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PubMed1998

X-ray studies on cross-linked lysozyme crystals in acetonitrile-water mixture.

Tetragonal crystals of hen egg white lysozyme were cross-linked and subjected to X-ray diffraction study in acetonitrile-water media with different acetonitrile concentrations. Crystals in neat acetonitrile did not scatter X-ray well. Structures of crystals in neat water, in 90% and 95% acetonitrile, and crystal back-soaked from acetonitrile to water, were determined to about 2 A resolution. For crystals in both 90% acetonitrile, and crystal back-soaked from acetonitrile to water, were determined to about 2 A resolution. For crystals in both 90% and 95% acetonitrile, only one protein-bond acetonitrile molecule is found in the active site cleft, and its location and binding-protein mode is similar to the C subunit of polysaccharide. The alteration in conformation and hydrogen-bond pattern involving water as solvent causes the reduction of the protein's flexibility in organic media. The back-soaked crystal regained its ordinary three-dimensional structure in water.

Acetonitriles↗
PubMed2000

An occlusal and cephalometric analysis of lower first and second premolar extraction effects.

This study was designed to examine lateral cephalometric and arch dimensional changes that occur in the mandibular arch during orthodontic treatment involving the extractions of various premolars. Pretreatment and posttreatment records of 73 patients were chosen at random from completed cases in the practice of one experienced orthodontist. Eighteen involved the extraction of lower first premolars, and 55 involved the extraction of lower second premolars. Of these 55, 29 involved the extraction of upper first premolars and 26 involved the extraction of upper second premolars. In the lower first premolar group, however, all 18 involved the extraction of upper first premolars. Males and females were evenly represented in the 3 subgroups. Pretreatment factors that suggested a basis for the extraction choice in this group of patients were found to include incisal overjet, molar relationship, and underlying vertical facial pattern. A wide variety of arch dimensional changes was found with different lower premolar extraction patterns. There was evidence, however, of more intermolar arch width reduction after the extraction of lower second premolars than lower first premolars. Orthodontic treatment with the extraction of premolars did not consistently cause a retrusive effect on the incisors. In fact, instances of proclination of the incisors occurred within all of the extraction groups. A large amount of individual variation in incisor and molar changes accompanied treatment involving all lower premolar extraction patterns.

Adolescent↗
PubMed1995

The proximal ligand variant His93Tyr of horse heart myoglobin.

The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for bovine liver catalase. The 1D 1H-NMR spectrum of the variant confirms the five-coordinate nature of the heme iron center and exhibits a broad resonance at 112.5 ppm that is attributable to the meta protons of the phenolate ligand. This result indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is unchanged from that of the wild-type protein (pH 8) while the midpoint reduction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 mV lower. The three-dimensional structure of the variant determined by X-ray diffraction analysis confirms the five-coordinate nature of the heme iron center and establishes that the introduction of a proximal Tyr ligand is accommodated by a shift of the F helix (residues 88-99) in which this residue resides away from the heme pocket. Additional effects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hydrogen bonding interactions with the heme prosthetic group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Involvement of serine 96 in the catalytic mechanism of ferredoxin-NADP+ reductase: structure--function relationship as studied by site-directed mutagenesis and X-ray crystallography.

The crystal structure of ferredoxin-NADP+ reductase (FNR) suggests that Ser96 is directly involved in hydride transfer between the isoalloxazine moiety of FAD and the nicotinamide ring of NADP(H). To probe its role, Ser96 has been mutated to valine (S96V) and glycine (S96G). These mutations primarily affected the interaction of the nicotinamide ring with the flavin. Absorbance, fluorescence, and circular dichroism spectra and the crystal structure of FNR-S96V indicate that this mutant folds properly. FNR-S96V shows only 0.05% of wild-type activity, while the affinities for both ferredoxin and NADP+ are virtually unchanged. However, spectral perturbations induced by NADP+ binding to FNR-S96V strongly resemble those elicited by the binding of 2'-monophosphoadenosine-5'-diphosphoribose, a substrate analog lacking the nicotinamide ring, both to the mutant and wild-type enzymes. Rapid reaction studies on the valine mutant failed to detect charge-transfer intermediates during flavin reduction by NADPH. In addition, no semiquinone formation was seen during photoreduction of FNR-S96V. The three-dimensional structure of the valine mutant shows small, albeit definite, changes only in the isoalloxazine microenvironment. The glycine mutant of FNR displays behavior intermediate between that of wild-type enzyme and that of the valine mutant. It maintains ca. 2% of the wild-type activity as well as the ability to form the charge-transfer species between reduced FNR and NADP+. In photoreduction experiments, the same degree of flavin semiquinone stabilization was observed with FNR-S96G and with the wild-type enzyme. NADP+ binding to the glycine mutant was very similar to that observed in the case of the valine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Allosteric activation of protein phosphatase 2C by D-chiro-inositol-galactosamine, a putative mediator mimetic of insulin action.

