The putative role of singlet oxygen ( I O P ) in the respiratory burst of neutrophils has remained elusive due to the lack of reliable means to study its quantitative production. To measure I O P directly from biological or chemical reactions in the near infrared region, we have developed a highly sensitive detection system which employs two InGaAs/InP pin photodiodes incorporated with a dual charge integrating amplifier circuit. Using this detection system, we detected light emission derived from a myeloperoxidase (MPO)-mediated reaction in physiological conditions: pH 7.4, 1^30 nM MPO, 10^100 W WM H P O P and 100^130 mM Cl 3 in place of Br 3 without the use of deuterium oxide. The MPO-H P O P -Cl 3 system exhibited a single emission peak at 1.27 W Wm with a spectral distribution identical to that of delta singlet oxygen. Our results suggest physiological production of I O P in the MPO-H P O P -Cl 3 system at an intravacuolar neutral pH. The MPO-mediated generation of I O P , which may have an important role in host defense mechanisms, is discussed in connection with previous results.z 1999 Federation of European Biochemical Societies.

A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the superoxide (O2-)-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Chronic granulomatous disease (CGD) is characterized by recurrent bacterial infection caused by a defect of the oxidase. Both subunits are absent from phagocytes in typical X-linked recessive CGD patients who are primarily defective in gp91-phox. We report here an atypical case of X-linked CGD in which neutrophils showed a complete absence of O2--forming NADPH oxidase activity, but a small amount (about 10% of control) of both subunits was detected by immunoblot analysis. Spectrophotometric studies of the neutrophils with a recently developed sensitive method gave no evidence for the heme spectrum in the cytochrome b558, of this CGD. Reverse transcription/polymerase chain reaction and sequence analysis revealed a C to T transition replacing histidine at amino acid position 101 (His101) by tyrosine in gp91-phox. These results provide evidence that His101 of gp91-phox is the one of the heme-binding ligands of cytochrome b558.

ABSTRACT. The aim of this study was to examine renal hemodynamics at the hypovolemic and recovery phases in two different hypovolemic shock models using Doppler ultrasonography, and to compare this with systemic hemodynamics. In experiment 1, the hypovolemic phase was induced in 6 mongrel dogs by removing arterial blood at 30 ml/kg for 60 min. In the recovery phase, this blood was reinfused at 30 ml/kg over 60 min. In experiment 2, hypovolemia was induced in 12 beagle dogs by rapid blood removal until blood pressure decreased to 40 mmHg and was maintained at this pressure for 30 min. Six of the dogs were then infused with 20 ml/kg hydroxyethyl starch over 5 min, and the other 6 were infused with 60 ml/kg lactated Ringer's solution also over 5 min. Parameters for systemic and renal hemodynamics were measured by using a polygraph and the Doppler method, respectively. The decrease of diastolic blood flow, resulted in an increase of vessel resistance, and was detected in the hypovolemic kidney by the Doppler method. The rapid and large volume infusion of resuscitation fluids was effective for the recovery of both systemic circulation and renal blood flow, however this induced an increase of kidney vessel resistance, a result of the autoregulation mechanism of the kidney. The changes in these parameters at the main renal artery and interlobar artery were similar. MATERIALS AND METHODSExperimental animals: Six adult mongrel dogs (4 males and 2 females; 2-9 years old), weighing 9-13 kg were used in experiment 1. Twelve adult beagles (7 males and 5 females; 2-7 years old), weighing 7-12 kg were used in experiment 2. These dogs were evaluated as clinically normal.Anesthesia: After induction of the general anesthesia with atropine sulfate (0.03 mg/kg, i.m.), flunitrazepam (0.03 mg/ kg, i.v.) and thiamylal sodium (approximately 15 mg/kg, i.v.), the dogs were endotracheally intubated, and anesthesia was maintained with isoflurane. The breathing movements were arrested using vecronium bromide (0.05 mg/kg, i.v., every 30 min), and were changed to mechanical ventilation with a ventilator (KV-2N, Kimura Ikakiki Co., Tokyo, Japan) at an inspired oxygen concentration (FIO 2 ) of 1.0 and a frequency of 16 breaths/min to achieve an arterial carbon dioxide tension (PaCO 2 ) of 35-45 mmHg. Heparin (500 U/kg, i.v.) was given every 30 min.Hemorrhagic shock model: Experiment 1: Hypovolemia was induced in 6 mongrel dogs during a 60 min period by removing arterial blood at 30 ml/kg/hr into a CPD blood transfusion bag (SC-207, TERUMO Co., Tokyo, Japan). The dogs were then observed over a 60-min period in the hypovolemic state. After that, blood was reinfused from the sterile bags at 30 ml/kg/hr for 60 min. Following reinfusion these dogs were again observed for 60 min.Experiment 2: Hypovolemia was induced in 12 beagles by removing blood until the mean arterial pressure (MAP)The primary causes of acute renal failure can be categorized as ischemic or nephrotoxic [4]. Prerenal azotemia is relatively common, particularly in dogs that have und...

