The three-dimensional structure of trypsin-treated Staphylococcus aureus alpha-toxin.

Trypsin treatment of staphylococcal alpha-toxin cleaves the molecule into two roughly equally sized parts, which results in inactivation of the toxin. Tetragonal arrays of oligomers, closely resembling the native ones, can however be formed on lipid layers. From tilted views of negatively stained crystals a 3D structure to 23 A resolution has been determined by electron microscopy and image processing. On comparison with the 3D structure of the native alpha-toxin (Olofsson et al., J. Mol. Biol. 214, 299-306, 1990) the subdomains are more separated, confirming the differences found when comparing the projection maps (Olofsson et al., J. Struct. Biol. 106, 199-204, 1991). The tryptic cleavage takes place in a postulated hinge region. The results are consistent with the hypothesis that the conformational change required for inducing the membrane permeabilizing property takes place in this region. Furthermore, we present a refined projection map at approximately 10 A resolution based on the analysis of a large number of crystals using unbending methods.     

The structure of Staphylococcus aureus α-toxin: Effects of trypsin treatment

Staphylococcus aureus alpha-toxin was treated with trypsin, which inactivates the toxin. Two-dimensional crystals of the modified protein were produced on preformed lipid layers. The projection structure obtained by electron crystallographic analysis of a large number of crystals showed tetragonal p4 symmetry and a resolution of approximately 12 A. The fragments of the toxin, 17 and 18 kDa large, were arranged in a way resembling those observed earlier for the native protein (Olofsson et al., J. Mol. Biol. 214, 299-306, 1990). However, after trypsin treatment the stain-deficient region corresponding to one alpha-toxin monomer shows two separated subdomains of similar size. This separation is probably related to the inability of the modified toxin to undergo the conformational change thought to be essential for the membrane-damaging effect.     

Three-Dimensional Reconstruction of the Mammalian Centriole from Cryoelectron Micrographs: The Use of Common Lines for Orientation and Alignment

The microtubule organizing center of the animal cell (S. D. Fulleret al.,1992,Curr. Opin. Struct. Biol.2,264–274; D. M. Gloveret al.,1993,Sci. Am.268,62–68; E. B. Wilson, 1925), (The Cell in Development and Heredity) comprises two centrioles and the pericentriolar material. We have completed several three-dimensional reconstructions of individual centrioles from tilt series of cryoelectron micrographs. The reconstruction procedure uses minimization of the common lines residual to define the orientation of the centriolar ninefold symmetry axis and then uses this symmetry to generate a structure by weighted backprojection to 28-nm resolution. Many of the features of these reconstructions agree with previous, conventional transmission electron microscopy studies (M. Paintrandet al.,1992,J. Struct. Biol.108,107–128). The microtubule barrel of the centriole is roughly 500 nm long and 300 nm in diameter and the microtubule bundles appear to taper toward the distal end. In addition, we see a handedness to the pericentriolar material at the base (distal end) of the centriole which is opposite to the skew of the microtubule triplets. The region at which the microtubule barrel joins this base is intriguingly complex and includes an internal cylindrical feature which is a site of γ tubulin localization.     

Amino acid sequence of squid troponin C

The complete amino acid sequence of squid Todarodes pacificus troponin C (TnC), which was shown to bind only 1 mol Ca²⁺/mol, was determined by both the Edman and cDNA methods. The squid TnC is composed of 147 amino acids including an unblocked Pro at the N-terminus and the calculated molecular weight is 17 003.9. Among the four potential Ca²⁺-binding sites, namely sites I–IV from the N-terminus, only site IV completely satisfied the consensus amino acid sequence for the active Ca²⁺-binding loop. This indicates that squid TnC possesses a single Ca²⁺-binding site at the site IV as scallop TnCs [Nishita et al., J. Biol. Chem. 269 (1994) 3464–3468; Ojima et al., Arch. Biochem. Biophys. 311 (1994) 272–276). The sequence homology of squid TnC to TnCs of scallop, arthropods, and rabbit was 61%, 31–38%, and 31%, respectively. In the sequence of the central D/E-helix region of squid and scallop TnCs, a deletion of three amino acids was required to maximize the homology with the other TnCs.     

