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Background: Extracellular vesicles (EVs) are thought to exert protective effects after ischemic and remote ischemic preconditioning. It is not well understood which EV content factors are most relevant for protective effects. We hypothesize that ischemic preconditioning leads to qualitative changes in EV mRNA content and quantitative changes in EV size and number. Methods: Using an in vivo porcine ischemic preconditioning model, EVs were collected from coronary venous blood, and isolated by differential ultracentrifugations. The presence and purity of EV were verified by electron microscopy and Western blot, and EV number was assessed by nanoparticle tracking analysis. The mRNA EV was identified by microarray. Results: Gene ontology analysis showed enrichment of EV mRNA coding for proteins associated with regulation of transcription, translation, extracellular matrix, morphogenic development and feeding behavior. There were 11,678 different mRNA transcripts detected in EV, where a total of 1103 was significantly increased or decreased after preconditioning, of which 638 mRNA sequences were up-regulated and/or emerged due to preconditioning. Several of them have known association with ischemic preconditioning. There was no significant difference in EV quantity or size before and after preconditioning. Conclusions: These findings demonstrate in an in vivo model that myocardial ischemic preconditioning influences the composition of mRNA in EV, including gene transcripts for proteins associated with the protective effect of ischemic preconditioning. The finding that preconditioned parental cells release EV containing mRNA that is qualitatively different from those released by non-preconditioned cells shows the importance of the external milieu on parental cell EV production.

Background Prostasomes are extracellular vesicles. Intracellularly they are enclosed by another larger vesicle, a so called “storage vesicle” equivalent to a multivesicular body of late endosomal origin. Prostasomes in their extracellular context are thought to play a crucial role in fertilization. Methods Prostasomes were purified according to a well worked-out schedule from seminal plasmas obtained from human, canine, equine and bovine species. The various prostasomes were subjected to SDS-PAGE separation and protein banding patterns were compared. To gain knowledge of the prostasomal protein systems pertaining to prostasomes of four different species proteins were analyzed using a proteomic approach. An in vitro assay was employed to demonstrate ATP formation by prostasomes of different species. Results The SDS-PAGE banding pattern of prostasomes from the four species revealed a richly faceted picture with most protein bands within the molecular weight range of 10–150 kDa. Some protein bands seemed to be concordant among species although differently expressed and the number of protein bands of dog prostasomes seemed to be distinctly fewer. Special emphasis was put on proteins involved in energy metabolic turnover. Prostasomes from all four species were able to form extracellular adenosine triphosphate (ATP). ATP formation was balanced by ATPase activity linked to the four types of prostasomes. Conclusion These potencies of a possession of functional ATP-forming enzymes by different prostasome types should be regarded against the knowledge of ATP having a profound effect on cell responses and now explicitly on the success of the sperm cell to fertilize the ovum. General significance This study unravels energy metabolic relationships of prostasomes from four different species.

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.

During ischaemia, ATP depletion leads to insufficient fuelling for Na+/K+ ATPase, decreased electrochemical potential and increased influx of calcium ions. This study demonstrated a means to assess ...

Human prostasomes, exosome-like microvesicles secreted by acinar cells of the prostate gland, contain chromosomal DNA. Agarose gel electrophoresis of DNA from seminal prostasomes displayed fragments of over 12 kb and smaller, with a distinct band around 1 kb that was excised, cloned, and sequenced. The sequences showed 8 out of 25 clones (32%) originating from genes. We elaborated the concept further by carrying out a genome-wide DNA copy number analysis of prostasomal DNA, hypothesizing that human prostasomes contain fragments of DNA randomly selected from the entire genome. Acridine orange-stained prostasomes were incubated with freshly prepared sperm for different times, and a transfer of acridine orange-stained prostasomal DNA to sperm (preferentially the head region) was observed. Fluorescence microscopy of slices in the center of 14 optical slides of the sperm head displayed an even fluorescence rather than a halo-like one, indicating DNA-uptake rather than just binding along the sperm head membrane.

Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (≤6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.

Background Prostasomes are suggested to be produced in the prostate gland. Although biochemical studies support this, some immunohistochemical findings indicate that also the seminal vesicles could be a source of prostasomes. Therefore, we have compared the secretion of the vesicles with that of the prostate using biochemical and ultrastructural techniques. Methods Ultracentrifuged pellets of substance from seminal vesicle secretions were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and flow cytometry. The secretory cells of the seminal vesicles were examined with transmission electron microscopy. These findings were then compared with published results from similar studies of the prostate secretory cells. Results In SDS-PAGE, the seminal vesicle pellets lacked the three prostasome-characteristic CD-markers, namely CD10, CD13, and CD26, but expressed two proteins of about 55 kDa and 70 kDa, corresponding to clusterin and heat shock protein (HSP70). Flow cytometry showed the presence of secretion particles in the seminal pellet, although of a smaller size than that of the prostasomes. Electron microscopy of the luminal part of the cells in the seminal vesicles demonstrated many secretion granules, each enclosed in a vesicle with a size of about 1 μm. Conclusions Pelleted seminal vesicle secretion is different to prostate secretion in several ways. No prostasome characteristics were detected in the pelleted seminal vesicle secretion.

