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Biomedical subjects

W A Samsonoff

Publications and source records attributed to W A Samsonoff.

At least 19 recordsLinked to original sources
PubMed2002

Crystine: fibrous biomolecular material from protein crystals cross-linked in a specific geometry.

Cysteine substitutions were engineered on the surface of maltose binding protein to produce crystine fibers, linear polymers of folded protein formed within a crystal. Disulfide bond formation between adjacent protein molecules within the lattice was monitored by X-ray crystallography. The cross-linked crystals were resistant to dissolution in water or neutral buffer solutions, even though the cross-linking was one-dimensional. However, crystine fibers were observed by transmission electron microscopy to dissociate from the crystals in acidic solutions. Some fibers remained associated as two-dimensional bundles or sheets, with a repeat unit along the fibers consistent with the packing of the individual protein molecules in the crystal. Neutralization of the acidic solutions caused the fibers to re-associate as a solid. Crystine threads were drawn out of this solution. In scanning electron microscopy images, many individual fibers could be seen unwinding from the ends of some threads. Crystine fibers are a new type of biomolecular material with potential applications wherever the use of proteins in a fibrous form is desirable, for example, the incorporation of enzymes into cloth or filtration material.

Carrier Proteins↗
PubMed2001

Biliproteins and phycobilisomes from cyanobacteria and red algae at the extremes of habitat.

This review considers the properties of biliproteins from cyanobacteria and red algae that grow in extreme habitats. Three situations are presented: cyanobacteria that grow at high temperatures; a red alga that grows in acidic conditions at high temperature; and an Antarctic red alga that grows in the cold in dim light conditions. In particular, the properties of their biliproteins are compared to those from organisms from more usual environments. C-phycocyanins from two cyanobacteria able to grow at high temperatures are found to differ in their stabilities when compared to C-phycocyanin from mesophilic algae. They differ in opposite ways, however. One is more stable to dissociation than the mesophilic protein, and the other is more easily dissociated at low temperatures. The thermophilic proteins resist thermal denaturation much better than the mesophilic proteins. The most thermophilic cyanobacterium has a C-phycocyanin with a unique blue-shifted absorption maximum which does not appear to be part of the adaptation of the cyanobacterium to high temperature. The C-phycocyanin from the high-temperature red alga is able to resist dissociation better than mesophilic C-phycocyanins. Electron micrographs show the phycobilisomes of these algae. The Antarctic alga grows under ice at some distance down the water column. Its R-phycoerythrin has a novel absorption spectrum that gives the alga an improved ability to harvest blue light. This may enhance its survival in its light-deprived habitat.

Cold Temperature↗
PubMed2001

Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects.

In Treponema denticola, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola, and double-crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects, including highly condensed chromosomal DNA, compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover, chains of cells are formed by the cytoplasmic filament-deficient mutant, and those cells show reduced spreading in agarose, which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure, segregation, or the cell division process in Treponema.

Bacterial Proteins↗
PubMed1999

Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks.

The treponemal fla operon is comprised of numerous motility-related genes; however, the initial gene of this operon, tap1, has no known function. A recently developed system to generate specific mutants in Treponema denticola was utilized to determine if Tap1 was essential for motility. T. denticola tap1 and flanking DNA were identified, cloned, and sequenced, and a suicide plasmid that contained tap1 interrupted with an erythromycin resistance cassette (ermF and ermAM) was constructed. Because of potential polar effects from this cassette, a second plasmid that contained tap1 interrupted with a modified erythromycin resistance cassette that lacked the putative ermF transcription terminator was constructed. Electroporation-mediated allelic exchange incorporated the interrupted tap1 genes into the T. denticola chromosome, creating Tap1-deficient mutants. Reverse transcriptase PCR revealed that the erythromycin resistance cassette within tap1 did not terminate fla operon transcription in either mutant. Moreover, the phenotypes of the two mutants were indistinguishable. These mutants lacked motion in liquid culture, were unable to spread on agar plates, and lacked flagellar filaments as determined by electron microscopy. Immunoblots revealed a marked reduction in detectable FlaB flagellar filament protein compared to that of wild type; however, flaB RNA was easily detectable, and transcription levels did not appear to be altered. The basis for the lack of filament protein expression is unknown. Immunoblotting also showed that the flagellar hook protein (FlgE) was synthesized in the Tap1-deficient mutant; however, electron microscopy revealed that the mutant possessed unusual elongated hooks of variable lengths. We propose that treponemal Tap1 is analogous to FliK, which is involved in monitoring the flagellar hook length of Salmonella typhimurium.

