Effects of UV-B radiation and nitrogen starvation on enzyme activities in isolated plasma membranes of Euglena gracilis

Circadian rhythms are characteristic of many physiological and biochemical processes in the freshwater flagellate Euglena gracilis. Earlier, we found that the rhythms of photosynthesis, phototaxis and cell shape followed the same pattern in control organisms, but were differently affected by stress such as UV-B irradiation and nitrogen deficiency. Here we extend our studies to use isolated plasma membranes to characterize the rhythms of some plasma membrane-bound enzymes. Also, we wanted to see whether stress-induced changes of these rhythms could be detected at the subcellular level and possibly be coupled to the changes seen in photosynthesis, phototaxis and cell shape. The isolation of plasma membranes using aqueous polymer two-phase partitioning was successful, as judged by the large enrichment of the plasma membrane-marker 5′-nucleotidase, and the difference in the polypeptide pattern compared with the microsomal fraction from which it was prepared. Two other enzymes were analyzed, K⁺, Mg²⁺-ATPase, and adenylyl cyclase. The specific activities of all three enzymes were decreased by UV-B radiation by ca 30–50%, compared with the control cultures. On the other hand, nitrogen deficiency not only reduced the activity of the K⁺.Mg²⁺-ATPase but also increased the activities of the 5′-nucleotidase and adenylyl cyclase. The different treatments also resulted in differences in polypeptide pattern, e.g., a polypeptide around 30 kDa seemed to be specific to plasma membranes of nitrogen-deficient cultures and one at 39 kDa for the UV-B radiated ones. All three enzymes showed diurnal rhythms that were affected by UV-B radiation. The peak in the rhythm of the ATPase was shifted by UV-B radiation, the rhythm of the 5′-nucleotidase nearly eliminated. The first peak of adenylyl cyclase activity was delayed, so that it looked more like a broad peak between 2 and 11 h after the onset of light. The rhythm of ATPase activity could be correlated with that of photosynthesis in both control and UV-B irradiated cultures. Also, the rhythms of adenylyl cyclase activity and cell shape changes showed some similarities.     

Constrained C-terminal hexapeptide neurotensin analogues containing a 3-oxoindolizidine skeleton

Summary In order to enforce different spatial orientations in the C-terminal hexapeptide of neurotensin (NT_8–13) and to gain information about the importance of the 10–11 peptide bond for binding to NT receptors, the Pro¹⁰-Tyr¹¹ fragment has been replaced with (2R,8S,8aR)-, (2S,8S,8aR)-, (2S,8S,8aS)-, (2S,8R,8aS)- and (2R,8R,8aS)-8-amino-2-benzyl-3-oxoindolizidine-2-carboxylic acid. Molecular dynamics calculations and energy minimization studies have shown that, contrarily to the Pro-Tyr moiety, none of these indolizidines display a tendency to adopt type I and III -turns, but those having (8S,8aR) or (8R,8aS) stereochemistry essentially adopt extended conformations and the (8S,8aS) stereoisomer prefers a nonstandard folding. The four diastereomeric NT_8–13 analogues incorporating (8S,8aR) or (8R,8aS) indolizidines displayed binding affinities for the brain NT receptor similar to that of [Ala¹¹]-NT_8–13 and only five- to ninefold lower than that of the corresponding analogue, [Phe¹¹]NT_8–13. Although this slight decrease could be attributed to differences in conformational behavior between these constrained NT_8–13 analogues and [Phe¹¹]NT_8–13 or NT_8–13, it is not clear whether the -turn around Pro¹⁰-AA¹¹ (AA=Phe, Tyr) is conserved upon receptor binding. An excessive restriction in the motions of the aromatic side chain, imposed by the highly steric constraint of the indolizidine moiety, emerges as an alternative explanation. The findings reported here demonstrate the possibility of replacing the Pro¹⁰-Tyr¹¹ dipeptide in NT_8–13 with a non-peptide residue without affecting considerably the affinity for brain NT receptors.     

Catecholamine-synthesizing enzymes in the rat stomach

Local production of catecholamines in the stomach of the rat was studied by immunohistochemical demonstration of tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), the enzymes catalyzing the formation of dopamine, noradrenaline and adrenaline, respectively. A rich innervation of TH- and DBH-immunoreactive nerve fibers was seen in the muscular layers and the myenteric plexus, in the submucosa and in the walls of submucosal blood vessels and in the lamina propria at the base of the epithelial layer. In addition, TH-, but not DBH-immunoreactive nerve fiber networks surrounding ganglion cells in the myenteric plexus were frequently observed, indicating dopaminergic preganglionic innervation of the myenteric plexus. In the oxyntic epithelium, single TH- and DBH-immunoreactive fibers extended in the strands of lamina propria as far as the middle portion of the gastric glands. A small population of single angulate cells in the oxyntic epithelium showed TH-, but not DBH-immunoreactivity. No specific PNMT immunoreactivity was observed.     

Synthesis of acetylenic sugars and uronic acids via two-carbon chain extension of d-ribose

Treatment of methyl β-d-ribofuranoside with acetone gave methyl 2,3-O-isopropylidene-β-d-ribofuranoside (1, 90%), whereas methyl α-d-ribofuranoside gave a mixture (30%) of 1 and methyl 2,3-O-isopropylidene-α-d-ribofuranoside (1a). On oxidation, 1 gave methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside (2), whereas no similar product was obtained on oxidation of 1a. Ethynylmagnesium bromide reacted with 2 in dry tetrahydrofuran to give a 1:1 mixture (95%) of methyl 6,7-dideoxy-2,3-O-isopropylidene-β-d-allo- (3) and -α-l-talo-hept-6-ynofuranoside (4). Ozonolysis of 3 and 4 in dichloromethane gave the corresponding d-allo- and l-talo-uronic acids, characterized as their methyl esters (5 and 6) and 5-O-formyl methyl esters (5a and 6a). Ozonolysis in methanol gave a mixture of the free uronic acid and the methyl ester, and only a small proportion of the 5-O-formyl methyl ester. Malonic acid reacted with 2 to give methyl 5,6-dideoxy-2,3-O-isopropylidene-β-d-ribo-trans-hept-5-enofuranosiduronic acid (7).     