Background: The secretory epithelial cells of the prostate gland use sophisticated vehicles named prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. Aim: The aim of this thesis was to examine different characteristics of prostasomes, especially those derived from malignant prostate cells, linked to their potential role in diagnosis and prognostication of prostate cancer. Results: Serum samples of prostate cancer patients contained autoantibodies against seminal prostasomes in a higher concentration than did control sera. These autoantibodies were most frequently directed against 25 prostasome-associated proteins, but no one was prostate specific. Clusterin was one of the most frequently occurring prostasomal proteins. Elevated titers were however seen in both patients´ and control sera. Clusterin turned out to be a major antigen of seminal prostasomes. No prostate specific or prostate cancer specific protein was discovered upon proteomic analysis of prostasomes deriving from malignant cells of vertebral metastases of prostate cancer patients. Human chromosomal DNA was identified in both seminal prostasomes and PC-3 cell prostasomes and strong evidence existed that the DNA was localized inside the prostasomes. Four out of 13 DNA clones of seminal prostasomes featured gene sequences (31%). The corresponding figures for PC-3 cell prostasomes were 4 out of 16 clones (25%). Conclusions: Prostasomes are immunogenic and give rise to serum autoantibodies. The most frequently occurring autoantibodies were directed against 25 prostasomal proteins but none of these was exclusively prostate specific. Thirty different proteins were identified in prostate cancer metastasis-derived prostasomes but none of these proteins was prostate cancer specific. Human chromosomal DNA was identified in prostasomes of both normal and malignant cell origin.

Few studies have examined energy metabolism during prolonged, strenuous exercise. We wanted therefore to investigate energy metabolic consequences of a prolonged period of continuous strenuous work with very high energy expenditure. Twelve endurance-trained athletes (6 males and 6 females) were recruited. They performed a 7-h bike race on high work-load intensity. Physiological, biochemical, endocrinological, and anthropometric muscular compartment variables were monitored before, during, and after the race. The energy expenditure was high, being 5557 kcal. Work-load intensity (% of VO(2) peak) was higher in females (77.7%) than in men (69.9%). Muscular glycogen utilization was pronounced, especially in type I fibres (90%). Additionally, muscular triglyceride lipolysis was considerably accelerated. Plasma glucose levels were increased concomitantly with an unchanged serum insulin concentration which might reflect an insulin resistance state in addition to proteolytic glyconeogenesis. Increased reactive oxygen species (malondialdehyde (MDA)) were additional signs of metabolic stress. MDA levels correlated with glycogen utilization rate. A relative deficiency of energy substrate on a cellular level was indicated by increased intracellular water of the leg muscle concomitantly with increased extracellular levels of the osmoregulatory amino acid taurine. A kindred nature of a presumed insulin-resistant state with less intracellular availability of glucose for erythrocytes was also indicated by the findings of decreased MCV together with increased MCHC (haemoconcentration) after the race. This strenuous energy-demanding work created a metabolic stress-like condition including signs of insulin resistance and deteriorated intracellular glucose availability leading to compromised fuelling of ion pumps, culminating in a disturbed cellular osmoregulation indicated by taurine efflux and cellular swelling.

Methylene blue (MB), generic name methylthioninium (C(16)H(18)ClN(3) S . 3H(2)O), is a blue dye synthesized in 1876 by Heinrich Caro for use as a textile dye and used in the laboratory and clinically since the 1890s, with well-known toxicity and pharmacokinetics. It has experimentally proven neuroprotective and cardioprotective effects in a porcine model of global ischemia-reperfusion in experimental cardiac arrest. This effect has been attributed to MB's blocking effect on nitric oxide synthase and guanylyl cyclase, the latter blocking the synthesis of the second messenger of nitric oxide. The physiological effects during reperfusion include stabilization of the systemic circulation without significantly increased total peripheral resistance, moderately increased cerebral cortical blood flow, a decrease of lipid peroxidation and inflammation, and less anoxic tissue injury in the brain and the heart. The last two effects are recorded as less increase in plasma concentrations of astroglial protein S-100beta, as well as troponin I and creatine kinase isoenzyme MB, respectively.

The about 400 million individuals worldwide suffering from a hereditary deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD) may experience different degrees of haemolytic anaemia. Haemoglobin is present in very high concentrations in the erythrocyte cytoplasm, at risk of falling out of solution if the internal environment or the haemoglobin itself is changed. G6PD is a crucial enzyme producing reduced glutathione in the erythrocyte cytoplasm for the purpose of protecting haemoglobin against oxidative damage. The presence of unopposed oxidizing agents leading to oxidation of the sulfhydryl bridges between parts of the haemoglobin molecule decrease the solubility of haemoglobin, leading to precipitations called Heinz bodies. The laboratory investigation of G6PD deficiency is commonly done by a quantitative spectrophotometric analysis or by a rapid fluorescent spot test detecting the generation of NADPH from NADP. Genetic tests based on polymerase chain reaction detect specific mutations and may be used for population screening, family studies, or prenatal diagnosis.

Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process.Keywords: extracellular ATP; prostate cancer; extracellular vesicles; exosomes; prostasomes; ATPase; glycolysis; energy metabolism; exosome uptake(Published: 7 March 2016)Citation: Journal of Extracellular Vesicles 2016, 5: 29877 - http://dx.doi.org/10.3402/jev.v5.29877

The prostate is a principal accessory genital gland that is vital for normal fertility. Epithelial cells lining the prostate acini release in a defined fashion (exocytosis) organellar nanosized structures named prostasomes. They are involved in the protection of sperm cells against immune response in the female reproductive tract by modulating the complement system and by inhibiting monocyte and neutrophil phagocytosis and lymphocyte proliferation. The immunomodulatory function most probably involves small non-coding RNAs present in prostasomes. Prostasomes have also been proposed to regulate the timing of sperm cell capacitation and induction of the acrosome reaction, since they are rich in various transferable bioactive molecules (e.g. receptors and enzymes) that promote the fertilising ability of sperm cells. Antigenicity of sperm cells has been well documented and implicated in involuntary immunological infertility of human couples, and antisperm antibodies (ASA) occur in several body fluids. The propensity of sperm cells to carry attached prostasomes suggests that they are a new category of sperm antigens. Circulating human ASA recognise prostasomes, and among 12 identified prostasomal antigens, prolactin- inducible protein (95 %) and clusterin (85 %) were immunodominant at the expense of the other 10 that were sporadically occurring.