Amino Acid Sequence↗
PubMed1999

Genetic and structural analyses of cytoplasmic filaments of wild-type Treponema phagedenis and a flagellar filament-deficient mutant.

Unique cytoplasmic filaments are found in the treponeme genus of spirochete bacteria. Their function is unknown, but their location underneath the periplasmic flagellar filaments (PFF) suggests a role in motility and/or cell structure. To better understand these unique structures, the gene coding for the cytoplasmic filaments, cfpA, was identified in various treponemal species. Treponema phagedenis cfpA was 2,037 nucleotides long, and the encoded polypeptide showed 78 to 100% amino acid sequence identity with the partial sequence of CfpA from T. denticola, T. vincentii, and T. pallidum subsp. pertenue. Wild-type T. phagedenis and a PFF-deficient isolate were analyzed by electron microscopy to assess the structural relationship of the cytoplasmic filaments and the PFF. The number of cytoplasmic filaments per cell of T. phagedenis (mean, 5.7) was compared with the number of PFF at each end of the cell (mean, 4.7); the results suggest that there is no direct one-to-one correlation at the cell end. Moreover, a structural link between these structures could not be demonstrated. The cytoplasmic filaments were also analyzed by electron microscopy at different stages of cell growth; this analysis revealed that they are cleaved before or during septum formation and before the nascent formation of PFF. A PFF-deficient mutant of T. phagedenis possessed cytoplasmic filaments similar to those of the wild type, suggesting that intact PFF are not required for their assembly and regulation. The extensive conservation of CfpA among pathogenic spirochetes suggests an important function, and structural analysis suggests that it is unlikely that the cytoplasmic filaments and the flagellar apparatus are physically linked.

Bacterial Proteins↗
PubMed1998

Analysis of constructed E gene mutants of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly.

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.

Amino Acid Sequence↗
PubMed1997

Intracellular location of thymidylate synthase and its state of phosphorylation.

Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis, was found to be associated mainly with the nucleolar region of H35 rat hepatoma cells, as determined both by immunogold electron microscopy and by autoradiography. In the latter case, the location of TS was established through the use of [6-3H]5-fluorodeoxyuridine, which forms a tight ternary complex of TS with 5-fluorodeoxyuridylate (FdUMP) and 5, 10-methylenetetrahydrofolylpolyglutamate within the cell. However, with H35 cells containing 50-100-fold greater amounts of TS than unmodified H35 cells, the enzyme, although still in the nucleus, was located primarily in the cytoplasm as shown by autoradiography and immunohistochemistry. In addition, TS was also present in mitochondrial extracts of both cell lines, as determined by enzyme activity measurements and by ternary complex formation with [32P]FdUMP and 5,10-methylenetetrahydrofolate. Another unique observation is that the enzyme appears to be a phosphoprotein, similar to that found for other proteins associated with cell division and signal transduction. The significance of these findings relative to the role of TS in cell division remains to be determined, but suggest that this enzyme's contribution to the cell cycle may be more complex than believed previously.

Animals↗
PubMed1994

Genetic and biochemical analysis of the flagellar hook of Treponema phagedenis.

