Concave Face Research Articles

Single and Double Diastereoselection in Azomethine Ylide Cycloaddition Reactions with Unsaturated Chiral Bicyclic Lactams

Double diastereoselectivity data were analyzed to provide insight into the structural features that influence π-facial selectivity in 1,3-dipolar cycloadditions of chiral and achiral azomethine ylides to chiral, unsaturated bicyclic lactams. Three major steric contributions to the differences in stability (ΔΔG⧧) between competing cycloaddition transition states were identified. The first major set of steric interactions involve that between the dipoles and the substituents on the left hemisphere (R2) and concave faces of the bicyclic lactams. This effectively hindered both α- and β-approaches in the nonextended transition states shown in Figure 1. The second major steric interaction was provided by the nonbonded interactions (i) between the R1 angular substituent on the bicyclic lactam and the π-system of the dipole as shown in Figures 3 and 4. This interaction was shown to be very significant, causing reversal in π-facial attack of chiral and achiral dipoles when the angular substituent is changed from p...

CBI-CDPBO 1 and CBI-CDPBI 1: CC-1065 analogs containing deep-seated modifications in the DNA binding subunit

The synthesis and preliminary examination of CBI-CDPBO 1 ( 2) and CBI-CDPBI 1 ( 3), CBI analogs of CC-1065 ( 1) and the duocarmycins incorporating the 3-carbamoyl-1,2-dihydro-3 H-pyrrolo[3,2- e] benzoxazole-7-carboxylate (CDPBI) DNA binding subunits, are detailed. The agents contain deep-seated modifications in the DNA binding subunits of the natural products with incorporation of a nitrogen capable of functioning as a hydrogen bond acceptor (CDPBO, CDPBI) or hydrogen bond donor (CDPBI) on their inside concave face which is in intimate contact with the minor groove floor. The CDPBO subunit was prepared through use of a novel and effective MnO 2-mediated oxidative coupling of 2-(benzyloxy)ethylamine with 5-hydroxyindole ( 4) to directly provide 2-[(benzyloxy)methyl]pyrrolo [3,2- e]benzoxazole ( 6, 48%) in a reaction cascade that initially proceeds with amine regioselective C4 nucleophilic addition to the in situ generated p-quinone monoimine 13. Subsequent conversion of 6 to 8 (debenzylation; MnO 2-NaCN, CH 3OH) and selective reduction of the fused pyrrole (Et 3SiH-CF 3CO 2H) completed the synthesis of the 1,2-dihydro-3 H-pyrrolo[3,2- e]benzoxazole-7-carboxylate ring system. The CDPBI subunit was prepared through selective C4 nitration of 22 followed by reduction of the nitro group and acid-catalyzed closure to the corresponding 2-[(benzyloxy)methyl]pyrrolo[3,2- e]benzimidazole 25. The final conversion of 25 to the 1,2-dihydro-3 H-pyrrolo[3,2- e] benzimidazole-7-carboxylate ring system (CDPBI) followed the same protocols introduced for CDPBO. The DNA alkylation efficiencies of 2 and 3 were identical and both were substantially diminished relative to that of CBI-CDPI 1 ( 40). Thus, the introduction of a single nitrogen atom in the DNA binding subunit of 40 has a pronounced and detrimental effect on the relative efficiency (100 ×) of DNA alkylation. Consistent with these observations, the in vitro cytotoxic activity of (+)- 2 and (+)- 3 were comparable (IC 50 = 200 pM, L1210) and 40 × less potent than (+)- 40 (IC 50 = 5 pM, L1210). In contrast to the large impact these small structural changes had on the efficiency of DNA alkylation, the selectivity of DNA alkylation by 2 and 3 was unperturbed and both agents were found to alkylate the same major sites as CBI-CDPI 1 ( 40). The potential origin of these effects is discussed.

Expected displacements and stresses in the encasing concrete of a francis turbine scroll case

The finite element stress analysis is used to determine the expected displacements and stresses in the encasing concrete of the scroll case of a Francis turbine. The minimum principal stress in all cases examined is compressive and has higher concentrations close to the stay ring supports. The maximum principal stress indicates tensile stress areas at the inside concave faces of the cross sections, which are extended into most of the central area of the cross section, around the horizontal plane of symmetry of the turbine. The displacements are within the allowed width of the asphalt cork liner of the scroll case.

