Crystals of an antibody FV fragment against an integral membrane protein diffracting to 1.28 Å resolution

The F_v fragment of a monoclonal antibody, 7E2 (IgG₁, κ, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli. Crystals suitable for highresolution X-ray analysis were obtained by microdialysis under low salt conditions. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 51.51 Å, b = 56.15 Å, c = 99.86 Å (1 Å = 0.1 nm) and contain one F _v fragment per asymmetric unit. Using synchrotron radiation diffraction data were collected up to 1.28 Å resolution. This high resolution is very unusual for a heterodimeric protein. The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics. © 1995 Wiley-Liss, Inc.     

Conversion of a racemate into a single enantiomer in one step by chiral liquid chromatography: Studies with rac-2,2′-diiodobiphenyl

The enantiomers of rac-2,2′-diiodobiphenyl were separated by liquid chromatography on microcrystalline triacetylcellulose. The conformational lability, a large separation factor α, and a suitable capacity factor k′(+) of this biphenyl allowed us to convert the racemate into 90% of enantiomerically pure (-)-2,2′-diiodobiphenyl and 10% of pure (+)-2,2′-diiodobiphenyl, respectively, by a series of in situ racemization-elution cycles. The much better retained (+)-enantiomer was racemized on the chromatographic column at 50°C after the less retained (-)-enantiomer has already been eluted at 8°C. © 1995 Wiley-Liss, Inc.     

Deletion mutagenesis of Tn10 Tet repressor — localization of regions important for dimerization and inducibility in vivo

The gene for the Tn 10 Tet repressor (TetR) was subjected to deletion mutagenesis. Screening for a transdominant operator-binding negative phenotype yielded 10 mutants with internal deletions. Three deletions extend from residue D5 to residues L41, W75, or Q76, respectively, and two contain deletions of the α-helix-turn-α-helix DNA-binding motif. Five deletions range from residue K84 to residues between R87 and K98. Since residues from the N-terminus up to position 98 are not necessary for dimerization, this must take place in the C-terminal half of the protein. Ability to dimerize was probed by introducing ochre non-sense codons (oc) at residues G138, H151, E159, l174, or K202. Koc202 shows wild-type in vivo operator-binding and inducibility by tetracycline indicating that the six C-terminal residues of TetR are not important for activity. Mutants with longer C-terminal truncations are inactive and not transdominant. They show reduced steady-state protein levels and are probably impaired in folding and degraded in vivo . Two mutants (Δ151–166, Δ164–166) with deletions in a region variable in primary structure and length among Tet repressers from different resistance determinants bind tet operator efficiently, but are not inducibie by tetracycline. This result indicates that these residues are not important for dimer formation in the operator-binding form.     

Chlorosphaeropsis epidemica,spec. nova,eine neue Grünalge mit endophytischer Lebensweise

The new parasitic algaChlorosphaeropsis epidemica is a member of theChlorosarcinales and lives endophytically in the leaf-tissue ofPotamogeton species. It forms stacks of cells and appears in great numbers, especially in dying leaf-blades. Sexual reproduction is described for the first time in this genus. Apart from its endophytic way of life,Chl. epidemica is characterized by mononuclear cells with a parietal chloroplast of irregular thickness and with a few perforations, sometimes of complicated reticulate shape, and with 1–5 pyrenoids. The usually sphaerical gametes (respectively zoospores) are provided with two equal flagella, a parietal plastid, one pyrenoid and one stigma. In connection with the asexual reproduction the terms vegetative division and desmoschisis are critically discussed.

     

Immunofluorescence of somatostatin-producing sites in the hypothalamus of the tadpole,Alytes obstetricans Laur.

Summary Three sites of somatostatin-synthesizing perikarya, or a related antigen, were determined by immunofluorescence in the hypothalamus of the tadpole, Alytes obstetricans (Amphibia, Anura). Two sites of neurosecretory perikarya were localized in the preoptic nuclei of the anterior hypothalamus; the axons extended either to the anterior diencephalon or to the median eminence and the pituitary. The third site was found in the posterior hypothalamus. These neurosecretory cells showed a strong immunofluorescent reaction; their axons all terminated at the level of the median eminence. Somatostatin cells were only found in intact or hypophysectomized tadpoles given somatotropin (STH). The strong reaction observed in hypophysectomized tadpoles was possibly due to the loss of the terminal portion of the neurosecretory pathway (median eminence and pituitary) by which the agent is transported to the site of discharge.     