The periplasmic flagellum of Treponema phagedenis consists of the flagellar filament and hook-basal body. We report here a characterization of the hook gene and flagellar hook of T. phagedenis, and in the process of this analysis we found evidence that the hook polypeptide is likely cross-linked in situ. A T. phagedenis genomic library was screened with a Treponema pallidum antiserum, and the DNA segments from several positive plaques were subcloned and sequenced. DNA sequencing of two overlapping segments revealed a 1,389-nucleotide (nt) open reading frame (ORF) with a deduced amino acid sequence that was 36% identical to that of FlgE, the hook polypeptide of Salmonella typhimurium. This gene was designated T. phagedenis flgE. Beginning at 312 nt downstream from flgE was a partial ORF of 486 nt with a deduced amino acid sequence that was 33% identical to that of MotA of Bacillus subtilis, a polypeptide that enables flagellar rotation. Upstream of flgE, separated by 39 nt, was a partial (291-nt) ORF with a deduced amino acid sequence that was homologous to that of ORF8, a polypeptide of unknown function located in an operon encoding polypeptides involved in motility of B. subtilis. The T. phagedenis flgE gene was cloned into an Escherichia coli protein expression plasmid, and the purified recombinant protein was used to prepare a FlgE antiserum. Western blots (immunoblots) of whole-cell lysates probed with this antiserum revealed a 55-kDa polypeptide and a ladder of polypeptide bands with increasing molecular masses. T. phagedenis hooks were then isolated and purified, and electron microscopic analysis revealed that the morphology of the hooks resembled that in other bacteria. The hooks were slightly curved and had an average length of 69 +/- 8 nm and a diameter of 23 +/- 1 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots of purified hook preparations using the FlgE antiserum also revealed a polypeptide ladder, suggesting that the hooks are composed of a covalently cross-linked polypeptide.

Amino Acid Sequence↗
PubMed1994

Monoclonal antibody to native P39 protein from Borrelia burgdorferi.

We have produced, by using a sonicate of Borrelia burgdorferi, a monoclonal antibody (MAb), NYSP39H, that is specific for the P39 protein band. This MAb reacted with 13 isolates of B. burgdorferi but not with eight different spirochetes (four borrelias, two leptospiras, and two treponemas). Surface labeling of B. burgdorferi with biotin and subsequent treatment with Nonidet P-40 showed that P39 was not biotinylated but was extracted with Nonidet P-40, indicating that it is present within the outer membrane, but not on the surface of the spirochete. Immunoelectron microscopy revealed the immunogold probe primarily at the cytoplasmic membrane region of the spirochete. The MAb detected B. burgdorferi in the indirect fluorescent-antibody test only when the spirochetes from a culture or in a tick homogenate were fixed with polylysine and not with acetone. NYSP39H appears to be an appropriate probe for use in the specific detection of B. burgdorferi.

Animals↗
PubMed1994

Subcellular distribution of adenosine deaminase and adenosine deaminase-complexing protein in rabbit kidney: implications for adenosine metabolism.

We evaluated the age-related distribution of adenosine deaminase (ADA) and adenosine deaminase-complexing protein (CP) in rabbit kidney by immunohistochemical staining procedures. Paraffin- or resin-embedded tissue from rabbits < 1 week-4 years of age were stained by the peroxidase-anti-peroxidase (PAP) method for ADA and CP. With the exception of neonates, the qualitative staining pattern of each protein remained generally constant with age. In the cortex, distal tubules, blood vessels, histiocytes, and epithelial cells lining Bowman's capsule stained for ADA. Proximal tubules and glomeruli were positive for CP. In contrast to the segregated pattern in the cortex, staining for ADA and CP overlapped in the corticomedullary junction. ADA and CP co-localized on the brush border of tubule cells of the S3 segment. In the cytoplasm of these cells, staining for ADA was characterized by scattered punctuate deposits of peroxidase reaction product. In some instances these punctuate deposits also appeared to be positive for CP. In medulla, epithelial cells of the thin limb were positive for both ADA and CP, whereas papillary collecting ducts stained only for CP. These results document the age-related, tissue-specific expression and localization of ADA in renal tissue, features that probably reflect the crucial role played by the enzyme in adenosine/deoxyadenosine catabolism. In addition, colocalization of ADA and CP on the brush border of cells in the S3 segment of proximal tubules provides support for the hypothesis that one function of CP may be to position ADA on the plasma membrane of specific cell populations, further expanding the enzyme's utility in nucleoside metabolism.