S layer regeneration in Methanococcus voltae protoplasts

The regeneration of the paracrystalline proteinaceous cell wall (S layer) of the methanogen Methanococcus voltae was examined by electron microscopy. The S layer was removed from the cell surface by exposing the cells to a protoplasting buffer but was completely regenerated by 60-80 min resuspension in a regeneration medium by some repair mechanism (normal cell generation time is 16 h). The protoplast surface appeared in freeze-fracture to be initially smooth and featureless and remained so for the first 20 min in regeneration medium. Thereafter, nascent S layer appeared on the surface in the form of paracrystalline patches of various sizes that were distributed over the entire surface. The patches were closely associated with disturbances of the cell surface curvature which may have been caused by electrostatic interactions. The steps leading to the complete regeneration of the S layer could not be followed due to the sampling times used, but presumably the patches grew large enough to contact neighbouring patches and somehow fused into a coherent layer. Other ultrastructural changes occurred to the cell envelope during S layer regeneration, most notably the recurrence of the intramembrane particles of the concave fracture face of the plasma membrane. Flagella remained attached to the protoplasts and were apparently still functional. The major changes to the protein profile of untreated cells, protoplasts and regenerating protoplasts included the loss and resynthesis of polypeptides at the 76 kDa (S layer polypeptide) and 60 kDa (unknown polypeptide) positions. SDS-PAGE evidence indicated that new S layer polypeptides were synthesized 20 min prior to their appearance as patches on the cell surface. The S layer polypeptide may have been shed from the surface during the early stages of regeneration to form the extracellular sheet-like material observed in large quantity in the regeneration medium. The response of the cell to changes that occur at the cell surface indicates that some feedback mechanism gears the synthesis of S layer and associated envelope components.

The taxonomy and ultrastructure of Chrysochromulina simplex (Prymnesiophyceae)

The type description of Chrysochromulina simplex Estep et al. (Prymnesiophyceae) is emended, based on clonal cultures from the southern Indian Ocean. Cells possess a single scale type (0.3–0.58 μm in diameter) with faint concentric markings on the distal face and 21–36 radiating ridges with an excentric cross on the proximal face. The long, coiling haptonema (max. 79 μm) and the two heterodynamic flagella (11–16 μm) are inserted on the concave face of the saddle-shaped cells. The flagellar apparatus consists of four microtubular roots (R1 of 6+4, a single-stranded R2, a bifurcated R3 of 2/2+1, a single-stranded R4) and various fibrous connectives (distal band with extensions to the R 1 and over the R2, a proximal band, three accessory bands). The ultrastructure of the flagellar apparatus shares features with both C. apheles Moestrup et Thomsen and C. acantha Leadbeater et Manton, and intra-and intergeneric comparisons are made.

Solution structure of the epithelial cadherin domain responsible for selective cell adhesion.

Cadherins are calcium-dependent cell adhesion molecules containing extracellular repeats of approximately 110 amino acids. The three-dimensional structure of the amino-terminal repeat of mouse epithelial cadherin was determined by multidimensional heteronuclear magnetic resonance spectroscopy. The calcium ion was bound by a short alpha helix and by loops at one end of the seven-stranded beta-barrel structure. An exposed concave face is in a position to provide homophilic binding specificity and was also sensitive to calcium ligation. Unexpected structural similarities with the immunoglobulin fold suggest an evolutionary relation between calcium-dependent and calcium-independent cell adhesion molecules.

High-frequency scattering of a whispering gallery mode by a cylindrically curved surface with second-order generalized impedance boundary conditions

A high-frequency solution is derived for the diffraction of electromagnetic waves by a cylindrically curved metallic sheet whose concave and convex faces are coated by dielectric layers of the same physical properties and thicknesses. After simulating the effects of the grounded dielectric layer by second-order generalized impedance boundary conditions, a canonical problem is formulated in an infinitely extended angular space from which explicit asymptotic expressions for the diffraction coefficients are obtained through the asymptotic solution of a Hilbert problem.

The three-dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum.

