DNA condensation at freestanding cationic lipid bilayers.

We describe a previously unreported coil-globule transition of DNA electrostatically bound to a freestanding fluid cationic lipid membrane. The collapse of a DNA coil into a compact globule takes place after the DNA molecule attaches in an extended conformation to the membrane. DNA condensation is favored at a higher cationic lipid content, while at lower membrane charge densities coexistence of DNA random coils, partially collapsed conformations, and globules is observed.

Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy.

In the bacterium Escherichia coli, selection of the division site involves pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation mechanisms based on cooperative effects between Min-proteins and on the exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane have been proposed. The parameters characterizing the dynamics of the Min-proteins in vivo are not known. It has therefore been difficult to compare the models quantitatively with experiments. Here, we present in vivo measurements of the mobility of MinD and MinE using fluorescence correlation spectroscopy. Two distinct timescales are clearly visible in the correlation curves. While the faster timescale can be attributed to cytoplasmic diffusion, the slower timescale could result from diffusion of membrane-bound proteins or from protein exchange between the cytoplasm and the membrane. We determine the diffusion constant of cytoplasmic MinD to be approximately 16 microm(2) s(-1), while for MinE we find about 10 microm(2) s(-1), independently of the processes responsible for the slower time-scale. The implications of the measured values for the oscillation mechanism are discussed.

Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede.

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.

Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting.

Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.

Analyzing single protein molecules using optical methods.

Studies on single protein molecules have advanced from mere proofs of principle to insightful investigations of otherwise inaccessible biological phenomena. Recent studies predict a tremendous number of possible future applications. The long-term vision of biologists to watch single molecular processes in real time by peering into a cell with three-dimensional resolution might finally be realized. Another fascinating perspective is the identification and selection of single favorable variants from complex libraries of diverse biomolecules.

Metabolic consequences of orthotopic pancreaticoduodenal transplantation with preservation of near normal physiology.

BACKGROUND: Several case reports suggested the use of pancreaticoduodenal allotransplantation alone or in combination with multivisceral transplants to treat exocrine and endocrine deficiency after pancreatectomy for chronic pancreatitis, upper abdominal malignancies, and cystic fibrosis. Our objective was to establish the metabolic consequences of this technique. METHODS: Inbred rats, which either underwent pancreaticoduodenectomy before receiving an orthotopic duodenopancreas transplant (Tx, n= 18) or laparotomy (sham, n=18), were subjected 3 months postoperatively to oral and "isoglycemic" i.v. glucose tolerance tests with arterial blood sampling (n=12) or oral glucose tolerance test with additional portal blood sampling (n=6). Fecal fat and chymotrypsin were evaluated in the 11th postoperative week as indicators of pancreatic exocrine function in eight animals of each group. RESULTS: The incremental arterial plasma glucose integrated over a 90-min period was similar after oral and i.v. glucose in the respective groups, but was significantly lower in Tx versus sham rats after oral glucose. Incremental portal glucose was also lower after oral glucose, while hepatic glucose extraction remained unchanged. The incremental response of arterial glucose-dependent insulinotropic peptide, and of arterial and portal insulin, was comparable in Tx and sham rats; also in both groups the arterial response was significantly greater with oral versus i.v. glucose, and the incretin effect for insulin was intact after transplantation. Fecal fat and chymotrypsin levels did not differ between the two groups. CONCLUSIONS: 1) In the Tx rat lower incremental plasma glucose after oral glucose intake likely results from decreased intestinal glucose uptake; 2) preservation of a normal entero-insular axis of insulin together with the absence of intestinal malabsorption of lipids suggest that orthotopic transplantation of a duodeno-pancreas preserved endocrine and exocrine pancreatic function and therefore qualifies as treatment modality for the above named indications.

Impact of arterialization on hepatic oxygen supply, tissue energy phosphates, and outcome after liver transplantation in the rat.