Adenosine Deaminase↗
PubMed1994

17-beta-Estradiol induced alterations of cell-matrix and intercellular adhesions in a human mammary carcinoma cell line.

The MCF-7 human mammary carcinoma cell line undergoes morphological differentiation in vitro when treated with 17-beta-estradiol. A prominent feature of this process is the postconfluent development of multicellular, three-dimensional nodules that rise above the surrounding monolayer. Formation of the nodules suggests that changes in cellular adhesion occur during this cellular overgrowth. Therefore changes in the distribution of cell-matrix and cell-cell adhesion plaque proteins were examined with respect to estradiol induction of nodule development. Estradiol treatment of the carcinoma cell line had the following effects: (1) vinculin- and talin-rich cell-matrix adhesion plaques were reduced in overall number and size in confluent and postconfluent cultures. No overt change in distribution or morphology of adhesion plaques was observed in subconfluent cultures. (2) Staining for vinculin was reduced in cell-cell adhesions situated at the apical region of subconfluent, confluent and postconfluent monolayers. Staining for F-actin and plakoglobin was retained at this region in estradiol-induced cells. (3) vinculin was not detected in intercellular adhesions of nodule cells although intense labelling for both F-actin and plakoglobin was observed. In addition, in untreated monolayer cells, both F-actin and plakoglobin were concentrated in a subapical/basolateral location, as a vesicle-like pattern, which corresponded to intercellular spaces observed with phase-contrast microscopy. Treatment with estradiol caused the rearrangement of subapical/basolateral F-actin and plakoglobin staining into a more uniform pattern. The findings of this study show that estradiol induces changes in both cell-matrix and cell-cell adhesions in an estrogen-responsive carcinoma cell line. The gradual loss of vinculin from cell-matrix and cell-cell adherens junctions of the monolayer could be a potential factor in the capacity of these cells to form multilayers or nodules in postconfluent growth. Furthermore, the development of the nodules in response to estradiol may provide a useful system in which to study steroid hormone regulation of adhesion and the cytoskeleton in responsive tumor cells.

Actins↗
PubMed1993

Three-dimensional reconstruction of Coxiella burnetii-infected L929 cells by high-voltage electron microscopy.

Previous examination of thin sections of L929 cells heavily infected with the Q fever Priscilla isolate by conventional transmission electron microscopy indicated that the rickettsiae resided within multiple vacuoles. The present study using high-voltage electron microscopy and three-dimensional reconstruction revealed that, in heavily infected cells, the rickettsiae, in fact, reside in one multilobed vacuole. As a result of asymmetric cell division, the multilobed vacuole containing the rickettsiae apparently segregates into one daughter cell, while the companion daughter cell emerges parasite free. This likely explains the appearance of naive uninfected cells in long-term-infected (i.e., ca. 2 years) cell populations that had not been supplemented with uninfected L929 host cells.

Animals↗
PubMed1992

Adherence and entry of Borrelia burgdorferi in Vero cells.