Peptide:N-glycosidase F (PNGase F) is an enzyme that catalyzes the complete removal of N-linked oligosaccharide chains from glycoproteins. Often called an endoglycosidase, it is more correctly termed an amidase or glycosylasparaginase as cleavage is at the asparagine-sugar amide linkage. The enzyme is widely used in structure-function studies of glycoproteins. We have determined the crystal structure of PNGase F at 1.8 A resolution. The protein is folded into two domains, each with an eight-stranded antiparallel beta jelly roll configuration similar to many viral capsid proteins and also found, in expanded form, in lectins and several glucanases. Two potential active site regions have been identified, both in the interdomain region and shaped by prominent loops from one domain. Exposed aromatic residues are a feature of one site. The finding that PNGase F is based on two jelly roll domains suggests parallels with lectins and other carbohydrate-binding proteins. These proteins either bind sugars on the concave face of the beta-sandwich structure (aided by loops) or amongst the loops themselves. Further analysis of the function and identification of the catalytic site should lead to an understanding of both the specificity of PNGase F and possibly also the recognition processes that identify glycosylation sites on proteins.

Ab initio molecular orbital calculations on the neutral hydrolysis and methanolysis of azetidinones, including catalysis by water. Relationship to the mechanism of action of β-lactam antibiotics

The docking of penicillins to a computer model of the active site of the penicillin-binding protein of Streptomyces R61 produces structures that exhibit a four-centred interaction between the O-H of a serine residue and the (O)C-N of the β-lactam ring. If such a structure is a stationary point on the reaction coordinate for the acylation of the serine OH by the β-lactam carbonyl group, the acylation mechanism would appear to consist of O—C and H—N bond formation, concerted with the cleavage of the C—N bond. The existence of this "N-protonation" mechanism, and the energetics of this mechanism in comparison to the usual "O-protonation" pathway in which proton transfer to oxygen and addition to the carbonyl group precede C—N bond fission, have been examined by ab initio MO calculations on the neutral hydrolysis and methanolysis of N-methylazetidinone, penam, and penam-3α-carboxylate. The geometries of reactants and transition structures have been optimized fully at the 3-21G or 3-21G* levels, the existence of transition structures has been confirmed by vibrational analysis, thermochemical data have been computed, and the one-point energies of all structures have been determined at the MP2/6-31G* level. The N-protonation mechanism is found to exist with all substrates, and to be preferred over the O-protonation mechanism by over 5 kcal/mol. The activation energies are 5 kcal/mol lower in the bicyclic penam than in the monocyclic N-methylazetidinone, and attack from the convex face is preferred over attack from the concave face. The introduction of a 3α-carboxylate group, as in penicillin itself, results in an additional 5 kcal/mol decrease in activation energy. The origins of these trends are discussed. Since the active sites of some penicillin-recognizing enzymes contain at least one water molecule, catalysis of the foregoing reactions by one water molecule has also been examined. The catalysis amounts to over 10 kcal/mol in both the N- and O-protonation mechanisms, and the preference for the N-protonation mechanism is maintained. It is concluded that penicillin complexes to its receptor in such a manner as to allow acylation of the active site serine residue to proceed via the energetically most favourable mechanism.

A semiempirical molecular orbital study of the methanolysis of complex azetidinones. A combined MM and QM analysis of the interaction of Δ2- and Δ3-cephems with the penicillin receptor

