Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.     

Molecular forms of cholecystokinin in human plasma during infusion of bombesin

Bombesin is a tetradecapeptide with stimulatory actions on several gastrointestinal functions. Infusion of bombesin (60 pmol/kg. 20 min) into 7 normal subjects induced significant increases in plasma cholecystokinin (CCK) as measured with 2 sequence-specific radioimmunoassays. Employing antibody 1703, specific for carboxyl-terminal CCK-peptides containing at least 14 amino acid residues, plasma CCK concentrations rose from 0.8 +/- 0.2 pmol/l to 9.9 +/- 1.7 pmol/l (p less than 0.005), while using antibody T204, specific for the sulfated tyrosine region of CCK, plasma CCK levels increased from 2.9 +/- 0.5 pmol/l to 12.4 +/- 1.3 pmol/l (p less than 0.005). Plasma samples obtained from 3 subjects during bombesin infusion were fractionated by Sephadex column chromatography. Fractionation revealed 4 molecular forms of CCK: peak I eluted in the void volume and comprised 0-7% of CCK-like immunoreactivity, peak II eluted at 35% and comprised 8-41% of CCK-like immunoreactivity, peak III eluted at 50% and comprised 44-61% of CCK-like immunoreactivity, and peak IV eluted at 75% and comprised 15-27% of CCK-like immunoreactivity. Radioimmunoassay with a carboxyl-terminal CCK-antibody fully cross-reacting with gastrin did not reveal additional molecular forms of CCK. Since both the carboxyl-terminus and the sulfated tyrosine region are required for biological activity of CCK, it is likely that all these molecular forms of CCK possess biological activity.     

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl₂, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P₂) resulted in the formation of [³H-]InsP ₃. GTP-gamma-S (125 µM) stimulated the production of [³H-]InsP ₃. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [³H-]Ptdlns(4,5)P ₂ hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca²⁺. Addition of phorbol ester (100 nM PMA) enhanced the ³²P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca²⁺, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P ₂-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate -adrenoceptor mediated Ptdlns(4,5)P ₂ hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.List of abbreviations ATP Adenosine 5-Trphosphate - CSU Catalytic Subunit of cyclic AMP-dependent protein kinase - DG Diacylglycerol - DMSO Dimethylsulfoxide - DTT DL-dithiothreitol - EDTA Ethylenedinitrilotetraacetic Acid - EGTA Ethyleneglycol-0,0-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - GTP-gamma-S Guanosine 5-O-(3-thiotriphosphate) - HPTLC High Performance Thin Layer Chromatography - InsP ₃ Inositol monophosphate - InsP ₂ Inositol bisphosphate - InsP ₃ Inositol trisphosphate - MES 2-Morpholinoethanesulfonic acid - MOPS 3-[N-Morpholino]Propanesulfonic acid - PAGE Polyacrylamide-gel Electrophoresis - PKC Protein Kinase C - PLase C Phospholipase C - PMA Phorbol 12-Myristate 13-Acetate - PMSF Phenylmethylsulfonyl Fluoride - PtdSer Phosphatidylserine - PtdIns Phosphatidyl inositol - PT Pertussis Toxin - Ptdlns(4)P Phosphatidylinositol 4-monophosphate - Ptdlns (4,5)PZ-Phosphatidylinositol4,5-bisphosphate - SDS-Sodium Dodecyl Sulfate Tris-Tris(hydroxymethyl) aminomethane     

Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: ontogenesis of the responsiveness to hormones

Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is present as soon as the cells become recognizable as hepatocytes. Immunochemical staining of the cultures shows that hepatocytes do not acquire or express the capacity to accumulate CPS at high rates synchronously. The average levels of CPS per hepatocyte that are observed upon hormone treatment are approx 50-fold lower in embryonic than in adult hepatocytes, corresponding with an approx 10-fold lower synthetic capacity (per gram hepatocytes) and an approx 5-fold smaller size of embryonic compared to adult hepatocytes. Carbamoylphosphate synthetase levels are therefore a good parameter in studies that aim to establish the mechanisms that underly the ontogenesis of the hepatic phenotype.     

