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Biomedical subjects

G Kamp

Publications and source records attributed to G Kamp.

At least 19 recordsLinked to original sources
PubMed2003

Experimental approaches to study vascularization in tissue engineering and biomaterial applications.

The success of tissue engineering and biomaterial applications is not only dependent on the growth and functioning of the organ- or tissue-specific cells on the biomaterial but is entirely dependent in most cases on a successful vascularization after implantation. The process of vascularization involves angiogenesis; the formation of new blood vessels which spread into the implant material and supply the existing cells with the nutrients to survive. We have established in vitro methods using human microvascular endothelial cells to evaluate novel biomaterials for endothelial cell attachment, cytotoxicity, growth, angiogenesis and the effects on gene regulation. These in vitro studies can be used to rapidly evaluate the potential success of a new biomaterial and for the development of matrix scaffolds which will promote a physiological vascularization response.

Journal Article↗
PubMed2003

Energy metabolism and intracellular pH in boar spermatozoa.

The effect of energy metabolism on intracellular pH was studied in boar spermatozoa using nuclear magnetic resonance (NMR) spectroscopy and confocal microscopy with the pH-sensitive dye seminaphthorhodafluor (SNARF-1). Freshly ejaculated spermatozoa had a high adenylate energy charge (AEC=0.8), which decreased to 0.6 under aerobic conditions and to 0.2 under anaerobic conditions. Correspondingly, no ATP resonances but high AMP resonance were visible in (31)P-NMR-spectra of the spermatozoa. When an artificial oxygen buffer (Fluosol) and a purpose-built air supply system were used during (31)P-NMR data acquisition, ATP resonances reappeared whereas the AMP resonance disappeared. Boar spermatozoa kept under aerobic conditions have intracellular compartments that differ markedly in pH, as demonstrated by both (31)P-NMR spectroscopy and confocal microscopy. Using confocal microscopy, the midpiece of the flagellum in which all mitochondria are located was identified as an acidic compartment (pH(i-mp) 6.7). The intracellular pH of both the head (pH(i-h)) and the long principal piece of the flagellum (pH(i-pp)) were 7.2 and, thus, only slightly below the extracellular pH (pH(e) 7.3). Storage of spermatozoa in a glucose-free medium at 15 degrees C when they are immotile slowly shifted the pH(i-mp) from 6.7 to 6.9 within 20 h, whereas pH(i-h) and pH(i-pp) remained unchanged (pH 7.1-7.2). When glucose was present in the medium, all visible compartments of the spermatozoa as well as the medium were acidified to pH 6.2 within 20 h. Under these conditions a resonance at 4.8 mg kg(-1) appeared representing glycerol 3-phosphate.

Adenosine Diphosphate↗
PubMed2000

Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro.

The effects of the male antifertility agent ornidazole on glycolysis as a prerequisite for fertilization were investigated in rats. Antifertility doses of ornidazole inhibited glycolysis within mature spermatozoa as determined from the lack of glucose utilization, reduced acidosis under anaerobic conditions and reduced glycolytic enzyme activity. As a consequence, cauda epididymidal spermatozoa from ornidazole-fed rats were unable to fertilize rat oocytes in vitro, with or without cumulus cells, which was not due to transfer of an inhibitor in epididymal fluid with the spermatozoa. Under IVF conditions, binding to the zona pellucida was reduced in spermatozoa from ornidazole-fed males and the spermatozoa did not undergo a change in swimming pattern, which was observed in controls. The block to fertilization could be explained by the disruption of glycolysis-dependent events, since reduced binding to the zona pellucida and a lack of kinematic changes were demonstrated by control spermatozoa in glucose-free media in the presence of respiratory substrates. The importance of glycolysis for binding to, and penetration of, the zona pellucida, and hyperactivation in rats is discussed in relation to the glycolytic production of ATP in the principal piece in which local deprivation of energy may explain the reduced force of spermatozoa from ornidazole-fed males.

