Karlheinz Voigt

Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine

Low-molecular-weight polyethylenimine (LMW-PEI) was synthesized by the acid-catalyzed, ring-opening polymerization of aziridine and compared with commercially available high-molecular-weight PEI (HMW-PEI) of 25 kDa. Molecular weights were determined by size-exclusion chromatography in combination with multi-angle laser light scattering. The weight average molecular weight (M(w)) of synthesized LMW-PEI was determined as 5.4+/-0.5 kDa, whereas commercial HMW-PEI showed a M(w) of 48+/-2 kDa. DNA polyplexes of LMW-PEI and HMW-PEI were characterized with regard to DNA condensation (ethidium bromide fluorescence quenching), size (photon correlation spectroscopy) and surface charge (laser Doppler anemometry). Compared with HMW-PEI, DNA condensation of LMW-PEI was slightly impaired at lower N/P ratios. Complexes with plasmid DNA at a N/P ratio of 6.7 showed significantly increased hydrodynamic diameters (590+/-140 vs. 160+/-10 nm), while the zeta-potential measurements were similar (23+/-2 vs. 30+/-3 mV). The cytotoxicity of LMW-PEI in L929 fibroblasts was reduced by more than one order of magnitude compared with HMW-PEI, as shown by MTT assay. LMW-PEI exhibited increased transfection efficiency in six different cell lines. Reporter gene expression was found to be increased by a factor of 2.1-110. The pharmacokinetics and biodistribution of 125I-PEI in mice were similar for both molecular weights with an AUC of ca. 330+/-100% ID/ml min. Approximately half of the injected dose accumulated in the liver. LMW-PEI proved to be an efficient gene delivery system in a broad range of cell lines. Due to differences in polyplex structure, as well as its relatively low cytotoxicity, which makes the application of high N/P ratios possible, LMW-PEI appears to possess advantageous qualities with regard to transfection efficiency over PEI of higher molecular weight.

Characterisation of the processing by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and comparison of the substrate specificity of the enzyme for other glucagon-like peptides.

The post-secretory processing of the potent insulinotropic peptide hormone, GLP-1(7-36)amide, probably involves one or more of a small group of membrane-bound ectopeptidases. Reported here, is the characterisation of the endoproteolysis of human GLP-1(7-36)amide by the recombinant human form of neutral endopeptidase (NEP) 24.11, which is one of the best characterised and widely-distributed of ectopeptidases and is involved in the processing of other peptide hormones. The products of the limited endoproteolysis were characterised by mass and primary structure following fractionation using high performance liquid chromatography. The rate of this endoproteolysis by NEP 24.11 was estimated and compared to that of GLP-1(7-36)amide-related peptides. GLP-1(7-36)amide appears to be good substrate for NEP 24.11 with most, but not all potential target bonds being cleaved. Also, the structurally-related peptides, secretin and glucagon appear to be good substrates whereas GIP and exendin-4 are very poor substrates. That the GLP-1(7-36)amide super-agonist, exendin-4 is a poor substrate for NEP 24.11 is significant for the possible use of this peptide as a prototype for the development of clinically-useful peptide agonists. Further studies should reveal whether NEP 24.11 is important for the metabolic clearance of GLP-1(7-36)amide and will be highly relevant for the attempts to realise the suggested therapeutic value of GLP-1(7-36)amide.

The Structure of PEG-Modified Poly(Ethylene Imines) Influences Biodistribution and Pharmacokinetics of Their Complexes with NF-κB Decoy in Mice

AbstractPurpose. To study the relationship between structure of poly(ethylene imine-co-ethylene glycol), PEI-PEG, copolymers and physicochemical properties as well as in vivo behavior of their complexes with NF-κB decoy. Methods. A variety of copolymers of PEG grafted onto PEI as well as PEI grafted onto PEG were synthesized and their complexes with a double stranded 20mer oligonucleotide were examined regarding size, surface charge, biodistribution and pharmacokinetics. Results. Polyplexes of copolymers were smaller compared to polyplexes formed by non-PEGylated PEI 25 kDa (58 - 334 nm vs. 437 nm for a nitrogen/phosphate ratio of 3.5 and 85 - 308 nm vs. 408 nm for N/P 6.0) and showed reduced zeta potential (−2.5 - 6.4 mV vs. 14.5 mV for N/P 6.0). IV injection into mice revealed liver (35-76 % of injected dose), kidney (3 - 22 %) and spleen (2 - 16 %) to be the main target organs for all injected complexes. Complexes formed by copolymers with few PEG blocks of higher molecular weight (5 kDa and 20 kDa) grafted onto PEI 25 kDa did not show different blood levels from PEI 25 kDa. In contrast, a copolymer with more short PEG blocks (550 Da) grafted onto PEI showed elevated blood levels with an increase in AUC of 62 %. Conclusions. A sufficiently high density of PEG molecules is necessary to effectively prevent opsonization and thereby rapid clearance from blood stream.

Human memory and neurohypophyseal hormones: Opposite effects of vasopressin and oxytocin☆

A classical task of experimental psychology, the retention of lists of words, was given twice to three groups of subjects treated with lysine vasopressin (LVP), oxytocin or saline. From a baseline session (no treatment) to a second session with treatment, the LVP and placebo groups showed an enhancement of the number of words remembered correctly, whereas the oxytocin group did not. Rather, oxytocin impaired memory performance. However, we cannot claim a memory enhancing effect of LVP, because placebo treatment enhanced memory performance to the same extent.