Insulin-stimulated glucose disposal in skeletal muscle proceeds predominantly through a nonoxidative pathway with glycogen synthase as a rate-limiting enzyme, yet the mechanisms for insulin activation of glycogen synthase are not understood despite years of investigation. Isolation of putative insulin second messengers from beef liver yielded a pseudo-disaccharide consisting of pinitol (3-O-methyl-d-chiro-inositol) beta-1,4 linked to galactosamine chelated with Mn(2+) (called INS2). Here we show that chemically synthesized INS2 has biological activity that significantly enhances insulin reduction of hyperglycemia in streptozotocin diabetic rats. We used computer modeling to dock INS2 onto the known three-dimensional crystal structure of protein phosphatase 2C (PP2C). Modeling and FlexX/CScore energy minimization predicted a unique favorable site on PP2C for INS2 in a surface cleft adjacent to the catalytic center. Binding of INS2 is predicted to involve formation of multiple H-bonds, including one with residue Asp163. Wild-type PP2C activity assayed with a phosphopeptide substrate was potently stimulated in a dose-dependent manner by INS2. In contrast, the D163A mutant of PP2C was not activated by INS2. The D163A mutant and wild-type PP2C in the absence of INS2 had the same Mn(2+)-dependent phosphatase activity with p-nitrophenyl phosphate as a substrate, showing that this mutation did not disrupt the catalytic site. We propose that INS2 allosterically activates PP2C, fulfilling the role of a putative mediator mimetic of insulin signaling to promote protein dephosphorylation and metabolic responses.

Amino Acid Substitution↗
PubMed1998

N-glycan structures of a recombinant mouse soluble Fcgamma receptor II.

N-glycans of a recombinant mouse soluble Fcgamma receptor II (sFcgammaRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226:139-46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was alpha-(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and alpha-(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry. Carbohydrate sequence [see text]

Animals↗
PubMed1986

Selective gene expression in focal cerebral ischemia.

Regional patterns of protein synthesis were examined in rat cortex made ischemic by the occlusion of the right common carotid and middle cerebral arteries. At 2 h of ischemia, proteins were pulse labeled with intracortical injections of a mixture of [3H]leucine, [3H]isoleucine, and [3H]proline. Newly synthesized proteins were analyzed by two-dimensional gel fluorography, and the results correlated with local CBF, measured with [14C]iodoantipyrine as tracer. Small blood flow reductions (CBF = 50-80 ml 100 g-1 min-1) were accompanied by a modest inhibition in synthesis of many proteins and a marked increase in one protein (Mr 27,000). With further reduction in blood flow (CBF = 40 ml 100 g-1 min-1), synthesis became limited to a small group of proteins (Mr 27,000, 34,000, 73,000, 79,000, and actin) including two new polypeptides (Mr 55,000 and 70,000). Severe ischemia (CBF = 15-25 ml 100 g-1 min-1) caused the isoelectric modification of several proteins (Mr 44,000, 55,000, and 70,000) and induced synthesis of another protein (Mr 40,000). Two polypeptides (Mr 27,000 and 70,000) dominated residual protein synthesis in severe ischemia. The changes in protein synthesis induced by different grades of ischemia most likely comprise a variation of the so-called "heat shock" or "stress" response found in all eukaryotic cells subjected to adverse conditions. Since heat shock genes are known to confer partial protection against anoxia and a variety of other noxious insults, their induction may be a factor in limiting the extent of ischemic tissue damage.

Animals↗
PubMed1989

An analysis of the stopped-flow kinetics of gaseous ligand uptake and release by adult mouse erythrocytes.

