Effects of ascorbate on leucocytes: Part II. Effects of ascorbic acid and calcium and sodium ascorbate on neutrophil phagocytosis and post-phagocytic metabolic activity.

The effects of ascorbic acid and calcium and sodium ascorbate at a concentration range of 10(-6)M - 10(-1)M on polymorphonuclear leucocyte (PMN) phagocytosis of Candida albicans and post-phagocytic nitroblue tetrazolium (NBT) reduction, hexose monophosphate shunt (HMS) activity and myeloperoxidase-mediated iodination of ingested protein were investigated. Phagocytosis of C. albicans was unaffected by ascorbate concentrations of 10(-6)M - 10(-2)M; however, progressive inhibition was observed at concentrations of 10(-2)M upwards. Enhancement of resting and stimulated HMS activity and NBT reduction was evident at ascorbate concentrations of 10(-5) M - 10(-2)M. The stimulations of HMS activity and NBT reduction was independent of myeloperoxidase iodination of ingested protein and this latter function was strongly inhibited by ascorbate. Concentrations of ascorbic acid and calcium and sodium ascorbate which caused inhibition of phagocytosis and HMS activity were the same as those which mediated stimulation of cell motility, indicating that independent cellular mechanisms may govern motility and phagocytosis.     

Plasma lipolytic activity. Relationship to postheparin lipolytic activity and evidence for metabolic regulation

Lipolytic activity was measured in human plasma without prior administration of intravenous heparin. Eluted from heparin-Sepharose in a barbital buffer containing 6 mg/ml heparin, plasma lipolytic activities in 20 subjects were distributed between hepatic triglyceride lipase (HTGL, mean +/- SE 60.6 +/- 4.6%) and extrahepatic lipoprotein lipase (LPL, 39.4 +/- 4.6%). Confirmation of the identities of HTGL and LPL was provided by inhibitory antisera. Preheparin LPL activity was absent in plasma from a patient with type I hyperlipoproteinemia. Both preheparin HTGL and LPL activities correlated with the respective activities measured in plasma obtained 15 min after intravenous injection of heparin (rs = + .774 and + .685, respectively; n = 12). Evidence for the metabolic regulation of preheparin lipases was provided by measurement of significant increases in LPL and HTGL activities after oral glucose ingestion. Overall, preheparin plasma HTGL and LPL activities may reflect ongoing lipoprotein lipolytic activity in tissue beds, and because these measurements do not require the administration of intravenous heparin, they should prove useful for additional studies of short-term regulation of the lipases.     

Intraoperative metabolic monitoring of the heart: II. Online measurement of myocardial tissue pH.

Under conditions of ischemia, the hydrogen ion [H+] accumulates in the myocardial tissue in proportion to the magnitude of the ischemic insult. The accumulation of [H+] is the result of both increased anaerobic production of [H+] secondary to decreased substrate and decreased washout of [H+] secondary to decreased coronary perfusion. The Khuri tissue pH electrode/monitoring system has been developed and validated over the past two decades. Its scientific basis and correlates have been established, and it is the only system that has been approved for use in humans. Myocardial tissue pH has been monitored in the anterior and posterior walls of the left ventricle in more than 700 patients undergoing major cardiac surgery. An understanding of the relationship between pH and temperature and between the pH and [H+] in tissues is important for the proper interpretation of the myocardial pH data generated in the course of an operation. Intraoperative monitoring of myocardial pH is the only modality available to the cardiac surgeon for online assessment and improvement of the adequacy of myocardial protection. By defining myocardial protection in terms of protection from myocardial tissue acidosis, this technology provides a new tool with which the comparative efficacy of the various myocardial protection techniques can be assessed. It also provides an online tool for assessing the adequacy of coronary revascularization, and has the potential of improving procedures and outcomes for off-pump coronary artery bypass grafting.     

Nucleons, I: A model for studying the mechanism of sperm nucleus swelling in vitro.