The importance of arterial reconstruction in experimental orthotopic rat liver transplantation is widely acknowledged in the literature. Non-rearterialization of the graft leads to impaired microcirculation and, in chronic models, to severe hepatobiliary damage, together with bile duct proliferation and fibrosis in such livers. The aim of the current study was to investigate the impact of rearterialization on hepatic oxygen tension (pO2), hepatic tissue content of adenine nucleotides, early graft function, and postoperative outcome. Orthotopic liver transplantation was performed in 27 male inbred rats. Ten rats underwent rearterialization and while 17 did not. A group of sham-operated animals (n = 6) served as controls. After reperfusion, liver grafts without arterial reconstruction showed significantly reduced levels of oxygen tension (mean +/- SD, 3.79 +/- 2.20 vs. 10.03 +/- 2.84 mmHg; P < 0.05) and a clear shift toward lower pO2 values in the pO2 histograms, as compared with arterialized grafts. Without arterialization, the level of liver ATP was 65% of that in sham animals, compared with 84% in arterialized livers. Without arterialization, bile secretion was reduced (0.42 +/- 0.04 vs. 0.71 +/- 0.06 mg/min x g liver; (P < 0.001), and the postoperative course of serum alanine transaminase, bilirubin, and alkaline phosphatase revealed severe hepatobiliary damage. These findings allow us to conclude that graft rearterialization is essential to ensure both an adequate oxygen supply and maintenance of tissue ATP. Arterialization may thus be a necessary part of liver transplantation models in this animal species, and should be considered when designing studies on the biochemical, microcirculatory, and histopathological status of the graft.

Fluorescence correlation spectroscopy and its potential for intracellular applications.

Fluorescence correlation spectroscopy (FCS) is a time-averaging fluctuation analysis of small molecular ensembles, combining maximum sensitivity with high statistical confidence. Among a multitude of physical parameters that are, in principle, accessible by FCS, it most conveniently allows to determine local concentrations, mobility coefficients, and characteristic rate constants of fast-reversible and slow-irreversible reactions of fluorescently labeled biomolecules at very low (nanomolar) concentrations, under equilibrium conditions and without physical separation. Its presently most popular instrumentation by confocal-microscope setups allows for a spatial resolution of fractions of femtoliters for the measurement volumes, containing sparse or even single molecules at any time, and encourages the adaptation of the solution-based technique for cellular applications. The scope of this review is thus, to introduce the FCS technique in particular to the reader with biological background, searching for new methods for a precise quantification of physical parameters governing cellular mechanisms and dynamics, especially if high sensitivity and fast dynamic resolution are required. After a short theoretical introduction, examples are given for the so far most important experimental applications, with respect to their implementation in cellular systems. As an interesting alternative to the confocal instrumentation, two-photon excitation will be introduced, offering a number of important advantages especially in cellular systems with high-noise and low-signal levels.

Enzyme assays for confocal single molecule spectroscopy.

During the last years confocal techniques have become increasingly popular for probing biological enzymatic reactions. In this paper we will summarize some of these methods, mainly focusing on measurement techniques suitable for analysis of freely diffusing molecules, where either the substrates or the enzymes are fluorescently labeled. The different approaches are classified according to their basic principles and algorithms. Several examples of enzymatic studies involving correlation strategies for data processing, like auto- and cross correlation analysis, will be presented. In addition, other evaluation schemes like coincidence analysis and fluorescence intensity distribution analysis are introduced and discussed. With respect to assay development, the fluorescence energy transfer principle is addressed as far as it is has been applied for investigating biocatalysis in solution. Finally, one part of this review addresses the aspects of bioconjugation and the basic requirements for proper labeling dyes in order to be compatible with single molecule fluorescent spectroscopy.

Clofibrate feeding to Sprague-Dawley rats increases endogenous biosynthesis of oxalate and causes hyperoxaluria.

The effects of clofibrate feeding (5 g/kg diet) on oxalate metabolism were investigated in male and female rats. Following clofibrate feeding, 24-hour urinary excretion of oxalate increased until 4 days and then reached a plateau. Whereas the contribution of dietary oxalate (1.4 g/kg diet, as potassium salt) to urinary oxalate was less than 5% in both control and clofibrate-treated male rats, the contribution of dietary glycolate (1.0 g/kg diet, as sodium salt) to urinary oxalate was six times higher in clofibrate-treated male rats compared with controls, indicating that the clofibrate-induced hyperoxaluria is due to increased endogenous biosynthesis of oxalate. This was supported by the increased lactate dehydrogenase (LDH) activity observed in liver supernatants of clofibrate-treated rats compared with controls, and the increased rate of conversion of glycolate and glyoxylate to oxalate by clofibrate-treated male rat liver supernatants. Female rats had lower excretion of urinary oxalate and lower levels of liver glycolic acid oxidase (GAO) as compared with males. Clofibrate-treated female rat liver supernatants had higher LDH levels and produced more oxalate from glyoxylate. Thus, it can be concluded that the increase in LDH activity may be the cause of the increased endogenous biosynthesis of oxalate leading to increased urinary excretion of oxalate in male and female rats treated with clofibrate.

The influence of progressive hyperinsulinemia on duodenal calcium absorption in the rat.

