Studies on growth of Pasteurella multocida on BTB agar

It has been reported that the Pasteurella multocida does not grow on the BTB agar. Therefore, this medium has been used as selective and differential medium for Pasteurella multocida. However, we have experienced that some of the Pasteurella multocida from the patient's materials grew on the BTB agar. Here, we will report on the studies of the growth of the Pasteurella multocida strain on the BTB agar. Ten strains of Pasteurella multocida from humans and animals were used as the test strains. Those were adjusted to McFarland No. 5 by the sterilized physiological saline and inoculated on the agars. We compared commercially prepared BTB agars from 3 companies and BTB agars prepared by our-self from dehydrated culture medium. Blood, Chocolate, Nutrient and MacConkey agar were also used in this study. As for the growth of the Pasteurella multocida, we checked the pH of each agar and the temperature during the cultivation. The results are as follows: 1) Pasteurella multocida was confirmed to grow on all of the BTB agar. 2) Pasteurella multocida grew most heavily at 37 degrees C and pH of 7.4 to 8.2. 3) The difference of the growth on each agar was considered to be the difference of the pH and nutritional condition of the agar.     

Nontuberculous mycobacterial infection followed for 12 years]

A 67-year-old woman presented in September 1985 with productive cough, bloody sputum, and dyspnea on exertion. Productive cough and bloody sputum had developed when the patient was 55 years old. Sputum culture and radiologic findings yielded a diagnosis of nontuberculous mycobacteriosis (NTM). Antituberculous therapy with INH, RFP, and EB was initiated in November 1987 because of the development of a cavity in the right upper lobe, and led to resolution of the lesion and clinical symptoms. Despite progression of bronchiectatic changes in both lungs and a relapse of her clinical symptoms during the following 10 years, the patient retained enough pulmonary function to be able to maintain an active daily life until she died of advanced gastric cancer at the age of 79. Autopsy revealed cystic bronchiectasis accompanied by bronchial wall thickening in both lungs, with some granuloma and acid-fast-bacteria observed in lung tissue. In this report, we concluded that patients with NTM usually experience a gradual progression of symptoms and radiographic changes during their clinical course, and that their pulmonary function may be conserved well enough to maintain an active daily life.     

Short Report: Normalization of low biotinidase activity in a child with biotin deficiency after biotin supplementation

We report findings in a Japanese boy with severe skin rash attributable to biotin deficiency. The patient had an intracranial malformation and developed biotin deficiency due to tube feeding with a single formula for over one year. Results of urinary organic acid analysis were consistent with multiple carboxylase deficiency, and low biotinidase activity was also observed. After biotin supplementation, the skin rash improved and biotinidase activity normalized. We speculate that biotin is one regulating factor in the biosynthesis of biotinidase.     

Isolated stenosis of left anterior descending or right coronary artery: Relation between site of stenosis and ventricular dysfunction and therapeutic implications

