Dynamics of cyanide binding to ferrous Scapharca inaequivalvis homodimeric hemoglobin

Flash photolysis experiments have been carried out for the first time on a hemoglobin ferrous cyanide adduct with an 8 ns laser pulse. A 95% nonexponential rebinding process occurs within 2 micros after full photolysis in ferrous cyanide dimeric Scapharca inaequivalvis hemoglobin (HbI), indicating that once photolyzed the cyanide anion is not able to escape from the protein matrix and rebinds to the heme iron. The resonance Raman spectrum of the 10 ns photoproduct is identical to that of the fully relaxed deoxy derivative, indicating that in the ferrous cyanide HbI adduct protein relaxation occurs within 10 ns after photolysis. This behavior is at variance with that of the carbonmonoxy HbI derivative in which very little geminate rebinding is observed and the photoproduct relaxes with a lifetime of 1 micros. The fast relaxation of the cyanide HbI photoproduct can be accounted for by the small perturbation of the heme structure induced by cyanide binding to ferrous HbI. This is consistent with a deoxy-like conformation of the HbI ferrous cyanide adduct and implies that the pathway for relaxation involves only minor local rearrangements of the heme moiety. Photolysis experiments carried out on ferrous cyanide horse myoglobin, which can be saturated only partially, show a qualitatively similar behavior in ligand rebinding, indicating that the geminate process of the cyanide anion is a general phenomenon in hemoproteins.     

Hydroxide rather than histidine is coordinated to the heme in five-coordinate ferric Scapharca inaequivalvis hemoglobin

The ferric form of the homodimeric Scapharca hemoglobin undergoes a pH-dependent spin transition of the heme iron. The transition can also be modulated by the presence of salt. From our earlier studies it was shown that three distinct species are populated in the pH range 6-9. At acidic pH, a low-spin six-coordinate structure predominates. At neutral and at alkaline pHs, in addition to a small population of a hexacoordinate high-spin species, a pentacoordinate species is significantly populated. Isotope difference spectra clearly show that the heme group in the latter species has a hydroxide ligand and thereby is not coordinated by the proximal histidine. The stretching frequency of the Fe-OH moiety is 578 cm-1 and shifts to 553 cm-1 in H218O, as would be expected for a Fe-OH unit. On the other hand, the ferrous form of the protein shows substantial stability over a wide pH range. These observations suggest that Scapharca hemoglobin has a unique heme structure that undergoes substantial redox-dependent rearrangements that stabilize the Fe-proximal histidine bond in the functional deoxy form of the protein but not in the ferric form.     

Ligand binding and conformation change in the dimeric hemoglobin of the clam Scapharca inaequivalvis

The reaction with carbon monoxide of the cooperative dimeric hemoglobin from Scapharca inaequivalvis has been examined by flash photolysis. In the nanosecond time range, geminate rebinding of 5% of dissociated CO occurs with a rate constant of 1.4 x 10(7) s-1. There is a change in absorbance of deoxyhemoglobin following photolysis at a rate of 1.2 x 10(6) s-1, consistent with a shift in the position of the Soret band to longer wavelengths. The amplitude of the change is proportional to the population of deoxydimer. In much of the Soret region this change is greater than the absorbance excursion associated with geminate recombination. There is at least one other slower change associated with the singly liganded species. Geminate rebinding of NO has components of 50, 8, and 0.035 ns-1, accounting for 75%, 25%, and less than 1% of the total reaction observed after a 35-ps photolysis flash. Simulation of diffusion of NO by molecular dynamics shows the ligands moving from the heme pocket to a subsidiary space between the edge of the heme and the surface of the protein.     