Ligand uptake and release by the haemoglobin contained within adult mouse erythrocytes was studied by using dual-wavelength stopped-flow techniques. The rate of O2 uptake is very much lower than that expected for an equivalent concentration of haemoglobin in free solution. The O2-concentration-dependence found in uptake experiments is greater than first-order. CO uptake shows the same pattern of reactivity as does O2, but the associated rates of uptake are lower and the concentration-dependence of the CO rates is first-order. O2 release from the adult erythrocytes was measured by stopped-flow mixing with Na2S2O4. Under these circumstances the deoxygenation of intracellular haemoglobin shows accelerating time courses. The apparent rate-constant-dependence on dithionite concentration shows a rate limit at high reductant concentrations. Computer simulations of both ligand uptake and release processes were carried out by using a three-dimensional model. The simulations clearly indicate that in rapid-mixing experiments the rather slow experimentally observed O2 uptake rate is due to rate-limiting diffusion through an extracellular stagnant solvent layer. In the case of O2 release, however, the major rate-controlling process is the rate of O2 dissociation from the haemoglobin molecules, which accelerates during the deoxygenation process.

Animals↗
PubMed1995

Halothane reduces focal ischemic injury in the rat when brain temperature is controlled.

BACKGROUND: Previous work has demonstrated that rats anesthetized with halothane during focal cerebral ischemia have better histologic and neurologic outcome than do rats undergoing the same insult when awake. The purpose of this experiment was to determine whether this difference persists when brain temperature is held similar in halothane-anesthetized and awake experimental groups. METHODS: Two ischemia experiments were performed. In both, the middle cerebral artery was occluded for 90 min. Temperature was monitored from a radiotelemetered thermistor implanted in the cerebral cortex. Four days after ischemia, infarct volume and neurologic function were assessed. In experiment 1, brain temperature was not controlled in awake rats. Temperature in rats anesthetized with halothane, approximately 1 minimum alveolar concentration, was regulated by servomechanism by surface heating or cooling to replicate the temperature profiles generated by awake animals. To address methodologic issues regarding infarct volume analysis, a subset of nine rats was examined for the effect of the histologic staining technique and the mathematical modeling algorithms used for computation of infarct volume values. In experiment 2, the brain temperature of awake and halothane-anesthetized rats was maintained normothermic (38.0 degrees C) throughout ischemia and early recirculation. RESULTS: In experiment 1 no difference between groups was observed for cortical (halothane 146 +/- 95 mm3 and awake 126 +/- 108 mm3; P = 0.64) or subcortical (halothane 110 +/- 48 mm3 and awake 100 +/- 66 mm3; P = 0.66) infarct volume. Neurologic function was also similar between groups. Total infarct volume was approximately 11% greater when histologic sections were stained with hematoxylin and eosin than when they were stained with nitro blue tetrazolium, although volumes correlated closely between the two techniques (r2 = 0.996). Analysis by orthogonal or frustum projection from two-dimensional planimetric areas to three-dimensional volumes resulted in nearly identical values (r2 = 0.999). In experiment 2, halothane-anesthetized rats experienced a 46% reduction in cortical infarct volume (halothane 106 +/- 97 mm3 and awake 197 +/- 103 mm3; P = 0.03). The incidence of hemiparesis was reduced in the anesthetized group (P = 0.03). CONCLUSIONS: When brain temperature was maintained normothermic throughout the focal ischemic insult, a neurologic and histologic protective effect for halothane anesthesia was observed. This effect of halothane was not sufficient to persist when large variations in brain temperature were allowed. Regulation of brain temperature is a critical factor in the determination of the effects of anesthetics on focal ischemic brain damage.

Animals↗
PubMed1998

Crystallization of the alanine dehydrogenase from Phormidium lapideum.

Amino-acid dehydrogenases catalyse the interconversion of their respective amino acids to the corresponding keto acid, with concomitant reduction of NAD or NADP. The enzymes phenylalanine, glutamate, leucine and valine dehydrogenase all share a similar three-dimensional subunit structure and a high degree of sequence similarity, indicating that they belong to an enzyme superfamily related by divergent evolution. In contrast, alanine dehydrogenase shows no sequence similarity with any of these enzymes despite catalysing a reaction with the same chemistry and thus it is predicted that it possesses a different three-dimensional structure. The alanine dehydrogenase from Phormidium lapideum has been crystallized in space group R32, cell dimensions a = b = 123.1 and c = 184.8 A, with a monomer in the asymmetric unit. The structure determination of this enzyme will shed light on how nature has evolved two different systems to carry out the same reaction.

Alanine Dehydrogenase↗
PubMed1987

Isolation and characterization of a monoclonal anti-P30 antibody resistant mutant of Toxoplasma gondii.

