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Muscle precursor cell (myoblasts) transplantation is considered as a potential approach to restore dystrophin expression in Duchenne muscular dystrophy (DMD) patients. The study purpose was to verify the implication of hypoxia in the myoblast death observed after their transplantation and also to evaluate the potential beneficial effects of vascular endothelial growth factor (VEGF) overexpression on myoblast engraftment in a murine model. Pimonidazole hydrochloride (hypoxyprobe-1) was used to mark selectively myoblasts to evaluate their hypoxia in vivo. In vitro, hypoxia was induced by culturing human myoblasts in hypoxic environment. In vitro effects of VEGF(165) on survival of human cells was assessed by Hoescht-PI labeling. Tibialis anterior (TA) female mouse muscles were electroporated with a plasmid containing the VEGF(165) or with an empty vector. Circulating VEGF concentration was assessed by ELISA. After 2 weeks of electroporation, severe combined immunodeficient (SCID) mice were transplanted with 800 000 human male myoblasts labeled with radioactive thymidine. Mouse muscles were harvested 2 and 4 days later and myoblast survival and proliferation were evaluated by scintigraphy and Y chromosome quantitative PCR. The long-term graft success was evaluated using gamma-radiograph imaging and by counting the dystrophin positive muscle fibers. Hypoxyprobe labeling has shown that most of the transplanted myoblasts were hypoxic. The transplantation of radioactive male myoblasts in female mice electroporated with the VEGF(165) plasmid demonstrated that VEGF reduced their death by 10% but did not improve their proliferation. VEGF(165) enhanced human myoblast survival in vitro under hypoxic conditions. Electroporation of TA muscles of SCID mouse with the vector coding for VEGF(165) promoted angiogenesis and improved by 1.5-fold the success of myoblast transplantation in comparison with the control mice that were electroporated with the empty vector. These results indicate that hypoxia is partially responsible for the death of the transplanted myoblasts. VEGF can be used to improve myoblast survival and the graft success.

Current therapeutic options for myocardial infarction include medical therapy, of proven but limited benefit, and various surgical options, which have either restricted applicability or unproven benefit. New cellular-based therapeutic strategies are being developed in response to the shortcomings of available treatments. These include attempts to reinitiate cardiomyocyte proliferation in the adult, conversion of fibroblasts to contractile myocytes and transplantation of myocytes into injured myocardium. The development and current status of these techniques and their relative advantages, problems remaining to be solved and potential clinical applications are reviewed.

Free radical lipid peroxidation contributes to the abnormal metabolism and ventricular function frequently seen after cardiac operations. Antioxidants may improve metabolic and functional recovery.A prospective, randomized, double-blind clinical trial was conducted to determine the effects of vitamin E (alpha-tocopherol) (n = 14) or a corn oil placebo (n = 14) in patients undergoing elective coronary bypass operations. The RRR-alpha-tocopheryl acetate doubled the alpha-tocopherol levels in the heart. Myocardial metabolism and ventricular function were assessed after the operation.Atrial pacing induced myocardial lactate production in the control patients but lactate consumption in the alpha-tocopherol-treated patients on bypass 25 minutes after crossclamp release. Left ventricular stroke work indices were higher, at similar ventricular volumes, in the alpha-tocopherol-treated group, which indicates improved preload recruitable stroke work, and diastolic compliance was greater 4 hours after the operation. The postoperative creatine kinase cardiac isoenzyme levels were lower in the patients who received alpha-tocopherol.Pretreatment with alpha-tocopherol sufficient to double the myocardial concentrations had a small but significant metabolic and functional effect after elective coronary bypass operations when compared with placebo. These results do not justify pretreatment of low-risk patients, but they do justify an evaluation in high-risk patients.