Localization of a Cell Adhesion Site on Collagen XIV (Undulin)

Cell adhesion to collagen XIV is implied to be mediated by proteoglycans as cellular receptors (T. Ehniset al.,1996,Exp. Cell Res.229, 388–397). In order to define the cell binding region(s), fusion proteins expressed inEscherichia coliand covering the large noncollagenous domain NC3 of collagen XIV were used as substrates for the adhesion of skin fibroblasts. A prominent cell binding site could be localized in the N-terminal fibronectin type III repeat of collagen XIV and its immediate C-terminal extension. Since this region also mediates the binding of the small chondroitin/dermatan sulfate proteoglycan decorin (T. Ehniset al.,1997,J. Biol. Chem.272, 20414–20419), our finding could provide the molecular basis for the observation that decorin serves as inhibitor and potential modulator of cellular interactions with collagen XIV.     

The compactness of ribonuclease A and reduced ribonuclease A

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small-angle X-ray scattering. The R_g values and the Kratky plots showed that non-reduced ribonuclease A maintain a compact shape with a R_g value of about 17.3 Å in 8 M urea. The reduced ribonuclease A is more expanded, its R_g value is about 20 Å in 50 mM Tris-HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.     

Germicide capacity of macrophages (MØ) in the Antarctic fish Notothenia coriiceps (Richardson, 1844) at 0°C

The germicide capability of the macrophage (MØ) of the Antarctic fish Notothenia coriiceps is demonstrated using fluorescence microscopy for the first time. The MØs were able to kill microorganisms by intracellular mechanism and this killing can be stimulated by oyster-derived glycogen. Although the phagocytosis index is lower than in temperate water fish species, this work demonstrates that non-specific defence mechanism plays an important role in the polar environment. There are some studies on inflammation in N. coriiceps [Silva et al. (1998) Polar Biol 20:206–212], parasite–host relation [Silva et al. (1999) Polar Biol 22:417–424] and phagocytosis [Silva et al. (2002) J Fish Biol 60:466–478]. These previous studies have shown that the MØ were able to identify biotic and abiotic factors. However, it can be of interest to study the activity of MØ in microorganism killing, and this work adds new insights of this fundamental process under Antarctic temperatures.     

Estimation of evolutionary distances between nucleotide sequences

A formal mathematical analysis of the substitution process in nucleotide sequence evolution was done in terms of the Markov process. By using matrix algebra theory, the theoretical foundation of Barry and Hartigan's (Stat. Sci. 2:191–210, 1987) and Lanave et al.'s (J. Mol. Evol. 20:86–93, 1984) methods was provided. Extensive computer simulation was used to compare the accuracy and effectiveness of various methods for estimating the evolutionary distance between two nucleotide sequences. It was shown that the multiparameter methods of Lanave et al.'s (J. Mol. Evol. 20:86–93, 1984), Gojobori et al.'s (J. Mol. Evol. 18:414–422, 1982), and Barry and Hartigan's (Stat. Sci. 2:191–210, 1987) are preferable to others for the purpose of phylogenetic analysis when the sequences are long. However, when sequences are short and the evolutionary distance is large, Tajima and Nei's (Mol. Biol. Evol. 1:269–285, 1984) method is superior to others.     