Effect of pancreatic enzyme supplementation on postprandial plasma cholecystokinin secretion in patients with pancreatic insufficiency

To test the hypothesis, based on studies in healthy man and dog, that patients with impaired digestion due to severe pancreatic insufficiency have impaired postprandial cholecystokinin (CCK) secretion that can be improved by the addition of pancreatic enzymes, we have studied plasma CCK responses to a test meal with and without addition of pancreatic enzymes in 10 patients with pancreatic insufficiency and steatorrhea, in 8 patients with chronic pancreatitis without steatorrhea, and in 6 healthy subjects. The patients with steatorrhea had a significantly (P less than 0.001) lower integrated plasma CCK response to the meal (177 +/- 23 pM.150 min) than the healthy subjects (468 +/- 41 pM.150 min), while patients with chronic pancreatitis without steatorrhea had an intermediate integrated postprandial CCK secretion (327 +/- 101 pM.150 min). Addition of pancreatic enzymes to the meal significantly augmented the integrated CCK response in both the patients with steatorrhea to 483 +/- 72 pM.150 min (P less than 0.01) and in those without steatorrhea to 480 +/- 85 pM.150 min (P less than 0.05). These values were not significantly different from those in the healthy subjects (521 +/- 86 pM.150 min). Integrated CCK secretion in the three groups during bombesin infusion was similar (patients with steatorrhea 134 +/- 23 pM.20 min, patients without steatorrhea 131 +/- 33 pM.20 min, and healthy subjects 146 +/- 28 pM.20 min), indicating a normal capacity to secrete CCK in response to a humoral stimulus. These data are in agreement with the suggestions from previous studies that digestion of nutrients by pancreatic enzymes plays an important role in the regulation of plasma CCK secretion after feeding.     

Phototrophic pigment production with microalgae: biological constraints and opportunities

There is increasing interest in naturally produced colorants, and microalgae represent a bio‐technologically interesting source due to their wide range of colored pigments, including chlorophylls (green), carotenoids (red, orange and yellow), and phycobiliproteins (red and blue). However, the concentration of these pigments, under optimal growth conditions, is often too low to make microalgal‐based pigment production economically feasible. In some Chlorophyta (green algae), specific process conditions such as oversaturating light intensities or a high salt concentration induce the overproduction of secondary carotenoids (β‐carotene in Dunaliella salina (Dunal) Teodoresco and astaxanthin in Haematococcus pluvialis (Flotow)). Overproduction of all other pigments (including lutein, fucoxanthin, and phycocyanin) requires modification in gene expression or enzyme activity, most likely combined with the creation of storage space outside of the photosystems. The success of such modification strategies depends on an adequate understanding of the metabolic pathways and the functional roles of all the pigments involved. In this review, the distribution of commercially interesting pigments across the most common microalgal groups, the roles of these pigments in vivo and their biosynthesis routes are reviewed, and constraints and opportunities for overproduction of both primary and secondary pigments are presented.     

Distinct Patterns of HIV-1 Evolution within Metastatic Tissues in Patients with Non-Hodgkins Lymphoma

Despite highly active antiretroviral therapy (HAART), AIDS related lymphoma (ARL) occurs at a significantly higher rate in patients infected with the Human Immunodeficiency Virus (HIV) than in the general population. HIV-infected macrophages are a known viral reservoir and have been shown to have lymphomagenic potential in SCID mice; therefore, there is an interest in determining if a viral component to lymphomagenesis also exists. We sequenced HIV-1 envelope gp120 clones obtained post mortem from several tumor and non-tumor tissues of two patients who died with AIDS-related Non-Hodgkin''s lymphoma (ARL-NH). Similar results were found in both patients: 1) high-resolution phylogenetic analysis showed a significant degree of compartmentalization between lymphoma and non-lymphoma viral sub-populations while viral sub-populations from lymph nodes appeared to be intermixed within sequences from tumor and non-tumor tissues, 2) a 100-fold increase in the effective HIV population size in tumor versus non-tumor tissues was associated with the emergence of lymphadenopathy and aggressive metastatic ARL, and 3) HIV gene flow among lymph nodes, normal and metastatic tissues was non-random. The different population dynamics between the viruses found in tumors versus the non-tumor associated viruses suggest that there is a significant relationship between HIV evolution and lymphoma pathogenesis. Moreover, the study indicates that HIV could be used as an effective marker to study the origin and dissemination of lymphomas in vivo.     

Basic strategies for valid cytometry using image analysis

The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetics in difficult imaging situations, such as low fluorescence signals and high absorbance signals. This revised version was published online in November 2006 with corrections to the Cover Date.