The semiempirical molecular orbital procedures MINDO/3, MNDO, MNDO/M, AM1, and PM3 have been used to examine possible mechanisms and modes of attack of methanol on penam, the bicyclic ring system of penicillin. These mechanisms include a one-step process in which C—O bond formation from the convex face of the molecule and proton transfer to the β-lactam nitrogen are concerted with the cleavage of the N—C(O) bond (the N-protonated pathway), and two O-protonated pathways, viz., addition to the β-lactam carbonyl group from the convex face and from the concave face of the bicyclic ring system. Only MINDO/3 reproduces the ab initio MP2/6-31G*//3-21G* trends, that the N-protonated pathway is energetically preferred over either O-protonated pathway, and, in the latter, attack from the convex face is slightly preferred over attack from the concave face. MINDO/3 has, therefore, been used for a systematic examination of the reactions of many substituted and unsubstituted β-lactam-containing ring systems with methanol. The N-protonated structure created by attack of the alcohol from the convex face of the molecule is always preferred, and structure–reactivity relationships are observed that parallel structure–activity relationships of β-lactam antibiotics. MINDO/3 has also been used to compute a scaled quantum mechanical force field for Δ2- and Δ3-cephems. This has allowed parameters to be proposed for Δ2- and Δ3-cephems in the force field of Allinger's MMP2(85) programme that complement earlier parameterizations of the program for penicillins and peptides. The availability of these parameters has led to the development of a protocol for the analysis of the differences in the antibacterial activities of penicillins and cephalosporins. The analysis is based on a combination of "fit" and "reactivity" at the site of action. Fit is determined from the geometry that results when the β-lactam compound is complexed to a hexapeptide model of the penicillin receptor, in particular, the four-centred interaction between C-O-H of the active site serine residue and (O)C-N of the azetidinone ring. Reactivity is derived from MINDO/3 calculations of ΔE≠ for the reaction of methanol with the different ring systems via the N-protonated pathway. Δ3-Cephalosporins are suggested to be inherently less active than penicillins because they are inherently less reactive. Δ2-Cephalosporins containing a 4α-oriented carboxyl group are much less active than Δ3-cephalosporins because they have a much poorer fit. The 4β epimers of these Δ2-cephalosporins are much less active than Δ3-cephalosporins because they are much less reactive.

Escherichia coli-derived rat intestinal fatty acid binding protein with bound myristate at 1.5 A resolution and I-FABPArg106—>Gln with bound oleate at 1.74 A resolution.

Rat intestinal fatty acid binding protein (I-FABP) is a 131-residue protein composed of two short alpha-helices (alpha I and alpha II) and 10 anti-parallel beta-strands organized into two nearly orthogonal beta-sheets. The structure of crystalline I-FABP with bound tetradecanoate (myristate) has been refined to a resolution of 1.5 A and compared to the 1.2 A structure of apo-I-FABP, the 1.9 A structure of I-FABP:hexadecanoate (palmitate) and the 1.75 A structure of I-FABP:9Z-octadecanoate (oleate) to determine how this model fatty acid receptor accommodates changes in the length of its fatty acid ligand. Myristate is located in the interior of the protein. A highly ordered, electrostatic network containing 7 hydrogen (H)-bonds links the OE1 and OE2 atoms of myristate's carboxylate group, the indole nitrogen of Trp82, NH1, and NH2 of Arg106, NE2, and OE1 of Gln115, and 2 interior ordered waters. The hydrocarbon chain of the bound fatty acid is slightly bent. Its convex face lies in a crevice, forming van der Waals contacts with the side chains of several hydrophobic and aromatic residues. Its concave face is exposed to an array of 8 interior ordered waters whose positions are stabilized by H-bond interactions with other waters, H-bond interactions with the side chains of polar/ionizable residues, and van der Waals contacts with the surface of the fatty acid. Addition of 2 or 4 methylenes to myristate produces remarkably little change in the positions of I-FABP's main chain and side chain atoms and interior ordered waters. The principal alterations are in the conformation of a surface opening (portal) connecting external and internal solvent and in the position of the benzene side chain of Phe55. Changes in the conformation of the portal reflect movement of two of its components: the backbone of alpha II and a type I turn (Ala73, Asp74) connecting two beta-strands. The positions of the main chain atoms of Ala73 and Asp74 appear to be determined by their ability to form van der Waals contacts with the omega-terminus of the fatty acid. The side chain of Phe55 appears to function as an adjustable aromatic lid, located over the portal, whose position is dependent on an ability to form van der Waals contacts with a fatty acid's omega-terminus.(ABSTRACT TRUNCATED AT 400 WORDS)

Edge waves on nonplanar bathymetry and alongshore currents: A model and data comparison

