An Incubator for the Simulation of a Fluctuating Light Climate in Studies of Planktonic Primary Productivity

A laboratory system for the quantification of phytoplankton photosynthesis under fluctuating light climate conditions is described. It consists of 2 temperature-controlled incubators with a variable light supply, an algal batch culture in incubation bottles with appropriate stirrers and a set of oxygen electrodes to monitor algal photosynthesis. By the rotation of special grey filters between the incubator and the light source, a regular up and down movement in the water column is simulated in up to 7 parallel bottles. Different ratios of euphotic depth to mixing depth and different velocities can be applied. Simultaneously, 8 bottles can be incubated under constant light. The system is demonstrated in experiments with Chlamydomonas sp. Further possibilities of application are proposed.     

Effects of ice duration on plankton succession during spring in a shallow polymictic lake

1. Long-term records of air temperature and ice phenology (ice duration), and phyto- and zooplankton time series (1979–1997) were used to study the effects of ice duration on the successional pattern within plankton communities during spring in a shallow polymictic lake. 2. Water temperature in March was significantly lower after cold winters when compared to average or mild winters. Mean water temperature in April was not significantly different after mild, average or cold winters, but showed an overall significant negative correlation with ice duration. 3. Ice duration affected the timing and the magnitude of the peak abundance of diatoms, rotifers and daphnids during spring, but had no direct effects on the timing and maximum of chlorophytes, cryptophytes, cyanobacteria, bosminids and cyclopoid copepods. 4. Plankton groups which appeared first in the seasonal succession (i.e. diatoms, rotifers and daphnids) reached maximum abundance earlier after mild and average winters. The peak abundance of diatoms was negatively correlated with ice duration, whereas that of rotifers and daphnids was independent of the conditions during the preceding winter. 5. Temperature alone was generally a poor predictor of the timing and magnitude of both phyto- and zooplankton maxima. Turbulence may be important in the timing and the magnitude of peaks in diatoms, while total algal biomass was the most important determinant for the timing of the rotifer maximum. The magnitude of the daphnid maxima were significantly influenced by water temperature in March and April, and by rotifer abundance. The magnitude of the bosminid maximum was correlated with food availability and predation, whereas the timing of the maximum was more closely related to water temperature in May. 6. We conclude that, as a result of the low heat storage capacity of shallow lakes, the effects of winter on planktonic communities are short lived, and soon overtaken by the prevailing weather and by biotic interactions.     

Insulin stimulates fatty acid transport by regulating expression of FAT/CD36 but not FABPpm

Because insulin has been shown to stimulate long-chain fatty acid (LCFA) esterification in skeletal muscle and cardiac myocytes, we investigated whether insulin increased the rate of LCFA transport by altering the expression and the subcellular distribution of the fatty acid transporters FAT/CD36 and FABPpm. In cardiac myocytes, insulin very rapidly increased the expression of FAT/CD36 protein in a time- and dose-dependent manner. During a 2-h period, insulin (10 nM) increased cardiac myocyte FAT/CD36 protein by 25% after 60 min and attained a maximum after 90-120 min (+40-50%). There was a dose-dependent relationship between insulin (10(-12) to 10(-7) M) and FAT/CD36 expression. The half-maximal increase in FAT/CD36 protein occurred at 0.5 x 10(-9) M insulin, and the maximal increase occurred at 10(-9) to 10(-8) M insulin (+40-50%). There were similar insulin-induced increments in FAT/CD36 protein in cardiac myocytes (+43%) and in Langendorff-perfused hearts (+32%). In contrast to FAT/CD36, insulin did not alter the expression of FABPpm protein in either cardiac myocytes or the perfused heart. By use of specific inhibitors of insulin-signaling pathways, it was shown that insulin-induced expression of FAT/CD36 occurred via the PI 3-kinase/Akt insulin-signaling pathway. Subcellular fractionation of cardiac myocytes revealed that insulin not only increased the expression of FAT/CD36, but this hormone also targeted some of the FAT/CD36 to the plasma membrane while concomitantly lowering the intracellular depot of FAT/CD36. At the functional level, the insulin-induced increase in FAT/CD36 protein resulted in an increased rate of palmitate transport into giant vesicles (+34%), which paralleled the increase in plasmalemmal FAT/CD36 (+29%). The present studies have shown that insulin regulates protein expression of FAT/CD36, but not FABPpm, via the PI 3-kinase/Akt insulin-signaling pathway.     

