Depletion of metal in the rat hippocampal mossy fibre system by intravital chelation with dithizone

Summary Adult albino rats were given dithizone by repeated intraperitoneal injections followed by a standard test dose. After a standard interval of 15 or 60 minutes the animals were killed by vascular perfusion with a buffered acidified formaldehyde solution. The brains were immediately removed, frozen, and cut at a constant thickness of 160 microns on a freezing microtome.The red stain of metal dithizonate known to be particularly marked in the hippocampal mossy fibre system was observed and recorded by a technique based on photography of sections. It was found that dithizone in doses of 25 mg per kg body weight, injected hourly for 12 hours prevented the dithizone staining of the mossy fibre system caused by the test dose in control animals. The result is interpreted as evidence of true depletion of metal.This study was supported in part by U.S.P.H.S. Grant NS 07998. This aid is gratefully acknowledged. Einar Hansen, Thorkild Nielsen, Birgit Örum, Albert Meier, Karin Sörensen and Inger Madsen rendered valuable technical assistance.     

Important structural features of 15-residue lactoferricin derivatives and methods for improvement of antimicrobial activity.

This review focuses on important structural features affecting the antimicrobial activity of 15-residue derivatives of lactoferricins. Our investigations are based on an alanine-scan of a 15-residue bovine lactoferricin derivative that revealed the absolute necessity of two tryptophan residues for antimicrobial activity. This "tryptophan-effect" was further explored in homologous derivatives of human, caprine, and porcine lactoferricins by the incorporation of one additional tryptophan residue, and by increasing the content of tryptophan in the bovine derivative to five residues. Most of the resulting peptides display a substantial increase in antimicrobial activity. To identify which molecular properties make tryptophan so effective, a series of bovine lactoferricin derivatives were prepared containing non-encoded unnatural aromatic amino acids, which represented various aspects of the physicochemical nature of tryptophan. The results clearly demonstrate that tryptophan is not unique since most of the modified peptides were of higher antimicrobial potency than the native peptide. The size and three-dimensional shape of the inserted "super-tryptophans" are the most important determinants for the high antimicrobial activity of the modified peptides. This review also describes the use of a "soft-modeling" approach in order to identify important structural parameters affecting the antimicrobial activity of modified 15-residue murine lactoferricin derivatives. This QSAR-study revealed that the net charge, charge asymmetry, and micelle affinity of the peptides were the most important structural parameters affecting their antimicrobial activity.     

Hepatocyte growth factor is upregulated by low-density lipoproteins and inhibits endothelin-1 release

Low-density lipoproteins (LDL) are known to cause endothelial injury and to promote the development of atherosclerotic lesions. This study demonstrates a significant concentration-dependent stimulatory effect of LDL on hepatocyte growth factor (HGF) synthesis (maximum release: 423 +/- 16% of control) and HGF receptor mRNA expression in cultured human coronary artery endothelial cells (HCAEC). HGF is a potent mitogen for endothelial cells but does not affect smooth muscle cell proliferation. In contrast, endothelin-1 (ET-1) acts as a mitogen on vascular smooth muscle cells and seems to be upregulated in coronary atherosclerosis. In this study, the basal ET-1 synthesis in HCAEC was concentration-dependently reduced by HGF (minimum: 54 +/- 3% of control). This inhibitory effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met, since it was antagonized by the tyrosine kinase inhibitor lavendustin A. In addition, HGF also significantly reduced the LDL-stimulated ET-1 release. The LDL-induced upregulation of HGF synthesis in HCAEC and the inhibitory effect of HGF on ET-1 synthesis suggest a protective role of HGF in coronary atherosclerosis.     

Toward standardized high-throughput serum diagnostics: multiplex-protein array identifies IL-8 and VEGF as serum markers for colon cancer

Development and progression of colon cancer may be related to cytokines. Cytokines with diagnostic value have been identified individually but have not been implemented into clinical praxis. Using a multiplex protein array, the authors explore a panel of cytokines simultaneously and compared its performance to carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). Serum concentrations of 12 cytokines were simultaneously determined by multiplex biochip technology in 50 colon cancer patients and 50 healthy controls. Serum levels of interleukin-8 (IL-8) and CEA were significantly higher in cancer patients than in healthy controls. Areas under the receiver operating characteristic curves (AUCs) were largest for IL-8, followed by CEA, vascular endothelial growth factor (VEGF), and CA 19-9. Analyses regarding marker combinations showed an advantage over single marker performance for CEA, VEGF, and CA 19-9 but not for IL-8. Multiplex biochip array technology represents a practical tool in cytokine and cancer research when simultaneous determination of different biomarkers is of interest. The results suggest that the assessment of IL-8, CEA, VEGF, and possibly CA 19-9 serum levels could be useful for colon cancer screening with the potential of also detecting early stage tumors. Further validation studies using these and additional markers on a multiplex array format are encouraged.     

Characterization of the tyrosine recombinase MrpA encoded by the <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2) plasmid SCP2*

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to significantly increase the stability of SCP2*-derived plasmids in Streptomyces lividans. The present report gives a functional characterization of MrpA. A sequence alignment revealed that MrpA shares highly conserved residues with members of the tyrosine recombinase family. After overexpression and Strep-tag purification, a DNase I footprint analysis and a gel mobility shift assay allowed for the identification of the 36-bp MrpA binding site mrpS. The mrpS site shows the configuration typical for tyrosine recombinases and contains two MrpA binding sites. The activity of MrpA was explored in vivo in E. coli cells and in vitro using purified MrpA. Depending on the position and orientation of the mrpS sites, three activities were detected: integration, resolution, and inversion. No accessory sites or proteins were required. Substitution of the conserved tyrosine (Y354F) by site-directed mutagenesis resulted in a complete loss of recombination activity but it still allowed the binding of MrpA to mrpS. The results define MrpA as a new site-specific tyrosine recombinase that acts with mrpS. In addition, we suggest that Y354 provides the nucleophile for DNA cleavage during recombination.     