The numerical solutions for the dispersion of linear, shallow‐water edge waves are compared with observed alongshore wavenumber‐frequency spectra of edge wave variance to examine their prediction of the effects of nonplanar bathymetry and moderate mean alongshore currents. Field observations were made at the two Southern California Nearshore Sediment Transport Study field sites, Torrey Pines (1978) and Leadbetter (1980) beaches. On Torrey Pines beach, where the bathymetry is very planar except for a concave (steepening landward) beach face, observed departures from the analytical plane beach dispersion solution, attributed to the concave face, are predicted by numerical dispersion solutions using the measured cross‐shore depth profile. On both beaches, asymmetry in observed dispersion curves of upstream and downstream progressing edge waves are well predicted by numerical dispersion solutions using the cross‐shore profile of measured mean alongshore current. Spectra of the alongshore velocity component from both beaches show changes in edge wave mode dominance with increased frequency. Oltman‐Shay and Guza (1987) demonstrated that mode dominance transition with frequency, as observed from a fixed offshore location, is associated with the seaward decay of edge waves and that the approximate frequency of mode transition is predicted by the profile solutions for edge wave variance and the assumption of equal shoreline elevation variance amongst modes. A submerged concave beach face at Torrey Pines beach is shown to alter the predicted cross‐shore scaling of the edge wave profiles from that predicted when mean water is below the concave beach face, and therefore to alter the mode transition frequency predictions. Different mode transition frequencies are observed in spectra from data runs with mean water levels both above and below the concave beach face, and the differences are shown to be reasonably predicted using edge wave profile solutions for the measured beach profile. For these data, observed and predicted mode transition frequencies did not change appreciably in the presence of mean alongshore current, implying that large changes in cross‐shore scaling of the edge wave profiles did not occur. However, as has been previously shown by Howd et al. (1992), there are also local edge wave profile shape changes because of the cross‐shore shear of the mean alongshore current. Verification of these predicted local shape changes can not be made with these data. Confidence in cross‐shore profile solutions is of practical importance for estimates of shoreline variances from offshore measurements. On these two beaches, estimates using both numerical solutions for the measured depth and current profiles, and analytical solutions for plane beach approximations typically differ by only 10 to 40%.

Refinement of the structure of Escherichia coli-derived rat intestinal fatty acid binding protein with bound oleate to 1.75-A resolution. Correlation with the structures of the apoprotein and the protein with bound palmitate.

The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:1) has been refined with x-ray diffraction data to a resolution of 1.75 A. The protein contains 10 anti-parallel beta strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation with C1-C12 more ordered than C13-C18. Two of the eight ordered waters in I-FABP:oleate are part of a hydrogen bond network that includes the carboxylate of oleate, the guanidinium group of Arg106, the nitrogen of the indole group of Trp82, and the side chain of Gln115. Most of the methylenes of bound oleate reside in a crevice formed by hydrophobic and aromatic side chains. Tyr70 and Tyr117 envelop the acyl chain from C3 to C8 forming contacts with both the convex and concave faces of its van der Waals surface. The hydroxyls of each phenolic side chain hydrogen bond to ordered water molecules. Two ordered waters make van der Waals contact with the concave face of the bound fatty acid. The omega-terminal methyl of oleate is oriented so that it points toward the center of the benzene of Phe55 allowing it to form van der Waals interactions with its component methylenes. Comparison of the structure of I-FABP:oleate with a recently refined 1.19-A model of apoI-FABP and an earlier 2.0-A model of I-FABP:palmitate revealed a remarkable degree of similarity in the positions of their main chain and side chain atoms and in the conformations of the bound oleate and palmitate. The principal differences were confined to a few discrete regions of the protein. The helical domain, the type I turn between beta strands C and D, and the ring of Phe55 together form a solvent-accessible portal to the interior of the protein. They are repositioned in I-FABP:oleate (and I-FABP:palmitate) so that the binding cavity is even more accessible to solvent and its volume is increased. The side chain of Phe55 which shows discrete disorder in the apoprotein functions as an omega-terminal "sensing device": moving progressively outward toward the surface as the chain length of the bound fatty acid increases by 2 methylenes. Tyr70 and Tyr117 which also show discrete disorder in the apoprotein structure due to rotation around their C alpha-C beta bonds, are stabilized in a single, well ordered position in the holoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Low frequency acoustic microscopy and pattern recognition for studying damaged and anisotropic composites and material defects