The nucleon, a highly organized chromatin structure, was studied to learn if its swelling takes place by the action of heparin/GSH, without the participation of any mechanism provided by sperm membranes, subcellular organelles, or other proteins foreign to the sperm nucleus. Sperm suspensions of guinea pigs and rats were incubated with 9 mM DTT and 1% CTAB. The nucleons obtained from washed epididymal spermatozoa appear under a phase-contrast microscope to preserve their original nucleus shape and to completely lack the acrosome, middle piece, and tail. In an electron microscope, nucleon thin sections show a slight nuclear chromatin decompressed from the periphery toward the center. An outstanding result was that the nucleon swelling pattern by heparin/GSH showed the same classic organization into hub-like nuclear bodies joined by a network of chromatin fibers ranging in thickness from 25 to 1.5 nm. Under the conditions of this study there was no need of any membrane or subcellular structure. At stage IV, all the thick fibers disappear, leaving only thin bead fibers on a string. With respect to nuclear swelling there is no doubt that the sperm chromatin is organized in a special form that decides a specific required pattern of unpacking.     

Pressure-induced ischemia. II. A metabolic study in hamster cheek pouch

The effects of pressure-induced ischemia on energy metabolism, measured as ATP and glucose content, is studied in hamster cheek pouch. Metabolic deterioration during ischaemia is studied and after 2 h the glucose content was significantly reduced but not the ATP content, which was significantly reduced after 4 h of ischemia. Restoration of glucose levels in the cheek pouch tissue was achieved within 30 min of recirculation after release of pressure. The cellular energy metabolism is unable after 4 h of ischemia to restore ATP levels in the tissue during the 120-min of postischemic observation time. There is a difference in ability to resume normal energy metabolism after 2 and 4 h of pressure-induced ischemia.     

Nutrition and somatomedin. II. Serum somatomedin activity and cartilage growth activity in streptozotocin-diabetic rats.

Since diabetes mellitus is a condition in which poor growth occurs despite elevation of plasma GH, we have attempted to determine if poor growth in diabetes, as in malnutrition, could be associated with a decrease in somatomedin activity. Young male rats were rendered diabetic with intravenous streptozotocin (STZ). The growth activity of their cartilage was estimated by 35SO4 incorporation in vitro, and somatomedin (SM) activity in their serum was determined by the stimulation of SO4 incorporation by cartilage from hypophysectomized rats or normal young pigs. Cartilage growth activity was significantly decreased 24 hours after STZ and fell to hypopituitary levels after 48 hours. The decreased growth activity could not be attributed to decreased cartilage responsiveness to SM, since incubation of diabetic cartilage with normal rat serum (normal SM) resulted in significant stimulation of cartilage SO4 incorporation. SM in diabetic serum decreased to hypopituitary levels 24 hours after STZ, and decreased further after 48 hours. The decrease in SM and cartilage growth activity was not prevented by the administration of high doses of bovine GH. The fall in bioassayable SM appeared to be due in part to the presence of an SM inhibitor in the diabetic serum, since addition of diabetic serum to normal serum decreased to measurable SM in the normal serum. Administration of insulin to diabetic rats 48 hours after STZ led to significant increases in SM and cartilage growth activity, and insulin therapy 24 hours after STZ prevented the decreases in SM and cartilage growth activity which occurred without insulin. Thus, acute STZ-induced diabetes in rats was associated with a significant decrease in both serum SM and cartilage growth activity; these changes were not ameliorated by administration of GH, and insulin therapy could both prevent and reverse the fall in SM and cartilage growth activity. From these observations, we conclude that (1) that fall in somatomedin activity and cartilage growth activity associated with STZ-induced diabetes appears to be due to insulin deficiency and (2) growth failure in diabetes, as in malnutrition, may be due to decreased somatomedin activity.     

Murine oocyte cryopreservation: comparison between fertilization success rates of fresh and frozen metaphase I and II oocytes

The surplus of oocytes obtained during in vitro and gamete intrafallopian transfer creates the need to either (1) cryopreserve (196 degrees C) or (2) fertilize these surplus oocytes and then freeze the resulting embryos. During this pilot study, C57BL6 X CBA female mice (F1 generation) were superovulated by means of hormone stimulation, resulting in adequate numbers of murine oocytes. Oocytes thus obtained were classified according to their nuclear status. The maturation stage of oocytes was evaluated and classified as metaphase I and metaphase II. The success of oocyte cryopreservation in each group was evaluated according to fertilization rate after the insemination procedure. Metaphase II oocytes had a fertilization rate of 33% (frozen) and 84% (fresh), whereas metaphase I oocytes had a 24% (frozen) and a 79% (fresh) fertilization rate. Metaphase I oocytes can mature to the metaphase II stage after having been frozen, metaphase II oocytes may be more vulnerable to chromosome damage, and the nuclear stage prior to freezing has no significant effect on the fertilizability of the frozen-thawed oocytes.     