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40–50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and ¹³C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. ¹³C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20–24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.     

Effects of cortisol, 17beta-estradiol and thyroliberin on prolactin and growth hormone production, cell growth and cell cycle distribution in cultured rat pituitary tumour cells.

Prolactin and growth hormone production were measured in a rat pituitary tumour cell strain (GH₃) after treatment with cortisol (5 × 10^?6 M), thyroliberin (2.5 × 10^?6 M) and 17β-estradiol (10^?6 M). The changes in hormone production were related to alterations in cell growth rate and cell cycle distribution. Cortisol inhibited prolactin production, stimulated growth hormone production and reduced the cellular growth rate measured two days after start of treatment (maximum about 40% inhibition). Flow-micro fluorometric analysis of DNA distributions showed that cortisol treatment reduced the relative number of cells in S phase (maximum effect about 50%) with a compensatory increase of the proportion of cells in G₁ phase. The lack of inhibition of prolactin production after three days of cortisol treatment may partly be related to the increased number of cells in G₁ phase. Thyroliberin and 17β-estradiol did not significantly affect cell growth after six days of treatment, although the fraction of cells in S phase was reduced by approximately 40% with a corresponding increase of cells in G₁ phase. For thyroliberin and 17β-estradiol, the stimulatory effect on prolactin production and the inhibitory effect on growth hormone production witin a period of treatment of six days cannot be explained by a shift in cell cycle distributions. None of the three hormones influenced the growth fraction which was equal to unity. In conclusion, thyroliberin and 17β-etradiol are able to change prolactin and growth hormone production without altering the cell cycle distribution. However, the effects of cortisol on prolactin and growth hormone production may partly be due to an alteration in cell cycle traverse resulting in an increased number of cells in the G₁ phase.     

Dilatory and inotropic effects of corticotropin-releasing factor (CRF) on the isolated heart. Effects on atrial natriuretic peptide (ANP) release.

The effects of CRF administration on cardiac performance, coronary flow and ANP release were investigated in the rat heart. Isolated hearts were perfused at a constant filling pressure according to working heart model with a Krebs-Henseleit solution containing glucose and insulin, saturated with a gas mixture containing 95% O2 and 5% CO2. Administration of CRF via a cannula into the left atrium elicited a prolonged increase in the coronary flow rate and a transient increase in the aortic pressure resulting in an overall increase in the pressure-volume work. The oxygen consumption, after the administration of CRF, increased in accordance with the cardiac effort. No changes were observed in the spontaneous heart rate. Furthermore, administration of CRF induced a short-term increase of ANP release into the coronary perfusate. Our experiments suggest that administration of CRF produces a prolonged dilatory effect on the coronary arteries while producing a transient positive inotropic effect and a transient increase of ANP release on the isolated rat heart.     

Invertebrate drift in a glacial river and its non-glacial tributary

1. Invertebrate drift was studied in a glacially fed river and a non-glacial tributary in western Norway. Samples were taken during two consecutive 24-h periods in May, July and October 1997. The 3 months are characterized by snowmelt, ice melt and rainfall runoff, respectively. The main glacial river has colder, more turbid water, especially during the period of maximum ice melt during summer.
2. Chironomidae, especially the genus Diamesa , dominated the drift in the main river in May and October, constituting 97 and 99% of total numbers, respectively. Simuliidae, Plecoptera, Ephemeroptera and Trichoptera were the other main components.
3. A comparison of drift and benthos data revealed that the tributary was of little significance for colonization of the main glacial river. Only some additional species in very low numbers were recorded downstream of the confluence.
4. During July significant differences in diel drift pattern of Chironomidae and Simuliidae existed between the glacial and non-glacial reaches. There was a mid-day peak independent of discharge in the glacial river, but this peak was not noted in the tributary. Species of the genus Diamesa appear to be adapted for daytime drift, possibly evolved through the absence of predators and competitors that are typical of rhithral systems where nocturnal drift is more usual.     

Analysis of the electrical responses of antennal thermo- and hygroreceptors of Antheraea (Saturniidae,Lepidoptera) to thermal,mechanical, and electrical stimuli

Summary Antennal styloconic thermo-hygro sensilla of Antheraea were studied with DC-coupled transepithelial recordings. — The transepithelial voltage changed by about 2 mV · °C^–1. The spike frequency of the cold cell reached 300 Hz at the onset of negative temperature steps, but only 30 Hz at static temperatures (as with metal electrodes). The cold cell spikes showed a brief afterhyperpolarization that increased with temperature. The spikes of the cold- and warm-stimulated cells facilitated each other at low temperature. Mechanical stimuli (push against the sensillum, hydrostatic pressure of < ± 50 kPa, ultrasonic vibrations 120 kHz) modified the responses of the cold- and the warm-stimulated cells. Latency of cold cell responses to ultrasonic stimulation was occasionally less than 3 ms. — The impulse frequencies of the warm and the cold cells depend on the temperature and the magnitude of temperature change. When the firing rate is high enough by either or both of these parameters, it can be forced still higher by application of clamp current (outside positive). The higher the firing rate prior to clamping, the greater the effect of the current. — By analogy with sensilla for other modalities, this relationship between frequency and clamp current strongly suggests that stimulus-dependent changes in the conductance of dendritic membranes control the excitation of the warm and cold cells.Abbreviations DC direct voltage - TER transepithelial slope resistance between recording electrode and reference electrode in the hemolymph - NTC thermistor with negative temperature coefficient - TEV transepithelial voltage between electrodes - THS thermo-hygro sensillum