Heteronuclear correlation experiments for the determination of one-bond coupling constants

Spin-state selective experiments, HSQC-/ and CT-HMQC-/, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional,¹ H-detected ¹⁵N-¹H or¹³ C-¹H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra.     

Membranotropic effects of peptides from the V3 loop of HIV-1

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell–cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the pH and the of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an -helix/-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus–cell fusion.     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

CD studies of peptides that mimic the four helicoidal sequences of the uteroglobin monomer

Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P2₁ and C222₁) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P2₁). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process.     

Polymerization of the Actin Component of the Cytoskeleton and Formation of the Geometry of a Cultured Nerve Cell (a Model Study)

Using mathematical modeling, we studied the biophysical aspects of the growth of the cell membrane and the growth of the actin network of the cytoskeleton of a neuron cultured on the rigid sublayer and the correlation between these processes. To describe the dynamics of the growth of the cytoskeleton limited by the cell membrane, we used the model of the thermal ratchet. Using the approaches of theoretical biophysics, we obtained a simple biophysical criterion that governs the selection of an alternative scenario of the formation of the cell, either growth of a single neurite or growth of a number of neurites. This criterion depends on the value of the adhesion between the cell and the substrate, the dimension of the actin monomer, and the thermal energy determining the frequency of thermal fluctuations of the cell membrane.     

Field experiments on the optical orientation of pelagic rotifers

Avoidance of shore by pelagic rotifers is considered to be the result of an optical orientation. Field experiments show that the spatial light distribution in the shore region determines the preferred direction of migration. The behaviour of Eudiaptomus gracilis was tested in comparison to that of rotifers.This publication is dedicated to Pater Dr. Josef Donner on the occasion of his 70th birthday     

Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions

Summary Nitrogen indole protection of the -methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for -methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions.     

The state of the environment of the Loosdrecht lakes

The Loosdrecht lakes are a system of shallow, interconnected, peat lakes in the centre of The Netherlands. The main environmental functions of the Loosdrecht lakes are nature and recreation. From the point of view of the Dutch policy, a Specific Environmental Quality (Bijzondere Milieukwaliteit) should be set for these lakes.The most serious environmental problem of the area is eutrophication. The Loosdrecht lakes have, by increasing external phosphorus loading, changed, from clear lakes with few macrophytes, followed by a period of abundant characean growth, to turbid lakes dominated by cyanobacteria and detrital matter. Eutrophication was counteracted by use of sewerage systems and dephosporization of the supply water. The resultant decrease in external phosphorus loading did not result in a decrease of turbidity by suspended particles.The eutrophication of the lake ecosystems was described as a series of phases. One of those phases, the status around 1940, has been used as an ecological reference system.By means of a graphical presentation technique, the so-called AMOEBE-approach, the state of the environment of the Loosdrecht lakes has been visualized. Thirty-two ecological parameters, including both biotic and abiotic factors, have been selected and quantified. Concrete target values for these parameters have been derived from historical reports and from Lake Western Loenderveen, located close to the Loosdrecht lakes, but less eutrophic.The general conclusion is that the state of the environment of the Loosdrecht lakes is far from what is required with respect to a Specific Environmental Quality, as many of the selected parameters, like water transparency, total phosphorus, mineral nitrogen, cyanobacteria, bream, pike, macrophytes, birds and otter, deviate by over an order of magnitude from their desired levels.     

Modifications of the EEG Activity upon Lateralized Stimulation of the Visual Inputs to the Right and to the Left Brain Hemispheres by Light with Different Wavelengths

In 80 healthy humans, we studied changes in the frequency spectrum and values of the relative spectral coefficients for subsequent 1-Hz-wide frequency bands under conditions of simultaneous lateralized stimulation of the retinal zones, which form receptive fields for the right- and for the left-brain hemispheres; light of different colors was used for stimulation. We found that reactions of the right and left hemispheres to such stimulations demonstrated obvious specificity; spatial characteristics of these reactions were obtained. Changes in the values of most corresponding spectral coefficients in the hemispheres usually had similar directions and demonstrated similarity in their intensities, which is indicative of a complementary pattern of the interhemispheric interaction. Reciprocal changes in the corresponding spectral coefficients in the hemispheres were observed more rarely. Modifications of EEG upon red-green stimulations of different polarity were found to be similar, which can be a manifestation of the moderating influence of the right hemisphere on the left one.     