Of the possible iodine-labelled Toxoplasma gondii surface proteins, P30 (apparent Mr 30,000) is the principal one recognized by acute and convalescent anti-toxoplasma sera. This protein which comprises from 3 to 5% of the total parasite protein was used to raise a panel of parasiticidal monoclonal anti-P30 antibodies. One of these monoclonal antibodies was able to select a resistant mutant from a large population of chemically mutagenized wild-type P strain parasites. This mutant retained the wild type sensitivity to other non-P30 parasiticidal monoclonal antibodies as well as polyclonal anti-P30 rabbit sera. Analysis of surface radioiodinated wild type and mutant parasites showed that the mutant had a quantitative reduction in the amount of P30. A comparison of surface biotin labelled wild type and resistant parasites by two dimensional electrophoresis showed that the mutant lacked one and possibly two of several proteins that make up wild type P30. Western blot analysis indicated that the mutant was devoid of antigenically reactive P30. These findings further support the hypothesis that antigenic variants of T. gondii can be induced and may involve the major surface membrane antigens of the parasite.

Animals↗
PubMed2000

Diagnostic and prognostic use of stress echo in acute coronary syndromes including emergency department imaging.

Early treatment of acute myocardial infarction (AMI) can improve the rate of coronary patency, salvage myocardium, and ultimately save lives; thus, rapid recognition of patients at a higher risk of developing AMI is very important. The clinical history in patients with documented AMI is sometimes atypical, and the initial cardiac enzyme levels often are within the normal range. Moreover, the typical ST-segment elevation is often absent on the initial electrocardiogram in patients who subsequently sustain an AMI. Stress-induced segmental wall motion abnormalities (SWMAs) in coronary artery disease patients can be readily detected by conventional two-dimensional echocardiography. Moreover, echocardiography is the only technique available that allows real-time assessment of stress-induced reduction in systolic wall thickening, a highly specific sign of myocardial ischemia. Echocardiography for the diagnosis of acute ischemia is most helpful in subjects with a high clinical suspicion but nondiagnostic electrocardiograms. Under these circumstances, reversible SWMA confirms the diagnosis of acute coronary syndrome. The location of regional SWMAs correlates well with the distribution of the artery involved and pathological evidence of infarction. A trained eye can easily recognize cardiac causes of acute chest pain other than coronary diseases such as aortic stenosis, hypertrophic cardiomyopathy, mitral valve prolapse, pericarditis, and aortic dissection. When echocardiography is performed soon after the patients arrival at the emergency department (ED) or during a chest pain episode, SWMAs are detected in 90-95% of transmural infarctions and in 80-90% of nontransmural or subendocardial infarctions, and the specificity of echocardiography is approximately 80-90%. Although stress echocardiography performed in the ED and interpreted at a distance through the use of telemedicine has the potential of being convenient, in our opinion, any form of stress echocardiography should be performed in the echocardiography laboratory only after an AMI has been completely ruled out. The detection of jeopardized myocardium early after AMI can identify patients at a higher risk to develop subsequent events. In conclusion, echocardiography is cost effective in the triage of patients presenting with acute chest pain when performed soon after ED admission or during a chest pain episode. However, echocardiography must be readily available, expeditiously performed, and skillfully interpreted. The clinical use of stress echocardiography in acute coronary syndromes has been greatly improved with the introduction of digital and second harmonics technology and further enhanced by the availability of contrast agents.

Diagnosis, Differential↗
PubMed2000

A cellular solid model for modulus reduction due to resorption of trabeculae in bone.

In osteoporotic trabecular bone, bone loss occurs by thinning and subsequent resorption of the trabeculae. In this study, we compare the effects of density reductions from uniform thinning of struts or from removal of struts in a random, open-cell, three-dimensional Voronoi structure. The results of this study, combined with those previous studies on other regular and random structures, suggest that the modulus and strength of trabecular bone are reduced more dramatically by density losses from resorption of trabeculae than by those from uniform thinning of trabeculae.

Aged↗
PubMed1982

Rhizobium japonicum mutants defective in symbiotic nitrogen fixation.

Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads.

Bacterial Proteins↗
PubMed1974

Percutaneous needle localization of clustered mammary microcalcifications prior to biopsy.

Mammography occasionally reveals the presence of suspicious appearing clustered microcalcifications without an associated mass. Clinical localization of these microcalcifications within the breast is difficult, even using the 2 dimensional effect of a mammogram. Percutaneous needle localization of these microcalcifications is recommended for its accuracy, patient acceptance, and reduction in size of the biopsy specimen. No complications of this procedure have been encountered.

Biopsy↗
PubMed2000

C1-C2 rotary subluxation following posterior stabilization for congenital atlantoaxial dislocation.