The apolar distal histidine mutant (His69-->Val) of the homodimeric Scapharca hemoglobin is in an R-like conformation

The effect of the apolar mutation of the distal histidine (His69-->Val) has been studied in the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis. Absorption, circular dichroism, and resonance Raman spectroscopy point to a more symmetric heme structure of the deoxy derivative, which is indicative of an R-like conformation of the deoxy heme. Resonance Raman spectroscopy also brings out alterations in the geometry and interactions of the bound CO molecule. The iron-carbon stretching frequency is decreased by about 30 cm-1 with respect to the native protein, while the diatomic ligand stretching frequency is increased by about the same degree. Consistent with the structural changes, the ligand binding properties are significantly altered. In the mutant the overall rate and the affinity for CO binding are increased about 100-fold with respect to the native protein, and cooperativity is abolished. In addition, the amplitude and the rate of the geminate rebinding process increase significantly. This finding may be correlated to the longer average residence time of the photolyzed CO molecule within the heme pocket of the H69V mutant, as indicated by molecular dynamics simulations.     

Solution 1H NMR investigation of the heme cavity and substrate binding site in cyanide-inhibited horseradish peroxidase

Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.     

Orientation of the heme vinyl groups in the hydrogen sulfide-binding hemoglobin I from Lucina pectinata

Hemoglobin I (HbI) from the claim Lucina pectinata is a unique heme protein that binds and transfers hydrogen sulfide (H2S) to a symbiotic bacteria. The metcyano, metaquo, carbon monoxy, oxy, and deoxy complexes of HbI were studies by resonance Raman (RR) spectroscopy, and the metacyano and carbon monoxy complexes were also studied by 1H-NMR. The results indicate a unique orientation of the heme 2-vinyl group relative to other heme proteins. The RR spectra of the HbICO, metHbICN, metHbIH2O, HbIO2 and deoxyHbI heme derivatives show a band at 1621 cm-1 and a shoulder at 1626 cm-1, indicative of an out-of-plane position for one of the vinyls relative to the other one. Spin-lattice relaxation properties of protons in the metHbICN complex also suggest a unique orientation for the heme 2-vinyl group of HbI. The longitudinal relaxation time (T1) for the 2-H alpha, 2-H beta c, and H beta t protons are 120 ms, 115 ms, and 135 ms, respectively. The data from both techniques suggest an out-of-plane and trans-oriented 2-vinyl group, and an in-plane and cis-oriented 4-vinyl group for the low-spin complexes of HbI. These results imply that the electron withdrawing character of the out-of-plane vinyl group contributes to the stability of the heme Fe+3 oxidation state, facilitates the binding of the H2S ligand, and promotes the stability of this ferric H2S complex.     

Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin

Residue Phe97, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding. This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity. Kinetic measurements of ligand binding to the Leu97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states. Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure. Increased oxygen affinity results from the absence of the Phe97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation. The absence of the Phe97 side chain is also associated with diminished cooperativity, since Leu97 packs in the heme pocket in both states. Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin.     

Mutational destabilization of the critical interface water cluster in Scapharca dimeric hemoglobin: structural basis for altered allosteric activity

We determined the proportion of survival variability explained by the usual prognostic factors in childhood acute lymphoblastic leukaemia (ALL) during a prognostic study of 1552 patients enrolled in three consecutive Fralle group protocols (Fralle 83, Fralle 87 and Fralle 89). The event-free survival rates at 5 years were 54.8% (SD 1.9), 43.1%) (SD 2.7) and 55.6% (SD 2.2), respectively. In the univariate analysis the following variables were predictive of poor outcome: male gender, elevated leucocytosis (> 50 x 10(9)/l), circulating blastosis. haemoglobin >12 g/dl, platelet count <100 x 10(9)/l, age under 1 year or over 9 years, enlarged mediastinum, nodes, spleen and liver, T phenotype, absence of CD10+ cells; testicular and meningeal involvement, poor response to induction therapy (CCSG M3), and LDH >400 U/l. Among the cytogenetic features, hyperdiploidy had a protective effect, whereas hypodiploidy, translocation and other structural abnormalities had a negative influence, particularly in cases of t(9;22) or t(4;11). Multivariate analysis summarized the prognostic information in terms of four variables: age, gender, leucocytosis and cytogenetic features. Missing data had little influence on the results. However, despite their significance in the multivariate analysis, these four variables each had very low predictive power (1.1% for gender, 2.0% for age, 3.5% for leucocytosis, and 1.6% for cytogenetic features). Thus, the most significant prognostic factors in childhood ALL each explain no more than 4% of the variability in prognosis. This may explain the disappointing practical value of these factors and underlines the need for prognostic tools in childhood ALL.     

Asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): the issue of valency exchange

A new framework for hemoglobin cooperativity was proposed by Ackers and colleagues on the basis of the hyper thermodynamic stability and deoxy (T) quaternary structure of one of diliganded deoxy-cyanomet hybrid hemoglobins, (alpha+CN-beta+CN-)(alpha beta), studied by hybridization of the equimolar mixture of deoxyhemoglobin and cyanomethemoglobin through a long (70-100 h) dimer exchange reaction [Daugherty et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. Recently, we reported that the published hyperstability of (alpha+CN-beta+CN-)(alpha beta) is incorrect due to the occurrence of valency exchange between the heme sites of both parental hemoglobins during the long deoxy incubation [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We also noted a difficulty in maintaining both anaerobicity and excess free cyanide of the sample during the long incubation, which led to formation of cyanide-unbound aqometheme in the original deoxyhemoglobin resulting from the electron transfer to cyanometheme. This paper is a response to a recent argument against our work [Ackers et al. (1997) Biochemistry 36, 10822-10829]. Ackers et al. have claimed that no appreciable formation of aqomethemoglobin with their methods ensures their sample integrity, based on a supposition that our observed valency exchange may have occurred via aqometheme. In this paper, however, we demonstrate that appreciable (>27%) valency exchange really occurs between deoxy and cyanometheme sites during 72 h incubation under conditions where both anaerobicity and excess free cyanide of the sample solution are maintained by a continuous flow of humidified N2 with HCN. This confirms our view that previous experimental data on (alpha+CN-beta+CN-)(alpha beta) obtained by the long incubations should be subject to reexamination while our earlier estimation of a lower limit of free energy of (alpha+CN-beta+CN-)(alpha beta) (i.e., >/= -10.1 kcal/mol) by a rapid method (35 min) is still valid. We also suggest a possibility that the T quaternary structure of (alpha+CN-beta+CN-)(alpha beta) assigned by Ackers and colleagues using the long incubations is an artifact arising from the valency exchange. These results suggest that the putative mechanistic picture for hemoglobin cooperativity inferred from studies on deoxy-cyanomet hybrids is without foundation.     

Effect of thiol reagents on functional properties and heme oxidation in the hemoglobin of Geochelone carbonaria

The reaction of thiol reagents with G. carbonaria hemoglobin was studied, and the oxygen equilibrium and kinetic of oxidation of derivatives determined. The oxygen affinity and kinetic of oxidation of hemoglobin derivatives were modified to various extents depending on the nature of thiol reagents used. Diamide yielded approximately 80% polymeric hemoglobin, although the oxidation kinetic, and the functional properties, were practically invariant (T1/2 = 10.0 min.; P50 = 5.0 mm Hg at pH 7.4; alkaline Bohr effect = -0.64). Iodoacetamide did not modify the electrophoretic pattern significantly, although all the free SH groups of hemoglobin were alkylated. A P50 of 2.5 mmHg at pH 7.4 and the Bohr effect of -0.15 were obtained; the T1/2 of about 6.4 min. was shorter than that for un-modified Hb. Similar T1/2 were obtained for Hb treated with oxidized glutathione, which produced polymeric Hb and glutathionyl-Hb. The oxygen binding characteristics showed that both of Hb derivatives, glutathionyl-Hb and polymeric Hb, maintain the capacity to transport the gas.     