In recent years, acoustic microscopy has been found to be very useful for characterizing engineering as well as biologic materials. With the present state of knowledge on acoustic microscopy, one can obtain the surface wave velocity of a homogeneous specimen or coating thickness of a coated material and produce images of near surface internal defects and inhomogeneities in a specimen. Applications of acoustic microscopy for obtaining material properties of anisotropic specimens and detecting material defects at a greater depth are meager because commercially available acoustic microscopes are insensitive to direction-dependent material properties and they, in general, have poor penetration properties because of high operating frequencies. Recently at the University of Arizona an unconventional low frequency (0.5–2.5 MHz) acoustic microscope has been fabricated where the microscope lens has been replaced by two ultrasonic transducers with cylindrical concave faces; one works as a transmitter and the other one works as a receiver. Using this arrangement, it has been found that it is possible to detect internal damages in a material and identify material anisotropy in fiber-reinforced composite plates. These capabilities of the microscope are demonstrated in this paper by presenting some experimental results along with theoretical justifications. Then pattern recognition techniques are used to solve the inverse problem, that is, to predict the type of material defect from reflected acoustic signals.

Analysis of outer membrane ultrastructure of pathogenic Treponema and Borrelia species by freeze-fracture electron microscopy

We analyzed the outer membrane (OM) ultrastructure of four pathogenic members of the family Spirochaetaceae by freeze fracture. The OM of Treponema pallidum subsp. pertenue contained a low intramembranous particle concentration, indicating that it contains few OM transmembrane proteins. The concave OM fracture faces of Treponema hyodysenteriae and Borrelia burgdorferi contained dense populations of particles, typical of gram-negative organisms. A relatively low concentration of particles which were evenly divided between a small and a large species was present in the concave OM fracture face of Borrelia hermsii; the convex OM fracture face contained only small particles. As for gram-negative bacteria, the convex OM fracture face particle concentrations of these pathogens were low. These spirochetes cleaved preferentially within the OM, in contrast to typical gram-negative bacteria, which tend to fracture within the inner membrane. The OM ultrastructure of T. pallidum subsp. pertenue provides an explanation for the lack of antigenicity of the treponemal surface and may reflect a mechanism by which this pathogen evades the host immune response.

Pleurostomella concava:a new benthic foraminifer in Oligocene and Miocene sediments from the Pacific and Indian Oceans

The speciesPleurostomella concavais a new benthic foraminifer recognized in Oligocene and Miocene sediments from the Indian and Pacific Oceans. The first mentioned finding of this species is in the material recovered from the experimental Mohole drilling near Guadalupe Island off Baja California. The morphology ofPleurostomella concavadiffers distinctively from other species of the genusPleurostomellaby its concave apertural face and keel-like rim around the ultimate chamber.

Organization of mitochondria in olfactory bulb granule cell dendritic spines

In contrast to dendritic spines with only postsynaptic functions, the spines of olfactory bulb granule cells subserve both pre- and postsynaptic roles. In single sections these spines were previously seen to contain mitochondria, most likely needed to provide energy for presynaptic functions, but their frequency and distribution were unknown. In order to understand the organization of mitochondria in these specialized dendritic appendages, we have studied the geometry and cytoplasmic organization of granule cell spines with computer-assisted reconstructions of serial electron micrographs. The spine heads were seen to be elliptical in shape with a single pair of reciprocal synapses on the concave face apposed to the mitral/tufted cell dendrite. Mitochondria were found localized in the spine neck as well as the spine head and often extended between the two compartments. Based on their variable distribution it seems reasonable to suggest that these mitochondria are motile and move in and out of spine compartments from the parent dendrite. Spine apparatus was apparent in most of the spines as membrane bound cisterns of smooth endoplasmic reticulum located close to mitochondria. The possible role of spine apparatus in facilitating the movement of mitochondria in the necks and heads of granule cell spines in the absence of microtubules is discussed.