To study the metabolic effects in vivo of L-2, 4 diaminobutyric acid (DAB) administered by retrograde microdialysis in glioblastoma and to evaluate the feasibility of the technique.In 10 patients with glioblastoma, a stereotactic biopsy was performed followed by implantation of microdialysis catheters. One or two catheters were implanted in tumor tissue and two reference catheters were implanted in normal brain tissue and subcutaneous abdominal tissue, respectively. Tumor catheters were perfused with 80 or 120 mmol/l DAB and reference catheters were perfused with a Ringer solution, all with a flow rate of 2.0 microl/min. Treatment was given for at mean 9.1 (5-19) days.The treatment was well tolerated by the patients with the exception of two patients in whom a transient brain edema appeared. No complications related to the technique were encountered. During treatment, an increase in the extracellular amino acids alanine, glycine, glutamate, aspartate, serine, threonine, and taurine was found demonstrating a significant influence on the intracellular pool of free amino acids induced by DAB. No change in glucose metabolism or glycerol was evident. The metabolism in normal brain was unaffected during treatment.Retrograde microdialysis is a feasible method for intracerebral administration of drugs to tumor tissue in patients with glioblastoma. We found it possible to deliver DAB to glioblastoma tumors in fully mobilized patients and to assess the metabolic effects induced by the treatment. The changes in extracellular amino acids were in concordance to what was expected from in vitro studies. Elevation of glutamate and taurine may be regarded as markers for an induced cellular toxicity while the unchanged level of glycerol may indicate no direct effects on phospholipase activity and membrane phospholipid composition. The effects were restricted to the tumor compartment. Although an improved survival could possibly be suspected no dramatic effect on outcome could be detected. However, the series was small and, most probably, the time for treatment was too short.

Prostasomes are extracellularly occurring submicron, membrane-surrounded organelles produced by the epithelial cells of the prostate and present in semen. Their precise physiological role is not known, although some of their properties assign them to important physiological and patho-physiological functions. In this thesis, some new properties of seminal and malignant cell line (DU145, PC-3 and LNCaP) prostasomes have been identified. Differences in the expressions and activities of prostasomal CD59, ATPase, protein kinases and tissue factor (TF) have been characterized. The transfer of prostasomal CD59 to CD59-deficient erythrocytes (rabbit and human PNH erythrocytes) has been established. CD59, protein kinases and TF were overexpressed by malignant cell prostasomes. ATPase activity was highest on seminal prostasomes with minimal expression by malignant cell prostasomes resulting in more residual ATP available for phosphorylation reactions. Several proteins were phosphorylated by prostasomal protein kinases, viz. complement component C3, fibrinogen, vitronectin and E-cadherin. Furthermore, TF was identified as the main endogenous phosphorylation substrate on prostasomes. In addition, prothrombotic effects of prostasomes were established. DU145 and PC-3-derived prostasomes exerted a higher clotting effect on whole blood and plasma compared to LNCaP and seminal prostasomes.In conclusion, malignant cell prostasomes showed higher ability to interact with the biological system in favor of prostate cancer cell promotion and survival. The roles played by prostasomes in this context may improve the understanding of the mechanisms that help the prostate cancer cells to avoid the complement attack (CD59 transfer and phosphorylation of C3), to promote angiogenesis (TF) and to metastasize. It may also provide a better understanding of some of the complications usually seen in some terminal prostate cancer patients like thrombotic events and tendency to develop disseminated intravascular coagulation.

Prostasomes are secretory granules synthesized, stored, and secreted by normal and neoplastic human prostate epithelial cells. In prostate cancer, they are anticipated to be released into the blood circulation where they may be immunogenic. The aim of our study was to examine whether prostasome antibody presence in serum bears any prognostic significance for men with prostate cancer. We developed a sensitive and specific immunoassay (enzyme-linked immunosorbent assay) to establish the presence of antiprostasome antibodies in serum. The antiprostasome antibody titer in serum, sampled before any kind of therapy for prostate cancer, was examined together with clinicopathologic variables and outcome over a median follow-up of 350 days in 218 patients with verified prostate cancer. We detected these antibodies in 191 (88%) of these patients. This antibody titer did not correlate to serum values of prostate-specific antigen. Significant, inverse relationships were registered for antiprostasome antibody titer, and metastases to bone and/or lymph nodes (P = 0.035) and pT (P = 0.025). These results indicate that the antiprostasome antibody titer in serum may be a novel marker for prostate cancer liability to metastasize.