Indications for coronary arterial bypass surgery in single vessel coronary artery disease are unresolved. To determine the extent of myocardium at risk with stenosis (70 percent or more) of a single coronary artery, left ventricular angiograms of 200 patients with stenosis confined to either the left anterior descending or right coronary artery and of 15 normal control subjects were assessed. Among patients without myocardial infarction, ejection fraction was unchanged (p > 0.05 versus normal values) in (1) those with stenosis of the proximal (above first septal branch, n = 19), mid (between septal and first diagonal branches, n = 14) and distal (within 2 cm distal to diagonal branch, n = 15) left anterior descending coronary artery, and (2) those with stenosis of the proximal (above acute marginal branch, n = 16) and distal (between acute marginal and posterior descending branches, n = 16) right coronary artery. In contrast, ejection fraction was depressed (p < 0.001 versus normal values) In left anterior descending arterial stenosis with anterior myocardial Infarction: proximal (38 ± 10 percent, n = 33), mid (46 ± 12 percent, n = 24; p < 0.01 versus proximal), and distal (56 ± 9 percent, n = 15; p < 0.01 versus mid). Ejection fraction was similar with proximal and distal stenosis of the right coronary artery and inferior Infarction: 54 ± 11 percent versus 55 ± 9 percent, p > 0.05; both p < 0.05 versus normal value. Shortening velocity was assessed in three anterior (I to III, base to apex) and three inferior (IV to VI, apex to base) equidistant hemichords perpendicular to the long axis, 30 ° right anterior oblique view. With anterior Infarction and left anterior descending stenosis, shortening of hemichords I to V, I to IV and II to III with proximal, mid and distal stenosis, respectively, was depressed (p < 0.05 versus normal value). Septal excursion and thickening on M mode echocardiography with proximal left anterior descending stenosis and infarction were depressed (p < 0.05 versus mid and distal stenosis with infarcts). Hemichordal shortening with Inferior infarction was similarly depressed (p > 0.05) with proximal and distal stenoses.In conclusion, stenosis of the left anterior descending coronary artery is a heterogenous disease, the extent of jeopardized myocardium is highly dependent on the site of stenosis, and the criteria for surgery cannot be applied uniformly. When the surgical goal is myocardial preservation, these data provide an objective rationale for bypass of stenosis of the proximal left anterior descending coronary artery. In stenosis confined to the right coronary artery, left ventricular preservation alone should not be considered an indication for coronary bypass grafting.     

Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N₂O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate (I), a key state to promote N–N bond formation and N–O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I. TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe–NO coordination in I, with an elongated Fe–NO bond length (Fe–NO = 1.91 Å, Fe–N–O = 138°) in the absence of NAD⁺. TR-infrared (IR) spectroscopy detects the formation of I with an N–O stretching frequency of 1,290 cm⁻¹ upon hydride transfer from NADH to the Fe³⁺–NO enzyme via the dissociation of NAD⁺ from a transient state, with an N–O stretching of 1,330 cm⁻¹ and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe³⁺–NHO^•− radical. The current findings provide conclusive evidence for the N₂O generation mechanism via a radical–radical coupling of the heme nitroxyl complex with the second NO molecule.

