Decreased incidence of mycorrhizal root tips associated with soil heavy-metal enrichment

Mycorrhizal incidence was studied at two forested locations in south-central Virgina. At each location, one site with soil naturally enriched in copper, lead and zinc was designated as mineralized, and an adjacent site, with significantly lower levels of these metals, was used as a control. A total of 223 soil samples were collected during the summer of 1984 and assayed for active mycorrhizal tips. A reduced active mycorrhizal root tip count was found in those samples collected from the mineralized sites at both locations (P≤0.001).     

Purification of proteins from polyacrylamide gels, free of detergent or dye

A two step procedure recovers proteins from sodium dodecyl sulfate polyacrylamide gels. The proteins are eluted by electrophoretic dialysis. The eluent is then passed through an Amberlite CG-400 anion-exchange resin. The recovery of protein is nearly total. The recovered proteins have no detectable sodium dodecyl sulfate contamination. With gels that have been stained with Coomassie Brilliant Blue R, the procedure recovers the proteins free of the dye. We have used this procedure successfully during the purification of epidermal glycoproteins.     

A continuous noncontact method for measuring in situ vascular diameter with a video camera

A new, continuous, on-line, video diameter-measuring technique, utilizing a video camera mounted on the sidearm of a stereo microscope, is described. Vessel diameter is derived from changes in the video output signal of the camera or a video recorder when the vessel of interest is displayed horizontally on a monitor and well contrasted with its background. A comparator threshold is set on the filtered video output signal and generates an output pulse that is used to gate horizontal video sync pulses to a digital counter-timer. The number of pulses counted for each video field (no. of horizontal video lines) is proportional to the vessel diameter. The video-derived diameter is calibrated using known standards and correlates well with sonomicrometer-derived diameters of the carotid artery and jugular vein during increasing pressure ramps (r greater than 0.999). The diameter update rate is 60 Hz, and the resolution of the system is one horizontal video line, independent of the vessel size. With suitable magnification and contrast both arteries and veins as small as 200 micron have been measured using this system.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Initiation and characterization of primary mouse kidney epithelial cultures

Summary Primary cultures of murine renal epithelial cells were established from a preparation of proximal tubule fragments. Confluent cultures exhibited multiple dome formation, indicating the presence of tight junctions and an intact transcellular transport process. Ultrastructural analysis revealed a monolayer of polarized cells, with a sparse but clearly defined microvillar surface facing the growth medium and a basolateral surface attached to the substratum. Cultures grown on collagen gels did not show domes. The epithelial monolayer exhibited several differentiated functions of the proximal tubule: a) parathyroid hormone (PTH)-stimulated cAMP synthesis; b) production of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃; c) high alkaline phosphatase activity; and d) Na⁺-dependent transport of phosphate (Pi) and α-methylglucoside (α-MG). The sugar uptake was selectively inhibited by phlorizin, a competitive inhibitor of glucose uptake at the luminal membrane. Kinetic analysis revealed independent transport systems for Pi and α-MG, with Km values corresponding to the high affinity systems identified in brush border membrane vesicles derived from the proximal tubule. Pi uptake by the epithelial monolayers was regulated by the concentration of Pi in the growth medium. Phorbol esters and PTH did not exert an effect on Pi and α-MG transport in mouse primary cultures. The present study demonstrates that primary cultures provide a useful in vitro preparation to investigate renal proximal tubular function. Cindy Bell was the recipient of an MRC Studentship Award. This work was supported by the MRC (Group in Medical Genetics). This is publication number 88011 of the McGill University-Montreal Children's Hospital Research Institute.     

Mapping of FMR1, the gene implicated in fragile X-linked mental retardation, on the mouse X chromosome

A genetic map of the Cf-9 to Dmd region of the mouse X chromosome has been established by typing 100 offspring from a Mus musculus x Mus spretus interspecific backcross for the four loci Cf-9, Cdr, Gabra3, and Dmd. The following order and genetic distances in centimorgans were determined: (Cf-9)-2.4 +/- 1.7-(Cdr)-2.0 +/- 1.4-(Gabra3)-4.1 +/- 2.0-(Dmd). Six backcross offspring carrying X chromosomes with recombination events in the Cdr-Dmd region were identified. These recombination events were used to define the position of Fmr-1, the murine homologue of FMR1, which is the gene implicated in the fragile X syndrome in man, and that of DXS296h, the murine homologue of DXS296. Both Fmr-1 and DXS296h were mapped into the same recombination interval as Gabra3 on the mouse X chromosome. These findings provide strong support for the concept that the order of loci lying in the Cf-9 to Gabra3 segment of the X chromosome is highly conserved between human and mouse.     

Identification of a CA repeat at the TCRA locus using yeast artificial chromosomes: a general method for generating highly polymorphic markers at chosen loci.

The creation of a comprehensive genetic map in human has been limited by the lack of highly polymorphic markers spaced evenly throughout the human genome. We have utilized yeast artificial chromosomes (YAC) containing large human DNA inserts to help identify highly polymorphic (CA)n repeats at a chosen locus. The DNA of a YAC containing the locus was subcloned in M13 vectors, and the recombinants were screened at high stringency to detect preferentially long (CA)n repeats (n greater than 20). These repeats, which are the most likely to be highly polymorphic, were then studied to confirm both the level of polymorphism and their precise genetic location. This strategy has permitted the identification of a new, highly polymorphic CA repeat (77% heterozygosity) at the T cell receptor alpha chain (TCRA) locus on chromosome 14q. It provides a powerful marker for assessing the role of this locus in the susceptibility to autoimmune and infectious diseases. This approach should permit the development of highly polymorphic markers at any targeted locus and rapidly improve the current human genetic map.     

Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification.

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.     

The problem of movelength and turn definition in analysis of orientation data.

This study examines the analysis of arthropod orientation data. Three problems are discussed: (1) dealing with time as it applies to spatial data, (2) determining the appropriate movelength to be used in collecting and in analyzing data, and (3) defining a turn, to discriminate between "gait noise" and course changes. The main objective is to determine the solution to defining the most appropriate movelength for comparisons between variables and between species. The technique described here for selecting the appropriate movelength that has relevance to both the locomotory rate of the animal and its body length, reduces variation resulting from lateral translational movements, prevents the use of movelengths that lead to artifactual or unrealistic turning values per move, and permits comparisons of species and individuals under various stimulus conditions.     

DNA sequence analysis of revertants of the hisD3052 allele of Salmonella typhimurium TA98 using the polymerase chain reaction and direct sequencing: application to 1-nitropyrene-induced revertants

We have used the polymerase chain reaction (PCR) to speed the DNA sequence analysis of revertants of Salmonella typhimurium TA98. Briefly, a crude DNA extract from a single colony was prepared and used in an asymmetric PCR to amplify a 328-bp fragment containing the hisD3052 mutation approximately in the center. Following ultrafiltration, the ssDNA was sequenced using an end-labeled probe and dideoxy sequencing. The most frequent mutation among the revertants was a -2 deletion of GC or CG within the sequence CGCGCGCG, which is upstream of the hisD3052 mutation. This deletion occurred in 38% (6/16) of the spontaneous (-S9) revertants and in 94% (15/16) of a set of 1-nitropyrene-induced revertants. Other mutations, mostly deletions but also some complex mutations (i.e., single mutational events involving a combination of insertions, deletions, and substitutions), occurred within quasipalindromic regions of DNA. Possible mutational mechanisms are discussed, and the results with 1-NP are compared to those obtained in other systems.