Retrograde delivery of warm blood cardioplegia may improve nutrient cardioplegic flow beyond coronary obstructions, but may not adequately perfuse the right ventricle and the posterior left ventricle. To determine the optimal flow rate for warm retrograde cardioplegia, we assessed 62 patients undergoing elective coronary artery bypass in two studies. In the low flow study, administration of 50 ml/min (n = 9), 75 ml/min (n = 11), or 100 ml/min (n = 7) was associated with high lactate production and oxygen extraction during cardioplegic administration. At 50 minutes of cardioplegic arrest, the coronary venous effluent pH was low in all groups. In the high flow study, 30 patients all received flow rates of 100, 200, and 300 ml/min in randomized order during the crossclamp period. In addition, five patients received cardioplegia at a rate of 500 ml/min for the duration of the crossclamp period. Administration of 200 ml/min or higher minimized lactate production and maintained coronary venous pH within the physiologic range, but flows of 300 ml/min or higher did not increase oxygen use or reduce lactate or acid production. Patients in the low flow groups had significantly greater myocardial lactate release during cardioplegic infusion and after removal of the crossclamp than the high flow group. Warm retrograde cardioplegia should be delivered at flow rates of at least 200 ml/min during elective coronary artery bypass operations.

Warm cardioplegia produced by essentially continuous infusion has been used as an alternative to traditional cold intermittent infusion techniques during cardiac surgery, but its effects on postoperative left ventricular function have not been defined. We performed a randomized clinical trial to assess the effects of warm and cold blood cardioplegia on load-independent indices of ventricular function. Fifty-three patients were randomized to warm (n = 27) or cold (n = 26) cardioplegia. Myocardial oxygen consumption, lactate production, adenine nucleotides, and adenine nucleotide degradation products were measured during cardioplegia and reperfusion. In 13 patients per group, pressure-volume loops were constructed and ventricular function was assessed 3 hours after the operation. Warm cardioplegia resulted in greater myocardial lactate production but improved recovery of oxygen consumption during reperfusion. Depletion of adenosine triphosphate was similar between groups, but total adenine nucleotides (adenosine triphosphate + adenosine diphosphate + adenosine monophosphate) fell further during warm cardioplegia. Cold cardioplegia was associated with an accumulation of adenosine diphosphate and adenosine monophosphate. Creatine kinase MB isoenzyme release was reduced in the warm group. Three hours after the operation, end-systolic elastance and preload-recruitable stroke work index were increased after warm cardioplegia, and early diastolic relaxation was also increased. Increased systolic function after warm cardioplegia may have been related to improved myocardial protection, elevated arterial lactate concentrations, or increased circulating catecholamine levels. Altered diastolic compliance in the warm group may reflect greater active relaxation during early diastolic filling.

Although normothermic cardioplegia has been used with acceptable clinical results, no studies have previously been performed to determine the metabolic consequences of these various techniques of myocardial protection. Therefore, we have performed a randomized clinical trial to assess the effects of three cardioplegic techniques on myocardial metabolic recovery.Seventy-four patients undergoing coronary artery bypass graft surgery were randomized to receive normothermic antegrade blood cardioplegia (n = 25), normothermic retrograde blood cardioplegia (n = 23), or intermittent cold antegrade blood cardioplegia (n = 26). Myocardial oxygen consumption and lactate production, adenine nucleotides, and adenine nucleotide degradation products were measured during the operation, and cardiac creatine kinase isoenzyme (CK-MB) release was assessed after surgery. Warm antegrade cardioplegia maximized myocardial oxygen consumption during cardioplegic delivery. Postoperative CK-MB release was less after warm antegrade cardioplegia, but the difference was not statistically significant. Warm retrograde cardioplegia resulted in the greatest degree of anaerobic lactate production but did not increase morbidity and mortality. Perioperative myocardial infarctions and postoperative low-output syndrome were most common after cold cardioplegia, but this trend was not statistically significant. During warm antegrade cardioplegia, adenosine triphosphate (ATP) was metabolized to diffusible precursors, which were washed out during cardioplegic infusion. Warm retrograde cardioplegia produced a breakdown of ATP to inosine and hypoxanthine, small molecules that accumulated during the cross-clamp period and were not washed out, perhaps because of inadequate perfusion with retrograde delivery. During cold cardioplegia, ATP was dephosphorylated, and adenosine diphosphate, adenosine monophosphate, and adenosine accumulated. These compounds were not regenerated to ATP but were not washed out of myocytes because they are large anionic molecules.Intermittent cold cardioplegia inhibited mitochondrial function but prevented the degradation of adenine nucleotides. Warm antegrade cardioplegia had the greatest myocardial oxygen consumption, and warm retrograde cardioplegia had the greatest anaerobic lactate production. There were no differences in clinical outcomes between cardioplegic groups.

