Role of the membrane-associated folate binding protein (folate receptor) in methotrexate transport by human KB cells

The uptake of methotrexate by KB cells was observed to be dependent on time, temperature, and concentration of extracellular methotrexate. The Kd for methotrexate surface binding to KB cells was approximately 200 nM. Following exposure of KB cells to trace quantities of [3H]methotrexate for periods ranging from 6 min to 24 h, the cellular methotrexate was progressively formed into methotrexate polyglutamates and was bound to dihydrofolate reductase as well as to a particulate folate binding protein. To further study the mechanism of methotrexate uptake in KB cells, the N-hydroxysuccinimide ester of methotrexate was used to covalently label the surface of KB cells and to inhibit transport of methotrexate. The N-hydroxysuccinimide ester of methotrexate was bound to a species of protein with an apparent molecular weight of 160,000 in 1% (v/v) Triton X-100 that bound folic acid and was specifically precipitated by antiserum raised against the previously purified high-affinity folate binding protein (the folate receptor) from human KB cells. In addition, trypsin was utilized to remove surface-accessible covalently bound methotrexate. The amount of covalently bound methotrexate that could be released by trypsin initially decreased on incubation at 37 degrees C, suggesting that the methotrexate and binding protein were internalized. However, with time, trypsin could again release the covalently bound methotrexate, suggesting that the binding protein cycles from the external cell surface to the inside of the cell and out again.     

Formation of heteroduplex DNA during mammalian intrachromosomal gene conversion.

We have studied intrachromosomal gene conversion in mouse Ltk- cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The gene intended to serve as the recipient in gene conversion differs from the donor sequence by virtue of a palindromic insertion that creates silent restriction site polymorphisms between the two genes. While selection for gene conversion at a XhoI linker insertion within the recipient gene results in coconversion of the nearby palindromic site in more than half of the convertants, 4% of convertant colonies show both parental and nonparental genotypes at the polymorphic site. We consider these mixed colonies to be the result of genotypic sectoring and interpret this sectoring to be a consequence of unrepaired heteroduplex DNA at the polymorphic palindromic site. DNA replication through the heteroduplex recombination intermediate generates genetically distinct daughter cells that comprise a single colony. We believe that the data provide the first compelling genetic evidence for the presence of heteroduplex DNA during chromosomal gene conversion in mammalian cells.     

Identification and characterization of an {alpha}-mannosidase from Trypanosoma cruzi

In this report we describe the first purification and characterizationof the acid -mannosidase from the human parasite Trypanosomacruzi. The purified enzyme exhibited a native mol. wt of 240000 Da and is apparently composed of four identical subunitsof mol. wt 58 000 Da. Each of the four subunits contains oneN-linked high-mannose-type oligosaccharide. The -mannosidaseexhibited a pH optimum of 3.5 and a pI of 5.9. This low pH optimumand the ability of swainsonine to inhibit its activity suggestthat the -mannosidase is a lysosomal enzyme. Antibodies againstthe T.cruzi enzyme did not react with mammalian lysosomal -mannosidaseand, conversely, antibody against a rat lysosomal -mannosidasedid not react with the T.cruzi enzyme. Thus, the T.cruzi enzymeappears to be distinct from its mammalian counterpart. -mannosidase lysosomal enzyme Trypanosoma cruzi     

The effect of grazing intensity on phosphorus spiralling in autotropic streams

The effect of grazing on primary productivity and phosphorus cycling in autotrophic streams was studied using the snail Goniobasis clavaeformes. Snails were added to each of three replicate laboratory stream channels, receiving once-through flow of groundwater, in densities of 2.1, 3.0, and 4.2 g ash free dry mass (AFDM)/m². A fourth channel received no snails and served as an ungrazed control. Presence of snail grazers resulted in a large reduction in aufwuchs biomass, primary productivity, and biotic phosphorus uptake; a modest reduction in fine particulate organic matter (FPOM); and an increase in the fraction of stream particulate organic matter (POM) exported as seston. Although primary production and aufwuchs biomass continued to decline with increasing snail density, phosphorus uptake increased. This increased phosphorus uptake is attributed to abiotic sorption to inorganic surfaces exposed as a result of efficient removal of aufwuchs at high snail densities. Although snail densities were chosen to bracket the density measured in a natural stream, the experimental densities may result in considerably higher grazing pressure on aufwuchs due to the absence of alternate food sources (e.g., coarse particulate organic matter) usually found in natural streams. Presence of snail grazers increased the spiralling length of phosphorus, primarily by reducing aufwuchs biomass and consequently reducing uptake of phosphorus from the water. Presence of snails also increased downstream transport velocity of phosphorus bound to organic particles. These results follow the patterns predicted in a previous theoretical analysis for mildly phosphorus-limited streams.     

The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse.

Homozygosity for a null mutation in the scl gene causes mid-gestational embryonic lethality in the mouse due to failure of development of primitive hematopoiesis. Whilst this observation established the role of the scl gene product in primitive hematopoiesis, the death of the scl null embryos precluded analysis of the role of scl in later hematopoietic development. To address this question, we created embryonic stem cell lines with a homozygous null mutation of the scl gene (scl-/-) and used these lines to derive chimeric mice. Analysis of the chimeric mice demonstrates that the scl-/- embryonic stem cells make a substantial contribution to all non-hematopoietic tissues but do not contribute to any hematopoietic lineage. These observations reveal a crucial role for the scl gene product in definitive hematopoiesis. In addition, in vitro differentiation assays with scl-/- embryonic stem cells showed that the scl gene product was also required for formation of hematopoietic cells in this system.     

Characterization of Mhc genes in a multigenerational family of ring-necked pheasants

Little is known about the major histocompatibility (Mhc) genes of birds in different taxonomic groups or about how Mhc genes may be organized in avian species divergent by evolution or habitat. Yet it seems likely that much might be learned from birds about the evolution, organization, and function of this intricate complex of polymorphic genes. In this study a close relative of the chicken, the ring-necked pheasant (Phasianus colchicus), was examined for the presence and organization of Mhc B-G genes. The patterns of restriction fragments revealed by chicken B-G probes in Southern hybridizations and the patterns of pheasant erythrocyte polypeptides revealed in immunoblots by antisera raised against chicken B-G polypeptides provide genetic, molecular, and biochemical data confirming earlier serological evidence for the presence of B-G genes in the pheasant, and hence, the presence of a family of B-G genes in at least a second species of birds. The high polymorphism exhibited by the pheasant B-G gene family allowed genetic differences among individuals within the small experimental population in this study to be detected easily by restriction fragment patterns. Further evidence was found for the organization of the pheasant Mhc class I and class II genes into genetically independent clusters. Whether these gene clusters are fully comparable to the B and Rfp-Y systems in the chicken or whether yet another organization of Mhc genes has been encountered in the pheasant remains to be determined.     

Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of α Gal(1–4)β Gal binding in virulence of a wild-type strain

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap⁺, sfa⁺, pil⁺, hly⁺ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were sub-cloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (Nal^R) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA⁺ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or α Gal(1–4)β Gal-coated latex beads. CBA mice (N =100) were challenged transurethrally with 10⁵, 10⁶, 10⁷, or 10⁹ cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uro-pathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.