (Comparative Molecular Field Analysis). The addition of hydrophobicity appears to offer increased chemical interpretability of CoMFA models. An example is given using the steroid model reported by Cramer et al. (J. Am. Chem. Soc., 110 (1988) 5959). While addition of the HINT field did not improve statistical parameters in this model, the CoMFA coefficient contours from the hydrophobic field unambiguously define the most active steroid molecules in the chemical terms of hydrophobic and polar substituents.

The design, synthesis, and testing of several halomethyl analogues of choline and acetylcholine as potential cholinotoxins is described. The compounds were evaluated for their ability to inhibit high-affinity choline transport and their affinity toward postsynaptic muscarinic receptors. Among the analogues tested, bromomethyl and iodomethyl analogues of choline were found to be the most potent inhibitors of the high affinity choline transport system. Introduction of a beta-methyl group in the halomethyl analogues drastically reduced their potencies. The bromomethyl and iodomethyl analogues were further investigated for their effects on choline acetyltransferase activity, acetylcholinesterase activity and QNB binding. Neither compound possesses significant ability to alter any of the above cholinergic markers, except at very high concentrations. These results suggest that the bromomethyl and iodomethyl choline analogues may be used as specific inhibitors of the presynaptic high-affinity choline transport system.

C18H17NO6.2H2O, Mr = 379.386, monoclinic, P21/c, a = 7.161 (5), b = 12.997 (2), c = 20.290 (3) A, beta = 99.22 (2) degrees, V = 1861.4 A3, Z = 4, Dx = 1.354 g cm-3, Cu K alpha radiation, lambda = 1.5418 A, mu = 9.2 cm-1, F(000) = 800, T = 293 K, final R(F) = 0.051 for 2262 observed reflections. The structure was solved by direct methods and compared to the previously determined structure of the analogous drug molecule, bezafibrate. The structural characteristics of the two compounds are then correlated to their relative activities as allosteric effectors of hemoglobin. The molecule adopts a synplanar-antiplanar conformation unlike other phenoxyacetic acids.

The CD2 receptor on T-lymphocytes plays a major part in mediating adhesive interactions via the LFA-3 ligand and in transducing signals for lymphocyte activation. In this study the expression, function, and internalization of the CD2 receptor was investigated in resting and activated murine T-cells. Surface iodination of intact lymphocytes showed that both types of cell expressed this antigen as a single polypeptide of 63 KDa, and flow cytometry analysis demonstrated that there was four times as much CD2 on lymphoblasts as on resting cells. Moreover, the CD2 receptor had a more prominent role in the adhesion of the activated lymphocytes to extravascular cells than in the binding of resting cells. Only activated lymphocytes internalized CD2, in the presence or absence of the anti-CD2 monoclonal antibody (mAb) 12-15, more than 80% of the 12-15/CD2 complex being removed from the cell surface within 24 hr. Application of 125I-labelled mAb 12-15 followed by subcellular fractionation on Percoll gradients showed that the complex was internalized initially into a low-density compartment and subsequently transported to heavy-density organelles, in which it was degraded. Immunogold electron microscopy revealed that immediately after the initial binding of mAb 12-15 to the lymphoblasts, the gold particles were localized in clusters exclusively at the plasma membrane. After a short period of culture, the mAb 12-15/CD2 complex was detected in small vesicles near the cell surface. Immunogold staining for a lysosomal enzyme beta-glucuronidase (Gus), for the lysosomal membrane protein LAMP-1, and for the mannose 6-phosphate targetting receptor (MPR) showed that the complex was transported from the endosomal compartment to lysosomal organelles in the activated T-cell. Although mAb 12-15 bound to CD2 in resting T-lymphocytes, in these cells the complex remained associated with the plasma membrane compartment only, even after prolonged culture. These data show that activated but not resting lymphocytes endocytosed the receptor, thereby regulating the expression of this antigen at the plasma membrane. This suggests that the endocytic and lysosomal compartments of lymphocytes have major roles in immune functions, by controlling the level of receptors at the lymphocytes cell surface and thus their response to cytokines and inflammatory mediators as well as their direct interaction with other cells.

The hemoglobin binding site of the antisickling agent 12C79 has been determined by x-ray crystallography. 12C79 is recognized as one of the first molecules to reach clinical trials that was designed, de novo, from x-ray-determined atomic coordinates of a protein. Several previous attempts to verify the proposed Hb binding sites via crystallographic studies have failed. Using revised experimental procedures, we obtained 12C79-deoxyhemoglobin crystals grown after reaction with oxyhemoglobin and cyanoborohydride reduction to stabilize the Schiff base linkage. The difference electron-density Fourier maps show that two 12C79 molecules bind covalently to both symmetry-related N-terminal amino groups of the hemoglobin alpha chains. This is in contrast to the original design that proposed the binding of one drug molecule that spans the molecular dyad to interact with both N-terminal alpha-amino groups.

The protein-bound conformations of six new allosteric effectors similar to bezafibrate that markedly decrease the oxygen affinity of hemoglobin have been determined by X-ray crystallography. Comparisons are made with the bound conformations of three urea analogues reported by Lalezari, Perutz, and co-workers. All six new molecules bind at the same site previously observed for bezafibrate and exhibit a wide range of allosteric activity. Unlike the urea derivatives, which show two binding sites for the most potent derivatives, only one of the six new molecules (one with moderate allosteric activity) exhibits a second binding site. A new computer program, HINT (hydrophobic interactions), has been created and utilized to identify the major interactions between small molecules and the protein. The three strongest interactions identified by HINT involve Arg 141 alpha with the acid of the analogues, Lys 99 alpha with the bridging amide carbonyl, and the amide NH of the side chain of Asn 108 beta with the halogenated aromatic ring.