The effects of normothermic systemic perfusion (35 degrees to 37 degrees C; n = 73) were compared with those of moderately hypothermic systemic perfusion (25 degrees to 29 degrees C; n = 73) with respect to blood loss, transfusion requirements, and platelet levels in 146 patients undergoing isolated, primary coronary artery bypass grafting. In addition, most patients were given an antifibrinolytic medication during operation as follows: tranexamic acid (10 gm intravenously; n = 63), epsilon-aminocaproic acid (15 gm intravenously; n = 63), or no drug as a control. (n = 20). Normothermic patients tended to bleed less at 24 hours (warm, 864 +/- 42 ml and cold, 918 +/- 68 ml), but these differences were not statistically significant. Patients receiving either tranexamic acid or epsilon-aminocaproic acid, regardless of perfusion temperature, bled less after 6, 12, and 24 hours than did cold control patients (p less than 0.05). Warm control patients also bled less than did cold control patients after 6 or 12 hours (p less than 0.05), and neither drug further reduced blood loss in these patients. Circulating platelet levels were better preserved in patients receiving either tranexamic acid or epsilon-aminocaproic acid and in patients with warm perfusion and no drug than in cold control patients. Normothermic systemic perfusion, tranexamic acid, and epsilon-aminocaproic acid each reduced postoperative blood loss and preserved platelets.

A prospective randomized controlled trial was performed to determine optimal flow rates and hemoglobin concentrations for continuous normothermic blood cardioplegia and to compare warm heart surgery with standard intermittent cold blood cardioplegia. Thirty-five patients received intermittent cold blood cardioplegia, low hemoglobin low flow, low hemoglobin high flow, high hemoglobin low flow, or high hemoglobin high flow warm blood cardioplegia (seven patients per group: low hemoglobin, 50 g/l; high hemoglobin, 80 g/l; low flow, less than 80 ml/min; high flow, greater than 80 ml/min). Hypothermia resulted in a significantly greater accumulation of ADP and AMP during cross clamp, consistent with impaired mitochondrial function. Low hemoglobin low flow warm blood cardioplegia increased myocardial oxygen consumption and coronary sinus blood flow after cross clamp release, and also decreased lactate consumption. Postoperative myocardial performance and diastolic compliance were reduced in low hemoglobin low flow warm patients, and diastolic compliance was increased with high hemoglobin high flow warm blood cardioplegia when compared with cold patients. In this study, continuous normothermic cardioplegia was safe when delivered at 80 ml/min or greater, with a hemoglobin concentration of at least 80 g/l, affording myocardial metabolic and functional recovery comparable to that found after intermittent cold blood cardioplegia.

