Genetically modified Lactococcus lactis producing a green fluorescent protein-bovine lactoferrin fusion protein suppresses proinflammatory cytokine expression in lipopolysaccharide-stimulated RAW 264.7 cells.

Lactoferrin (LF), an iron-binding glycoprotein distributed widely in the biological fluids of mammals, is believed to play an important role in host defenses against infection. Previous studies in animal models and humans demonstrated that combined administration of LF and probiotic lactic acid bacteria (LAB) can prevent sepsis. In this study, we genetically engineered a probiotic LAB strain, Lactococcus lactis, to produce recombinant bovine LF based on the green fluorescent protein (GFP)-fused expression system. Western blotting confirmed that the genetically modified L. lactis strain (designated NZ-GFP-bLF) produced a protein corresponding to a fusion of GFP and bLF in the presence of nisin, an inducer of target gene expression. The protein synthesized by NZ-GFP-bLF was fluorescent and thus we monitored the time-dependent change in the production level of the recombinant protein using fluorometric analysis. The utility of NZ-GFP-bLF in preventing sepsis was determined by investigating its anti-inflammatory property in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Pretreatment of RAW 264.7 cells with NZ-GFP-bLF significantly attenuated the LPS-induced mRNA expression and protein production of 3 proinflammatory cytokines (IL-1α, IL-6, and tumor necrosis factor-α) compared with pretreatment with a vector control strain of L. lactis. Our results suggest that NZ-GFP-bLF holds promise for the development of a new prophylaxis for sepsis.

The effects of halothane and isoflurane on the phosphoenergetic state of the liver during hemorrhagic shock in rats: an in vivo 31P nuclear magnetic resonance spectroscopic study.

We studied the effects of halothane versus isoflurane on the phosphoenergetic state and intracellular pH (pHi) of the rat liver using in vivo 31P nuclear magnetic resonance (NMR) spectroscopy during and after hemorrhagic shock. Seventeen rats were anesthetized with 1 minimum alveolar anesthetic concentration of halothane or isoflurane. The mean arterial blood pressure was reduced to 40 mm Hg and maintained at this level for 45 min by withdrawing blood from the common carotid artery. The shed blood was then returned slowly. In vivo 31P NMR spectra were consecutively collected throughout the study. The phosphoenergetic state of the liver was evaluated from the changes in adenosine triphosphate (ATP) and inorganic phosphate (P(i)) levels. pHi was calculated from the chemical shifts of P(i) and alpha-ATP peaks. During hemorrhagic shock, beta-ATP decreased to 35% and 45%, and P(i) increased to 300% and 230% of their initial values in the halothane and isoflurane groups, respectively. Intracellular acidosis was more severe in the halothane group. The recoveries of beta-ATP and P(i) were better in the isoflurane group. Halothane showed a more detrimental effect than isoflurane on the hepatic phosphoenergetic level during and after hemorrhagic shock.

Relationship between gluconeogenesis and phosphoenergetics in rat liver assessed by in vivo 13C and 31P NMR spectroscopy.

The relationship between the phosphoenergetic state and gluconeogenesis in the liver after ischemic damage was investigated using living rats. The ATP level was determined with in vivo 31P nuclear magnetic resonance spectroscopy, and gluconeogenesis was evaluated with in vivo 31C NMR spectroscopy using L-[3-13C]alanine as a tracer. These two measurements were alternated repeatedly. The rats were divided into three groups: without ischemia (group A); with 10 min ischemia (group B); and with 30 min ischemia (group C). ATP was depleted to 20% of the preischemic state after 10 min ischemia and this level was maintained during 30 min ischemia. After reperfusion, the ATP level was partially restored, but the recovery was smaller in group C. Infusion of [3-13C]alanine was started immediately after the reperfusion. In vivo 13C NMR disclosed changes in the alanine C3, glutamine/glutamate C2 and C3, glucose C1-6, and glycogen C1 signals in the liver. After 60 min infusion of [3-13C]alanine, the ATP level correlated negatively with the signal intensity of alanine (r = -0.664, p = 0.008) and positively with those of glucose and glyogen (r = 0.586, p = 0.023, and r = 0.643, p = 0.011, respectively). These results suggest that the ATP level participates in gluconeogenesis and glycogenesis in the liver. Such multinuclear in vivo NMR observations might uncover new aspects of the metabolic function of the liver in the in vivo state.

[Anesthetic management of a child with pheochromocytoma using sevoflurane, diltiazem and continuous epidural blockade].

A 13-year-old girl with pheochromocytoma was anesthetized for the resection of the tumor. We selected sevoflurane as an inhalation anesthetic in combination with continuous epidural blockade. We administered diltiazem mainly to control hypertension but severe hypertension during the manipulation of the tumor was difficult to control. However, systemic vascular resistance was maintained at a low level and there was no arrhythmia during surgery. This report suggests that the use of diltiazem provides a stable hemodynamic condition for the removal of pheochromocytoma.

[A reused suction catheter was broken and produced a tracheobronchial foreign body].