A Qualitative and Quantitative Study of a Sensory Epithelium in the Inner Ear of a Fish (Colisa labiosa; Anabantidae)

The macula lagenae of the anabantide fish Colisa labiosa was studied with light and transmission electron microscopy. (1) The sensory area is naturally divided in a central area (A) surrounded by a peripheral part (B). (2) Generally the central hair cells are separated by supporting cells, while the peripheral hair cells are found in groups. The cells of a group are not separated by supporting cells. (3) Tubuli-like structures, hexagonal in cross section, are found in all cells. In peripheral hair cells the longitudinally oriented tubuli-like structures are aggregated in thick bundles. (4) Variation in shape, electron density, stereocilia arrangement and size of mitochondria was found in different hair cells. (5) The central hair cells contain large accumulations of presynaptic bodies (10–44). Contrarily, the peripheral hair cells contain only a few pre-synaptic bodies (1–3). (6) The central hair cells are innervated by thick afferent (6–15 μm) and fine presumed efferent (less than 1 μm nerve fibres, while the peripheral hair cells are innervated by thin (1–6 μm) afferent nerve fibres only.     

Structural studies of the polysaccharide antigen of Eubacterium saburreum, Strain L 32

The structure of the polysaccharide antigen produced by Eubacterium saburreum, strain L 32, has been investigated. The principal methods used were methylation analysis, graded hydrolysis with acid, and n.m.r. spectroscopy. The polysaccharide, which contains the unusual sugar 3,6-dideoxy-D-arabino-hexose (tyvelose, Tyv), is composed of trisaccharide repeating-units having the following structure:

     

Chronic Copper Poisoning in Sheep

Four cases of experimental copper poisoning in sheep were examined. Light microscopical and ultrastructural alterations of erythrocytes were observed a few hours before a significant haemolysis was evident. Heinz body formation in otherwise unchanged red cells was the first morphological alteration observed. The Heinz bodies were predominantly membrane-attached. During the haemolytic crisis severe erythrocytic distortion, structural membrane alteration and Heinz body-containing ghost cells were observed. Erythrophagocytosis was mainly located to the RE cells of the spleen. Intrafollicular necroses were found in all histological sections from the spleen. Pathological changes in liver and kidney were comparable to those of earlier reports, comprising hepatocellular and renal tubular necrosis.     

Histochemical observations on uptake of L-dopa into endocrine cells of the rat pituitary gland during the postnatal development

The uptake of L-dopa into the cells of the adenohypophysis of the rat was studied during the postnatal development and at adult age using the formaldehyde-induced fluorescence method (FIF). The cells taking up L-dopa were classified by Alcian blue-PAS-Orange G staining. The correlation between the cells taking up L-dopa and those containing tryptophyl-peptide was estimated during the postnatal period and in adult rats. The cells containing tryptophyl-peptide were demonstrated using fluorescence induced by treatment with combined formaldehyde and acetyl chloride vapour. The following observations were made: 1) Great majority of the cells taking up L-dopa did not contain tryptophyl-peptide. Thus the accumulation of L-dopa into the cells of pars distalis is not due to accumulation of L-dopa into the cells by the same transport mechanism as the amino acids for tryptophyl-peptide. 2) Of the cells taking up L-dopa in the adult rats 96% were chromophobes, 2.0% acidophilic cells (somatotrophs and cells producing prolactin), 0.9% R-mucoid cells (corticotrophs), and 1.2% S1- and S2-mucoid cells (gonadotrophs and thyrotrophs). At 10 and 25 days' age the relative numbers of the cells taking up L-dopa were about the same. 3) Pretreatment with nialamide caused only a slight increase in the number of the cells taking up L-dopa. The decrease in the number of the cells uptaking L-dopa of the pars distalis, which takes place after 5 weeks' age is thus not caused by the increased MAO-activity. 4) Strongly chromophilic cells did not take up L-dopa. At the light of our results it seems evident that L-dopa is taken up by the chromophobic cells when these differentiate into chromophilic cells. The accumulation of L-dopa may be a sign of an active transport of amino acids into the cells. The accumulation of L-dopa into the chromophobic stellate and follicular cells may reflect their metabolic activity. These cells probably have an important role in the production of the hormones of the pars distalis.     

Family studies of hereditary hemochromatosis in Denmark and the Faroe Islands

Summary Pedigree studies were performed based on one Faroese and four Danish probands with overt idiopathic hemochromatosis (IH). The study consisted of HLA typing and determination of biochemical iron status indicators (serum transferrin saturation, serum ferritin). In total, 130 persons were evaluated. The screening identified 6 homozygous (h/h) subjects with preclinical IH, 46 heterozygous (h/n), and 8 normal (n/n) subjects, while 39 subjects were classified as normal or heterozygous (n/h?). One family demonstrated both a homozygous x heterozygous as well as a heterozygous x heterozygous mating. Recombination between the HLA region and IH locus occurred possibly in three subjects in three different families. The significance of detailed screening in families with probands with IH is discussed.