— [1-14C]Arachidonic acid was incorporated into brain lipids with a half-life of approx. 5 min. Within 40 min after intra-cerebral injection, radioactivity was distributed mainly among the diacyl-sn-glycero-3-phosphorylcholine (45 per cent), diacyl-sn-glycero-3-phosphorylinositol (22 per cent), diacyl-sn-glycero-3-phosphorylethanolamine (14 per cent) and triacylglycerols (9 per cent). At comparable times, the proportions of radioactivity distributed in diacyl-sn-glycero-3-phosphorylserines and alkenylacyl-sn-glycero-3-phosphorylethanolamines were relatively small. Radioactivity was initially incorporated into the phosphatidio acids and diacylglycerols before labelling of the triacylglycerols and other phosphogly-cerides. The relative specific activity of diacylglycerols was maximum between 3–6 min after injection. Due to the small level of diacyl-sn-3-phosphorylinositol present in brain, its relative specific radioactivity was higher than other types of brain phosphoglycerides. Results of the experiment thus indicate that labelled arachidonic acid is an excellent precursor for metabolic studies with regard to acyl groups present in the 2-position of the phosphoglyceride molecules. Furthermore, this labelled precursor is specially useful in studies related to metabolism of diacyl-sn-glycero-3-phosphorylinositol in brain.

—Age-related changes in acyl group composition of diacyl-glycerophosphorylethanolamine (GPE), alkenylacyl-GPE and diacyl-glycerophosphorylcholine (GPC) were examined in myelin and microsomal fractions of mouse brain during development. In general, the phosphoglycerides in the myelin fraction showed an increase in the proportions of 18:1 and 20:1 and a decrease in the proportions of 16:0, 20:4(n-6) and 22:6(n-3) with increasing age. These changes were especially obvious with the acyl groups of alkenylacyl-GPE. The acyl group profiles of phosphoglycerides in the microsomal fraction were different from those in the myelin fraction. During development, there was an increase in 22:6 and a decrease in 20:4 in the phosphoglycerides of microsomes. These changes were especially obvious with the diacyl-GPE. Starting from 2 weeks of age, there was also an increase in the proportions of 18:1 and 20:1 in alkenylacyl-GPE in the microsomal fraction but this change was not as dramatic as that in the myelin fraction. In general, the acyl groups of diacyl-GPC in both myelin and microsomal fractions showed only little age-related changes as compared to the ethanolamine phosphoglycerides. Results suggest an induction in the synthesis of monoenoic fatty acids in brain during development. The monoenoic fatty acids synthesized during this period are rapidly and preferentially incorporated into the ethanolamine plasmalogen for further utilization in synthesis of the myelin membranes.

The incorporation of [1-14C]-oleic acid into the neutral glycerides and phosphoglycerides of adult mouse brain was examined between 1 and 80 min after intracerebral injection. Radioactivity of the free oleic acid in brain decreased rapidly with a half-life of ca. 5 min. The specific radioactivity of the phosphatidic acids was highest at 1 min after injection. This was followed by the diacylglycerols and triacylglycerols which attained a maximum specific radioactivity at 3 and 20 min after injection, respectively. Specific radioactivities of the neutral glycerides were higher than the phosphoglycerides. A larger proportion of the radioactivity in the diacylglycerols was transferred to the phosphoglycerides than to the triacylglycerols. Among the phosphoglycerides, radioactivity was actively incorporated into the inositol phosphoglycerides. The specific radioactivity of the inositol phosphoglycerides was higher than the diacylsn-glycero-3-phosphorylcholines, and the kinetics of incorporation of radioactivity into these lipids was also different. A water soluble material was found which showed maximum specific radioactivity at 6–10 min after injection. The properties of this water soluble material suggested that it may be an intermediate involved in the acyl group metabolism of phosphoglycerides in brain.

