Selenocysteine tRNA-specific elongation factor SelB is a structural chimaera of elongation and initiation factors

In all three kingdoms of life, SelB is a specialized translation elongation factor responsible for the cotranslational incorporation of selenocysteine into proteins by recoding of a UGA stop codon in the presence of a downstream mRNA hairpin loop. Here, we present the X-ray structures of SelB from the archaeon Methanococcus maripaludis in the apo-, GDP- and GppNHp-bound form and use mutational analysis to investigate the role of individual amino acids in its aminoacyl-binding pocket. All three SelB structures reveal an EF-Tu:GTP-like domain arrangement. Upon binding of the GTP analogue GppNHp, a conformational change of the Switch 2 region in the GTPase domain leads to the exposure of SelB residues involved in clamping the 5' phosphate of the tRNA. A conserved extended loop in domain III of SelB may be responsible for specific interactions with tRNA(Sec) and act as a ruler for measuring the extra long acceptor arm. Domain IV of SelB adopts a beta barrel fold and is flexibly tethered to domain III. The overall domain arrangement of SelB resembles a 'chalice' observed so far only for initiation factor IF2/eIF5B. In our model of SelB bound to the ribosome, domain IV points towards the 3' mRNA entrance cleft ready to interact with the downstream secondary structure element.     

A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.     

Compatibility of antihuman lymphocyte globulin (AHLG). Possible side effects and complications]

The results in the antihuman lymphocytic globulines (AHLG) therapy of 25 patients with predominantly haematological and neurological diseases are reported. Extent and scope of the side-effects observed are discussed. A careful clinical, clinico-chemical and immunological observation of the patients during the AHLG therapy is indispensable for performing this biological immunosuppression and a strict selection of patients is also required. Under these conditions there are no higher risk and responsibility in an AHLG therapy than in other intensive kinds of therapy.     

Reabsorption of inorganic sulfate in the frog kidney: saturation of transport mechanism

1. Clearance experiments were performed to study reabsorption of inorganic sulfate (SO4) in the frog kidney. 2. During stepwise elevation of the SO4 concentration in plasma by i.v. sulfate administration (SO4 titration), the absolute reabsorption of SO4 did not increase but kept constant during mild SO4 loading. 3. The maximal reabsorptive capacity for SO4 (TmSO4) was about 0.37 mumol/30 min in this species. 4. The present results do not indicate renal net secretion of SO4 in the frog.     

Erythrocyte-associated plasma proteins, a further aspect of erythrocyte function?

The amount of plasma proteins associated to erythrocytes was determined or calculated by using three different techniques. Thus, there are 3.48 X 10(12) g of protein in a single erythrocyte or 94.12 g of protein respectively in the total of all erythrocytes in a healthy man with a body weight of 70 kg. The mass of plasma protein associated to erythrocytes will decrease in case of immunocomplex aggregates or otherwise denatured plasma proteins being fixed to the erythrocyte surface so that from a purely calculating point of view protein amounts on a scale of up to 46% of all plasma proteins may enter the free blood plasma under extreme conditions. In this, an immediately efficient possibility of compensation is seen by the authors in case of an acute consumption of blood proteins.     

Incomplete factorial search for conditions leading to high quality crystals of Escherichia coli cytidine deaminase complexed to a transition state analog inhibitor

We have used an incomplete factorial design (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) to find conditions for growing high quality crystals of Escherichia coli cytidine deaminase (EC 3.5.4.5). Crystals grow at pH 6.0 in hanging or sitting drops with either 1.6 M ammonium sulfate or 2.4-2.5 M sodium phosphate as precipitant. Both conditions produce crystals with identical morphologies and unit cell constants. The space group is P3(1)21 (or its enantiomorph P3(2)21), and the unit cell constants are a = b = 120.3 A, c = 78.4 A. The asymmetric unit is most reasonably one dimer of 66,000 Mr. The crystal size is very dependent on the supersaturation ratio, S = [initial protein concentration]/[equilibrium protein concentration], exhibiting a maximum at S = 7.7. The largest crystals diffract to at least 2.5 A and have a lifetime of 4 to 5 days in the x-ray beam at room temperature. The enzyme in these crystals is complexed with the transition state analog inhibitor 1-(beta-D-ribofuranosyl)-5-fluoropyrimidin-2-one (5-fluoropyrimidin-2-one riboside). We have collected data from parent crystals and from a heavy atom derivative in which the transition state analog is replaced by the active site directed inhibitor 5-(chloromercuri)cytidine.     

Direct Detection of Fungal Siderophores on Bats with White-Nose Syndrome via Fluorescence Microscopy-Guided Ambient Ionization Mass Spectrometry

White-nose syndrome (WNS) caused by the pathogenic fungus Pseudogymnoascus destructans is decimating the populations of several hibernating North American bat species. Little is known about the molecular interplay between pathogen and host in this disease. Fluorescence microscopy ambient ionization mass spectrometry was used to generate metabolic profiles from the wings of both healthy and diseased bats of the genus Myotis. Fungal siderophores, molecules that scavenge iron from the environment, were detected on the wings of bats with WNS, but not on healthy bats. This work is among the first examples in which microbial molecules are directly detected from an infected host and highlights the ability of atmospheric ionization methodologies to provide direct molecular insight into infection.