Quadriceps and hamstring muscle activity during cycling as measured with intramuscular electromyography.

PURPOSE: The aim of this study was to describe thigh muscle activation during cycling using intramuscular electromyographic recordings of eight thigh muscles, including the biceps femoris short head (BFS) and the vastus intermedius (Vint). METHODS: Nine experienced cyclists performed an incremental test (start at 170 W and increased by 20 W every 2 min) on a bicycle ergometer either for a maximum of 20 min or to fatigue. Intramuscular electromyography (EMG) of eight muscles and kinematic data of the right lower limb were recorded during the last 20 s in the second workload (190 W). EMG data were normalized to the peak activity occurring during this workload. Statistical significance was assumed at p ≤ 0.05. RESULTS: The vastii showed a greater activation during the 1st quadrant compared to other quadrants. The rectus femoris (RF) showed a similar activation, but with two bursts in the 1st and 4th quadrants in three subjects. This behavior may be explained by the bi-articular function during the cycling movement. Both the BFS and Vint were activated longer than, but in synergy with their respective agonistic superficial muscles. CONCLUSION: Intramuscular EMG was used to verify muscle activation during cycling. The activation pattern of deep muscles (Vint and BFS) could, therefore, be described and compared to that of the more superficial muscles. The complex coordination of quadriceps and hamstring muscles during cycling was described in detail.

Prostasomes are mediators of intercellular communication: from basic research to clinical implications.

Prostasomes are nanosized microvesicles secreted by acinar epithelial cells of the prostate gland. Furthermore, they are intracellular microvesicles inside another larger vesicle, a so-called storage vesicle, equivalent to multivesicular bodies of late endosomal origin. Prostasomes are thought to play an important role in intercellular communication by direct interaction primarily between the immobile acinar cells of the prostate gland and the mobile spermatozoa. Prostasomes transfer not only membrane components but also genetic material to spermatozoa. They are rich in various transferable bioactive molecules (e.g., receptors and enzymes) that promote the fertilizing ability of spermatozoa. In this review, the pleiotropic biological effects of prostasomes that are relevant for successful fertilization will be discussed. The ability to synthesize and export prostasomes to the extracellular space is observed not only in normal prostate epithelial cells but also in malignant prostate cells. Release of prostasomes by prostate cancer cells suggests a role in malignant cell growth and proliferation. These findings may provide new therapeutic and diagnostic strategies.

High levels of cathepsins B, L and S in human seminal plasma and their association with prostasomes.

Semen is a heterogenous and complex fluid with different functions, some of them well known, others still obscure. The aim of this study was to investigate the presence of cathepsins B, S and L in human seminal plasma and their possible associations with other semen variables. Cathepsin B, L and S concentrations were measured in seminal plasma from 99 men utilising commercial ELISA kits. Seminal plasma cathepsin B was approximately 70 times higher, while the cathepsin L values were approximately 500 times higher and the cathepsin S values approximately 40 times higher in seminal plasma than in a group of serum samples. The study shows that seminal plasma contains high levels of cathepsins B, L and S. All three cathepsins were also bound to the surface of prostasomes.

Association of cystatin C with prostasomes in human seminal plasma.

It was recently elucidated that cystatin C, a protein targeted to the classical secretory pathway by its signal peptide sequence, can also be secreted in association with exosomes. Accordingly, we wanted to investigate whether there is a secretory link between cystatin C and prostasomes in human seminal plasma. Cystatin C concentrations in seminal plasma from 50 men including 6 vasectomized men were measured by turbidimetry on an Architect Ci8200. Some of the seminal plasma samples were also analysed utilizing an Epics Profile XL-MCL cytometer. We found high concentrations of cystatin C in seminal plasma. The 2.5-97.5 percentiles, performed by bootstrap estimation, were 25.8 [95% confidence interval (CI): 22.3-29.4] to 77.0 mg/L (95% CI: 71.9-82.1). Cystatin C is present in approximately 50 times higher concentration in seminal plasma compared with blood plasma. There was no clear difference as regards seminal plasma content of cystatin C between vasectomized men and the rest of the group. Immunoblot analysis with chicken anti-cystatin C antibody revealed a firm association of cystatin C with prostasomes. Flow cytometric analysis demonstrated that cystatin C was linked to prostasomes also meaning an at least partial prostasomal membrane surface localization.

Ischaemic pre-conditioning means an increased adenosine metabolism with decreased glycolytic flow in ischaemic pig myocardium.