Depressed modulation of oxygen consumption by endogenous nitric oxide in cardiac muscle from diabetic dogs

Our previous study indicated that nitric oxide (NO)-dependent coronary vasodilation was impaired in conscious dogs with diabetes. Our goal was to determine whether modulation of O(2) consumption by NO is depressed in canine cardiac muscle after diabetes. Diabetes was induced by injection of alloxan (40-60 mg/kg iv), dogs were killed after diabetes was induced (4-5 wk), and the cardiac muscle from the left ventricle was cut into 15- to 30-mg slices. O(2) uptake by the muscle slices was measured polarographically with a Clark-type O(2) electrode. S-nitroso-N-acetylpenicillamine decreased O(2) consumption in normal and diabetic tissues (10(-4) M, 61 +/- 7 vs. 61 +/- 8%, P > 0.05). Bradykinin (10(-4) M)- or carbachol (CCh, 10(-4) M)-induced inhibition of O(2) consumption was impaired in diabetic tissues (51 +/- 6 vs. 17 +/- 4% or 48 +/- 4 vs. 19 +/- 3%, respectively, both P < 0.05 compared with normal). The inhibition of O(2) consumption by kininogen or kallikrein was depressed in diabetic tissues as well. In coronary microvessels from diabetic dogs, bradykinin or ACh (10(-5) M) caused smaller increases in NO production than those from normal dogs. Our results indicate that the modulation of O(2) consumption by endogenous, but not exogenous, NO is depressed in cardiac muscle from diabetic dogs, most likely because of decreased release of NO from the vascular endothelium.     

Denervation provokes greater reductions in insulin-stimulated glucose transport in muscle than severe diabetes

We have examined the independent and combined effects of insulin insufficiency (streptozotocin (STZ)-induced diabetes, 85 mg/kg i.p.) and reduced muscle activity (denervation) (7 days) on basal, insulin-stimulated and contraction-stimulated glucose transport in rat muscles (soleus, red and white gastrocnemius). There were four treatments: control, denervated, diabetic, and denervated + diabetic muscles. Contraction-stimulated glucose transport was lowered (~ 50%) (p < 0.05) to the same extent in all experimental groups. In contrast, there was a much smaller reduction insulin-stimulated glucose transport in muscles from diabetic animals (18-24% reduction, p < 0.05) than in denervated muscles (40-60% reduction, p < 0.05) and in denervated + diabetic muscles (40-60% reduction, p < 0.05). GLUT-4 mRNA reduction was greatest in denervated + diabetic muscles (~ -75%, p < 0.05). GLUT-4 protein was decreased (p < 0.05) to a similar extent in all three experimental conditions (~ -30-40%). In conclusion, (1) muscle inactivity (denervation) and STZ-induced diabetes had similar effects on reducing contraction-stimulated glucose transport, but (2) muscle inactivity (denervation), rather than severe diabetes, produced a 2-fold greater impairment in skeletal muscle insulin-stimulated glucose transport.     

Impact of peptides on the recognition of HLA class I molecules by human HLA antibodies

MHC class I molecules expressed on cell surfaces are composed of H chain, beta2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.     

Functional evaluation of Dent’s disease-causing mutations: implications for ClC-5 channel trafficking and internalization

ClC-5 is a member of the ClC family of voltage-gated chloride channels. Loss-of-function mutations of its corresponding gene (CLCN5) cause Dents disease, an X-linked kidney disorder, characterized by low-molecular weight proteinuria, hypercalciuria, nephrocalcinosis/nephrolithiasis, and progressive renal failure. Here, we examined the effect of different mutations on function and cellular trafficking of the recombinant protein. Mutant CLCN5 cDNAs were generated by site directed mutagenesis for two premature stop codon variants (R347X and M517IfsX528), and several missense mutations (C221R, L324R, G462 V, and R516 W). We also tested L521R (instead of L521RfsX526 observed) and mutants G506E and R648X (previously reported by others). After heterologous expression in Xenopus oocytes, ClC-5 channel activity and surface expression were determined by two-electrode voltage-clamp analysis and ClC-5 surface ELISA, respectively. Except for the R516 W and R648X variants, none of the mutated proteins induced functional chloride currents or reached the plasma membrane. This is readily understandable for the truncation mutations. Yet, the tested missense mutations are distributed over different transmembrane regions, implying that correct channel structure and orientation in the membrane is not only a prerequisite for proper ClC-5 function but also for Golgi exit. Interestingly, the R648X mutant although functionally compromised, displayed a significant increase in surface expression. This finding might be explained by the deletion of a ClC-5 carboxy-terminal PY-like internalization signal, which in turn impairs channel removal from the membrane. Our observations further imply that recruitment of ClC-5 to alternative routes (plasma membrane or early endosomes) in the trans-Golgi network is mediated via different signal sequences.Electronic Supplementary Material Supplementary material is available for this article at .     