Analysis of Variance↗
PubMed1997

Phosphorous metabolites in boar spermatozoa. Identification of AMP by multinuclear magnetic resonance.

Boar spermatozoa revealed three prominent resonances in the 31P-NMR spectrum of intact cells. Two of these are known to be GPC and Pi, the third is a phosphomononoester (PME), the identification of which was carried out by proton-detected 2D 1H,31P and 1H,13C chemical shift correlation experiments with gradient selection. The PME was unambiguously assigned to adenosine 5'-monophosphate (AMP). The identification was confirmed by an AMP consuming enzymatic assay. Other physiologically relevant PME's, in particular inosine 5-monophosphate (IMP) and sugar phosphates, were excluded. The intensity of the 31P signal of AMP in boar sperm extract was much higher than those of ADP and ATP, and in intact cells only AMP but no ATP was visible.

Adenosine Monophosphate↗
PubMed1997

Nuclear magnetic resonance studies of boar seminal plasma. Problems encountered in the identification of small molecules: hypotaurine and carnitine.

Two major components in boar seminal plasma were assigned by 1H and 13C nuclear magnetic resonance spectroscopy. The first, previously called substance X (see Ref. 1, Biochim. Biophys. Acta 1243, 101-109 (1995)), was identified with difficulty as hypotaurine. This pointed to general difficulties in the NMR assignments of small molecules in mixtures of substances, even at the highest magnetic fields. In contrast, the identification of the second component as carnitine was obtained in a straightforward manner by total correlation spectroscopy and proton-detected 13C chemical shift correlation methods (gradient-selected heteronuclear single quantum coherence and heteronuclear multiple bond correlation). Carnitine is known as a transporter of fatty acids through membranes. Both compounds were ultimately confirmed by addition of the authentic compounds.

Animals↗
PubMed1997

Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa.

Evidence is provided that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is covalently linked to the fibrous sheath. The fibrous sheath is a typical structure of mammalian spermatozoa surrounding the axoneme in the principal piece of the flagellum. More than 90% of boar sperm glyceraldehyde 3-phosphate dehydrogenase activity is sedimented after cell disintegration by centrifugation. Detergents, different salt concentrations or short term incubation with chymotrypsin do not solubilize the enzyme, whereas digestion with trypsin or elastase does. Short term incubation with trypsin (15 minutes) even resulted in an activation of glyceraldehyde 3-phosphate dehydrogenase. Purification on phenyl-Sepharose yielded a homogeneous glyceraldehyde 3-phosphate dehydrogenase as judged from gel electrophoresis SDS-PAGE and native gradient PAGE. The molecular masses are 41.5 and 238 kDa, respectively, suggesting native glyceraldehyde 3-phosphate dehydrogenase to be a hexamer. Rabbit polyclonal antibodies raised to purified glyceraldehyde 3-phosphate dehydrogenase show a high specificity for mammalian spermatozoal glyceraldehyde 3-phosphate dehydrogenase, while other proteins of boar spermatozoa or the muscle glyceraldehyde 3-phosphate dehydrogenase are not labelled. Immunogold staining performed in a post-embedding procedure reveals the localization of glyceraldehyde 3-phosphate dehydrogenase along the fibrous sheath in spermatozoa of boar, bull, rat, stallion and man. Other structures such as the cell membrane, dense fibres, the axoneme or the mitochondria are free of label. During the process of sperm maturation, most of the cytoplasm of the sperm midpiece is removed as droplets during the passage through the epididymis. The labelling of this cytoplasm, in immature boar spermatozoa and in the droplets, indicates that glyceraldehyde 3-phosphate dehydrogenase is completely removed from the midpiece during sperm maturation in the epididymis. The inverse compartmentation of the glycolytic enzyme and mitochondria in the mammalian sperm flagella suggests that ATP-production in the principal piece mainly occurs by glycolysis and in the midpiece by respiration.

Animals↗
PubMed1996

The Pasteur effect in facultative anaerobic metazoa.