Prostate cancer consistently remains a difficult clinical enigma. Therefore, the development of novel strategies for diagnosis and treatment (e.g. immunotherapy) of prostate cancer is essential. We tried to identify the prostasome-derived proteins that were immunogenic in prostate cancer patients. Prostate cancer patients' sera (n = 44) with high enzyme-linked immunosorbent assay (ELISA) titers against prostasomes were selected for immunoblotting against purified seminal prostasomes. The SDS-PAGE and immunoblotting experiments were performed with Bio-Rad systems. Twenty-five of the recognized proteins were isolated and analyzed by means of mass spectrometry. Out of 44 patients' sera, 31 (70%) demonstrated in immunoblotting experiments reactivity against several prostasomal protein bands in the molecular weight range of 10-200 kDa. Some of the bands (55, 70 and 170 kDa) were more frequently recognized by the patients' sera. Concomitantly run control sera generated only very weak or no bands at all. The most frequently occurring prostasomal proteins were identified as heat shock proteins (HSP 70, 71) and clusterin. This study identified the most important molecular targets of autoantibodies against prostasomes generated in connection with the development of prostate cancer in man. These immunogenic prostasomal proteins could be appropriate target molecules for specific immunotherapy of prostate cancer patients.

The exocrine secretion of the acinar gland cells in the human prostate consists of, among other components, a serous secretion and prostasomes. The prostasomes are functionally associated with both reproduction and prostate cancer development and are capable to raise autoantibodies at various pathologies. Therefore, we are trying to characterize prostasome antigens by analysing prostasome-producing cell lines of prostate cancers with the cDNA microarray technique. To obtain one state with synthesis of prostasomes and another state without synthesis, we checked whether the prostasome differentiation was influenced by the mode of growing the cells, that is, whether the cells had been growing on a solid support or on a flexible one. We studied the expression of prostasomes in the cell lines PC3, DU145 and LNCaP. We grew the cells with the following methods: Monocellular layers on microbeads, multicellular spheroids, single cells in suspension cultures, and xenotransplants in nude rats. The presence of prostasomes was examined by ELISA, immunocytochemistry or electron microscopy. The results showed that growing the cells on microbeads (solid support) produced a differentiation of prostasomes, while growing the cells in multicellular spheroids (flexible support) did not. Thus it should be possible to apply cDNAmicroarray analyses for characterizing the genes which are active at the cellular expression of prostasomes and then deduce the prostasome antigens.

The presence of naturally occurring anti-sperm anti- bodies (ASA) is a well-known cause of infertility in men and women, but the antigens for these antibodies are poorly characterized. We have previously shown that prostasomes adhere to sperm cells and that prostasomes are major targets for ASA associated with infertil- ity. These autoantigens have not been characterized. We used 2- dimensional electrophoresis, immunoblotting, and mass-spectrom- etry to identify the prostasome antigens for these autoantibodies. By these techniques, we revealed that prolactin-inducible protein (PIP) and clusterin were dominant prostasome immunogens for sperm- agglutinating autoantibodies of 20 patients with immunological infer- tility. PIP was identified by 19 of 20 (95%) patient sera and clusterin by 17 of 20 (85%). In addition, 10 sporadically occurring prostasomal antigens were identified in this context, viz alcohol dehydrogenase (NADP1), annexin I, annexin III, BRCA1-associated ring domain pro- tein 1, heat shock 27-kd protein, isocitrate dehydrogenase, lactoyl- glutathione lyase, NG,NG-dimethylarginine dimethylaminohydrolase 1, peroxiredoxin 2, and syntenin 1.

Antisperm antibodies (ASA) are present in patients with immunological infertility, but the antigens are poorly characterized. Prostasomes adhere to sperm cells and are recognized as antigens for ASA. This investigation aimed to study the prevalence of antiprostasome antibodies in ASA-classified sera. We studied the reactivity of ASA-positive sera from 116 immunoinfertile patients. Ninety-seven per cent (113 of 116) of the patients' sera contained IgG antibodies against seminal prostasomes. Accordingly, prostasomes are one of the major targets for ASA. An enzyme-linked immunosorbent assay based on prostasomes is simpler to perform than ASA tests presently in use. It is also easier to achieve reproducible and standardized results.

Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.

Prostasomes are submicron secretory granules synthesized, stored and secreted by the epithelial cells of the human prostate gland. They are membrane-surrounded also in their extracellular appearance and the membrane architecture is composite. They are believed to be life-giving and act as protectors of the spermatozoa in the lower and upper female genital tract on their way to the ovum. Hence, the prostasomes are immunosuppressive and inhibitory of complement activation. Further, they promote sperm's forward motility and have antioxidant and antibacterial capacities. The prostasomes with their many composite abilities seem to turn against the host cell after the age of 50 y being conducive to the transition of the normal prostate epithelial cell into a neoplastic cell and therewith lay the foundations of the very high prevalence of prostate cancer of men of more than 50 y of age.

We have characterized a stereospecific transport mechanism for L-dopa into human epidermal Langerhans cells (LCs). It is different from any other amino acid transport system. It is highly concentrative, largely pH-independent, and independent of exogenous Na+, glucose and oxygen, and fuelled by a renewable intracellular energy source inhibited by iodoacetate but not by arsenate. We propose that the mechanism is a unidirectional L-dopa/proton counter-transport system. We have recently demonstrated anaerobic glycolysis in human epidermis, which substantiates the need of proton pumps for resident LCs. The findings prompt a re-evaluation of the profound changes LCs undergo when exposed to oxygen in aerobic culture. L-dopa is not metabolized by LCs but can rapidly be dislocated to the intercellular space by certain extracellular amino acids, i.e. LCs can profit by L-dopa in a dualistic way, altogether a remarkable biological phenomenon.