Low-Dimensional Dynamics in Sensory Biology 1: Thermally Sensitive Electroreceptors of the Catfish

We report the results of a search for evidence of periodic unstableorbits in the electroreceptors of the catfish. The function of thesereceptor organs is to sense weak external electric fields. Inaddition, they respond to the ambient temperature and to the ioniccomposition of the water. These quantities are encoded by receptorsthat make use of an internal oscillator operating at the level of themembrane potential. If such oscillators have three or more degreesof freedom, and at least one of which also exhibits a nonlinearity,they are potentially capable of chaotic dynamics. By detecting theexistence of stable and unstable periodic orbits, we demonstratebifurcations between noisy stable and chaotic behavior using theambient temperature as a parameter. We suggest that the techniquedeveloped herein be regarded as an additional tool for the analysisof data in sensory biology and thus can be potentially useful instudies of functional responses to external stimuli. We speculatethat the appearance of unstable orbits may be indicative of a stateof heightened sensory awareness by the animal.

ENDOPROTEOLYSIS OF GLUCAGON-LIKE PEPTIDE (GLP)-1(7-36) AMIDE BY ECTOPEPTIDASES IN RINM5F CELLS

This study concerns whether the pancreatic beta cell expresses cell-surface ectopeptidases that are capable of proteolysis of peptide hormones and neuropeptides that modify glucose-dependent insulin release. These biochemical investigations of the RINm5F cell line found that these cells express ectopeptidases. We have characterized the limited endoproteolysis of GLP-1 (7-36) amide that occurs in the presence of RINm5F plasma membranes. The products and the sensitivity to specific peptidase inhibitors of the proteolysis is characteristic of neutral endopeptidase (NEP) 24.11. Vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), amylin, glucagon, glucose-dependent insulinotropic polypeptide (GIP), and exendin-4 also undergo proteolysis in the presence of RIN cell membranes. NEP 24.11-activity in RIN cell membranes was confirmed using a specific fluorogenic assay, by histochemistry, and by comparison with the recombinant enzyme with respect to the kinetics of proteolysis of GLP-1 (7-36) amide and of a fluorogenic substrate. Specific fluorogenic assays revealed the presence of aminopeptidase N and the absence of aminopeptidase A and of dipeptidylpeptidase IV.

Vasopressin but not oxytocin enhances cortical arousal: an integrative hypothesis on behavioral effects of neurohypophyseal hormones.

Behavioral changes after administration of the neurohypophyseal hormones vasopressin and oxytocin can be observed in animal and man. Several groups attempted to specify these changes in terms of memory or attention processing enhancement for vasopressin and amnesic properties for oxytocin. These interpretations, however, were targets for recent criticism. In a double-blind between-subject comparison with male volunteers receiving arginine-vasopressin (AVP), oxytocin or placebo intranasally prior to the experimental session, we tried to develop an alternative hypothesis on the basis of behavioral and EEG measures. At the beginning of the session subjects had to learn a list of 25 unrelated nouns within five trials. Recall was assessed 1 h later. Neither learning nor long-term recall were affected by peptide treatments. In a second vigilance task subjects had to covertly count eight series of tone pips. Averaged auditory evoked potentials to these tones showed the expected habituation during the course of the task within all three groups. Vasopressin-treated subjects, however, displayed significantly higher amplitudes of the vertex potential as compared to the other treatment groups. AVP effects were most prominent with the longest interstimulus interval. No influences on heart rate or blood pressure were found. Results indicate that vasopressin induces an enhancement of stimulus-related phasic cortical arousal, and that in this respect oxytocin has no effect.

Ability of corticotropin releasing hormone to stimulate cortisol secretion independent from pituitary adrenocorticotropin

Cortisol secretion by the adrenal cortex is thought to depend upon a preceding release of pituitary ACTH. This concept ignores a large number of observations suggesting important extrapituitary influences on adrenocortical function. The present study was designed to demonstrate the contribution of these extrapituitary mechanisms in the release of cortisol induced by human corticotropin releasing hormone (hCRH) in man. In patients with proven deficiency in pituitary ACTH the functional atrophy of the adrenals had been restored by pretreatment with long-acting ACTH. Fifty-eight hours after the second and last injection of ACTH a CRH test was performed (100 micrograms hCRH intravenously). Administration of hCRH induced a small but significant increase in plasma cortisol. Surprisingly, this rise was preceded by an increase in plasma ACTH similar to the ACTH response observed in the control group. It appeared that hCRH is able to stimulate cortisol release in the absence of pituitary ACTH, presumably by stimulating extrapituitary sources of ACTH.

Low-Dimensional Dynamics in Sensory Biology 2: Facial Cold Receptors of the Rat

We report the results of a search for evidence of unstable periodic orbits in the sensory afferents of the facial cold receptors of the rat. Cold receptors are unique in that they exhibit a diversity of action potential firing patterns as well as pronounced transients in firing rate following rapid temperature changes. These characteristics are the result of an internal oscillator operating at the level of the membrane potential. If such oscillators have three or more degree of freedom, and at least one of which also exhibits a nonlinearity, they are potentially capable of complex activity. By detecting the existence of unstable periodic orbits, we demonstrate low-dimensional dynamical behavior whose characteristics depend on the temperature range, impulse pattern, and temperature transients.

Neural Synchronization at Tonic-to-Bursting Transitions

We studied the synchronous behavior of two electrically-coupled model neurons as a function of the coupling strength when the individual neurons are tuned to different activity patterns that ranged from tonic firing via chaotic activity to burst discharges. We observe asynchronous and various synchronous states such as out-of-phase, in-phase and almost in-phase chaotic synchronization. The highest variety of synchronous states occurs at the transition from tonic firing to chaos where the highest coupling strength is also needed for in-phase synchronization which is, essentially, facilitated towards the bursting range. This demonstrates that tuning of the neuron’s internal dynamics can have significant impact on the synchronous states especially at the physiologically relevant tonic-to-bursting transitions.