The existence and the regulatory mechanisms of the Pasteur effect in facultative anaerobic metazoa are discussed. There are three reasons for the controversy surrounding this phenomenon. 1) The different definitions of the Pasteur effect, 2) the antagonistic effect of metabolic depression and its species specific response to hypoxia, as well as 3) the laboratory-specific differences in the experimental procedures for analyzing the Pasteur effect and its regulation. This review aims to clarify the confusion about the existence of the Pasteur effect in facultative anaerobic metazoa and to offer possible molecular mechanisms.

Adenosine Triphosphate↗
PubMed1996

Spermatozoa: models for studying regulatory aspects of energy metabolism.

Spermatozoa are highly specialized cells, and they offer advantages for studying several basic aspects of metabolic control such as the role of adenosine triphosphate-(ATP)-homeostasis for cell function, the mechanisms of fatigue and metabolic depression, the metabolic channelling through the cytoplasm and the organization and regulation of glycolytic enzymes. Spermatozoa of four species with different reproductive modes are introduced and the first results are presented: Spermatozoa of the marine worm Arenicola marina are well adapted to external fertilization in sea water with fluctuating oxygen tension: they are motile for several hours in oxygen-free sea water, even when the ATP level is dramatically reduced. Anaerobic ATP production occurs by alanine, acetate and propionate fermentation probably by the same pathways known from somatic cells of this species. Under aerobic conditions the phosphagen system might function like a shuttle for energy-rich phosphate from mitochondria to the dynein-ATPases. Storage of turkey and carp spermatozoa for several hours without exogenous substrates and oxygen results in the degradation of phosphocreatine and ATP to inorganic phosphate and adenosine monophosphate (AMP), respectively. Despite low energy charges, stored spermatozoa of both species are capable of progressive movements. In carp spermatozoa fatigue of motility is not accompanied by the dramatic acidosis one discusses as an important effect in muscle fatigue. Energy metabolism of boar spermatozoa is typically based on glycolysis consuming extracellular carbohydrates and producing lactate and protons. The sperm seem to tolerate low intracellular pH (< 6.5). The lack of a phosphagen system (no energy shuttle from mitochondria to the distal dynein-ATPases) is probably compensated by a high glycolytic ATP-production in the mitochondria-free piece of the flagellum.

Adenosine Triphosphate↗
PubMed1995

Multinuclear magnetic resonance studies of boar seminal plasma.

Multinuclear magnetic resonance studies were performed on aqueous solutions of lyophilisates of boar seminal plasma. 1H- and 13C-NMR spectral assignments were obtained by one- and two-dimensional experiments. Four prominent constituents were identified in the lyophilisate as well as in the original seminal plasma: inositol (95% myo-inositol, 5% scyllo-inositol), citrate, lactate and glycerophosphorylcholine (GPC). The concentrations of these compounds were evaluated from appropriate 1H- and 13C-NMR resonances using biochemically determined citrate as reference. 31P-NMR spectroscopy confirmed the presence of GPC and revealed phosphorylcholine, glycerophosphorylserine and glycerophosphorylethanolamine as further components.

Animals↗
PubMed1992

Partially phosphorylated glycogen phosphorylase in the lugworm Arenicola marina, its regulatory function during hypoxia.

Glycogen phosphorylase (GPase) from the body wall of the lugworm Arenicola marina (Annelida, Polychaeta) probably exists as a phospho-dephospho hybrid (GPase ab). The hybrid was identified by phosphorylation of purified lugworm GPase b (unphosphorylated form) with rabbit muscle GPase kinase and [gamma-32P]ATP. The completeness of phosphorylation was checked on DEAE-Sephacel. Only one GPase form was eluted. Its 32P incorporation was determined to 0.52 +/- 0.08 mol 32P/100,000 x g protein (n = 4). This GPase ab produced by in vitro phosphorylation has shown similar dependences on AMP and caffeine as GPase extracted from the body wall of the lugworm. Its reversible conversion with endogenous phosphatase and kinase to GPase b has also been demonstrated while a completely phosphorylated form (GPase a) was not detected neither in vivo nor in vitro. Lugworm GPase ab has shown a 2.4-fold higher specific activity as GPase b. The Km for P(i) was 16 mmol/l in absence and 13 mmol/l in presence of AMP. Half maximum activation by AMP was reached at 9 mumol/l. IMP up to 10 mmol/l did not activate and ATP up to 4 mmol/l did not inhibit GPase ab in absence of AMP.