Nitric oxide (NO) is a radical gas molecule exhibiting high reactivity, but it is synthesized in some cellular systems. We should know how biological systems manage the highly cytotoxic NO without damaging biological compounds, such as proteins, lipids, and nucleic acids. The nitrogen oxide–based anaerobic respiration of microorganisms, so-called denitrification, is one such biological NO-generating system, in which NO is generated from nitrite (NO₂⁻) with one-electron reduction catalyzed by NO₂⁻ reductase, but is immediately decomposed into nitrous oxide (N₂O) to avoid its cytotoxicity before diffusing into the cell. This NO decomposition is catalyzed by the iron-containing enzyme (i.e., NO reductases [NORs]) by following the reaction: 2 NO + 2 e⁻ + 2 H⁺ → N₂O + H₂O. Because the chemistry of this NOR reaction contains N–N bond formation and N–O bond cleavage steps (1), its mechanism has garnered broad interest to be established at the atomic and electronic levels. In environmental science, it is also believed that the molecular mechanism of NO decomposition in biological systems should be established, as the product N₂O of the NOR-catalyzed reaction acts as a greenhouse gas and an ozone-depleting substance on the global level (2), and ∼70% of the global N₂O emission is attributable to NO reduction by NORs in microorganisms in the soil. From such chemical, biological, and environmental points of view, the NOR reaction is highly attractive (3).NOR in a fungal denitrification system is a cytochrome P450-type enzyme (P450nor), which contains one Cys-coordinated heme at the active site (4). This protein catalyzes the reduction of NO using NAD(P)H as an electron donor: 2 NO + NAD(P)H + H⁺ → N₂O + H₂O + NAD(P)⁺. In the first step of this reaction, NO and NAD(P)H bind to P450nor in the resting ferric state to generate a ternary complex. Although the order of NO and NAD(P)H binding to P450nor is yet unknown, it has been presumed that NO binding precedes NAD(P)H binding because of the low affinity of NAD(P)H (5). The Fe³⁺–NO heme in the complex subsequently reacts with NAD(P)H to form a short-lived intermediate with a ∼100-ms half-life, designated as I (6). The resulting NAD(P)⁺ is proposed to then be released from the protein as the next step (7), and finally, I decays back to the resting state, whose rate depends on the NO concentration, suggesting that the process involves an attack of a second NO to produce N₂O. Thus, it is realized that I is a key state for N–N bond formation and N–O bond cleavage in the P450nor enzymatic reaction. Hence, the coordination and electronic structures of I in this reaction have been a central subject of the NO reduction mechanism in the last few decades.Because the Soret absorption peak of I is significantly different from those of the Fe³⁺–NO and Fe²⁺–NO complexes of P450nor, we proposed its electronic structure as a two-electron reduced form of the Fe³⁺–NO species, described as {Fe–NO}⁸ in the Enemark–Feltham notation, resulting from the direct hydride (H⁻) transfer from NAD(P)H (6). After our proposal, spectroscopic and theoretical studies have been conducted for the P450nor enzyme and model systems, and some possibilities for the detailed electronic structure of I have been proposed. A time-resolved (TR) resonance Raman study revealed that the N–O bond of the first NO is not cleaved in I and proposed transient hyponitrite (HON = NO⁻) formation by an attack of the second NO on the N atom of I (8). The pulse radiolysis experiment suggested that I could be a Fe³⁺–NHOH^• species (or an equilibrium between Fe³⁺–NHOH^• and Fe⁴⁺–NHOH⁻), because the generation of a hydroxylamine radical (NHOH^•) in the presence of ferric P450nor gave a Soret peak similar to that of I (5). Theoretically, two pathways for hyponitrite formation were first calculated via Fe²⁺–NHO or Fe⁴⁺–NHOH⁻, the latter of which was proposed to be I (9). By contrast, recent quantum mechanics/molecular mechanics (QM/MM) calculations combined with magnetic circular dichroism and Mössbauer spectroscopies suggested that I is more likely to be either Fe³⁺–NHO^•− or Fe³⁺–NHOH^• (10, 11). In parallel, model compound studies have also progressed. For example, the possibility of Fe³⁺–NHOH^• was supported by a study synthesizing Fe³⁺–NHOMe^• (12), whereas another study suggested the possibility of Fe²⁺–NHO by analyzing the reaction of ferric nitrosyl heme complexes with a H⁻ reagent (13, 14). As summarized, despite extensive efforts over the decades by multiple approaches, the electronic structure of I remains elusive, in that possible models (i.e., Fe²⁺–NHO, Fe³⁺–NHO^•−, Fe³⁺–NHOH^•, or Fe⁴⁺–NHOH⁻ state) have been proposed. To establish the NO reduction mechanism of P450nor, there is a need for conclusive experimental evidence for the coordination and electronic structures of the Fe–NO moiety of I. To address this issue, we have been trying to directly observe the intermediates in the P450nor reaction using the TR techniques.Most recently, we successfully characterized the coordination and electronic structures of the Fe³⁺–NO complex of P450nor with TR, serial femtosecond crystallography (TR-SFX) using an X-ray free electron laser (XFEL) (15). In TR-SFX, caged NO (16) was a useful tool to supply the substrate NO, as it can generate NO molecules quantitatively in the microsecond time domain upon ultraviolet (UV) pulse illumination, and it was possible to track the NO reduction reaction in a TR manner with a light trigger. Additionally, we also developed a new measurement system for TR-infrared (IR) spectroscopy. While conventional Fourier transform IR (FTIR) requires the enzyme sample in the form of a thin layer (<10 μm) of concentrated solution (>millimolar) to suppress the interference by bulk water absorption (17), our TR-IR measurements use a femtosecond laser-based TR-IR spectrometer (18, 19) with an improved design for microspectroscopy, allowing the use of our “microflow flash” system with a thick flow channel (≥15 µm) and relatively low-enzyme concentration (0.4 mM). The details of the TR-IR spectrometer are described in SI Appendix, Fig. S1. In this study, these two TR experimental techniques were applied to elucidate the electronic and coordination structures of the short-lived intermediate I in the P450nor enzymatic reaction, which would provide direct experimental evidence to establish the molecular mechanism.