提高冻存胚胎胰岛移植效果的实验研究

目的探讨三维组织细胞旋转培养系统(RCCS)对胚胎胰岛冻存复苏后质量的影响。方法将胚胎胰岛平均分为3组,实验组1、2为胚胎胰岛冻存前后分别用RCCS培养和普通培养,对照组新鲜胰岛经RCCS培养。切取胚胎胰腺,胶原酶V消化,纯化。然后进行标准冻存步骤,复苏后继续培养。并检测各组胰岛数量、活性、胰岛素刺激实验结果。结果纯化后收获胰岛最多每个胚胎5012.73IEQ,最少2432.68IEQ,平均(3548.07±273.46)IEQ。微重力培养组胰岛细胞存活率、胰岛素释放量、胰岛素刺激指数等均高于普通培养组。移植经过微重力培养的(2000±1)%IEQ新鲜胚胎胰岛或冻存胚胎胰岛在移植后1周内可达100%纠正糖尿病。结论微重力旋转培养有利于胰岛细胞的生长繁殖,使胰岛具有更好的胰岛素分泌能力,该方法同胰岛冻存相结合,可以进一步提高胰岛的冻存效果,为胰岛库的成功建立探索出一条新的途径。     

Successful cryopreservation of microvenous allografts.

Vein grafts are used in approximately 20% of microsurgical cases. Although autogenous veins currently form the major source, they are associated with increased operating time and donor site scars. Cryopreserved allograft veins may serve as an alternative source. To our knowledge, cryopreservation of veins (1 mm or less) has not been reported. In this article we have described the process of cryopreserving rat veins (less than 1 mm in diameter) and their preliminary use as interpositional vein grafts.     

Continuous overnight monitoring of bladder activity in vesicoureteral reflux patients: II. Bladder activity types

Continuous overnight monitoring of the bladder activity was undertaken in children with vesicoureteral reflux. The investigation succeeded in 46 of 56 patients and showed bladder activity in all cases. Three types of bladder activity were revealed. One consisting of single contraction episodes exhibited periodicity with intervals of approximately 90 min. The second type consisted of a complex of contractions with increasing amplitude. They frequently lead to voiding. This type of activity seemed to be related to the bladder fullness. The third type of bladder activity consisted of vigorous repetitive contractions, most frequently found in the nonenuretic part of the material. The contractions in type III most frequently occurred with a frequency of 3–6/min. The degree of reflux was related to the type of bladder activity. Thus, the degree of reflex was significantly increased in patients with the second and third type of bladder activity when compared to patients with type I. The study shows that bladder activity takes place outside provocational episodes, e.g., cystometry and micturition, and seems to be closely related to the severity of reflux.     

Functional activity and transmembrane signaling mechanisms after cryopreservation of human internal mammary arteries