Adenosine Monophosphate↗
PubMed1990

In vivo 13C-NMR studies on the metabolism of the lugworm Arenicola marina.

13C-NMR natural-abundance spectra of specimens of Arenicola marina obtained, showed seasonal changes in the concentration of some metabolites, with the osmolite alanine as well as triacylglyceride storage compounds present at high concentrations. Glycogen was sometimes only barely detectable due to the low natural abundance level of 13C. Glycogenic metabolism of the lugworm A. marina was studied in vivo by 13C-NMR spectroscopy using 13C-labelled glucose. During recovery from a hypoxic period [1-13C]glucose was incorporated into glycogen. [1-13C]Glucose was injected 5 h after the end of hypoxia to guarantee sufficient and reliable 13C labelling of glycogen. An earlier injection of [1-13C]glucose led to considerably diminished incorporation of 13C-labelled glucosyl units into glycogen, probably due to the consumption of the available glucose as fuel for ATP production. No scrambling of 13C into the C6 position of glycogen was observed, indicating a lack of gluconeogenic activity. 13C was also incorporated into the C3 positions of alanine and alanopine. To assign correctly this last 13C-NMR resonance, the compound was synthesized biochemically. No labelling of glycogen was observed when [3-13C]alanine was injected into the coelomic cavity with similar incubation conditions being used. The 13C of [1-13C]glucose, incorporated into glycogen, showed a very low turnover rate in normoxic lugworms as shown by two 13C(1H)-NMR spectra, one obtained 48 h after the other. On the other hand, in hypoxia lugworms the signal due to 13C-labelled glycogen decreased very rapidly proving a high turnover rate. The disappearance of 13C from glycogen during the first 24 h of hypoxia indicates that the last glycosyl units to be synthesized are the first to be utilized. Lugworms were quite sensitive to the 1H-decoupling field used for obtaining the 13C(1H)-NMR spectra, especially at 11.7 T. Using bi-level composite-pulse decoupling and long relaxation delays, no tissue damage or stress-dependent phosphagen mobilization, as judged by 31P-NMR spectroscopy, was observed.

Alanine↗
PubMed1989

Glycogenolysis during recovery from muscular work. The time course of phosphorylase activity is dependent on Pi concentration in the abdominal muscle of the shrimp Crangon crangon.

1) Glycogen is degraded in the abdominal muscle of the shrimp Crangon crangon (Decapoda, Crustacea) during the recovery period following work. The regulation of post-exercise glycogen breakdown and the properties of glycogen phosphorylase (EC 2.4.1.1) have been studied: 2) Glycogen phosphorylase exists as unphosphorylated b-form and phosphorylated a-form, the latter contains 1 molecule phosphate/subunit. Both forms of phosphorylase are dimers, isoenzymes have not been detected. 3) The purified b-form is inactive in absence of AMP and has very low affinities for AMP and Pi. For half-maximum activation 0.33 +/- 0.04 mM AMP is necessary, and the Km-value for Pi at 1 mM AMP is 48 +/- 5 mM. IMP does not affect the activity of the b-form. 4) The a-form is active without effectors, its Km-value for Pi is 5.3 +/- 1.5 mM. The proportion of phosphorylase a increases in vivo, from about 25% at rest, to approximately 90% upon work and remains at this high level during the first minutes of recovery. 5) It is concluded that the glycogenolytic flux in the abdominal muscle of the shrimp even during post-exercise periods depends on the level of the a-form the activity of which is restricted in time and extent by the cytoplasmic Pi concentration (Kamp, G. & Juretschke, H. P. (1987) Biochim. Biophys. Acta 929, 121-127).