Using an in situ loop technique, we studied the influence of moderate hyperinsulinemia on the bidirectional fluxes [lumen-to-plasma (LP), plasma-to-lumen (PL)] and on the net absorption of calcium (Ca) in the duodenum of the rat. Under hyperinsulinemic euglycemic clamp conditions, three different steady-state plasma insulin levels (66 +/- 16, 187 +/- 13, 263 +/- 24 microU/mL) were maintained by intravenous (IV) infusion of either 20, 40, or 60 mU/h of insulin. LP flux and net Ca absorption (CaA) increased significantly under all, while the PL flux was not changed by any of the three insulin doses. Under 40 and 60 mU/h of IV insulin, the individual plasma insulin levels and LP fluxes were positively and significantly correlated, suggesting a dose-dependent action of insulin on duodenal CaA. No significant changes were seen in plasma somatostatinlike immunoreactivity (SLI), parathyroid hormone (PTH), and serum Ca, while serum inorganic phosphate (Pi) and 1,25(OH)2 vitamin D levels fell with increasing doses of IV insulin. It is concluded for the rat that physiologic degrees of hyperinsulinemia enhance duodenal CaA by an as yet unknown mechanism, this action seems to be independent of PTH and, the role of 1,25(OH)2 vitamin D in this context is poorly understood.

Effect of venous drainage site on insulin action after pancreas transplantation in the rat--is there insulin resistance and a risk for atherosclerosis?

The aim of the present study was to determine the influence of the venous drainage site on insulin homeostasis and the possible risk for atherosclerosis development after pancreas transplantation. We studied inbred rats that received pancreas transplants with either systemic (STX) or portal (PTX) venous drainage after prior induction of diabetes with streptozotocin and sham-operated controls. The observation period was 6 months. Fasting plasma glucose and insulin levels were similar in all 3 groups, but fasting plasma glucagon levels were elevated in STX (mean +/- SEM, 282+/-35 ng/L) in comparison to PTX rats (119+/-9 ng/L, P < .05), although the difference versus the control group (191+/-31 ng/L) was insignificant. Glucose utilization and hepatic glucose production (HGP), assessed by a dose-response euglycemic-hyperinsulinemic clamp in combination with tritiated glucose infusion, were similar in all 3 groups. The groups were also similar with respect to the molar ratio of plasma C-peptide and insulin during basal steady state and the metabolic clearance rate (MCR) of insulin during the clamp studies, suggesting an unchanged hepatic insulin extraction (HIE) after transplantation with either technique. Factors known to be related to atherosclerosis, ie, blood pressure, intracellular magnesium, and fasting levels of plasma cholesterol, triglycerides, and high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, were similar in all 3 groups. Light microscopy of the aorta showed a slightly thicker intima in STX rats (24.3+/-0.5 microm, P < .05) versus PTX rats (21.4+/-0.7 microm) and control (21.4+/-0.6 microm); however, atherosclerosis-like lesions were absent in all 3 groups. In conclusion, in a rat model with streptozotocin-diabetes and pancreas transplantation but no need for immunosuppression, both systemic and portal venous drainage avoid peripheral and hepatic insulin resistance; also, there is no increased risk for atherosclerosis.

Preservation of the incretin effect after orthotopic pancreas transplantation in inbred rats.

To establish whether the incretin effect is under neural control, insulin, C-peptide, and glucose-dependent insulinotropic peptide (GIP) responses and hepatic insulin clearance were investigated after oral and "isoglycemic" intravenous glucose in 12 inbred rats after denervation of the pancreas by orthotopic transplantation with portal venous drainage (Tx group) and in 12 laparotomized controls (sham group). Effective pancreas denervation was documented by a decreased pancreatic polypeptide (PP) response to insulin-induced hypoglycemia and by decreased levels of norepinephrine and calcitonin gene-related peptide (CGRP) in pancreatic tissue. Basal and incremental arterial plasma glucose integrated over 180 minutes did not differ between oral and intravenous glucose, but the integrated insulin response (mean +/- SEM) was significantly greater with oral versus intravenous glucose (Tx group, 104.9 +/- 22.0 v 31.0 +/- 4.9 nmol x L(-1) x min, P < .01; sham group, 79.5 +/- 10.6 v 36.6 +/- 5.8 nmol x L(-1) x min, P < .01). The integrated response of C-peptide was similar during both tests (Tx group, 105 +/- 14 v 79 +/- 8 pmol x mL(-1) x min; sham group, 112 +/- 10 v 121 +/- 12 pmol x mL(-1) x min). Hepatic insulin clearance was significantly decreased in both groups by oral compared with intravenous glucose administration (Tx group, 1.3 +/- 0.2 v 3.3 +/- 0.6 mmol/mmol, P < .01; sham group, 1.6 +/- 0.1 v 3.9 +/- 0.6 mmol/mmol, P < .02). The incretin effects for insulin (Tx group, 5.6 +/- 2.7; sham group, 3.0 +/- 0.8) and C-peptide (Tx group, 1.4 +/- 0.2; sham group, 1.1 +/- 0.2), calculated as the ratio of the integrated oral response and integrated intravenous response, and GIP responses to oral and intravenous glucose were not significantly different between the two groups. We conclude that there is preservation of the incretin effect in rats with orthotopically transplanted and hence extrinsically denervated pancreas, thus ruling out the possibility that the autonomic nervous system substantially contributes. Hepatic insulin clearance and insulinotropic hormones such as GIP appear to be more important.