Crystallization and Preliminary X-Ray Diffraction Analysis of the 190-Å-Long Coiled-Coil Dimerization Domain of the Actin-Bundling Protein Cortexillin I fromDictyostelium discoideum

We have crystallized the ≈190-Å-long parallel two-stranded coiled-coil oligomerization domain of the actin-bundling protein cortexillin I fromDictyostelium discoideum. The orthorhombic crystals belong to the space group C222₁with unit cell dimensions ofa= 71.3 Å,b= 127.8 Å, andc= 91.6 Å. As both native and selenomethionine-substituted protein crystals diffract to 3.0 and 2.85 Å resolution, respectively, using synchrotron radiation, they are suitable for the first high-resolution structural analysis of a two-stranded coiled coil comprising more than six heptad repeats. Moreover, because the polypeptide chain fragment contains a recently identified two-heptad-repeat long sequence that is indispensable for the assembly of the cortexillin I coiled-coil oligomerization domain, its high-resolution structure should enable us to extend our knowledge on the molecular mechanisms underlaying coiled-coil formation and to establish the precise manner in which the two “trigger” sequences interact with one another in the dimer.     

Cloning and characterization of a third type of human α-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human α-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229–235; Tomita et al., Gene 76 (1989) 11–18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human α-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively.Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197–1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5′ region; hence the promoter lies far upstream relative to the other two AMY genes.     

New Crystal Forms of the Motile Major Sperm Protein (MSP) ofAscaris suum

We have obtained two new crystal forms of theAscarismajor sperm protein (MSP) that mediates amoeboid cell motility in nematode sperm. We obtained crystals with C2 symmetry from bacterially expressed α-MSP witha= 216.5 Å,b= 38.6 Å,c= 32.5 Å, γ = 93.1° and also crystals with P2₁symmetry from native β-MSP witha= 63.1 Å,b= 91.7 Å,c= 72.5 Å, γ = 91.3°. A full native data set has been collected for each crystal form using synchrotron radiation. Both crystal forms diffract to 2 Å and are suitable for high-resolution structural investigation.     

Changes of Skull Proportions in Ontogeny of Barbus canis from Lake Kinneret and of the Lake Tana large-mouthed barbs, Barbus intermedius Complex

Skull proportions of Barbus canis individuals drastically change during their life. Yearlings have skull proportions characteristic for barbs of the NEC phenetic group (sensu Mina et al. 2001 Environ. Biol. Fish. 61: 242–252), but with age become similar to those of the Lake Tana large-mouthed morphotypes which acquire their specific features at 3–7 years old while at earlier ages they have skull proportions of a NEC form identified as 'intermedius' sensu Nagelkerke et al. (1994 Environ. Biol. Fish. 39: 1–21). The large-mouthed Lake Tana barbs originated from an ancestral NEC form through peramorphosis, but in relation to B. canis they are paedomorphic in skull proportions. B. canis supposedly originated from a NEC form as a consequence of an adaptive radiation that took place in western Asia.     

Homologous IRCM-Serine Protease 1 from pituitary,heart atrium and ventricle: A common pro-hormone maturation enzyme?

IRCM-Serine Protease 1 (IRCM-SP1) has recently been isolated and characterized from porcine pituitary anterior and neurointermediate lobes (Cromlishet al., 1986a,J. Biol. Chem. 261:10850–10858; Cromlishet al., 1986b,J. Biol. Chem. 261:10859–10870). This pituitary serine protease was shown to selectively cleave human proopiomelanocortin (POMC)-derived peptides at both pairs of basic residues and C-terminal to specific Arg residues, all known to be cleavedin vivo. Here, a similar enzyme was isolated from rat heart atria and ventricles. Rat IRCM-SP1 was shown to be highly specific for the same cleavage sites in POMC, as the porcine pituitary homologue. Furthermore, the rat and the porcine enzymes cleave rat pro-Atrial Natriuretic Factor (pro-ANF 1–126) to yield ANF 103–126, 102–126 and 99–126 in that order of preference. This suggests thatin vitro the cleavage sites preferred in pro-ANF resemble those found in brain and hypothalamus. The enzyme is nine times more abundant in atria versus ventricles/mg protein. It is concluded that IRCM-SP1, could well represent a common pro-hormone maturation enzyme for POMC and Pro-ANF and possibly many other pro-hormones.     