Involvement of a lysine-specific cysteine proteinase in hemoglobin adsorption and heme accumulation by Porphyromonas gingivalis

The oral anaerobic bacterium Porphyromonas gingivalis, a major pathogen of advanced adult periodontitis, produces a novel class of cysteine proteinases in both cell-associated and secretory forms. A lysine-specific cysteine proteinase (Lys-gingipain, KGP), as well as an arginine-specific cysteine proteinase (Arg-gingipain), is a major trypsin-like proteinase of the organism. Recent studies indicate that the secreted KGP is implicated in the destruction of periodontal tissue and the disruption of host defense mechanisms. In this study, we have constructed a KGP-deficient mutant to determine whether the cell-associated KGP is important for pathophysiology of the organism. Although the mutant retained the strong ability to disrupt the bactericidal activity of polymorphonuclear leukocytes, its hemagglutination activity was reduced to about one-half that observed with the wild-type strain. More important, the mutant did not form black-pigmented colonies on blood agar plates, indicating the defect of hemoglobin adsorption and heme accumulation. Immunoblot analysis showed that the expression of a 19-kDa hemoglobin receptor protein, which is thought to be responsible for hemoglobin binding by the organism, was greatly retarded in this mutant. The mutant also showed a marked decrease in the ability to degrade fibrinogen. These results suggest the possible involvement of KGP in the hemoglobin binding and heme accumulation of the organism and in the bleeding tendency in periodontal pockets.     

Superoxide produced in the heme pocket of the beta-chain of hemoglobin reacts with the beta-93 cysteine to produce a thiyl radical

The role of the beta-93 cysteine residue in the hemoglobin autoxidation process has been delineated by electron paramagnetic resonance. At low temperatures (8 K) after incubation at 235 K, free radical signals were detected. An analysis of the free radical spectrum produced implies that, besides the superoxide radical expected to be formed during autoxidation, an isotropic free radical is produced with a giso of 2.0133. This g value is consistent with that expected for a sulfur radical. Blocking the beta-93 sulfhydryl group with N-ethylmaleimide was found to eliminate the formation of the isotropic radical, but not the superoxide. This finding confirms the assignment of the isotropic radical as a thiyl radical originating from the oxidation of the cysteine SH group. A kinetic analysis of the time course for the formation of both the superoxide and thiyl radicals is consistent with a reversible electron transfer process between superoxide in the heme pocket of the beta-chains and the cysteine residue. This reaction is expected to produce both a thiyl radical and a peroxide. Direct evidence for peroxide production comes from the detection of a transient Fe(III) heme peroxide complex. The significance of the electron transfer process producing a thiyl radical is discussed. It is shown that the formation of the thiyl radical decreases the rate of autoxidation for the beta-chain and reduces heme degradation attributed to the reaction of superoxide with the heme. The insights gained from these low-temperature studies are believed to be relevant to room-temperature autoxidation.     

Solution 1H-NMR structure of the heme cavity in the low-affinity state for the allosteric monomeric cyano-met hemoglobins from Chironomus thummi thummi. Comparison to the crystal structure

Solution 1H-NMR studies of the heme cavity were performed for the cyanomet complexes of monomeric hemoglobins III and IV from the insect Chironomus thummi thummi, each of which exhibit marked Bohr effects. The low pH 5, paramagnetic (S = 1/2) derivatives were selected for study because the large dipolar shifts provide improved resolution over diamagnetic forms and allow distinction between the two isomeric heme orientations [Peyton, D. H., La Mar, G. N. & Gersonde, K. (1988) Biochim. Biophys. Acta 954, 82-94]. The crystal structure for the low-pH form of the hemoglobin III derivative, moreover, has been reported and showed that the functionally implicated distal His58 side chain adopts alternative orientation, either in or out of the pocket [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309-338]. All heme pocket residues for the low-pH forms of the two hemoglobins were located, at least in part, and positioned in the heme cavity on the basis of nuclear Overhauser effects to the heme and each other, dipolar shifts, and paramagnetic-induced relaxation. The resulting structure yielded the orientation of the major axis of the paramagnetic susceptibility tensor. The heme pocket structure of the cyanomet hemoglobins III and IV were found to be indistinguishable, with both exhibiting a distal His58 oriented solely into the heme cavity and in contact with the ligand, and with two residues, Phe100 and Phe38, exhibiting small but significant displacements in solution relative to hemoglobin III in the crystal.     