Two-photon fluorescence cross-correlation spectroscopy.

Dual-color cross-correlation spectroscopy is a special kind of fluctuation analysis which selectively probes the formation or deletion of linkages between two different fluorescently labeled molecules at extremely low concentrations. Two-photon excitation can, under certain circumstances, significantly simplify this method if different probe molecules with distinct emission properties are accessible by a common IR excitation wavelength.

Gastric secretion and gastrin under progressive doses of somatostatin-14 and -28 administered intraperitoneally to the rat.

The actions of progressive doses of intraperitoneally (IP) administered somatostatin-14 (SS-14) and -28 (SS-28) on gastric secretion (acid, pepsin) and mucosal blood flow (MBF) were studied in conscious gastric fistula rats both under basal conditions and under additional administration of pentagastrin. Also, somatostatin-like immunoreactivity was measured in aortal blood in all groups as well as aortal gastrin levels under basal conditions. IP infusion of equimolar doses of SS-14 and SS-28 resulted in an equal and dose-dependent inhibition of basal as well as pentagastrin-stimulated gastric acid secretion. MBF was reduced by either peptide both in the basal and pentagastrin experiments. Under basal conditions pepsin secretion was significantly increased by infusion of SS-14 at the higher doses, by infusion of SS-28 only at the intermediate dose (3.1 nmole kg-1.hr-1). In the pentagastrin experiments, low and intermediate doses of SS-14 tended to lower pepsin outputs but the highest dose of SS-14 stimulated pepsin secretion, whereas SS-28 had no effect on pepsin. Administration of SS-28 inhibited gastrin only at the highest dose (12.3 nmole kg-1.hr-1), and SS-14 had no influence at all on gastrin. After IP infusion of both peptides, plasma SLI rose dose-dependently under basal and stimulated conditions. Gel chromatography indicated an in-vivo conversion of SS-28 to SS-14 or intermediate fragments. It is concluded that SS-14 and SS-28 delivered by IP infusion, inhibit basal and stimulated gastric acid equally in the rat without suppressing gastrin. The mechanism underlying SS-mediated pepsin stimulation is unknown.

Long-term pancreatic duct occlusion impairs the entero-insular axis in the dog--failure of plasma VIP to respond as "incretin".

The response of VIP to either an oral glucose load (OGT) or intravenous glucose (IV glucose), aimed at reproducing the plasma glucose level after OGT, was studied in trained, conscious, sham-operated (Sham; n = 6) dogs, and dogs having initially (12 months before the glucose experiments) undergone occlusion of the pancreatic duct by the prolamine glue technique (Occ; n = 5). As a result, prior to glucose studies, the exocrine pancreas function was found subtotally reduced, as indirectly evaluated by the para-aminobenzoic acid (PABA) test, but no signs of diabetes were detected. The two studies with glucose administration designed to demonstrate the release of insulin, VIP, somatostatin into plasma as modified by enteric signals (represented by the difference of plasma peptide concentration during OGT minus peptide concentration during IV glucose) revealed the following: (1) basal plasma glucose, insulin, VIP, somatostatin did not differ between Sham and Occ dogs; (2) after OGT in Occ dogs the plasma glucose was elevated, whereas plasma insulin was markedly reduced, and VIP, somatostatin were largely unchanged; (3) the integrated output of insulin only was impaired when considering the so-called entero-insulin axis, while integrated VIP, somatostatin were unaltered. It was concluded (a) the Occ procedure in the dog has the capacity to subtotal destruction of the pancreatic acinar tissue, and of the entero-insular axis of insulin, the latter through yet unknown pathways, (b) the Occ technique may be a useful tool for investigation of the nature of "incretin," (c) VIP and somatostatin do not respond to elevated blood glucose and may have no role in the "incretin" concept of enteric modulation of the B-cell.

Somatostatin and intestinal calcium transport in the rat.