Statistics of canonical RNA pseudoknot structures

In this paper we study canonical RNA pseudoknot structures. We prove central limit theorems for the distributions of the arc-numbers of k-noncrossing RNA structures with given minimum stack-size τ over n nucleotides. Furthermore we compare the space of all canonical structures with canonical minimum free energy pseudoknot structures. Our results generalize the analysis of Schuster et al. obtained for RNA secondary structures [Hofacker, I.L., Schuster, P., Stadler, P.F., 1998. Combinatorics of RNA secondary structures. Discrete Appl. Math. 88, 207–237; Jin, E.Y., Reidys, C.M., 2007b. Central and local limit theorems for RNA structures. J. Theor. Biol. 250 (2008), 547–559; 2007a. Asymptotic enumeration of RNA structures with pseudoknots. Bull. Math. Biol., 70 (4), 951–970] to k-noncrossing RNA structures. Here k2 and τ are arbitrary natural numbers. We compare canonical pseudoknot structures to arbitrary structures and show that canonical pseudoknot structures exhibit significantly smaller exponential growth rates. We then compute the asymptotic distribution of their arc-numbers. Finally, we analyze how the minimum stack-size and crossing number factor into the distributions.     

Transinhibition and Voltage-gating in a Fungal Nitrate Transporter

We have applied enzyme kinetic analysis to electrophysiological steady-state data of Zhou et al. (Zhou, J.J., Trueman, L.J., Boorer, K.J., Theodoulou, F.L., Forde, B.G., Miller, A.J. 2000. A high-affinity fungal nitrate carrier with two transport mechanisms. J. Biol. Chem. 275:39894–9) and to new current-voltage-time records from Xenopus oocytes with functionally expressed NrtA (crnA) 2H⁺-NO ₃ ^– symporter from Emericella (Aspergillus) nidulans. Zhou et al. stressed two Michaelis-Menten (MM) mechanisms to mediate the observed nitrate-induced currents, I _{NO 3} ^– . We show that a single straightforward reaction cycle describes the data well, pointing out that during exposure to external substrate, S = (2H⁺+NO ₃ ^– )_o, the product concentration inside, [P] = [H⁺] _i ² · [NO ₃ ^– _i, may rise substantially near the plasma membrane, violating the condition [P] [S] for MM kinetics. Here, [P] and its changes during experimentation are treated explicitly. K _1/2 20 µM for I _{NO 3} ^– at pH_o from Zhou et al. is confirmed. According to our analysis, NrtA operates between about 0.2 and 0.6 of the electrical distance in the membrane (outside 0, inside 1). In absence of thermodynamic gradients, the predominant orientation of the binding site(s) is probably inwards. The activity of the enzyme is sensitive to the transmembrane voltage, V, with an apparent gating charge of +1.0 ± 0.5 for inactivation, and transition probabilities of 0.3–1.3 s^–1 at V = 0. This gating mode impedes loss of cellular NO ₃ ^– during depolarization.     

Structural Features of Glycera dibranchiata Monomer Hemoglobins. Primary Sequences of Monomer Hemoglobin Components II and III

Primary sequences for the remaining two members (GMH2, GMH3) of the group of three major monomeric hemoglobins from the marine annelid Glycera dibranchiata have been obtained. Full sequences of each 147-amino acid globin were achieved with a high degree of confidence using standard Edman technology in combination with molecular mass determinations of the intact globins and of the cyanogen bromide cleavage fragments using electrospray ionization mass spectrometry. When minor assumptions concerning Q/E identities are made these new results indicate the likely correspondence of GMG2 with the protein represented by the first Glycera dibranchiata monomer hemoglobin complete sequence [Imamura et al., (1972), J. Biol. Chem. 247, 2785–2797]. When these new sequences are combined with the previously determined primary sequence for the third major monomer hemoglobin, GMH4 [Alam et al., J. Protein Chem. (1994), 13, 151–164], it becomes clear that these three (GMG2–4) are truly distinct proteins, contrary to previous suggestions. Surprisingly, our results show that none of these three primary sequences is identical to the published sequence of the refined monomer hemoglobin crystal structure protein; however, there is a strong correspondence to the GMG2 sequence. The present sequencing results, in combination with the published GMH4 sequence, confirm the presence of a distal Leu in place of the more commonly encountered distal His in all three of the major monomer hemoglobins isolated in this laboratory and indicate that the unusual B10 Phe occurs only in GMH4. Analysis of the sequences presented here, along with comparison of amino acid content for Glycera dibranchiata monomer hemoglobins isolated from three different laboratories, and comparison of NMR results from two laboratories suggest further correspondences which unify disparate published isolations.     