We present a rare case involving a broken suction catheter that became lodged in the tracheobronchial tree. An eight-month-old infant was scheduled for hernioplasty. Following intubation with a 4 French (Fr.) endotracheal tube, a 5 Fr. reused suction catheter was applied for suctioning a moderate amount of secretion. This catheter had been resterilized by ethylene oxide gas (EOG). Insertion of the catheter into the tube was not done smoothly, and we could not obtain any secretion. During the extraction of the suction catheter by force, the catheter broke. The distal fragment (20 cm length) seemed to have been lodged in the tracheobronchial tree. Prior to its removal by bronchoscopy, the endotracheal tube was extubated. Fortunately the remaining part of the catheter come out with the tube. Ten cm of the fractured catheter was included in the tube and 5 cm protruded from the tube. The catheter of smaller diameter is easy to be broken even by weaker force. After resterilization by EOG for once, there was no change in length and force at breaking point. Elongation of the broken catheter (85.5%) was less compared with the new sample (155%). At the breaking point, half of the cross section was very smooth and looked as if it had been cut by a razor, while the other half appeared to have been broken by pulling. The break may have started from the crack which had occurred at the insertion or resterilization. Therefore, we should restrict the reuse of small suction catheters, and should always utilize the catheter of the largest size possible.

Dissociation between lactate accumulation and acidosis in middle cerebral artery-occluded rats assessed by 31P and 1H NMR metabolic images under a 2-T magnetic field.

The relationships among tissue edema, lactate accumulation, and intracellular pH in middle cerebral artery (MCA)-occluded rats were investigated with multiecho 1H magnetic resonance imaging and spatially resolved metabolic images constructed by 1H and 31P nuclear magnetic resonance (NMR) chemical shift imaging (CSI). For the effective and sensitive detection of NMR signals from the brain, outer volume suppression (OVS), reduced k-space sampling and proton irradiation were incorporated into the CSI sequences. The consecutive three measurements of calculated T2 image, lactate image, and pH image which were required for 3.75 h were repeated for four cycles of 1-16 h after MCA occlusion. Tissue edema and lactate accumulation in the infarcted region were gradually and consistently increased during the 15-h observation period. In contrast, severe acidosis was already detected on the first pH image (2-4.7 h after MCA occlusion); thereafter, the degree of acidosis became milder and showed no further progression. The dissociation between the time courses of the lactate accumulation and pH decrease was clearly demonstrated by the NMR metabolic images. Acid-base balance in cerebral infarction might be affected not only by lactate production but also by complicated interactions with tissue edema and some other factors.

[The effect of intraocular lens (IOL) length and design on anterior chamber depth under contraction and relaxation of the ciliary muscle].

We compared the effect of intraocular lens (IOL) length and its design on the anterior chamber depth (ACD) change during contraction and relaxation of the ciliary muscle. We performed phacoemulsification or extra capsular cataract extraction using continuous circular capsulorhexis on 100 eyes, and implanted in 23 eyes a short one-piece IOL, in 22 eyes a short three-piece IOL, in 25 eyes a long one-piece IOL, and in 30 eyes a long three-piece IOL. All IOLs were fixed in the capsular bag. ACD changes after administration of 2% pilocarpine and 1% cyclopentolate were measured with a pachymeter between 3 and 6 months postoperatively. The compatibility of the IOLs with ciliary muscle contraction and relaxation was, in descending order, the short one-piece IOL, the short three-piece IOL, the long one-piece IOL, and the long three-piece IOL. The results indicated that short IOLs are more physiologically fixed in the capsular bag, with less stress on the zonules of Zinn.

[A case of benign epithelioma of the ciliary body].

A 34-year-old woman showed a partially pigmented, greyish-white mass at a position of 1 o'clock behind the iris of her left eye. The lens equator was compressed and focal opacity had developed. The adjacent ciliary processes and zonules were intact. Photocoagulation was not effective and dense cataract gradually developed, so we performed extracapsular cataract extraction and partial iridocyclectomy. The tumor was nonencapsulated, nodular and myxomatous in appearance, arising from the pars plicata. Histological examinations revealed that the tumor was composed of nonpigmented cells and a small number of pigmented cell clusters. The cells were arranged like cords or gland-like structure with intervening myxomatous, fibrillar, and partially hyalinous interstitium. There was no finding of malignancy. The tumor showed similar microscopic structure and findings of mucous stains of normal ciliary nonpigmented epithelium and internal basement membrane, so a diagnosis of benign epithelioma was made.

Effect of electrical stimulation on the rheological properties of rabbit skeletal muscle.

The effect of electrical stimulation on the rheological properties of rabbit skeletal muscle after death was investigated. The extensibility of electrically stimulated psoas muscles decreased more rapidly than that of non-stimulated muscles. For raw non-stimulated longissimus thoracis muscles excised from the carcasses immediately after slaughter, the penetration force required was greatest 24 h after slaughter and then decreased slightly after 168 h. The corresponding force for stimulated longissimus thoracis muscles increased to a maximum in 12 h and then decreased to values less than non-stimulated muscles. However, in the case of raw longissimus thoracis muscles whichhad been attached to the skeleton until measurement, there was no significant difference in penetration force between stimulated and non-stimulated muscles. In cooked muscles, electrical stimulation resulted in lower penetration forces at 24 h post mortem, but on further storage the differences decreased.