In intact rats we studied the influence of low doses of intravenously (i.v.) administered somatostatin (SRIF) on the net absorption and the bidirectional fluxes (lumen-to-plasma, LP; plasma-to-lumen, PL) of calcium in the duodenum, jejunum, ileum and caecum. In the duodenum SRIF inhibited the LP-flux and the net absorption of Ca significantly at infusion rates of 0.75 and 1.0 microgram SRIF . kg-1 . h-1. The PL-flux was not altered by any of the SRIF doses administered. In the other gut segments studied (jejunum, ileum, caecum) neither the net absorption nor the bidirectional Ca fluxes were changed by i.v. SRIF. It is concluded that SRIF in the plasma levels achieved in this study has an influence on the duodenal calcium absorption (CaA) of the rat; questions regarding the mechanisms of this action as well as the physiological significance of our findings are as yet unresolved.

Kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies.

This review demonstrates the large analytical and diagnostic potential of fluorescence correlation spectroscopy applied to freely diffusing biomolecules in solution. All applications discussed here in detail are based on changes in the diffusion characteristics of fluorescenctly labeled complementary strands of nucleic acids when they associate. However, the principle of the measurement can be extended to many different reactions with characteristic association times between several minutes up to several hours. If the reaction significantly affects the diffusion constants of at least one partner, single-color auto-correlation analysis is sufficient to extract kinetic parameters. If the observed binding process has only a moderate effect on diffusion coefficients, the detection selectivity and sensitivity can be improved by dual-color cross-correlation analysis. Finally, we show that diffusional analysis on the single-molecule level even opens up diagnostic applications, such as the detection of minute amounts of infectious agents like HIV-1 viruses in blood.

Proximal gastric vagotomy: effects of two surgical modifications on oral and intravenous glucose tolerance in the conscious rat.

Vago-vagal nervous links between different splanchnic organs, the stomach included, may modulate glucose metabolism. Therefore, the effect of highly selective (HSV, cutting nerve fibers and vessels) and superselective vagotomy (SSV, cutting nerve fibers only) on oral and intravenous (IV) glucose tolerance was studied in the rat. Gastric emptying was normal in HSV and SSV. After oral glucose, cumulative blood glucose and insulin were significantly lower in SSV than in controls, whereas in HSV, both parameters tended towards lower values. After IV glucose, cumulative blood glucose was significantly lower than in controls following both vagotomies, whereas cumulative insulin was lower in HSV and significantly higher in SSV. The former effect may be insulin-independent. The latter reflects enhanced insulin sensitivity in HSV and increased glucose-stimulated insulin release in SSV. The improvement of oral and IV glucose tolerance by both procedures may reflect the abolition of physiological vagal (SSV) or partial abolition of sympathetic (HSV) nervous links between the stomach and the pancreas, which modulate insulin secretion or organ sensitivity to insulin.

Acute effects of calcium sodium citrate supplementation of a test meal on mineral homeostasis, oxalate, and calcium oxalate crystallization in the urine of healthy humans--preliminary results in patients with idiopathic calcium urolithiasis.

Calcium, in the form of regular food supplementation, can improve bone metabolism, but it can also increase the risk for renal calcium stones, and may aggravate pre-existing calcium urolithiasis. To study the first of these two aspects, ten healthy volunteers were given a conventional test meal (breakfast; calcium content 28 mg) with or without two dosages of calcium (as calcium-sodium citrate, CSC 1, 680 mg; CSC 2 1,360 mg), taken after an overnight 12 h fast. To study the latter aspect, patients with idiopathic recurrent calcium urolithiasis (ICU) received a balanced test meal of fixed composition, containing 1,000 mg calcium either as CSC (Meal + CSC3; n = 6) or as calcium gluconate (Mcal; n = 8). In normals, CSC induced a dose-dependent increasing intestinal absorption of calcium, and a decrease in oxalate absorption; in serum, CSC increased calcitonin and suppressed parathyroid hormone, but left unchanged the markers of bone turnover, serum osteocalcin and bone alkaline phosphatase. In urine, CSC decreased bone resorption markers (collagen crosslinks) and phosphaturia increased citrate, created signs of metabolic alkalosis, and inhibited several parameters of CaOx crystallization. In ICU, the CSC3 load failed to promote the crystallization of CaOx and calcium phosphate. It was concluded that CSC supplementation of a meal: (1) is well tolerated by healthy subjects and ICU patients, renders calcium highly available to bone, and prevents post-prandial oxaluria from rising; and, (2) is followed by the inhibition of crystallization of renal stone forming calcium-containing substances. Long-term studies aimed at evaluating the usefulness of CSC in preserving healthy bone, and in the metaphylaxis of renal stones would appear justified.