Three isomeric series of 2-(aryloxy)-2-methylpropionic acids were prepared and studied for their ability to decrease the oxygen affinity of human hemoglobin A. The isomeric aryloxy groups included 4-[[(aryloyl)amino]methyl]phenoxy, 4-(arylacetamido)phenoxy, and 4-[[(arylamino)carbonyl]methyl]phenoxy. A total of 20 compounds were synthesized and tested. Structure-activity relationships are presented. Several of the new compounds were found to be strong allosteric effectors of hemoglobin. The two most active compounds are 2-[4-[[(3,5-dichloroanilino)carbonyl]-methyl]phenoxy]- 2-methylpropionic acid and the corresponding 3,5-dimethyl derivative. The latter two compounds have been compared to other known potent allosteric effectors in the same assay and show greater activity. Both compounds also exhibit a right shift in the oxygen equilibrium curve when incubated with whole blood. The new compounds may be of interest in clinical or biological areas that require or would benefit from a reversal of depleted oxygen supply (i.e., ischemia, stroke, tumor radiotherapy, blood storage, blood substitutes, etc.). They are also structurally related to several marketed antilipidemic agents.

The stereochemistry associated with the amobarbital N-glucoside diastereomers (1a and 1b) that are excreted by humans in urine is unknown. Using X-ray crystallography, the absolute configuration of 1b was determined to be S (C-5 position of the barbiturate ring). Following oral administration of amobarbital to Caucasians and Orientals, from 5 to 25% of the dose of amobarbital was excreted in the urine as 1b. The other diastereomer, 1a, accounted for less than 0.1 to 0.2% of the dose in four individuals, with none detected in nine individuals. The rate constants, kf,1b, determined from the urinary excretion of 1b were lower than those previously reported for unresolved amobarbital N-glucosides. However, based on the urinary excretion of 1b, the rate constants, K, for elimination of amobarbital in Caucasians and Orientals were similar to those previously determined from the serum levels of amobarbital and the urinary excretion of unresolved amobarbital N-glucosides. In previous studies of the N-glucosylation of amobarbital, it is likely that a single N-glucose diastereomer, 1b, was being observed.

This paper details comprehensive binding studies (solution and X-ray) of human hemoglobin A with a group of halogenated carboxylic acids that were investigated as potential antisickling agents. It is, to our knowledge, the first study to compare solution and crystal binding for a series of compounds under similar high-salt conditions used for cocrystallization. The compounds include [(3,4-dichlorobenzyl)oxy]acetic acid, [(p-bromobenzyl)oxy]acetic acid, clofibric acid, and bezafibrate. The location and stereochemistry of binding sites have been established by X-ray crystallography, while the number of binding sites and affinity constants were measured by using equilibrium dialysis. The solution binding studies were conducted with deoxygenated hemoglobin and carbonmonoxyhemoglobin with low (50 mM) and high (2 M) salt concentrations. It was concluded that the observed crystal structures are consistent with the binding observed in solution and that the number of binding sites is independent of salt concentration, while the binding constant increases with increasing salt concentration. The studies also reveal that relatively small changes in the chemical structure of a drug molecule can result in entirely different binding sites on the protein. Moreover, the X-ray studies provide a possible explanation for the multiplicity in function exhibited by these compounds as allosteric modulators and/or antisickling agents. Finally, the studies that these compounds bind differently to the R and T states of hemoglobin, an observation of special significance to the original design of these agents.

This paper correlates the X-ray structures of two 4-(beta-1-naphthylvinyl)pyridine analogues (one cis and one trans) with chemical and biological activity data for this class of cholineacetylase inhibitors. Our results suggest that one of the two proposed mechanisms for inhibition by this class of compounds better describes their efficacy. Previous arguments about coplanarity of the aromatic rings and nucleophilicty across the vinyl linkage need to be modified. Quantum calculations are also included and substantiate previous suggestions about the charge distribution across the vinyl linkages. An alternate new mechanism of inhibition is proposed to encompass the published data and more recent results discussed in this paper.

We have used a three-dimensional model of deoxyhemoglobin to design potential antisickling agents with an intended binding site in the vicinity of the beta-6 mutation (donor site). Two proline derivatives, (4S)-1-butyryl-4-[(carboxymethyl)amino]-L-proline (9a) and its 1-benzoyl analogue (9b), were designed to interact, via ionic or hydrogen bonds, with polar residues beta His-2, beta Thr-4, and beta Lys-132 of hemoglobin S (HbS). Two other proline derivatives containing a salicylate leaving group, (4S)-1-butyryl-4-[(carboxymethyl)methylamino]-L-proline, 2-ester with salicyclic acid (14a), and its 1-benzoyl analogue (14b), were designed to bind covalently to beta Lys-132, as well as to interact with beta His-2 and beta Thr-4 via ionic and hydrogen bonds. This paper describes the synthesis of these agents, beginning with natural L-hydroxyproline methyl ester, and the testing of their ability to increase or decrease the solubility of dHbS by using a standard solubility assay. The covalent derivatives 14a,b were found to be inactive, while the noncovalent compounds 9a,b showed weak antigelling activity, below that observed for phenylalanine. The presence of only weak activity does not invalidate this approach, since only one structural parameter has been investigated.