We have reported that epidermal Langerhans cells possess an H+-extruding mechanism signalling their existence in an anaerobic environment. This study highlights the energy metabolism of human epidermis. In their habitual state the keratinocytes contain more lactate than do most other cell types. Their lactate production in vitro is vigorous and independent of oxygen and most of it is released to the medium. Autoincubation of the epidermis under starved conditions resulted in a 30% increase of lactate, indicating ongoing glycogenolysis. Iodoacetate inhibited lactate production by > 90%. Energy charge values were low, approximately 0.82, and comparable with those previously reported for smooth muscle. Moreover, the overwhelming majority of the keratinocytic mitochondria had an appearance markedly deviating from those in the Langerhans cells, melanocytes and fibroblasts, and, above all, were characterized by an enormous reduction of the inner membrane. This structure is in all probability incompatible with an effective oxidative metabolism of glucose. We conclude that epidermal energy metabolism is predominantly anaerobic in spite of the formal presence of mitochondria. The high production of lactate obviously demands extracellular transport pathways for rapid elimination of this organic acid. An extracellular space complying with such a demand emerges on electron microscopy when an isotonic glutaraldehyde-based fixative is used. The prevailing view regarding the size of the extracellular space is based on the common use of hypotonic fixatives, such as Karnovski's fixative, which causes gross cellular swelling and concomitant near total elimination of the extracellular space, leaving interstices with a diameter significantly smaller than that allowing fluid flow. (Less)

Objectives: The validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism is not known. To address this question interstitial levels of energy-related metabolites (lactate, adenosine, inosine and hypoxanthine) obtained by the microdialysis technique were compared with corresponding metabolites from myocardial biopsies at given intervals in a porcine heart model using different protocols of ischaemia and reperfusion. Methods: In an open chest porcine heart model, interstitial levels of energy-related metabolites were monitored using the microdialysis technique. All animals (n = 23) were subjected to 120-min pretreatment followed by 40 min of regional ischaemia and 120 min of reperfusion. Tissue biopsies were obtained in the beginning, middle and at the end of the 40-min ischaemic period and at the end of the reperfusion period. Pretreatment consisted of either rest (group 1, n = 7), or rest for 90 min and one ischaemia/reperfusion (10 + 20 min) cycle (group 2, n = 9), or four ischaemia/reperfusion cycles (10 + 20 min each) (group 3, n = 7). Results: Interstitial levels of energy-related metabolites monitored by the microdialysis technique correlated with tissue biopsy levels of lactate (r = 0.90, P

Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.

Transfer of prostasomal CD59 to CD59-deficient red blood cells results in protection against complement mediated hemolysis

Exosomes are nanovesicles released from cells through exocytosis and are known to be mediators of proximal as well as distant cell-to-cell signaling. They are surrounded by a classical bilayered membrane with an exceptionally high cholesterol/phospholipid ratio. Exosomes were first described in 1977, then named prostasomes, and in 1987 the name exosome was coined. Exosomes contain surface proteins, some of which can act as labels in order to find their target cells. Exosomes also contain messages in the form of proteins and nucleic acids (RNA and DNA) that are transferable to target cells. Little is known and written about cardiac exosomes, although Gupta and Knowlton described exosomes containing HSP60 in 2007. It is now known that exosomes from cardiomyocytes can transfect other cells and that the metabolic milieu of the parental cell decides the quality of exosomes released such that they induce differential gene expression in transfected cells. Future clinical use of exosomes in diagnosis, monitoring disease progress, and treatment is promising.

Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.

The evaluation of patients with symptoms suggestive of an acute myocardial infarction (AMI) and a nondiagnostic electrocardiogram is a difficult clinical task. During the past decade, test systems for the more sensitive and specific myocardial markers, creatine kinase (CK-MB[mass]), troponin T, and myoglobin have been developed, making it possible to shorten the time needed for detection or exclusion of AMI.1‐ 4 In addition to the evaluation of each marker separately using a predefined decision limit, other marker strategies have been suggested. 4‐ 9 However, most previous studies evaluating these new markers have been hampered by either small sample sizes or inclusion of patients with ST elevation. The aims of the present study were therefore to compare the early diagnostic performance of these new biochemical markers and to evaluate different marker strategies in a large sample of patients with chest pain and a nondiagnostic elec

Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.

CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa

Background Prostasomes are a secretory product from the prostate. We aimed to investigate whether the distribution and amount of prostasomes in normal prostate epithelium were influenced by the dedifferentiation occurring in adenocarcinomas of the human prostate gland. Methods Transurethrally resected material from 11 patients with prostatic carcinoma of various malignancy grades, material from two lymph node metastases, and benign tissue from 10 total prostatectomies were subjected to immunohistochemical staining, using a mouse monoclonal antibody against human prostasomes (mAb78). Results Immunostaining of low-grade carcinoma was similar to that of normal prostate gland which displayed a cytoplasmic granular staining of the apical (luminal) aspects of the secretory epithelial cells. In moderately well and poorly differentiated adenocarcinoma, the amount of stained components decreased, and the staining pattern became more heterogeneous. In multilayered glandular structures, the staining was concentrated at the lumen, leaving most other cells negative. The neoplastic cells of lymph node metastases of prostate carcinoma differed in amount and distribution of immunostained prostasomes. Conclusions The antigen recognized in the prostasomes by mAb78 was expressed in benign prostate tissue, prostate cancer, and to a lesser degree in lymph node metastases. There was a tendency towards decreased expression with increasing tumor grade.