Cytoarchitectonic pattern of the hypothalamus in the cobra,Naja naja

Summary The distribution and cytoarchitectonic pattern of the magno- and parvocellular hypothalamic nuclei of the cobra, Naja naja, are described at the light-microscopic level. With respect to their tinctorial affinity to paraldehyde fuchsin (AF) as a representative of the Gomori-type of stains, the magnocellular neurons belong to the AF-positive and the parvocellular neurons to the AF-negative elements. In addition to the supraoptic and paraventricular nuclei proper, two accessory aggregations of magnocellular neurons, the nucleus retrochiasmaticus and nucleus circularis, can be identified. Although in a peculiar location, they may be regarded as subunits of the supraopticoparaventricular neurosecretory complex. As many as 22 AF-negative nuclear areas are identified in the hypothalamus of the cobra. The nucleus periventricularis hypothalami of earlier authors is subdivided into several circumscribed neuronal complexes. The nucleus arcuatus, nucleus hypothalamicus lateralis and nucleus lateralis recessus infundibuli are well developed. An attempt is made to interpret the significance of these nuclei on a comparative and phylogenetic basis.On leave from the Department of Zoology, Nagpur University, Nagpur, India     

The Activities of Six Exo- and Endopeptidases in the Substantia Nigra,Neostriatum, and Cortex of the Rat Brain

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and -Glutamyl transpeptidase (GTP), and two endopeptidases: Postproline endopeptidase (PEP) and Trypsin-like peptidase (T-L P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) GTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a Trypsin-like peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Low-level arsenite activates the transcription of genes involved in adipose differentiation

In this study we analyzed gene expression in 3T3-F442A pre-adipocyte cells that differentiate in the presence of micro-molar arsenate concentration. Two concentrations of arsenite (As2O3, 0.25 micromol/L and 0.5 micromol/L) were applied for three days with and without insulin (170 nmol/L) and gene expressions were evaluated by quantitative RT-PCR. The genes included genes of oxidative-stress responses: heme-oxygenase-1 (HO1) and the hypoxia inducible factor 1a (HIF1alpha), genes of cell-cycle: c-jun and Kruppel like factor 5 (KLF5), and genes that play important roles in adipose determination: a peroxisome proliferator-activated receptor (PPARgamma) and a CCAAT/ enhancer binding protein (C/EBPalpha). Arsenite induced the expression of HO1, HIF1alpha, KLF5, PPARgamma and C/EBPalpha. These results suggest that under condition of oxidative stress arsenite induces genes that are required for adipose differentiation.     

Folding versatility of the C-terminal tetrapeptide amide sequence of the lipopeptaibol antibiotics trichodecenin and trichogin

Summary An X-ray diffraction analysis ofZ-l-Leu-Aib-Gly-l-Ile-l-Leu-OMe, containing the N-acylated tetrapeptide amide sequence-l-Leu-Aib-Gly-l-Ile-, showed that in the crystal state the carbonyl group preceding thel-Leu¹ residue acts as the acceptor of two C=OH–N intramolecular H-bonds, which give rise to an-l-Leu¹-Aib²-type-III' -turn and an-l-Leu¹-Aib²-Gly³-l-Ile⁴--turn, respectively. A second (type-I') -turn encompasses the-Aib²-Gly³-sequence. This is the third type of folding motif known for that tetrapeptide sequence, considering also those already published for the C-terminal segment of the lipopeptaibol antibiotics trichodecenin I and trichogin A IV.     

Subunit Composition of Nicotinic Acetylcholine Receptors in the Neurons of Autonomic Ganglia

The distribution of subunits in different parts of the autonomic nervous system and the correlation between physiological functions of autonomic ganglion neurons and subunit composition of the nAChR are the subjects of their analysis. In particular, it was found that functionally different autonomic ganglia differ from each other in the subunit composition of their nAChR, and the sensitivity of the nAChR to specific antibodies is markedly variable even within the same ganglion.     