Three-dimensional reconstruction of cytochrome oxidase vesicle crystals prepared by cholate solubilization

Cytochrome oxidase vesicle crystals with long-range order have been obtained from cholate-solubilized, highly purified reconstitutively active preparations. These crystals, which are suitable for electron-microscopic structure investigation, show pgg symmetry in the 0 degree projection. Using Fourier reconstruction and modified back-projection methods, a three-dimensional reconstruction has been obtained at a resolution of 25 A. Our structural results are in agreement with the model of Henderson et al. [J. Mol. Biol. 112, 631 (1977)] obtained for their Triton-derived crystals.     

Galactosyltransferase activity is not localized to the brush border membrane of human small intestine

A recent report [Rothet al. (1985)J. Cell Biol. 100: 118–125], using immunocytochemical techniques, calimed that human duodenal galactosyltransferase is located predominantly on the external aspect of enterocyte brush border membranes. Analytical subcellular fractionation by sucrose density gradient centrifugation of human jejunum biopsy homogenates demonstrated that galactosyltransferase activity is localized to the Golgi fraction (equilibrium density of 1.14 g cm^–3) and is not found in significant amounts in the brush border membrane (equilibrium density of 1.22 g cm^–3).     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

Human thymus poly(A) polymerase (EC 2.7.7.19) activity has been investigated using poly(A) and oligo(A) as initiators. All obtained fractions reveal more than one polypeptide as detected by immunoblotting after SDS-PAGE. In addition to the homogeneously purified (Tsiapalis et al., J Biol Chem 250: 4486–1496, 1975 and Wahle, J Biol Chem 266: 3131–3139, 1991), about 60 kDa polypeptide, a larger polypeptide, about 80 kDa, that comigrates in the region of poly(A) polymerase activity was detected, enriched and partially characterized; it appears having similar size with bovine poly(A) polymerase cloned in E. coli. Polyclonal antiserum produced against recombinant bovine poly(A) polymerase reacts more efficiently with the about 80 kDa polypeptide upon immunoblotting, and can precipitate the poly(A) polymerase activity. This enzyme form, from human tissue, is novel in terms of size and may reflect intact or physiological form of poly(A) polymerase in human thymus, and supports and substantiates recent reports on the enzyme from other sources.     

Three-Dimensional Structure of the Porcine Gastric H,K-ATPase from Negatively Stained Crystals

A low-resolution three-dimensional model of membrane-bound H,K-ATPase from pig gastric mucosa has been reconstructed by electron microscopy and image processing of two-dimensional crystals in negative stain. The crystal formation is induced by magnesium and vanadate, which stabilize the E₂conformation of the enzyme. The unit cell, with a size ofa=b= 123 Å, γ = 90°, has tetragonal p4 symmetry. There are four separate αβ protomers within each unit cell. The high-contrast region is limited to the cytoplasmic part of the protein. The total volume of the observed asymmetric protein domain corresponds to a molecular mass of 80–90 kDa. It consists mainly of a large pear-shaped domain measuring 60 × 45 Ų, with a height of 50 Å as measured perpendicular to the membrane plane. A small stalk segment, 20 Å in length, forms a connection to the transmembrane region.