Mapping of the spectral density function of a Cα−Hα bond vector from NMR relaxation rates of a 13C-labelled α-carbon in motilin

Summary The peptide hormone motilin was synthesised with a ¹³C-enriched -carbon in the leucine at position 10. In aqueous solution, six different relaxation rates were measured for the ¹³C–H fragment as a function of temperature and with and without the addition of 30% (v/v) of the cosolvent d ₂-1,1,1,3,3,3-hexafluoro-2-propanol (HFP). The relaxation rates were analysed employing the spectral density mapping technique introduced by Peng and Wagner [(1992) J. Magn. Reson., 98, 308–322] and using the model-free approach by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570]. The fit to various models of dynamics was also considered. Different procedures to evaluate the overall rotational correlation time were compared. A single exponential time correlation function was found to give a good fit to the measured spectral densities only for motilin in 30% (v/v) HFP at low temperatures, whereas at high temperatures in this solvent, and in D₂O at all temperatures, none of the considered models gave an acceptable fit. A new empirical spectral density function was tested and found to accurately fit the experimental spectral density mapping points. The application of spectral density mapping based on NMR relaxation data for specific ¹³C–¹H vector is shown to be a highly useful method to study biomolecular dynamics. Advantages are high sensitivity, high precision and uniform sampling of the spectral density function over the frequency range.Abbreviations CD circular dichroism - NOE nuclear Overhauser enhancement - NOESY two-dimensional NOE spectroscopy - INEPT insensitive nuclei enhanced by polarisation transfer - DANTE delays alternating with nutations for tailored excitation - WALTZ-16 wideband, alternating phase, low-power technique for zero residual splitting - FID Free induction decay - ppm parts per million - TSPA 3-trimethylsilyl-(3,3,2,2-d)-propionic acid - HFP d ₂-1,1,1,3,3,3-hexafluoro-2-propanol - CPMG Carr-Purcell-Meiboom-Gill - TFD time-resolved fluorescence depolarisation - CSA chemical shift anisotropy Partly presented at the symposium Dynamics and Function of Biomolecules, Szeged, Hungary, July 31–August 2, 1993.     

Neural and smooth muscle development in the chicken gizzard

The development of the autonomic ganglia of Auerbach's plexus and gizzard smooth muscle was studied in chicken embryos. Nervous system and smooth-muscle-specific antibodies were employed in immunofluorescence stainings on tissue sections to investigate the temporal and spatial frame of neural and muscular differentiation in relation to each other. Subserosal clusters of neural cells were clearly demonstrable at embryonic day 5 (ED5), the earliest stage analysed, with the monoclonal antibody El (SGIII-1). Fine nerve fibres (ED6) and, later, large axon bundles projecting from subserosal neuron clusters towards the lumen were followed and found to reach the luminal border by ED11. Already in early development the area of the future laminar tendons on the ventral and dorsal surface of the gizzard was devoid of neuroblasts, and nerve fibres were not extending to the muscle-tendon borderline until ED16. Double stainings with antibodies to smooth muscle myosin (SMM) and El revealed that SMM expression, taken as an indicator for muscle differentiation, followed neural growth. It was first detectable in close apposition to the differentiating neuroblasts in the caudal and cranial portion of the gizzard at ED6. With further development, myosin expression proceeded inward towards the lumen in a wave which followed the ingrowth of E1-positive nerve fibres from the prospective Auerbach plexus. Neuromuscular differentiation deviated from this pattern in the lateral tendon area where nerve growth was delayed and myosin expression preceeded the arrival of E1-positive nerve fibres. The findings suggest that the gizzard could serve as a model system for the analysis of potential early nervous system imprints on smooth premuscle mesenchyme differentiation.     

Distribution of negatively charged liposomes on rat liver hepatocytes and non-hepatocytes in cell suspension

The in vitro interactions between negatively charged multilamellar liposomes and purified rat liver parenchymal and non-parenchymal cells were studied. The liposomes were labelled with [¹⁴C]cholesterol and contained [³H]methotrexate. For both cell types the time course of liposomal attachment to the cells slowed down gradually after a rapid initial phase lasting ca 90 min. The rate of attachment at 4 °C was 3–7 times lower than that at 37 °C, and the metabolic inhibitors dinitrophenol and iodoacetic acid caused reduction of 20–30%. Up to 45% of the cell-associated liposomal radioactivity could be detached within 1 h incubation with unlabelled liposomes. Whereas liver parenchymal cell suspension seemed to exhibit similar characteristics in vitro as in vivo, the non-parenchymal cells in vitro showed a 20–50-fold reduction in the rate of liposomal attachment compared to in vivo.     