Transformation of cooperative free energies between ligation systems of hemoglobin: resolution of the carbon monoxide binding intermediates

A strategy has been developed for quantitatively "translating" the distributions of cooperative free energy between different oxygenation analogs of hemoglobin (Hb). The method was used to resolve the cooperative free energies of all eight carbon monoxide binding intermediates. These parameters of the FeCOHb system were determined by thermodynamic transformation of corresponding free energies obtained previously for all species of the Co/FeCO system, i.e., where cobalt-substituted hemes comprise the unligated sites [Speros, P. C., et al. (1991) Biochemistry 30, 7254-7262]. Using hybridized combinations of normal and cobalt-substituted Hb, ligation analog systems Co/FeX (X = CO, CN) were constructed and experimentally quantified. Energetics of cobalt-induced structural perturbation were determined for all species of both the "mixed metal" Co/Fe system and also the ligated Co/FeCN system. It was found that major energetic perturbations of the Co/Fe hybrid species originate from a pure cobalt substitution effect on the alpha subunits. These perturbations are transduced to the beta subunit within the same dimeric half-tetramer, resulting in alteration of the free energies for binding at the nonsubstituted (Fe) sites. Using the linkage strategy developed in this study along with the determined energetics of these couplings, the experimental assembly free energies for the Co/FeCO species were transformed into cooperative free energies of the 10 Fe/FeCO species. The resulting values were found to distribute according to predictions of a symmetry rule mechanism proposed previously [Ackers, G. K., et al. (1992) Science 255, 54-63]. Their distribution is consistent with accurate CO binding data of normal Hb [Perrella, M., et al. (1990b) Biophys. Chem. 37, 211-223] and also with accurate O2 binding data obtained under the same conditions [Chu, A. H., et al. (1984) Biochemistry 23, 604-617].     

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase

A new ruthenium-labeled cytochrome c derivative was designed to measure the actual rate of electron transfer to the Trp-191 radical cation and the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(FeIV = O,R.+)}. The H39C,C102T variant of yeast iso-1-cytochrome c was labeled at the single cysteine residue with a tris (bipyridyl)ruthenium(II) reagent to form Ru-39-Cc. This derivative has the same reactivity with CMPI as native yCc measured by stopped-flow spectroscopy, indicating that the ruthenium group does not interfere with the interaction between the two proteins. Laser excitation of the 1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in electron transfer from RuII* to heme c FeIII with a rate constant of 5 x 10(5) s-1, followed by electron transfer from heme c Fe II to the Trp-191 indolyl radical cation in CMPI(FeIV = O,R*+) with a rate constant of k(eta) = 2 x 10(6) s-1. A subsequent laser flash led to electron transfer from heme c to the oxyferryl heme in CMPII-(FeIV = O,R) with a rate constant of k(etb) = 5000 s-1. The location of the binding domain was determined using a series of surface charge mutants of CcP. The mutations D34N, E290N, and A193F each decreased the values of k(eta) and k(etb) by 2-4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc-CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. A mechanism is proposed for reduction of the oxyferryl heme in which internal electron transfer in CMPII(FeIV = O,R) leads to the regeneration of the radical cation in CMPII-(FeIII,R*+), which is then reduced by yCcII. Thus, both steps in the complete reduction of CMPI involve electron transfer from yCcII to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant k(eta) with theoretical predictions indicate that the electron transfer pathway identified in the crystalline yCc-CcP complex is very efficient. Stopped-flow studies indicate that native yCcII initially reduces the Trp-191 radical cation in CMPI with a second-order rate constant ka, which increases from 1.8 x 10(8) M-1 s-1 at 310 mM ionic strength to > 3 x 10(9) M-1 s-1 at ionic strengths below 100 mM. A second molecule of yCcII then reduces the oxyferryl heme in CMPII with a second-order rate constant kb which increases from 2.7 x 10(7) M-1 s-1 at 310 mM ionic strength to 2.5 x 10(8) M-1 s-1 at 160 mM ionic strength. As the ionic strength is decreased below 100 mM the rate constant for reduction of the oxyferryl heme becomes progressively slower as the reaction is limited by release of the product yCcIII from the yCcIII-CMPII complex. Both ruthenium photoreduction studies and stopped-flow studies demonstrate that the Trp-191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.     