Purpose: Cryopreserved human blood vessels are important tools in bypass surgery. However, several in vitro studies have demonstrated diminished postthaw functional activity. Therefore the aim of this study was to investigate the consequences of various freezing/thawing protocols and the role of protein kinase C in the postthaw functional activity of cryopreserved human arteries. Methods: In vitro responses of frozen/thawed human internal mammary arteries (IMA) were used to investigate the functional activity after thawing at 15°, 30°, and 100° C/min and after different prefreezing equilibration times (10, 60, 120, 240 minutes) with the cryomedium (Krebs-Henseleit solution containing 1.8 mol/L dimethyl sulfoxide and 0.1 mol/L sucrose) at room temperature followed by cryostorage at -196° C. Results: Prefreezing equilibration for 10 to 120 minutes diminished maximal α-adrenoceptor-mediated responses to noradrenaline to approximately 60%, and equilibration for 240 minutes attenuated noradrenaline effects to less than 25% of that produced by unfrozen controls. Contractile responses were slightly better when thawing was performed at 15° C/min compared with 100° C/min. The postthaw sensitivity to direct activation of protein kinase C by phorbol 12,13-dibutyrate was enhanced. Compared with unfrozen tissues (pD₂ = 7.36 ± 0.07, n = 32) maximal sensitization to phorbol 12,13-dibutyrate was observed in IMA that had been frozen after 60 minutes of equilibration with the cryomedium (pD₂ = 8.31 ± 0.09, n = 30). Responses to phorbol 12,13-dibutyrate of cryopreserved IMA were highly susceptible to blockade of calcium influx by nifedipine, whereas those of unfrozen IMA were resistant to nifedipine. Against noradrenaline nifedipine was equipotent in cryopreserved (pD'₂ = 7.75 ± 0.15, n = 8) and unfrozen IMA (pD'₂ = 7.70 ± 0.10, n = 6). Endothelium-dependent relaxant responses to acetylcholine were significantly attenuated after cryopreservation (E_max = 26% ± 5%, n = 4) compared with unfrozen IMA (E_max = 71% ± 4%, n = 4, p < 0.001); endothelium-independent relaxant responses to sodium nitroprusside were unchanged. Conclusions: Cryopreservation of human IMA under the conditions applied in this study (1) attenuated endothelial cell function and (2) induced an activation of protein kinase C, thereby increasing calcium influx through dihydropyridine-sensitive calcium channels. These experimental data suggest that postoperative administration of calcium channel blockers alone or combined with long-acting nitrates should effectively prevent the development of spasms in arterial grafts. (J Vasc Surg 1998;27:528-37.)     

不同保存方式对嗅鞘细胞活性的影响

[目的]探索嗅鞘细胞最佳保存方式.[方法]20例对数生长期OECs,随机分为5组,分别加入10%DMSO、5%DMSOfi%HES、5%DMSO,冰箱降温或程控降温仪降温并液氮保存,定期复苏,通过观察细胞形态、MTT 比色法、台盼蓝染色法检测细胞活性.[结果]同种降温方式下,5%DMSO-6%HES组细胞活性优于另两组,差异显著;同种低温保护剂下,冰箱降温与程控降温仪降温并液氮保存相比,差异不显著;同种保存方式下低温保存半年,各组回收率差异不显著.复苏标本不洗涤,室温下放置不同时间,细胞活性均下降,其中10%DMSO组下降最大.[结论]推荐5%DMSO-6%HES作为OECs冻存低温保护剂;小样本保存应选用冰箱降温液氮保存方式,大样本保存可选用程控降温仪降温或冰箱降温,并液氮保存方式;OECs低温保存半年仍具有较好的细胞活性.     

Hormonal and associated metabolic alterations following burn injury: Part II. A comparative evaluation in surviving and non-surviving patients

In surviving burn patients the elevated circulating levels of catecholamines, cortisol and blood sugar start declining from week 1 onwards along with a gradual rise in insulin levels. On the other hand, in non-surviving burn cases these levels remain markedly elevated up to week 2 with a further reduction in insulin. Quite possibly, such a prolonged and marked increase in catecholamines, cortisol and suppression of insulin in non-surviving burn patients might be responsible for higher mortality, because of their known effects on various metabolic processes.     

Osteosarcoma cells in tissue culture: II. Characterization and localization of alkaline phosphatase activity

Although elevated alkaline phosphatase levels in osteosarcoma have been shown to be related to prognosis, the functional significance is unclear. Human osteosarcoma cells in tissue culture retain detectable amounts of alkaline phosphatase activity. In this study the specific activity of that enzyme was compared in 13 osteosarcoma tissue culture lines and 13 normal skin fibroblast lines derived from the same patients. Osteosarcoma cells had significantly higher baseline alkaline phosphatase levels and could be stimulated with hydrocortisone to produce more enzymatic activity than the fibroblast lines. Activity was localized ultracytochemically to the cell membrane and to many small intracellular vesicles in stimulated osteosarcoma cells. These observations aid in the differentiation of osteosarcoma and fibroblast lines in tissue culture and suggest an association between elevated alkaline phosphatase levels and metabolic abnormalities in patients with osteosarcoma.