On Paraptychostomum almae N. G., N. Sp., a Commensal Ciliate from the Digestive Tract of Oligochaetes of the Cameroons, in a New Subclass Hysterocinetia

ABSTRACT. Morphological and ultrastructural studies on a new ciliate, Paraptychostomum almae , from the digestive tract of an oligochaete ( Alma emini ) from the Cameroons are carried out. The flattened cell has a large size; its left lateral face bears an anterior thigmotactic zone that includes seven-nine short kinetal segments. The somatic cortex is composed of flattened alveoli, a thin epiplasm and a microfibrillar ecto-endoplasmic boundary. Kineties are made of monokinetids, each particularly characterized by a long anteriorly directed kinetodesmal fiber, and a hyperdivergent postciliary ribbon. The postero-ventral buccal apparatus consists of a short peristome and a deep longitudinal infundibulum. The paroral organelle is a long stichodyad. The three adoral organelles are of different types: ADI and AD3 are of the membranoid type, respectively with two and one rows of ciliated kinetosomes; AD2 is of the peniculus type with six-seven rows of ciliated kinetosomes. A microfibrillar network with nodes arises from all the buccal kinetosomes and extends under the naked wall. Mitochondria are small and numerous and dispersed throughout the whole cell. The existence of an AD2 with more than two rows of kinetosomes warrants the creation of the new genus Paraptychostomum and a new family, Ptychostomatidae. The presence of a distinct ecto-endoplasmic boundary and of somatic kinetids exclusive without transversal dense tractus, hyperdivergent postciliary ribbons, and dispersed numerous mitochondria, added to particularities of the stomatogenesis, allow us to clearly separate hysterocinetians from the scuticociliates and to set up for them the new subclass Hysterocinetia, within the class Oligohymenophorea, with a single new order Hysterocinetida.     

A genetic map of soybean (Glycine max L.) using an intraspecific cross of two cultivars: ‘Minosy’ and ‘Noir 1’

Genetic markers were mapped in segregating progeny from a cross between two soybean (Glycine max (L.) Merr.) cultivars: Minsoy (PI 27.890) and Noir 1 (PI 290.136). A genetic linkage map was constructed (LOD 3), consisting of 132 RFLP, isozyme, morphological, and biochemical markers. The map defined 1550cM of the soybean genome comprising 31 linkage groups. An additional 24 polymorphic markers remained unlinked. A family of RFLP markers, identified by a single probe (hybridizing to an interspersed repeated DNA sequence), extended the map, linking other markers and defining regions for which other markers were not available.     

Fine Structure and Cytochemistry of Tritrichomonas foetus and Rat Neutrophil Interaction

ABSTRACT. The fine structure of normal and antibody-coated Tritrichomonas foetus cells and their interaction with rat peritoneal neutrophils was studied. Peritoneal neutrophils were obtained by glycogen stimulation. The neutrophils readily associated with and killed the parasites, which were subsequently ingested. The process involved activation of the respiratory burst, as demonstrated by the use of cytochemical methods. Images were obtained indicating that binding of parasites to the neutrophil surface triggers an exocytic response with release of oxygen-derived products. Cytochemical localization of acid phosphatase and peroxidase activities showed that leukocyte granules fused with the parasite-containing phagocytic vacuoles. We also showed the cytochemical localization of alkaline phosphatase in the parasite-neutrophil interaction.     

Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants.

Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of randomly generated mutants of T4 lysozyme show that protein stability can be enhanced by relaxation of strain and by improved hydrogen bonding via bound solvent.

The structures of three mutants of bacteriophage T4 lysozyme selected using a screen designed to identify thermostable variants are described. Each of the mutants has a substitution involving threonine. Two of the variants, Thr 26-->Ser (T26S) and Thr 151-->Ser (T151S), have increased reversible melting temperatures with respect to the wild-type protein. The third, Ala 93-->Thr (A93T), has essentially the same stability as wild type. Thr 26 is in the wall of the active-site cleft. Its replacement with serine results in the rearrangement of nearby residues, most notably Tyr 18, suggesting that the increase in stability may result from the removal of strain. Thr 151 in the wild-type structure is far from the active site and appears to sterically prevent the access of solvent to a preformed binding site. In the mutant, the removal of the methyl group allows access to the solvent binding site and, in addition, the Ser 151 hydroxyl rotates to a new position so that it also contributes to solvent binding. Residue 93 is in a highly exposed site on the surface of the molecule, and presumably is equally solvent exposed in the unfolded protein. It is, therefore, not surprising that the substitution Ala 93-->Thr does not change stability. The mutant structures show how chemically similar mutations can have different effects on both the structure and stability of the protein, depending on the structural context. The results also illustrate the power of random mutagenesis in obtaining variants with a desired phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution.

The three-dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 A resolution. The structure has a Kunitz-type fold and is composed of 12 antiparallel beta-strands, 6 of which form a beta-barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441-3445). Two beta-mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the S gamma atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 A resolution and the structure refined to an R-factor of 13.8%. The binding of the more electron-dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 A from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.     

Impression Cytology In Patients With Keratoconjunctivitis Sicca

Impression cytology is a simple and painless procedure that allows the collection of the superficial layers of the conjunctival epithelium. Each sample is assigned a grade of epithelial metaplasia, and goblet cell density is calculated in each one. We have studied the superior and temporal bulbar conjuctiva of dry eyes and have compared it with that of normal controls. In normal and dry eyes we find a statistically significant difference both in goblet cell density and grade of metaplasia, between superior and temporal bulbar conjunctiva. the differences in grade of metaplasia and goblet cell density between normal and dry eyes are significant in the superior conjunctiva, but in the temporal conjunctiva we only find significant differences in grade of metaplasia.