BACKGROUND: Ischaemic pre-conditioning (IP) is a potent protective mechanism for limiting the myocardial damage due to ischaemia. It is not fully known as to how IP protects. The metabolism of adenosine may be an important mechanistic component. We study the role of adenosine turnover together with glycolytic flow in ischaemic myocardium subjected to IP. METHODS: An acute myocardial ischaemia pig model was used, with microdialysis sampling of some metabolites (lactate, adenosine, glucose, glycerol, taurine) of ischaemic myocardium. An IP group was compared with a control group before and during a prolonged ischaemia. ¹4C-labelled adenosine and glucose were infused through microdialysis probes, and lactate, ¹4C-labelled lactate, glucose, taurine and glycerol were analysed in the effluent. The glycogen content in myocardial biopsies was determined. RESULTS: The ¹4C-adenosine metabolism was higher as there was a higher production of ¹4C-lactate in IP animals compared with the controls. The glycolytic flow, measured as myocardial lactate formation, was retarded during prolonged ischaemia in IP animals. Myocardial free glucose and glycogen content decreased during the prolonged ischaemia in both groups, with higher free glucose in the IP group. We confirmed the protective effects of IP with lower myocardial concentrations of markers for cellular damage (glycerol). CONCLUSIONS: This association between increased adenosine turnover and decreased glycolytic flow during prolonged ischaemia in response to IP can possibly be explained by the competitive effect for the metabolites from both glucose and adenosine metabolism for entering glycolysis. We conclude that this study provides support for an energy-metabolic explanation for the protective mechanisms of IP.

Metabolic responses in ischemic myocardium after inhalation of carbon monoxide.

BACKGROUND: To clarify the mechanisms of carbon monoxide (CO) tissue-protective effects, we studied energy metabolism in an animal model of acute coronary occlusion and pre-treatment with CO. METHODS: In anesthetized pigs, a coronary snare and microdialysis probes were placed. CO (carboxyhemoglobin 5%) was inhaled for 200 min in test animals, followed by 40 min of coronary occlusion. Microdialysate was analyzed for lactate and glucose, and myocardial tissue samples were analyzed for adenosine tri-phosphate, adenosine di-phosphate, and adenosine mono-phosphate. RESULTS: Lactate during coronary occlusion was approximately half as high in CO pre-treated animals and glucose levels decreased to a much lesser degree during ischemia. Energy charge was no different between groups. CONCLUSIONS: CO in the low-doses tested in this model results in a more favorable energy metabolic condition in that glycolysis is decreased in spite of maintained energy charge. Further work is warranted to clarify the possible mechanistic role of energy metabolism for CO protection.

Cerebrospinal fluid markers of disturbed brain cell metabolism.

Reversibly and irreversibly disturbed brain cell metabolism may be monitored in an indirect way by the analyses of enzymes in the CSF according to the hypothesis of cell swelling induced by energy shortage. Adenylate kinase fulfils the criteria for an ideal CSF marker with the exception that it is not organspecific, which necessitates precautions to avoid influence of AK in erythrocytes and serum. When taking such limitating factors into account, AK determinations may be diagnostically useful in combination with radiological and clinical observations. Besides, it is possible that a combination of AK analyses and clinical signs are useful in the prognostication in individual patients suffering from global cerebral ischemia and cerebral infarction.

A Mg2+- and Ca2+-stimulated adenosine triphosphatase at the outer surface of Ehrlich ascites tumor cells.

A Mg2+- and Ca2+-stimulated adenosine triphosphatase (ATPase) at the outer surface of intact Ehrlich ascites tumor cells is described. A surface-bound adenosine triphosphate (ATP)-splitting activity at a lower rate was also demonstrated in the absence of Ca2+ but with Mg2+, Na+, and K+ present in the isotonic medium. Hence, when part of the Mg2+ was exchanged for Ca2+, a marked increase of the ATP-splitting activity was observed. The stimulatory effect of Ca2+ was seen only if both Na+ and K+ were present in the isotonic incubation medium. Thus, the enzyme activity was Mg2+- and Ca2+-dependent. Ca2+, together with the monovalent cations was inhibitory compared with Mg2+ under similar conditions. The apparent Km for ATP for the Mg2+-stimulated ATPase is 0.05 mM, while that of the Mg2+- and Ca2+-stimulated enzyme is 0.10 mM. The Vmax of the former is 0.8 mu-mole per 100 mg Schneider protein per 30 sec compared with 1.92 mu-moles per 100 mg Schneider protein per 30 sec for the latter. The calculated Km for the Mg2+- and Ca2+-stimulated ATPase after subtraction of the Mg2+-stimulated part is 0.22 mM. Ethacrynic acid and N-ethylmaleimide both inhibited the Mg2+- and Ca2+-stimulated ATPase by about 10 percent, while the ouabain inhibition was 15 percent. Cytochalasin B did not influence the enzyme activity, whereas La3+ had a slight stimulatory effect.

