Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or β Subunits: Circular Dichroism, 1H NMR, and Resonance Raman Studies

Native human adult hemoglobin (Hb A) has mostly normal orientation of heme, whereas recombinant Hb A (rHb A) expressed in E. coli contains both normal and reversed orientations of heme. Hb A with the normal heme exhibits positive circular dichroism (CD) bands at both the Soret and 260-nm regions, while rHb A with the reversed heme shows a negative Soret and decreased 260-nm CD bands. In order to examine involvement of the proximal histidine (His F8) of either α or β subunits in determining the heme orientation, we prepared two cavity mutant Hbs, rHb(αH87G) and rHb(βH92G), with substitution of glycine for His F8 in the presence of imidazole. CD spectra of both cavity mutant Hbs did not show a negative Soret band, but instead exhibited positive bands with strong intensity at the both Soret and 260-nm regions, suggesting that the reversed heme scarcely exists in the cavity mutant Hbs. We confirmed by 1H NMR and resonance Raman (RR) spectroscopies that the cavity mutant Hbs have mainly the normal heme orientation in both the mutated and native subunits. These results indicate that the heme Fe-His F8 linkage in both α and β subunits influences the heme orientation, and that the heme orientation of one type of subunit is related to the heme orientation of the complementary subunits to be the same. The present study showed that CD and RR spectroscopies also provided powerful tools for the examination of the heme rotational disorder of Hb A, in addition to the usual 1H NMR technique. Chirality 28:585–592, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.Embargo Period 24 month

Localized Talaromyces marneffei infection presenting as a tonsillar mass mimicking malignancy

Talaromyces marneffei is an opportunistic fungal infection seen in immunocompromised patients including those with HIV/AIDS. It is usually seen in patients who live in or are from tropical Asia. In HIV patients, oropharyngeal and laryngeal lesions are usually part of disseminated infection. We describe a case of 63-year-old Vietnamese male with history of HIV/AIDS who presented with localized T. marneffei tonsillar infection without disseminated disease. Imaging studies showed a right tonsillar mass with right cervical lymphadenopathy which was initially thought to be malignancy. The patient underwent biopsy of the mass and histology showed noncaseating granulomas on hematoxylin and eosin stain as well as yeast on Grocott methenamine silver stain. Fungal culture of the biopsy specimen grew suede-like grayish-white colonies with diffuse underlying deep red color pigment which was identified as Talaromyces marneffei. The patient was treated with intravenous liposomal amphotericin B and achieved resolution of symptoms and tonsillar mass. In HIV/AIDS patients who are either from endemic regions or with history of travel to endemic areas particularly Southeast Asia and China, T. marneffei infection should be considered in differential diagnoses of a tonsillar mass

Increased Synthesis of Anti-Tuberculous Glycolipid Immunoglobulin G (IgG) and IgA with Cavity Formation in Patients with Pulmonary Tuberculosis▿

Tuberculous glycolipid (TBGL) antigen is a cell wall component of Mycobacterium tuberculosis and has been used for the serodiagnosis of tuberculosis. We investigated correlations between the levels of anti-TBGL antibodies and a variety of laboratory markers that are potentially influenced by tuberculous infection. Comparisons between patients with cavitary lesions and those without cavitary lesions were also made in order to determine the mechanism underlying the immune response to TBGL. Blood samples were obtained from 91 patients with both clinically and microbiologically confirmed active pulmonary tuberculosis (60 male and 31 female; mean age, 59 ± 22 years old). Fifty-nine patients had cavitary lesions on chest X-rays. Positive correlations were found between anti-TBGL immunoglobulin G (IgG) and C-reactive protein (CRP) (r = 0.361; P < 0.001), between anti-TBGL IgA and soluble CD40 ligand (sCD40L) (r = 0.404; P < 0.005), between anti-TBGL IgG and anti-TBGL IgA (r = 0.551; P < 0.0000005), and between anti-TBGL IgM and serum IgM (r = 0.603; P < 0.00000005). The patients with cavitary lesions showed significantly higher levels of anti-TBGL IgG (P < 0.005), anti-TBGL IgA (P < 0.05), white blood cells (P < 0.01), neutrophils (P < 0.005), basophils (P < 0.0005), natural killer cells (P < 0.05), CRP (P < 0.0005), KL-6 (sialylated carbohydrate antigen KL-6) (P < 0.0005), IgA (P < 0.05), and sCD40L (P < 0.01). The observed positive correlations between the anti-TBGL antibody levels and inflammatory markers indicate the involvement of inflammatory cytokines and NKT cells in the immunopathogenesis of pulmonary tuberculosis

Left: The CD spectral changes due to the quaternary and tertiary structure transition for Hb A.

<p>The spectra are the quaternary structure transition (A: pink spectrum) and tertiary structure transition (B: blue spectrum) expected for Hb A and the observed deoxy-minus-oxy difference spectra of Hb A (C: black spectrum). <b>Right: Comparison of the deoxy-minus-oxy difference spectra of rHb(αH87G) and rHb(βH92G) with that of Hb A.</b> The difference spectra are Hb A (black spectrum), rHb(αH87G) (orange spectrum) and rHb(βH92G) (green spectrum).</p

Hill’s plots of oxygen binding by Hb A, rHb(αH87G), rHb(βH92G) and sperm whale Mb.

<p>Hb A (-IHP) (blue closed triangle: ▲), Hb A (+IHP) (pink closed square: ■), rHb(αH87G) (black closed circle: ●), rHb(βH92G) (orange closed circle: ●) and sperm whale Mb (light green closed diamond: ◆). <i>Y</i> and <i>p</i>O₂ are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135080#pone.0135080.g002" target="_blank">Fig 2</a>. The symbols are the observed points. The hemoglobin concentration was 60 μM on a heme basis in 0.05 M bis-Tris buffer (pH 7.4) containing 0.1 M Cl^-. In addition, rHb(αH87G) and rHb(βH92G) contained 5 mM imidazole and a metHb reducing system. The temperature was set at 25°C. IHP was added to a final concentration of 2 mM.</p

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α₂β₂ Tetramer

<div><p>Human hemoglobin (Hb), which is an α₂β₂ tetramer and binds four O₂ molecules, changes its O₂-affinity from low to high as an increase of bound O₂, that is characterized by ‘cooperativity’. This property is indispensable for its function of O₂ transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O₂-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O₂-binding with no cooperativity, indicating the coexistence of two independent hemes with different O₂-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O₂-binding curve with no cooperativity. Resonance Raman, ¹H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O₂-binding to rHb(αH87G), but it did partially occur with O₂-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O₂-binding, but its absence in the β subunit simply enhances the O₂-affinity of α subunit.</p></div