Calcium and sulphur in neurosecretory granules and calcium in mitochondria as determined by electron microscope X-ray microanalysis

Summary Sections of neurosecretory cells fixed in glutaraldehyde and osmium tetroxide were studied by means of an EMMA-4 analytical microscope. Secretory granules in neurosecretory cells of the corpus cardiacum and of the brain, both in the desert locust Schistocerca and in the blowfly Calliphora, as well as neurosecretory granules in posterior pituitaries of the frog Rana and of the albino rat all contain a high concentration of calcium. A distinct sulphur peak was also a constant feature.In neurosecretory cells of the corpus cardiacum of Schistocerca the chromatin contained a high concentration of calcium. The mitochondria also contained much calcium, but part of this disappeared during preparation except when fixative and wash contained calcium chloride. By block staining with uranyl acetate most calcium is displaced from the mitochondria, whereas most of the calcium remains in the neurosecretory granules. Since the calcium peaks in spectra from neurosecretory granules appear of similar size, regardless of variations in the preparative procedure, this calcium must be firmly bound. The possible role of the calcium bound to the neurosecretory substance is discussed.The presence of sulphur in insect neurosecretory granules indicates the presence of a protein besides the hormone, i.e., an insect neurophysin.We wish to thank Dennis Greer for the operation of the analytical microscope. This work was supported by a Wellcome-Carlsberg Travelling Research Fellowship awarded to T.N. The EMMA-4 facility is supported by a grant from the British Science Research Council     

Prostaglandin D2, another renal prostaglandin?

Prostaglandin (PG) D₂ was biosynthesized by rabbit renal papillae incubates in vitro. Quantification of the renal prostaglandins by gas chromatography-mass spectroscopy demonstrated that the concentration of PGD₂ generated by renal papillae was to the amount of PGE₂ or about 1 μg/g tissue/30 min. Infusion of the sodium salt of PGD₂ into the renal artery of the dog produced a dose related increase in renal blood flow and urine flow, free water clearance, sodium excretion and potassium excretion without changes in systemic hemodynamics. At low doses PGD₂ increased renal blood flow to all cortical zones. Higher concentrations of PGD₂ produced a shift in the intrarenal distribution of blood flow toward the juxtamedullary nephrons.     

Membrane fluidity in normal and cystic fibrosis fibroblasts

We have observed distinct differences in the polarization of fluorescence and temperature dependent emission intensity of the highly fluorescent phospholipid derivative (1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)--aminocaproyl phosphatidylcholine (NBD-PC), when incorporated in the plasma membranes of normal and cystic fibrosis fibroblasts. Fluorescence polarization measurements indicate that the fluorochrome has a much higher degree of rotational mobility in cystic fibrosis fibroblasts as compared with normal cells. Temperature dependent transitions in the emission intensity of NBD-PC incorporated in normal fibroblasts are indicated at 17.7 and 21.2° C while the abnormal cell membranes apparently undergo transitions at 8.7 and 13.5° C. These differences might be due to changes in plasma membrane composition and/or organization, in the case of the cystic fibrosis cells.     

Regulation of adenosine 5′-phosphosulfate sulfotransferase activity by H2S and cyst(e)ine in primary leaves of Phaseolus vulgaris L.

During chloroplast development in the primary leaves of Phaseolus vulgaris, the extractable activity of adenosine 5-phosphosulfate sulfotransferase increased ten-fold. When chloroplast development took place in air enriched with 3.5 l H₂S·l^-1 there was a decrease in adenosine 5-phosphosulfate sulfotransferase activity. Cyst(e)ine in concentrations up to 1 mM (in the external medium) did not affect the increase in adenosine 5-phosphosulfate sulfotransferase activity in intact plants. In plants with excised roots, 0.75 mM cyst(e)ine inhibited this increase. In green primary leaves, H₂S or cyst(e)ine treatment resulted in a decrease of extractable adenosine 5-phosphosulfate sulfotransferase activity. In intact plants, this effect of cyst(e)ine was observed at a concentration of 1 mM, and in plants with excised roots, 0.25 mM had a comparable effect.In developing plants, the extractable activities of O-acetyl-L-serine sulfhydrylase (EC 4.2.99.9) and ribulosebisphosphate carboxylase (EC 4.1.1.39.) were not affected by H₂S or cyst(e)ine.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5phosphosulfate sulfotransferase - BSA bovine serum albumin - DTE dithioerythritol - EDTA ethylenediaminetetra-acetic acid - OASSase O-acetyl-L-serine sulfhydrylase - PAPS adenosine 3-phosphate 5-phosphosulfate - POPOP 1,4 Di 2-(5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazol - RubP ribulose-bisphosphate - RubPCase ribulosebiphosphate carboxylase This is no. 8 in the series Regulation of Sulfate Assimilation in Plants. The term cysteine is used when it is clear that cystine is not involved; cyst(e)ine is used for an undefined mixture of cysteine and cystine. The concentrations are expressed in all cases relative to cysteine