Enzyme activities at the surface of intact Ehrlich tumor cells with albumin in the isotonic assay medium.

Glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase are, together with some other enzymes, present on the surface of intact Ehrlich tumor cells. Aldolase, on the contrary, represents cytoplasmic enzymes not present at all on the external surface, provided 2.5 percent of bovine albumin is included in the isotonic assay medium. A flux of aldolase from the cell interior to the cell exterior could be demonstrated in the absence of albumin. Therefore, any enzymatic activity monitored when keeping the Ehrlich tumor cells in the isotonic assay medium containing 2.5 percent albumin was considered to be primarily related to the outside of the plasma membrane. Of the total glyceraldehyde 3-phosphate dehydrogenase, 0.7 percent was located on the outer surface of the tumor cell, while the corresponding figure for 3-phospoglycerate kinase was 2.7 percent. Eighty percent of this surface-located 3-phosphoglycerate kinase was released into the assay medium during incubation, while the release of glyceraldehyde 3-phosphate dehydrogenase, at the same time, was minimal. A plasma membrane preparation of Ehrlich cells, mainly consisting of vesicles, showed the presence of 3-phosphoglycerate kinase but the absence of glyceraldehyde 3-phosphate dehydrogenase. Because of the vesicular nature of the membrane preparation, it was assumed that only one side of the membrane was exposed during assay. The specific binding properties of the two enzymes to the plasma membrane, as well as possible differences in their intramembranous location, are discussed.

The prostasome: its secretion and function in man.

An intact organelle, the prostasome, is secreted by the acinar epithelial cell of the human prostate gland. The ultrastructural location of the prostasome is within membrane-bound storage vesicles in the epithelial cells. Prostasomes are delivered into the glandular lumen by an exocytotic event, which is preceded by fusion of adjacent membranes belonging to the storage vesicle and the epithelial cell. Alternatively, the storage vesicle can be translocated in toto from the cell interior into the acinar lumen through the plasma membrane. This latter event has been designated diacytosis. Both phenomena seem to occur with approximately equal frequency in the human prostate gland. An ATPase system that is Mg2+ and Ca2+-dependent is firmly linked to the membranes encasing the prostasomes. The ATPase system may be the molecular basis for vectorial transport of calcium into these organelles. Also a protein kinase activity is located in the membranes. An increase in membrane thickness was observed on phosphorylation. The physiologic function of the prostasomes is not known. They may be important for promoting forward motility of spermatozoa.

Restoration of detergent-inactivated adenosine triphosphatase activity of human prostatic fluid with concanavalin A.

A stimulation by concanavalian A OF Mg2+ -and Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) of human prostatic fluid has been observed after the enzyme system had been inactivated by a detergent such as 0.05% deoxycholate. The concanavalin A effect was specific since the positive effect was abolished in the presence o alpha-methyl-D-mannoside. Furthermore, the positive effect of concanavalin A was obtained with a low lectin concentration, equal to the concentration reported for optimal stimulation of other membrane enzymes.

Regional differences in energy charge of the pregnant human uterus regardless of functional status in comparison with the non-pregnant uterus.

The energy status of the cell is mainly dependent on adenine nucleotides and can be expressed as energy charge (EC). EC is known to be kept at narrow limits near 0.90 under normal conditions in most cells. We recently reported remarkably low EC values in the human uterus under apparent steady-state conditions. The present paper is an extension of previous work. It shows that EC varies in different regions of the uterus in that the isthmic part in pregnant women displays a higher EC than the fundus of the uterus. There were no intergroup differences between non-pregnant and term pregnant women, nor between those who were in active normal labour, dysfunctional labour or those who were not in labour at all. On the other hand, EC in uterine muscle of post-menopausal women showed a significantly lower EC value. Human uterus seems to manage its metabolic requirements under different functional conditions in spite of low ATP and EC values. This suggests that ATP occurs in sufficient amounts to pertinent enzyme reactions, especially ATPases, which means Km values adapted for this unusually low ATP concentration.

Low energy charge in human uterine muscle.