The authors report a rare complication of C1-C2 rotary subluxation in two children following posterior stabilization for congenital atlantoaxial dislocation (AAD). A patient, with mobile AAD, underwent Brook's C1-C2 fusion while the other, with fixed AAD, underwent transoral decompression followed by Jain's occipitocervical fusion. A pre-existing ligamentous laxity associated with an asymmetrical wire tightening or slippage of the wires due to rotation of the neck in the former, and the drilling of the C1-C2 lateral joints during the transoral procedure in the latter, could have contributed to the rotary subluxation. Both patients presented with persistent torticollis due to fusion in an asymmetrical position with dislocated facet joints. Rotary C1-C2 subluxation, when coexisting with anterior dislocation, has the potential to cause severe and occasionally fatal cord compression. Well defined criteria to diagnose this entity by conventional radiology exist, however, due to the overlap of anatomy, the condition is often overlooked. In the present study, three dimensional reconstruction images using helical computerized tomography were very useful in delineating the subluxation and in planning its surgical reduction and arthrodesis.

Adolescent↗
PubMed1987

[Endoluminal mitral valvuloplasty in young adults and children. Apropos of 10 cases].

Between April 1 and May 15, 1986, transluminal dilatation of the mitral valve was performed at the Marie-Lannelongue Hospital, near Paris, in 10 patients aged from 12 to 48 years (mean 25,3 years) suffering from rheumatic mitral stenosis with supple valve leaflets. The procedure, carried out under local anaesthesia, included trans-septal catheterization followed by installation of one, then usually two balloons opposite the mitral orifice. The total diameter of the balloons was often greater than that of the mitral annulus. No haemorrhage, embolism or arrhythmia was observed. A significant left-to-right shunt, evaluated by radioisotope exploration with technetium pyrophosphate performed on the 8th day, was present in only one patient. The LV-LA end-diastolic gradient fell from 14.4 +/- 9.8 to 4.6 +/- 3.4 mmHg (p less than 0.01). The mean pulmonary arterial pressure was reduced less significantly from 39.9 +/- 18.4 to 27.2 +/- 11.3 mmHg (p less than 0.02). The cardiac output was only moderately increased from 2.52 +/- 0.48 to 2.88 +/- 0.66 l/min/m2. The mitral valve area, measured from the smaller axis on 2-dimensional echocardiography, more than doubled, reaching a mean value of 1.99 +/- 0.56 cm2. This figure was confirmed by the reduction, at Doppler velocimetry, of the gradient half-regression time from 267 +/- 62 to 118 +/- 46 ms (p less than 0.01). Similarly, the end-systolic gradient evaluated by Doppler, fell from 17.5 +/- 8.9 to 5.3 +/- 4.7 mmHg (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Craniofacial growth following rigid fixation: suture excision, miniplating, and microplating.

The effects of plating the coronal and nasofrontal sutures on skull growth were studied in New Zealand white rabbits. Removal of the underlying coronal suture and reduction in the size of the plate were also studied with regard to their effect on growth. Three-dimensional coordinate landmarks located on the skulls were digitized and analyzed to determine the differences in form between operated and unoperated animals using Euclidian distance matrix analysis. Interlandmark distances in three dimensions were compared to demonstrate changes induced by plating. Coronal suture plating (n = 8) resulted in local decreases in dimensions of the plated bones and contralateral and adjacent skull size increases. Removal of the coronal suture without plating (n = 6) resulted in an increase in the size of the adjacent frontal bone and an increase in the width of the suture. Placement of a plate after removal of the underlying suture (n = 8) resulted in decreases in dimensions of the plated bones, with distant length and width increases in a pattern similar to that of the skulls that were simply plated. Plating across the nasofrontal suture (n = 6) resulted in decreases in dimensions in the plated bone, with some distant width increases at the midfrontal bone, the coronal suture, and the posterior parietal bone. In this group, no contralateral increase in dimensions was detected. This is unlike the pattern observed for the animals with a coronal suture plate only. Placing a microplate across the coronal suture (n = 6) resulted in morphologies similar to those with the use of miniplates across the coronal suture. We propose that the varying morphologies in these groups represent compensatory changes accompanying growth restriction induced by plating. We suggest that the lack of contralateral growth increases with nasofrontal suture plating results from lesser effects on the underlying growing cranial contents at this location, as compared with the coronal suture. We find consistent results despite suture removal, change in plate location, and change in plate size.

Animals↗