REGIONAL CHANGES IN CEREBRAL GABA CONCENTRATION AND CONVULSIONS PRODUCED BY d AND BY l-ALLYLGLYCINE

Abstract— The convulsant action of allylglycine (2-amino-4-pentenoic acid) is due to the metabolic conversion of allylglycine to 2-keto-4-pentenoic acid, a more potent glutamic acid decarboxylase inhibitor and more potent convulsant than the parent compound. We report regional changes in cerebral GABA concentration in rats after administration of d - and l -allylglycine. d -Allylglycine (3.75 mmol/kg) induced convulsions in 95–115 min, characterised by repeated clonic limb movements and rapid rotation around the head to tail axis. GABA concentrations were only reduced in cerebellum and ponsmedulla during the pre and post-convulsive periods. The localised reduction of GABA concentration is consistent with the enzymic conversion of d -allylglycine to 2-keto-4-pentenoic acid catalysed by cerebral d -amino acid oxidase, an enzyme known to be localised to the hind brain and spinal cord. l -allylglycine (1.2mmol/kg i.p.) induced convulsions in 65 -90 min, characterised by violent running followed by tonic flexion and extension. During the pre-convulsive period, GABA concentrations were reduced in all brain areas studied except the globus pallidus and ventral midbrain. The widespread decreases in GABA concentration suggest that the enzyme(s) which catalyse the conversion of l -allylglycine to 2-keto-4-pentenoic acid are widely distributed within the brain.     

THE KINETICS OF CONTACT INHIBITION IN MAMMALIAN CELLS

To elucidate the process of contact inhibition in mammalian cells, we investigated the kinetics of growth arrest in [³H]thymidine labelled embryonic chinese hamster (Cricetulus griseus L.) cells after the addition of various concentrations of unlabelled cells. It was observed that after the contact inhibition concentration had been reached, the cells grew undisturbed for one more generation. In the following 24 hr the concentration fell back to the level at the beginning of the experiment and stayed there.     

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temperature influence on life table statistics of the chicken miteDermanyssus gallinae (Acari: Dermanyssidae)

An age-specific life table for the chicken miteDermanyssus gallinae (DeGeer, 1778) was based on various observations carried out at 25°C. The generation time was calculated to be 16.8 days; the intrinsic rate of natural increase was 0.12 per day, and the net reproductive rate was 7.2. A nonlinear function was found satisfactory to describe the developmental rate of the different immature lifestages at temperatures below 40°C. The stage-specific survival of the immature life-stages was generally high between 10 and 37°C, but decreased quickly outside this temperature range. Most of the eggs were laid during the first three weeks of the adult life. The proportion of surviving females rapidly decreases after moulting to the adult stage. At a temperature of 30°C, the highest number of eggs (3 eggs per day) was laid.     

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) with a total length of 3748 bp by screening of a human thyroid carcinoma lambda gt11 library using two heterologous oligonucleotides to conserved regions which derived from frog skin and bovine pituitary PAM sequences. Furthermore we have identified a sequence which differs in a 321 bp deletion. COS cells transfected with a truncated form of this cDNA (lacking the putative carboxyl-terminal transmembrane domain) generated a functional PAM that showed a 20-fold increase of the activity compared to the control and was visualized by immunoblotting.     

Activation of Retinal Tyrosine Hydroxylase In Vitro by Cyclic AMP-Dependent Protein Kinase: Characterization and Comparison to Activation In Vivo by Photic Stimulation

Abstract: Tyrosine hydroxylase in rat retina is activated in vivo as a consequence of photic stimulation. Tyrosine hydroxylase in crude extracts of dark-adapted retinas is activated in vitro by incubation under conditions that stimulate protein phosphorylation by cyclic AMP-dependent protein kinase. Comparison of the activations of the enzyme by photic stimulation in vivo and protein phosphorylation in vitro demonstrated several similarities. Both treatments decreased the apparent K _m of the enzyme for the synthetic pterin cofactor 6MPH₄. Both treatments also produced the same change in the relationships of tyrosine hydroxylase activity to assay pH. When retinal extracts containing tyrosine hydroxylase activated either in vivo by photic stimulation or in vitro by protein phosphorylation were incubated at 25°C, the enzyme was inactivated in a time-dependent manner. The inactivation of the enzyme following both activation in vivo and activation in vitro was partially inhibited by sodium pyrophosphate, an inhibitor of phosphoprotein phosphatase. In addition to these similarities, the activation of tyrosine hydroxylase in vivo by photic stimulation was not additive to the activation in vitro by protein phosphorylation. These data indicate that the mechanism for the activation of tyrosine hydroxylase that occurs as a consequence of light-induced increases of neuronal activity is similar to the mechanism for activation of the enzyme in vitro by protein phosphorylation. This observation suggests that the activation of retinal tyrosine hydroxylase in vivo may be mediated by phosphorylation of tyrosine hydroxylase or some effector molecule associated with the enzyme.