Background: Chronic granulomatous disease (CGD) is an inherited disease characterized clinically by severe recurrent bacterial infections from infancy. This disease is a disorder of the formation of superoxide (02-) b j the neutrophil NADPH oxidase system, mostly due to defects in cytochrome b,,, (cyt b,,,), which is one oj the oxidase components. Diagnosis of CGD has been performed by the assay of the 0,forming activity. immunological determination of defects in the oxidase components, and or spectrophotometry of cyt bS5, However, spectrophotometric analysis of the b-type heme is difficult with small amounts of blood from infant CGD patients, as the limited amounts of neutrophils are contaminated with a relatively high ratio of hemoglobin (Hb) that interferes with the heme spectrum of cyt b,,,. This report presents an accurate method fol the spectrophotometric analysis of cyt b,,, in a small amount of CGD neutrophils that were treated with CO gas in ; I safe procedure instead of the previously reported CO-bubbling method.Methalr and Results: The difference of the reduced minus oxidized cyt bS58 spectrum was measured undet no interference from oxy Hb at the a and bands and differentiated as d[AA]ldh ( h = wavelength) to obtain further evidence for the defects of the cyt b,,, heme spectrum. The interference from CO-insensitive met Ht was eliminated by subtracting the absorption peak at the Soret (y) band of the contaminating met Hb, which was estimated from the CO-treated and untreated spectra of the same, hemolyzed sample. ConclusWns: This spectrophotometric method is feasible for the determination of abnormality and heme content of cyt b,,, with a small amount of CGD neutrophils in 10-20 mL of blood even in the presence of contaminating Hb.Key words chronic granulomatous disease, cytochrome b,,,, oxyhemoglobin, methemoglobin, myeloperoxidase.Phagocyte specific cyt bSSX is an essential component of the membrane-bound NADPH oxidase that generates 0; in response to invading microorganisms.' The important role of the NADPH oxidase has been further emphasized by the demonstration of a major defect in the oxidase of neutrophils from patients with chronic granulomatous disease (CGD) who suffer from recurrent severe infectious diseases.IJ The absence of the heme spectrum of cyt bgSk in neutrophils of patients with X-linked recessive CGD was discovered in 1978., Over the past two decades, further detailed information on cyt b,,, has been obtained by extensive studies on this heme protein of patients with X-linked CGD. Cyt b,,, is a membrane-bound heterodimer of a 91 kDa glycosylated subunit (gp91-phox) and a 22kDa subunit (~22-phox);~,' both of the subunits are lacking in neutrophils of X-linked CGD. In addition to the classic X-linked CGD, patients with autosomal recessive CGD have normal levels of cyt h,,,, but lack cytosolic factors; that is. a 47 kDa protein (+7-phox) and a 67 kDa protein (p67-phox), which are both required for the activation of the NADPH oxidases8 The b type cytochrome is also absent in autosomal CG...