Increased superoxide leads to decreased flow-induced dilation in resistance arteries of Mn-SOD-deficient mice

The role of mitochondrial manganese-superoxide dismutase (Mn-SOD) in the maintenance of vascular function has not yet been studied. Thus we examined flow- and agonist-induced dilations in isolated mesenteric arteries (approximately 90 microm in diameter) of Mn-SOD heterozygous (Mn-SOD+/-) and wild-type (WT) mice. Increases in flow elicited dilations in all vessels, but the magnitude of the dilation was significantly less in vessels of Mn-SOD+/- mice than in those of WT mice (64 vs. 74% of passive diameter). N(omega)-nitro-L-arginine methyl ester inhibited the dilation in vessels of WT mice but had no effect on vessels of Mn-SOD+/- mice. Tempol or tiron (superoxide scavengers) increased flow-induced dilation in vessels of Mn-SOD+/- mice. Acetylcholine- and sodium nitroprusside-induced, but not adenosine-induced, dilations were also decreased in arteries of Mn-SOD+/- mice. Superoxide levels in the arteries of Mn-SOD+/- mice were significantly increased. Western blot analysis confirmed a 50% reduction of Mn-SOD protein in the vessels of Mn-SOD+/- mice. A 41% reduction in endothelial nitric oxide synthase (eNOS) protein and a 37% reduction in eNOS activity were also found in the vessels of Mn-SOD+/- mice. Whereas there was no difference in eNOS protein in kidney homogenates of WT and Mn-SOD+/- mice, a significant reduction of nitric oxide synthase activity was found in Mn-SOD+/- mice, which could be restored by the administration of tiron. We conclude that an increased concentration of superoxide due to reduced activity of Mn-SOD, which inactivates nitric oxide and inhibits eNOS activity, contributes to the impaired vasodilator function of isolated mesenteric arteries of Mn-SOD+/- mice. These results suggest that Mn-SOD contributes significantly to the regulation of vascular function.     

Impact of altered substrate utilization on cardiac function in isolated hearts from Zucker diabetic fatty rats

The goal of this study was to determine whether changes in cardiac metabolism in Type 2 diabetes are associated with contractile dysfunction or impaired response to ischemia. Hearts from Zucker diabetic fatty (ZDF) and lean control rats were isolated and perfused with glucose, lactate, pyruvate, and palmitate. The rates of glucose, lactate, pyruvate, and palmitate oxidation rates and glycolysis were determined during baseline perfusion and low-flow ischemia (LFI; 0.3 ml/min for 30 min) and after LFI and reperfusion. Under all conditions, ATP synthesis from palmitate was increased and synthesis from lactate was decreased in the ZDF group, whereas the contribution from glucose was unchanged. During baseline perfusion, the rate of glycolysis was lower in the ZDF group; however, during LFI and reperfusion, there were no differences between groups. Despite these metabolic shifts, there were no differences in oxygen consumption or ATP production rates between the groups under any perfusion conditions. Cardiac function was slightly depressed before LFI in the ZDF group, but during reperfusion, function was improved relative to the control group despite the increased dependence on fatty acids for energy production. These data suggest that in this model of diabetes, the shift from carbohydrates to fatty acids for oxidative energy production did not increase myocardial oxygen consumption and was not associated with impaired response to ischemia and reperfusion.     

Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy

The success of cellular therapies will depend in part on accurate delivery of cells to target organs. In dendritic cell therapy, in particular, delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. We show here that in vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. Autologous dendritic cells were labeled with a clinical superparamagnetic iron oxide formulation or (111)In-oxine and were co-injected intranodally in melanoma patients under ultrasound guidance. In contrast to scintigraphic imaging, magnetic resonance imaging (MRI) allowed assessment of the accuracy of dendritic cell delivery and of inter- and intra-nodal cell migration patterns. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.