Distribution of two types of lymphocytes (intraepithelial and lamina-propria-associated) in the murine small intestine

The intestine, which is exposed to nutrition and to food-derived antigens and microbes including viruses and bacteria, might be an important site for the immune response. Crucial structural and functional differences exist between the small and large intestine, regional differences even having been demonstrated within the small intestine. Accordingly, intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) might be heterogeneous among the different intestinal regions. The aim of this study has been to describe, as accurately as possible, the numbers and T-cell receptor (TCR) phenotypes of IELs and LPLs present in distinct regions of the murine small intestine under physiological conditions. Using an immunohistological technique to differentiate IELs from LPLs, the differential enumeration of IELs and LPLs in distinct regions of the murine small intestine, based upon their definition originally determined by their location, has been performed for the first time and has demonstrated that (1) there are more IELs than LPLs in the duodenum and jejunum, but more LPLs than IELs in the ileum, (2) in the duodenum and jejunum, TCR IELs account for 70%–75% of the total CD3⁺ IELs, a much greater percentage than previously reported, (3) the ratio of TCR to TCR IELs is inverted in the ileum, with more than 75% IELs being TCR-positive, (4) the lamina propria forms one functional unit throughout the small intestine in terms of the TCR subset components (TCR:TCR=3:1), and (5) the ileum is entirely different from other regions of the small intestine. To deepen our understanding of the functional significance of the small intestine as an immunologically competent organ, the precise distributions of IELs and LPLs, the ratio of their various subsets, and the strict distinction of IELs and LPLs, as described in this study, is indispensable.This work was in part supported by a Grand-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (07407066, 10470002, and 13670002 to T.I.)     

Reduction of Glutamate Uptake into Cerebral Cortex of Developing Rats by the Branched-Chain Alpha-Keto Acids Accumulating in Maple Syrup Urine Disease

In the current study we investigated the effect of the branched-chain alpha-keto acids (BCKA) co-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV), and alpha-ketoisovaleric (KIV) acids, which accumulate in maple syrup urine disease (MSUD), on the in vitro uptake of [3H]glutamate by cerebral cortical slices from rats aged 9, 21, and 60 days of life. We initially observed that glutamate uptake into cerebral cortex of 9- and 21-day-old rats was significantly higher, as compared to that of 60-day-old rats. Furthermore, KIC inhibited this uptake by tissue slices at all ages studied, whereas KMV and KIV produced the same effect only in cortical slices of 21- and 60-day-old rats. Kinetic assays showed that KIC significantly inhibited glutamate uptake in the presence of high glutamate concentrations (50 microM and greater). We also verified that the reduction of glutamate uptake was not due to cellular death, as evidenced by tetrazolium salt and lactate dehydrogenase viability tests of cortical slices in the presence of the BCKA. It is therefore presumed that the reduced glutamate uptake caused by the BCKA accumulating in MSUD may lead to higher extracellular glutamate levels and potentially to excitotoxicity, which may contribute to the neurological dysfunction of the affected individuals.     

The role of macrophages in the tissues regeneration stimulated by the biomaterials

Allogenic grafted tissues are subjected to biodegradation and replaced by the regenerate. To minimize the immune response and improve the rebuilding of tissues there was developed a technology to treat tissues with a cells elimination and dosed out extraction of proteoglycanes (Alloplant^®). With aim to clarify the role of macrophages in the tissues regeneration resulting implantation the biomaterials 112 rats were injected the allogenic and xenogenic (rabbits) pulverized biomaterials in the form of suspension. Injections were performed subcutaneously into the animals back by the base of the tail. The control group (14 rats) were injected a physiologic saline. Animals were killed by ether inhalation on day 2, 4, 7, 14, 30, 90 and 180 and tissue sections were studied by light and electron microscopy. The study showed the key role of the macrophages in resorption of the allogenic biomaterial and formation of the newly-formed tissue. Implantation of the biomaterial induced activity a great number of the mature macrophages, which completely lysed and resorbed the biomaterial particles. Expression TNF was significantly higher whereas expression TGF-1 was significantly lower. With xenogenic biomaterial implantation there were less macrophages, their activity was restricted. Macrophages containing large vacuoles with an active endo- and exocytosis were revealed in the allogenic biomaterial implantation and were named matrix-forming macrophages. We may suppose that these macrophages synthesize (or re-synthesize) proteoglycan component of the newly-formed collagen fibers. There was put forward a hypothesis about the two component mechanism of the collagen fibers formation.