Gaugement of the inner space of the apomyoglobin's heme binding site by a single free diffusing proton. I. Proton in the cavity

Time resolved fluorimetry was employed to monitor the geminate recombination between proton and excited pyranine anion locked, together with less than 30 water molecules, inside the heme binding site of Apomyoglobin (sperm whale). The results were analyzed by a numerical reconstruction of the differential rate equation for time-dependent diffusion controlled reaction with radiating boundaries using N. Agmon's procedure (Huppert, Pines, and Agmon, 1990, J. Opt. Soc. Am. B., 7:1541-1550). The analysis of the curve provided the effective dielectric constant of the proton permeable space in the cavity and the diffusion coefficient of the proton. The electrostatic potential within the cavity was investigated by the equations given by Gilson et al. (1985, J. Mol. Biol., 183:503-516). According to this analysis the dielectric constant of the protein surrounding the site is epsilon prot < or = 6.5. The diffusion coefficient of the proton in the heme binding site of Apomyoglobin-pyranine complex is D = 4 x 10(-5) cm2/s. This value is approximately 50% of the diffusion coefficient of proton in water. The lower value indicates enhanced ordering of water in the cavity, a finding which is corroborated by a large negative enthropy of binding delta S0 = -46.6 cal.mole-1 deg-1. The capacity of a small cavity in a protein to retain a proton had been investigated through the mathematical reconstruction of the dynamics. It has been demonstrated that Coulombic attraction, as large as delta psi of energy coupling membrane, is insufficient to delay a free proton for a time frame comparable to the turnover time of protogenic sites.     

Occurrence of free D-aspartate and aspartate racemase in the blood shell Scapharca broughtonii

In spite of the high prevalence of tuberculosis worldwide, there are few studies on its psychiatric complications. The mental state of 53 patients with pulmonary tuberculosis seen in a Nigerian chest clinic was examined using the 30-item General Health Questionnaire (GHQ-30), the Present State Examination (PSE), and a clinical evaluation based on the International Classification of Disease, tenth edition (ICD-10). Results were compared with two comparison groups: (1) a group of 20 long-stay orthopedic patients with lower limb fractures; and (2) a group of 20 apparently healthy controls. The sociodemographic characteristics of the groups were also compared. A significantly higher prevalence of psychiatric disorders was found in the tuberculosis group (30.2%) than in the orthopedic group (15%) and the apparently healthy controls (5%). The types of psychiatric disorders encountered included mild depressive episode, generalized anxiety disorder, and adjustment disorder (ICD-10). Psychiatric morbidity was higher in tuberculosis patients with low educational attainment, and did not show a statistically significant relationship with other sociodemographic parameters. Ways of improving the mental health of tuberculosis patients are discussed.     

On the tryptic peptides from hemoglobin chains of six carnivores

The amino acid compositions of the tryptic peptides of the following carnivore hemoglobin chains have been determined: gray fox (Urocyon cineroargenteus); raccoon (Procyon lotor); polar bear (Thalarctos maritimus); coati mundi (Nasua nasua) beta chain; coati mundi (Nasua narica) two beta chains; cat (Felis catus) alpha chain; and lion (Pantbera leo) beta chain. These provide a basis for future sequencing of these hemoglobins and construction of an evolutionary tree. The specific results are summarized in the following article (Stenzel and Brimhall, 1977).     

Simple bone cyst: investigation on the presence of gas in the cavity using computed tomography--review of 52 cases

BACKGROUND: Deep penetrating dermatofibroma is an unusual and histologically distinctive form of dermatofibroma that usually occurs on the lower extremities of adults. OBJECTIVE: The dermatologic surgeon and pathologist should be aware of this entity to prevent excessive treatment of a benign condition. METHODS: We are reporting a case of deep penetrating dermatofibroma with the results of histologic examination and immunohistochemical studies. RESULTS: The neoplasm consisted of a fibrohistiocytic tumor penetrating into the subcutis accompanied by scalloped extensions along the septae. Immunohistochemical stains were positive for Factor XIIIa and negative for CD34. CONCLUSION: Recognition of the histologic pattern is important in distinguishing deep penetrating dermatofibroma from dermatofibrosarcoma protuberans.