Adherence to and entry of the parasite into the host is one of the essential elements of microbial pathogenicity. We investigated the adherence to and entry into primate kidney epithelial (Vero) cells of Borrelia burgdorferi by radiolabelling techniques, immunofluorescence and electronmicroscopy. The attachment to and subsequent entry of both untreated and heat (50 degrees C)-treated B. burgdorferi into Vero cells occurred at cell-surface sites associated with aggregated coated pits. In contrast, there was minimal attachment of spirochaetes heated at 60 degrees C. Radiometric studies showed that, with untreated cells, there was incorporation of both 14C-glucose-1-phosphate and 14C-thymidine, whereas with the 50 degrees C-treated spirochaetes only glucose-1-phosphate was incorporated, and with the 60 degrees C-treated spirochates neither radionuclide was incorporated. Spirochaetes heated at 50 degrees C or 60 degrees C did not grow at 35 degrees C in culture medium. These results suggest that the presence of certain metabolic activities of the spirochaete but not viability (ability to grow) are necessary for the attachment process. After entry of untreated B. burgdorferi, most of the spirochaetes were either free in the cytoplasm or tightly bound to the host membrane. In contrast, 50 degrees C-treated spirochaetes remained bound to host membrane in large phagosome-like vesicles.

Animals↗
PubMed1991

Scanning electron microscopy of the conidia produced by the mycelial form of Paracoccidioides brasiliensis.

The conidia produced by the mycelial form of Paracoccidioides brasiliensis were examined by scanning electron microscopy for the first time. Several different conidial types were characterized. These included intercalary arthroconidia, several types of septate conidia that are formed from other conidia, pedunculate conidia, and terminal hyphal conidia. In addition, the ultrastructure of the supporting pedestal of the pedunculate conidium was found to be separated from the mother conidium by a septum in some instances, and at other times it was not.

Microscopy, Electron, Scanning↗
PubMed1991

Electron microscopic study of conidia produced by the mycelium of Paracoccidioides brasiliensis.

The ultrastructure of asexual spores (conidia) produced by the mycelial form of Paracoccidioides brasiliensis was studied for the first time with transmission electron microscopy, using thin sections of aldehyde-osmium-fixed and epoxy-resin-embedded samples. The various types of conidia observed in the sections correlated well with previous light-microscopic descriptions. These types were intercalary or apical conidia, depending on their location along the originating hyphae. As in previous studies they were characterized as arthroconidia, aleuriospores and sessile or pedunculate pyriform conidia. The sporogenous cells were clearly distinguished from hyphal cells by the thickness and appearance of their cell walls. Copious fibrillar material (glycocalyx) detected at the cell surface was stained with ruthenium red during the fixation process. Typical subcellular organelles (nucleus, nucleolus, mitochondria, ribosomes, etc) were found in most of the sections. It was concluded that the spores produced by the mycelial phase of P. brasiliensis possess all attributes of viable and physiologically competent eukaryotic cells.

Humans↗
PubMed1991

Analysis of Coxiella burnetii isolates in cell culture and the expression of parasite-specific antigens on the host membrane surface.

Coxiella burnetii isolates may be classified into several groups based on plasmid character. These groups may also be correlated with disease syndrome--chronic or short-term acute. L929 mouse fibroblast cells were exposed, independently, to two members of the three major C. burnetii groups, and their growth/morphological characteristics analysed by light and electron microscopy, including High Voltage Electron Microscopy. The fates of the isolates were followed. Two acute isolates [Nine Mile (RSA 493) and Henzerling (RSA 331), QpH1-type plasmids] and two chronic isolates (S Q217 and L Q216, plasmidless) readily infected L929 cells in static culture. Priscilla (Q177) and F (Q228) isolates (QpRS-type plasmid, and implicated in causing chronic Q fever) took longer to infect cells, and, unlike the members of the other two groups, gradually disappeared when shifted to suspension culture. Cells infected with Q177 and Q228 exhibited a higher degree of vacuolation than cells infected with the other isolates. Parasite-specific antigens on the surfaces of the host cells were analysed by immunofluorescence/flow cytometry. The acute and plasmidless isolates caused the display of significantly more C. burnetii-specific antigen on the host cell membrane than the two QpRS plasmid-containing isolates. This host cell model system clearly reveals biological differences among the C. burnetii groups.

Animals↗