Six pregnant and 10 non-pregnant patients undergoing Caesarean section and laparatomy, respectively participated in this study which was aimed at determining the energy status of human uterine muscle and comparing it to that of striated muscle from the same individual. Biopsies were taken from the fundus uteri between the ligamenta rotunda and from the rectus abdominis muscle. A low energy charge of 0.55 in pregnant patients and 0.64 in non-pregnant patients was observed in uterine biopsies, compared to the expected value of 0.90 found in rectus biopsies. The main determinant of this finding was a 4-8 times lower level of ATP in uterine biopsies compared to that in rectus biopsies. The same pattern was apparent for total nucleotide content and creatine phosphate. The ADP and AMP contents were of the same order of magnitude in both tissues. The lactate content in uterine biopsies from pregnant patients was significantly higher than that found in biopsies from rectus muscle and from uterine tissue of non-pregnant patients, indicating an increased glycogenolysis in pregnant uterus. The low energy charge and low level of phospho-compounds observed in uterine muscle, regardless of the functional state, are new and unexpected findings.

Human prostasome membranes exhibit very high cholesterol/phospholipid ratios yielding high molecular ordering.

Lipid analysis and ESR studies were carried out on prostasomes isolated from human semen. Cholesterol plus phospholipids amounted to approximately 0.80 mumol per mg protein with a striking quantitative domination of cholesterol over the phospholipids, the molar ratios of cholesterol/sphingomyelin/glycerophospholipids being 4:1:1. Saturated and monounsaturated fatty acids were dominating both in the glycerophospholipids and in sphingomyelin. The order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes were very high, viz. 0.75 for 5-doxylstearic acid and 0.30 for 16-doxylstearic acid at 25 degrees C. Slightly lower values were obtained for the spin-labelled fatty acids when they were incorporated into dispersions of extracted prostasome lipids or into synthetic lipid mixtures of similar composition. The highly ordered lipids in the prostasome membrane thus seemed to be minimally perturbed by proteins in the membrane and ESR spectra showed no signs of immobilized lipids.

Analysis of free inositol trisphosphate in heart tissue by FPLC and isotachophoresis.

Recent, increasing interest in inositol tris(1,4,5)phosphate (IP3) turnover and metabolism has led to a need for a fast and quantitative determination of this compound in various tissues. These requirements are fulfilled by separation in different steps (FPLC and Sephadex G-10) culminating in further separation and quantification by isotachophoresis. Isotachophoresis means a migration of ion species of the same sign in an electrical field with all ions moving with the same velocity. The migration takes place when an electrical field is applied to a system of electrolytes of specific design. Detection was carried out by monitoring conductivity changes. The method is highly sensitive and allows measurements of IP3 in the pmolar range. Linearity was demonstrated over a wide range, 50-4500 pmol. The total imprecision was low with a coefficient of variation of 3%. When determining in biological tissue the recovery was estimated to be close to 100%. The mean content of IP3 in 15 rat heart specimens was 44.3 pmol/mg dry weight corresponding to tissue samples in the order of 4 mg wet weight. By noradrenaline stimulation myocardial content of IP3 increased by 50%.

Comparison between strategies using creatine kinase-MB(mass), myoglobin, and troponin T in the early detection or exclusion of acute myocardial infarction in patients with chest pain and a nondiagnostic electrocardiogram.

Different strategies using creatine kinase-MB(mass), myoglobin, and troponin T were compared in 738 patients admitted because of chest pain and an electrocardiogram not diagnostic of acute myocardial infarction. We conclude that a combination of creatine kinase-MB and troponin T during the first 6 hours enables early detection or exclusion of acute myocardial infarction in this population.

Adverse effects on the brain in cardiac operations as assessed by biochemical, psychometric, and radiologic methods.

In order to describe subclinical brain injury in conjunction with cardiac operations 94 patients were prospectively studied with three brain injury assessment methods: CSF analyses 24 hours after bypass, psychometry, and computed tomography of the brain. Adenylate kinase (AK), a marker of ischemic brain cell injury, was measured in cerebrospinal fluid (CSF) and in serum. In 13% of the patients, a considerable increase in CSF-AK was seen, in 46% there was a moderate increase, and in 41% no or trivial increase. Psychometry measured as change between preoperative scores in a test battery (SS3) revealed a moderate decrease in intellectual function after operation. There was a significant inverse correlation between CSF-AK and SS3 (r = -0.46, p less than 0.001, r2 = 0.21, n = 71). Computed tomography (CT) of the brain was performed preoperatively and postoperatively in 54 patients. Two of these had cerebral infarctions visible on the CT, despite an essentially normal postoperative state. There was no correlation between indices of brain injury and patient diagnosis and length of perfusion. It is concluded that subclinical brain injury is often seen after cardiac operations. Most often the injury appears trivial and/or reversible, but in a minority of cases there is evidence that the brain injury is irreversible. Factor analysis favors the view that the microembolism theory might no longer be a valid concept in modern cardiopulmonary bypass (CPB). Instead, circumstances in the operative field seem more likely to be important causative factors. This interpretation calls for new principles in the search for an improved cerebral protection during cardiac operations.