Changes in the surface of Leptospira interrogans serovar grippotyphosa during in vitro cultivation

Surface components of virulent and attenuated Leptospira interrogans serovar grippotyphosa were compared by using Triton X-114 solubilization and phase partitioning, immunoprecipitation of intact organisms, and freeze-fracture electron microscopy. Removal of the leptospiral outer membrane by using 0.1% Triton X-114 was demonstrated by whole-mount electron microscopy and by essentially complete solubilization of a lipopolysaccharidelike substance (LLS) from the outer membrane. Triton X-114 (0.1%) did not solubilize subsurface proteins, such as endoflagellar filaments or penicillin-binding proteins, which are markers for the periplasmic space and inner membrane, respectively. Triton X-114 solubilized material from both the virulent and attenuated strains, which partitioned into the hydrophobic, detergent phase, contained LLS and major proteins of 41 and 44 kDa, which were also immunoprecipitable from intact organisms. The virulent strain contained greater amounts of an LLS component with an apparent molecular mass of 30 kDa (R(f) = 0.57), whereas the attenuated strain contained larger amounts of an LLS component with an apparent molecular mass of 20 kDa (R(f) = 0.74). Differences in protein components between virulent and attenuated organisms were also detected; whereas the 41- and 44-kDa proteins were immunoprecipitated in equal amounts from both the virulent and attenuated strains, a 33-kDa protein was immunoprecipitated in significantly greater amounts from the attenuated strain. Quantitation of outer membrane particle density by freeze-fracture electron microscopy showed that both strains had a low transmembrane outer membrane protein content compared with that of typical gram-negative bacteria. The virulent and attenuated strains had 443 and 990 particles (P less than 0.000001) per micron, respectively, in the concave outer membrane fracture face. These findings suggest that in vitro cultivation of L. interrogans is accompanied by quantitative and qualitative changes in both LLS and outer membrane-associated proteins.

Ultrastructural features of the developing eosinophils in bone marrow and spleen of the musk shrew, Suncus murinus.

Eosinophilopoiesis in the musk shrew, Suncus murinus, a representative of the order Insectivora, was studied by light and electron microscopy. To examine biochemical features of cytoplasmic granules, extraction with proteolytic enzymes was carried out on ultrathin sections of bone marrow. In this species, eosinophils are produced in the same manner in both spleen and bone marrow. Developing eosinophils were distinguished as belonging to four stages, recognized by ultrastructural changes in cytoplasmic organelles as well as the eosinophilic granules during maturation. Granulogenesis began by budding of vacuoles containing flocculent material from the concave face of the Golgi apparatus, in the promyelocyte to myelocyte stage. The matrix of developing granules transformed into a finely granular structure, and the large spherical granules of mature eosinophils were homogeneous without crystalline cores. It was shown by proteolytic enzyme extraction that the proteinaceous cores of mature granules were uniformly removed; there was no evidence that they contained crystalloid inclusions. These results indicate that shrew eosinophils can be regarded as cells that retain a prototype of eosinophil granules, probably like those of ancestral mammals rather than those of higher living Mammalia.

Demonstration of rare protein in the outer membrane of Treponema pallidum subsp. pallidum by freeze-fracture analysis.

The surface of Treponema pallidum subsp. pallidum (T. pallidum), the etiologic agent of syphilis, appears antigenically inert and lacks detectable protein, as judged by immunocytochemical and biochemical techniques commonly used to identify the outer membrane (OM) constituents of gram-negative bacteria. We examined T. pallidum by freeze-fracture electron microscopy to visualize the architecture of its OM. Treponema phagedenis biotype Reiter (T. phagedenis Reiter), a nonpathogenic host-associated treponeme, and Spirochaeta aurantia, a free-living spirochete, were studied similarly. Few intramembranous particles interrupted the smooth convex and concave fracture faces of the OM of T. pallidum, demonstrating that the OM of this organism is an unusual, nearly naked lipid bilayer. In contrast, the concave fracture face of the OM of S. aurantia was densely covered with particles, indicating the presence of abundant integral membrane proteins, a feature shared by typical gram-negative organisms. The concentration of particles in the OM concave fracture face of T. phagedenis Reiter was intermediate between those of T. pallidum and S. aurantia. Similar to typical gram-negative bacteria, the OM convex fracture faces of the three spirochetes contained relatively few particles. The unique molecular architecture of the OM of T. pallidum can explain the puzzling in vitro properties of the surface of the organism and may reflect a specific adaptation by which treponemes evade the host immune response.