Adenosine Monophosphate↗
PubMed1987

An in vivo 31P-NMR study of the possible regulation of glycogen phosphorylase a by phosphagen via phosphate in the abdominal muscle of the shrimp Crangon crangon.

31P-NMR spectroscopy has been used to determine the concentrations of Pi and phosphagen in the abdominal muscle of the shrimp Crangon crangon at rest and to follow the changes during their recovery after exhaustive work in vivo. Additionally, the effect of physiological Pi concentrations to the phosphorylase a activity has been examined. At rest, the cytoplasmic Pi and phosphagen concentrations were 1 mM and 38 mM, respectively. After exhaustive work the concentrations of both metabolites were in the same range (20 mM). During recovery, the time course of phosphagen regeneration correlates with the decrease of Pi, as the sum of the concentrations of these metabolites is constant. The ATP level was stable throughout the experiment. The phosphorylase a, which was already present in the resting muscle, was virtually inactive at the resting Pi level (1 mM). During work, however, Pi, which is probably generated by phosphagen-ATP splitting, might ensure a sufficient activation of phosphorylase a.

Adenine Nucleotides↗
PubMed1986

Features of glycogen phosphorylase from the body wall musculature of the lugworm Arenicola marina and the mode of activation during anoxia.

The activities of glycogen phosphorylases a and b from the body wall musculature of the marine worm Arenicola marina (Annelida, Polychaeta) were determined after various periods of anoxia. Already under normoxic conditions one third of the total activity was produced from the a form. During anoxia the ratio of both forms as well as the total activity did not change. The activity of soluble phosphorylase kinase was comparatively low in this tissue 4.3 +/- 1.2 nmol . min-1 . (g wet wt.)-1; the fast twitching tail muscle of shrimps, e.g., had a 10-fold higher phosphorylase kinase activity, whereas phosphorylase activities in both tissues were about the same 2.3 +/- 0.5 mumol . min-1 . (g wet wt.)-1. Glycogen phosphorylase b was purified from the body wall tissue of the marine worm in one step by 5'-AMP-Sepharose resulting in a single protein band in SDS-PAGE. This preparation was accepted as substrate by the phosphorylase kinase from rabbit muscle but a complete phosphorylation could not be achieved. The molecular mass of native phosphorylase was approximately 216 kDa, that of subunits 95 kDa indicating that the enzyme exists as a dimer. There were no isozymes in this preparation, the RF-value (0.17) of the single band in PAGE ranged between those of the isozymes from mice hearts. The activities of phosphorylases b and a were similarly dependent on pH and temperature but differed drastically in the affinities to phosphate and AMP. In presence of 1 mM AMP the app. Km of phosphorylase a for phosphate was 16 mM, that of phosphorylase b above 100 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1983

Comparative investigations of glutaminase development in cerebral cortex of chick embryo and in primary cultures of neurons and glial cells.

Glutaminase activity was determined in pure cultures of neurons, glial cells and in mixed cultures obtained from chick embryo brain. The development of this enzyme was observed periodically over time and compared to its evolution in corresponding cerebral hemispheres during embryonic and postnatal development. The specific activity of brain glutaminase increased between the twelfth and sixteenth day of embryogenesis. A similar increase was observed in cultures of neuroblasts during the corresponding period of time, although the activity in culture was about one-third lower than in vivo. In contrast to neurons, there was no significant increase of glutaminase activity in glial cells before the fifteenth day of culture. The enzyme level in glial cells between the thirteenth and fifteenth days of culture was approximately 25% of that in 7- and 8-day-old neurons. The different development of glutaminase activity in neurons and glial cells was demonstrated in both pure and mixed cultures. The results support the hypothesis that there is a glutamine shunt from glial cells to neurons.

Animals↗