Myocardial uptake of amino acids and other substrates in relation to myocardial oxygen consumption four hours after cardiac operations.

Myocardial metabolism seems to be markedly abnormal during the first hours of reperfusion after aortic crossclamping. Thus we previously demonstrated no uptake of carbohydrate or lipid substrates 1 hour after coronary operations. Amino acids were the only exogenous substrates taken up by the heart. The aim of the present study was to examine if this metabolic abnormality persisted a few hours later. This was done by measuring coronary sinus blood flow and arterial-coronary sinus differences of oxygen, glucose, free fatty acids, glycerol, lactate, beta-OH-butyrate, and amino acids in a similar group of 19 patients 4 to 5 hours after coronary operations. The results demonstrate a change toward normalization of myocardial free fatty acid use, although the threshold for free fatty acid uptake seemed elevated in comparison with that in the normal postabsorptive state. No correlation was found between free fatty acid uptake and myocardial oxygen consumption. Despite elevated arterial levels of glucose, lactate, pyruvate, and beta-OH-butyrate, no uptake was observed. Myocardial amino acid exchange demonstrated a pattern suggestive of postischemic metabolic adaptation. Several amino acids were extracted, glutamate and branched chain amino acids being the quantitatively most important. The uptake of glutamate and branched chain amino acids correlated with myocardial oxygen consumption, which suggests a direct link to myocardial energy metabolism. Myocardial glutamate uptake seemed to be limited by substrate availability.

Naloxone as an adjuvant in chemotherapy of an experimental tumor.

The antitumor effect of DAB (L-2,4 diaminobuturic acid) has been demonstrated in a previous study. Severe side-effects (especially weight loss and severe neurological symptoms) accompanying DAB treatment have raised the theory that DAB, through a direct effect on the hypothalamus, might cause a diabetes insipidus-like condition, which in turn would activate endogenous opiate systems. This study has verified this state of dehydration and hemoconcentration in a group of 41 mice treated daily with 0.5 ml 0,1 M DAB solution IP. A rise in the serum albumin concentration to 28.8 g/l (SD 2.04) was demonstrated, as against 23.7 g/l (SD 2.3) in a control series. Furthermore, to prevent the neurological side-effects, an adjuvant treatment with an opiate antagonist (Nalone; naloxone) was tried in a group of 19 tumor-bearing mice receiving DAB. This group was compared with a group of 19 tumor-bearing mice receiving DAB only. The mortality rate was significantly reduced in the group receiving Nalone together with DAB (2 dead out of 19) compared with the other group (9 dead out of 19). The tumor weight reduction was about the same in the two groups, 40.5% and 46%, respectively. Combined treatment with DAB and Nalone seems to indicate a possible way of reducing the severe side-effects hitherto accompanying DAB alone, making this unique amino acid a potentially useful antitumor agent.

Antitumor activity of L-2,4 diaminobuturic acid against mouse fibrosarcoma cells in vitro and in vivo.

Mouse fibrosarcoma cells were grown in vitro and incubated with L-2,4 diaminobuturic acid, a non-metabolizable amino acid. The tumor cells were irreversibly and totally damaged by incubation with 10 mM DAB for 24 h at 37 degrees C. The cell-destructive effect by DAB was probably due to an osmotic lysis induced by the non-saturated intracellular accumulation of DAB. The harmful effect of DAB could be abolished by concomitant incubation with L-alanine and L-methionine, that compete with DAB for the same transport system, while the D-forms of the same amino acids as well as sarcosine had a weak effect. The fibrosarcoma cells were also transplanted s.c. into mice that were subsequently treated with i.p. injections of an isotonic 0.1 M DAB solution. The neoplastic cells were transplanted into totally 90 animals. The mean tumor weight of 42 treated animals was 1.16 g (+/- 0.77 g) compared with the corresponding figures of the 27 untreated mice, that were 2.05 g (+/- 1.22 g), i.e., a 43.4% reduction of tumor growth. There were, however, 17 drug-related deaths. Treatment with DAB generally resulted in weight reduction, at least partly due to loss of appetite, in animals. In addition, neurological symptoms of a specific character could develop among several of the treated animals. The side effects apparently restrict the usefulness of DAB alone as an anti-tumor agent, but since the principle of action of DAB is unique and not shared by other known chemotherapeutics it might offer new possibilities in the combined treatment of neoplastic growth.