Background A hypothesis suggesting an inducible inability of the enteric bacteria to metabolize urea in infants, resulting in metabolic alkalosis and subsequent respiratory insufficiency, has been proposed as the cause of sudden infant death syndrome (SIDS). Methods Microbiological cultivation and determination of faecal urease activity and faecal urea content were carried out in 30 cases of unexpected infant deaths out of which 22 were considered to be due to SIDS and eight from other causes. The concentration of nitric oxide (NO) in sealed test tubes was determined after incubation of faeces in normal saline. Results The SIDS subjects differed significantly from the control cases in two respects: they had low or no sigmoid faecal urease activity and an unmetabolized sigmoid faecal urea content, whereas the control subjects had normal faecal urease activity and none, or very little, remaining faecal urea. The NO concentration in faeces was correlated with the faecal content of urea in the SIDS cases. Conclusion The present findings lend support to the hypothesis of an insufficient metabolism of enteric urea in infants with SIDS.

BACKGROUND The prostasomes are secreted into the gland ducts of the human prostate. At ejaculation, these native prostasomes are expelled with the secretions of the prostate and appear in the seminal plasma as seminal prostasomes, where they facilitate sperm function in various ways. We have designed methods for producing monoclonal anti-prostasome antibodies to be used for immunohistochemistry and sequencing analyses of the prostasomes. METHODS The immunogen applied was purified seminal prostasomes placed on small pieces of nitrocellulose membranes (prostasome blots) and deposited into the spleen of mice for immunization. For screening, both seminal and native prostasomes were used. RESULTS We obtained antibodies which detected native prostasomes both in prostatic secretions and in paraffin sections of the prostate. The immunostaining demonstrated that all prostate epithelial cells contained prostasomes. They were located in the apical parts of the secretory cells and in the gland ducts, while the nuclei and the corpora amylacea were unstained. CONCLUSIONS Using the methods described, monoclonal antibodies against native prostasomes were produced. In addition to their usefulness in structural and functional studies of prostasomes, specific monoclonal antibodies can be used to characterize prostasomes by sequencing analyses. Prostate 35:178–184, 1998. © 1998 Wiley-Liss, Inc.

Purpose The characteristics of an antigen corresponding to a monoclonal antibody (mAb 78) against human prostasomes were compared with prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP). The correspondence of Ag 78 to two other prostate-derived antigens, prostasin and peptide pGlu-Phe-Pro-NH 2 , were also considered. Materials and Methods The immunoreactivity of mAb 78 and the cross-reactivity of mAb 78 with PSA and PAP were studied with immunohistochemical and enhanced chemiluminescence (ECL) Western blotting methods. Results The mAb 78 did not bind to PSA blots, and anti-PSA antibody did not label prostasome blots. Neither did PSA and PAP impede the binding of mAb 78 onto prostasome blots nor to paraffin sections of prostate epithelium. After Western blots, mAb 78 bound diffusely to a band with a molecular weight of 35 kDa, but did not bind to PSA (33 kDa) or PAP (monomer 50 kDa, intact molecule 100 kDa) bands. From purified 35 kDa bands, three fractions were sequenced, which showed no similarities to PSA and PAP. Conclusions The Ag 78 is different from PSA and PAP, and probably also from prostasin and peptide pGlu-Phe-Pro-NH 2 . The mAb 78 can be used as a new marker for human prostasomes.

The chance of obtaining a fertilization and establishing a pregnancy increases with the number of motile spermatozoa that can reach and interact with the oocyte after the time of insemination. In an attempt to increase the recovery of freeze-thawed and motile spermatozoa, the swim-up medium was supplemented with prostasomes and some other effectors. Swim-up media supplemented with prostasomes were superior in comparison to the other effectors investigated in the recovery of motile spermatozoa for insemination. These results suggest that prostasome inclusion in swim-up medium might be of benefit in improving results in assisted reproductive technologies using freeze-thawed spermatozoa.

Adenine nucleotides, lactate and pyruvate were monitored by microdialysis in pig hearts, comprising four experimental groups. Two preconditioned groups, one beta-blocked by metoprolol (0.3 mg kg-1 body wt; n = 6) and the other (n = 7) without beta-blockade. Two groups were not preconditioned, one beta-blocked (n = 6) and one without beta-blockade (n = 7). Probes were inserted into ischaemic and non-ischaemic myocardium. Preconditioning consisted of four consecutive 10 min periods of ischaemia each separated by 20 min of reperfusion. All animals were subjected to 40 min of index ischaemia followed by 25 min of reperfusion. Myocardial cAMP content was determined in biopsies after the final reperfusion and was found low in the beta-blocked groups. Lactate levels during index ischaemia exceeded the basal level 4-6 fold in dialysate. Adenosine concentration reached 12 mumol L-1 during the first preconditioning period while an attenuation was typical for the following three preconditioning periods. The sum of the concentrations of adenosine inosine and hypoxanthine was significantly lower during index ischaemia in the preconditioned groups displaying a peak value of 115 mumol L-1. The corresponding value for unpreconditioned hearts was 230 mumol L-1. The part of adenosine was 5% and less than 1%, respectively. Pyruvate concentration decreased during each brief ischaemic period of preconditioning rising to a higher level of reperfusion. The decrease in pyruvate was smaller in the controls during index ischaemia. The effects of beta-blockade and preconditioning on ischaemic metabolism were comparable and the results of the two treatments were not additive in this respect.

Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in targets cells. We recently reported that cultured cardiomyocytes are able to release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the transcriptional content of the released exosomes. Exosomes were isolated from media collected from cultured cardiomyocytes (HL-1) with or without growth factor treatment (TGF-β2 and PDGF-BB), with a series of differential centrifugations, including preparative ultracentrifugation and separation with a sucrose gradient. The exosomes were characterized with dynamic light scattering (DLS), electron microscopy (EM) and Western blot and analyzed with Illumina whole genome microarray gene expression. The exosomes were rounded in shape and had an average size of 50–90 nm in diameter with no difference between treatment groups. Analysis of the mRNA content in repeated experiments conclusively revealed 505 transcripts in the control group, 562 in the TGF-β2-treated group and 300 in the PDGF-BB-treated group. Common transcripts (217) were found in all 3 groups. We show that the mode of stimulation of parental cells affects the characteristics of exosomes released. Hence, there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated, or not stimulated, with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system.Keywords: extracellular vesicles; exosomes; cardiomyocytes; growth factors; gene expression(Published: 17 May 2013)Citation: Journal of Extracellular Vesicles 2013, 2: 20167 - http://dx.doi.org/10.3402/jev.v2i0.20167To access the supplementary material to this article, please see Supplementary files under Article Tools online.

BACKGROUND. Prostasomes are prostate-derived organelles that exist extracellularly in human seminal plasma. METHODS. In this study, we have investigated and characterized human prostasomes with regard to their contents of synaptophysin, members of the chromogranin family, and some neuropeptides. RESULTS. By radioimmunoassay measurement and electron microscopy we show the presence of the neuroendocrine markers chromogranin B, neuropeptide Y, and vasoactive intestinal polypeptide in about equimolar amount in human prostasomes and chromogranin A in about 2% of that amount. To our knowledge, such a high ratio of chromogranin B to chromogranin A has never before been observed. The membrane-bound protein synaptophysin, a well-established immunocytochemical marker for neuroendocrine cells and neurones, was also detected. Hence, we show that synaptophysin could be used as a marker for intact prostasomes. CONCLUSIONS. The presence of synaptophysin has recently been shown in the serotonin-containing vesicles in platelets. A protein with a similar structure denoted granulophysin has been found in granulocytes and prostasomes. It is suggested that synaptophysin and granulophysin molecules are members of a family of proteins, maybe expressed in all cells that have regulated release of granule content. Our presented data indicate a neurotransmittor function of the prostasomes. The target cells are however not known but could be either the spermatozoa, the epithelial mucous cells of the uterus or tubas or perhaps the ovum.

Gastrointestinal degradation of urea might, according to a new hypothesis, have consequences for the regulation of acid-base balance as well as control of breathing during infancy. Thirteen infants were investigated from their first few days of life to the age of 6 months by collecting faecal samples at the age of 3 days, 2, 3, and 6 months, respectively. The faecal microflora was determined after aerobic and anaerobic cultivation and the faecal urease activity was assessed after 36 h aerobic and anaerobic preincubation. The infants were mostly breast fed and had a faecal microflora containing anaerobic bacteria such as Bifidobacteria, Bacterioides and Lactobacilli but also aerobics such as Escherichia coli, Enterococci and sometimes Klebsiella. The faecal pH increased from approximately 5.30 to 5.90, the pH after anaerobic preincubation being on an average 0.2 pH units lower than after aerobic preincubation. Simultaneously the nitric oxide production of the faecal specimens increased approximately 10-fold and the urease activity decreased by a factor of 3 to 5. We also found an inhibitory action of nitrate, nitrite (in mumolar concentration) and nitric oxide (in parts per million concentration) on the faecal urease activity. Hence, the present results warrant further research in order to determine more precisely the action of different concentrations of various nitrous oxides on individual bacterial species, and furthermore, to assay the faecal urease activity in victims of sudden infant death syndrome as well as in infants dead due to other causes.

A method is described for the quantitative determination of the cyanogenic glycoside linamarin. A preseparation procedure for urine samples was necessary to remove interfering substances. This was done by solid-phase extraction on a silica column containing cyclohexyl functional groups, which retained linamarin but allowed thiocyanate to pass unrestricted through the column. After elution of linamarin from the column by 35% (v/v) aqueous methanol, the glycoside was quantified following enzymatic hydrolysis, using the specified enzyme linamarase, and the free cyanide thus liberated was estimated spectrophotometrically. This method allowed quantification of linamarin in urine down to 10 mumol/l, with an estimated recovery of 91%. In 75 Tanzanian subjects consuming insufficiently processed cassava, the mean (+/- SD) urinary linamarin concentration was 104 (+/- 104) mumol/l (range 0 - 644 mumol/L), while that for thiocyanate was 486 (+/- 451) mumol/l (range 10-2,940 mumol/l), giving an approximate 1:5 molar concentration ratio between urinary linamarin and thiocyanate.

Background: Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions. Methodology/Principal Findings: Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls. Conclusions/Significance: Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted "cardiosomes", can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes. Ingår i projekt1?Ingår i projektOm publikationen ingår i ett projekt, ange projektets namn. För att ange flera projekt, klicka på Ytterligare projekt.x

We determined the concentrations of adenosine and some of its catabolic products in biopsy specimens from predetermined loci of human myometrium under different functional conditions to compare uterine muscle with rectus abdominis muscle from the same individuals. In order to achieve a good resolution in the separation of nucleosides and purine bases, a preseparation procedure was developed prior to analysis of these compounds on high performance liquid chromatography. Adenosine occurred in a nearly 70-fold higher concentration in smooth uterine muscle in comparison with striated skeletal muscle. Similarly, myometrial inosine and hypoxanthine were 7- and 2.4-times in excess over the rectus muscle, whereas xanthine was scarcely and rather evenly represented in the two types of muscles. The uterine content of adenosine and inosine was distinctly higher in pregnant women compared to non-pregnant ones. A regional difference existed for adenosine, with 3.3 times higher concentration in fundus uteri compared to the isthmic part. A reverse pattern was observed for hypoxanthine and inosine, being 2–3 times more frequent in the isthmic part. The orthophosphate concentration was not stoichiometrically related to the adenosine concentration in a simple way, being 2–3 times lower in uterine muscle compared to the skeletal muscle. A significant correlation existed between uterine contents of AMP and adenosine and similarly, significant inverse correlations were apparent between uterine ATP and ADP contents and energy charge on one hand and adenosine content on the other.