Simultaneous addition of RNA preparations from two picornaviruses to facilitator compound resulted in greatly reduced numbers of plaques as compared to the number produced by similar concentrations of interferee RNA (that whose plaque formation was not blocked in interference experiments). A similar phenomenon was demonstrated with RNA from biochemical mutants of picornaviruses and their corresponding wild-type viruses. A method of biochemical blocking was shown to be as effective as that using antiserum. Infectivity of RNA preparations was markedly reduced by ultraviolet irradiation, heat, and snake venom phosphodiesterase; however, these agents did not significantly affect interfering capacity of the RNA preparations.

the kinetics of peroxidation of fatty acid micelles promoted by ionizing radiation, hydrogen peroxide and ascorbate were compared. At the dose-rate range of ionizing radiation studied, the higher the dose-rate, the greater the total dose required to produce the same effect. With ascorbate, the rate of lipid peroxidation was dependent on the concentration of the promoter only up to 1 X 10(-4) M, beyond which a decreasing rate of peroxidation induction was observed. Higher concentration of ascorbate also suppressed the promoting effect of ionizing radiation. Formate, a hydroxyl radical scavenger, inhibited the peroxidation process promoted by these three agents. Caesium was found to be slightly inhibitory. EDTA and deoxycholate were also inhibitory, which may be attributed to iron-chelating and micelle-disrupting capacity, respectively. Addition of iron (Fe2+ or Fe3+) to EDTA-chelated fatty acid micelles re-initiated the peroxidation process. The ease of fatty acid oxidation at pH 7.5 was arochidonic (20:4) greater than linolenic (18:3) greater than linoleic (18:2). This order was reversed at pH 11.5. Similarities in the kinetics of peroxidation obtained suggest that certain biological sequelae encountered in cells treated with these seemingly dissimilar agents might arise through some common mechanism(s).

Although the nucleus appears to represent the primary target for the killing of cells by ionizing radiation, the cell membrane may be another important site for radiation injury and modification of the radiation response. In this paper, the effects of radiation on various membrane-associated parameters which are expected to affect cellular viability or function are reviewed. These include the effect of radiation on membrane transport, lectin or hormone receptor sites, cell surface electrokinetics, cell membrane fluidity, cell surface topography, etc. Alternative biochemical mechanisms that may account for the effects of radiation on cell membrane in general will also be discussed.

The percentage of uncoating of monkeypox virus (MPV) in CV-1 cells measured by sensitivity of viral DNA to deoxyribonuclease was 24, 46, 53, 48, and 54, at respective 2, 3, 4, 5, and 6 hours post infection. Cores began to appear 1/2 to 1 hour post infection and subsided 3–4 hours post infection to yield naked DNA. The uncoating process was not inhibited by cytosine arabinoside. In cells pretreated with actinomycin-D and infected with MPV in the presence of 2 μg/ml actinomycin-D virion and core peaks were observed but no naked DNA appeared. The time required for a single step growth curve was 12 hours. At 2 hours post infection RNA slightly smaller than 16 S was synthesized. At 4 hours post infection 10 S to 20 S RNA was synthesized; 22 S at 6 to 10 hours. Nuclear RNA synthesis was suppressed.

Monkeypox virus (MPV) replicated efficiently in CV-1 monolayer cells to reach maximal titer in 12 hours. Particles were initially purified in sucrose cushion and then ultimately purified in either sucrose, potassium tartrate or cesium chloride density gradient. Viral cores were also analyzed. The buoyant densities of virion and the cores were 1.28 and 1.32, respectively.

The cellular membrane has been recognized for some time as an important target for radiation injury (Myers, 1970; Alper, 1971). In addition to this, it is also believed to play an important role in repair processes, thus influencing the ultimate survival of the irradiated cells. In an attempt to elucidate these mechanisms further, we have examined the ability of certain “membrane-active” compounds to modify the radiation response of mammalian cells under various experimental conditions. In this report we present the radioprotective effect afforded by a local anaesthetic, procaine hydrochloride (PH), on L5178Y, a murine leukaemic cell line. Asynchronous L5178Y cells were grown in Fischer's Medium plus 10% horse serum. Cultures were harvested by centrifugation and resuspended in serum-free medium to final concentration of 2 × 105 cells/ml. Cells were irradiated at room temperature with a Picker 250 kV X-ray generator at a dose rate of 60 rad/min. An appropriate amount of 1 M PH solution, made up in saline, ...