A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Microsurgical forearm "cricket bat-transformer" phalloplasty.

Presently, the donor flap of choice for microsurgical phallic reconstruction is the radial forearm flap. The success of several different design modifications confirms the reliability of the radial and ulnar forearm flaps. Farrow et al. described their experience with the "cricket bat" concept in 1980. To the previous "cricket bat" design, we now wish to add modifications. These modifications utilize longitudinal and transverse rotations of the linear forearm tissues to create a phallus--much like the transformation of a toy robot into a truck. Deepithelialized flaps and a full-thickness skin graft coronoplasty complete glans reconstruction. The "cricket bat-transformer" flap appears to produce the most predictable results in subtotal phallic reconstructions and phallic constructions in the pediatric and transgender patient groups.     

Tick anticoagulant peptide: kinetic analysis of the recombinant inhibitor with blood coagulation factor Xa

Tick anticoagulant peptide (TAP) is a 60 amino acid protein which is a highly specific inhibitor of human blood coagulation factor Xa (fXa) isolated from the tick Ornithodoros moubata [Waxman, L., Smith, D. E., Arcuri, K. E., & Vlasuk, G. P. (1990) Science 248, 593-596]. Due to the limited quantities of native TAP, a recombinant version of TAP produced in Saccharomyces cerevisiae was used for a detailed kinetic analysis of the inhibition interaction with human fXa. rTAP was determined to be a reversible, slow, tight-binding inhibitor of fXa, displaying a competitive type of inhibition. The binding of rTAP to fXa is stoichiometric with a dissociation constant of (1.8 +/- 0.02) x 10(-10) M, a calculated association rate constant of (2.85 +/- 0.07) x 10(6) M-1 s-1, and a dissociation rate constant of (0.554 +/- 0.178) x 10(-3) s-1. Binding studies show that 35S-rTAP binds only to fXa and not to DFP-treated fXa or zymogen factor X, which suggests the active site of fXa is required for rTAP inhibition. That rTAP is a unique serine proteinase inhibitor is suggested both by its high specificity for its target enzyme, fXa, and also by its unique structure.     

Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast

Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.     

Isolation and identification of spirorenone metabolites from the monkey (Macaca fascicularis)

The plasma level of spirorenone was determined 3 h after 1, 8, 22 and 46 daily oral administrations of 20 mg/kg to two female and two male monkeys ($\text{Macaca fascicularis}$). A fifth animal, female, was treated with eight daily doses of tritium-labelled drug and was completely bled from the carotid vein 4 h after the last administration in order to isolate and identify plasma metabolites.After repeated daily doses of spirorenone the mean plasma level of unchanged drug was 711 ± 213 ng/ml. In the plasma of the fifth animal four radioactively labelled compounds could be detected after extraction and subsequent HPLC separation. Mass spectrometric identification of three of the substances indicated 1,2-dihydrospirorenone, hydroxy-1,2-dihydrospirorenone and the unchanged drug itself.     

Electrophoretic and biochemical studies of human aldehyde dehydrogenase isozymes in various tissues

Four major ALDH isozymes have been identified in human tissues using starch gel electrophoresis and isoelectric focusing. The isozyme bands have been termed as ALDH I, II, III and IV according to their decreasing electrophoretic migration and increasing isoelectric point. The isozymes have been partially purified via preparative isoelectric focusing. Kinetic characteristics of ALDH I and II were found to be quite similar to ALDH enzyme 2 and enzyme 1 described earlier by Greenfield and Pietruszko (Biochem Biophys Acta, $\text{483}$ 35–45 1977). ALDH III and IV showed a very high Km for propionaldehyde (1.0–1.5 mM at pH 9.5) and were not inhibited by disulfiram at pH 9.5. A variant phenotype of ALDH which lacked in isozyme I was detected in various tissues from Japanese individuals. Comparative kinetic properties of normal and variant enzyme are given.     

The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosis

In an organism (strain C 1/2 from Dr. P. R. Hayes, Leeds) regarded as a typical representative of the genus Flavobacterium, flexirubin-type pigments have been identified. The Flavobacterium pigments contain structural elements of both, the pigments of the genus Flexibacter and the pigments of the genus Cytophaga. As flexirubin-type pigments seem to have a rather restricted distribution among bacteria, and have formerly proved to be useful chemosystematic markers for the flexibacteria, this new observation may indicate that there is a relatively close phylogenetic relationship between this type of flavobacteria and the Cytophaga-Flexibacter group.