Human spermatozoa are associated with arachidonate 15-lipoxygenase activity. This activity could be due to 15-lipoxygenase in small organelles (prostasomes), which are known to bind hydrophobically to germ cells. This possibility was assessed by separating prostasomes from human spermatozoa by differential centrifugation and purifying them by gel filtration (Sephadex G-200). Purified prostasomes metabolized [1-14C]arachidonic acid to 15(S)-hydroxyeicosatetraenoic acid as determined by reverse phase and by chiral phase HPLC and by gas chromatography-mass spectrometry. Biosynthesis of prostaglandins could not be detected, but the prostasomes contained trace amounts of the four major E prostaglandins of human seminal fluid (3.6 nmol mg-1 of prostasomal protein). Arachidonic acid 15-lipoxygenase has recently been implicated in the acrosome reaction of bull spermatozoa and it may have a similar function in the acrosome reaction of human spermatozoa.

Membrane-bound glyceraldehyde 3-phosphate dehydrogenase activity was measured in erythrocytes from 43 patients with sickle cell anaemia, 24 heterozygous and 27 controls. A significant increase of the activity was found among the patients but no such difference was observed between the heterozygous and the control individuals. The patients showed nearly non Gaussian distribution of the enzyme activity and were subgrouped on the basis of these results, subgroup I had normal values and subgroup II showed markedly increased activities. The patients in subgroup II had significantly lower blood haemoglobin concentrations and significantly higher lactate dehydrogenase activities in serum than subgroup I. The subgroups did not differ in blood reticulocyte counts, serum bilirubin, serum iron concentrations or band 3 protein content of erythrocyte membranes.

Forty-three patients with homozygous sickle cell disease (haemoglobin SS), 24 heterozygous (AS) subjects and 24 controls (AA) from Sudan were investigated by haemolytic activation for complement function via the classical and the alternative pathways, respectively, and by determination of the plasma levels of C3, C4 and factor B as well as of immunoglobulins IgG, IgA and IgM. The study failed to reveal any direct involvement of the complement system in sickle cell disease and nor was any alteration of the immunoglobulin levels registered as a possible cause of increased susceptibility to infections in patients with homozygous sickle cell anaemia.

'Pre-treatment' with short repetitive periods of ischaemia (ischaemic preconditioning) has proved to be a powerful mechanism for modification of the extent of myocardial damage following acute coronary artery occlusion. The exact mechanism of protection induced by ischaemic preconditioning is not known. We herewith put forward a contributing component for protection with preconditioning involving a shift in the adenylate kinase (AK) equilibrium reaction in favour of adenosine triphosphate (ATP) formation.A coronary artery was occluded in anaesthetized thoracotomized pigs to induce ischaemic preconditioning as well as a longer period of ischaemia. Microdialysis probes were inserted in ischaemic and control myocardium and were infused with (14)C- adenosine with two different specific activities. (14)C-lactate was identified and measured in the effluent.(14)C-adenosine was taken up by non-preconditioned and preconditioned myocardium during ischaemia. Significantly increased levels of (14)C-lactate were recovered in preconditioned myocardium. (14)C-adenosine with high specific activity resulted in a specific activity of lactate that was 2.7 times higher than that of lactate after administration of (14)C-adenosine with low specific activity. Mass spectrography verified the identity of (14)C-lactate.Preconditioning up-regulates a new metabolic pathway (starting with 5'-nucleotidase and ending up with lactate) resulting in ATP formation in the micromolar range on top of another effect terminating in a useful shift in the AK equilibrium reaction in favour of ATP generation in the millimolar range. Although the up-regulation of the purine nucleoside phosphorylase pathway is clearly demonstrated, its biological relevance remains to be proved.

It is generally believed that a relationship exists between muscle fatigue and intracellular accumulation of lactate. This reasoning is relevant to obstetrical issues. Myocytes in uterus work together during labor, and the contractions need to be strong and synchronized for a child to be delivered. At labor dystocia, the progress of labor becomes slow or arrested after a normal beginning. It has been described that, during labor dystocia, when the force of the contractions is low, the uterus is under hypoxia, and anaerobic conditions with high levels of lactate in amniotic fluid dominate. The purpose of this study was to examine whether myometrial cells are involved in the production of lactate in amniotic fluid and whether there are differences in production and distribution of lactate in cells incubated under aerobic and anaerobic conditions. We also wanted to elucidate the involvement of specific membrane-bound lactate carriers. Women undergoing elective caesarean section were included. Myometrial biopsies from uteri were collected and subjected to either immunohistochemistry to identify lactate carriers or in vitro experiments to analyze production of lactate. The presence of lactate carriers named monocarboxylate transporters 1 and 4 was verified. Myometrial cells produced lactate extracellularly, and the lactate carriers operated differently under anaerobic and aerobic conditions; while being mainly unidirectional under anaerobic conditions, they became bidirectional under aerobic conditions. Human myometrial cells produced and delivered lactate to the extracellular medium under both anaerobic and aerobic conditions. The delivery was mediated by lactate carriers.