Cytokeratin provides a specific marker for human arachnoid cells grown in vitro

Cells from cranial and spinal arachnoid membranes of humans were grown in culture. Their growth characteristics, morphology and details of their cytoskeletal composition are described. Arachnoid membranes, obtained at autopsy, were finely minced and incubated in tissue culture medium. Monolayers of cells of homogeneous morphology grew from these tissue fragments. The cells were flat and polygonal. They divided slowly to form non-overlapping monolayers of low cell density. Electron microscopic examination of cultured arachnoid cells revealed numerous desmosome-like tight junctions and abundant intermediate filaments (tonofilaments). Both morphological features are characteristic of arachnoid cells in situ, but not of cells in the fibroblast-rich dura mater. Immunofluorescence microscopy with monoclonal antibodies demonstrated cytokeratin in the cytoplasm of primary cultures of arachnoid cells. Thus we demonstrated that these cultured cells retained certain of the specific differentiated properties of arachnoid cells in situ and that they are not fibroblasts (which lack tight junctions and cytokeratins). To our knowledge, there have been no previous reports of in vitro growth of arachnoid cells. This in vitro model should be useful in studying the response of arachnoid cells to a variety of substances thought to be involved in the chronic inflammatory condition of the meninges known as arachnoiditis.     

Purification and characterization of Rhodobacter sphaeroides acyl carrier protein

Acyl carrier protein (ACP) has been purified from the facultative phototrophic bacterium Rhodobacter sphaeroides. The ACP preparation was greater than 95% homogeneous as determined by native and disodium dodecyl sulfate (Na2DodSO4)-polyacrylamide gel electrophoreses and N-terminal amino acid analysis. Amino acid compositional analysis revealed that the protein contains approximately 75 amino acids, has a calculated minimum molecular weight of 8700, and lacks the amino acids tyrosine and tryptophan. The presence of the characteristic 4'-phosphopantetheine prosthetic group was indicated by the occurrence of equimolar quantities of beta-alanine and taurine in amino acid hydrolysates and was confirmed by independent chemical analysis. The protein displayed a pI of 3.8 and had a calculated partial specific volume of 0.732 mL/g. The primary structure of the protein has been determined for the first 46 amino acid residues from the N terminus of the molecule, and the region of the molecule encompassing the amino acids from residues 31 to 44 was found to have 100% homology with the identical residues in Escherichia coli ACP. In contrast to E. coli ACP, R. sphaeroides ACP migrated according to its molecular weight during Na2DodSO4 gel electrophoresis, was resistant to pH-induced denaturation, and comigrated with the cis-vaccenoyl-ACP derivative during native gel electrophoresis. It is proposed that the basis for these properties is the enhanced hydrophobic character of the protein.     

Aluminium bone disease in patients receiving plasma exchange with contaminated albumin.

Aluminium balance studies were carried out on eight patients with various immunological disorders who were receiving plasma exchange with albumin solutions known to be contaminated with aluminium. Four patients with impaired renal function (creatinine clearance less than 50 ml/min) retained between 60% and 74% of the aluminium infused during a single plasma exchange. Transiliac bone biopsy specimens from three patients in this group had a high content of aluminium and showed histological evidence of current or previous bone disease related to aluminium. Two of these patients suffered intermittent bone pain. The main route of excretion of injected aluminium was in urine, only a small proportion of the total input being removed in the "plasma bag" during plasma exchange. The extent of aluminium retention and bone deposition was not reflected by the plasma aluminium concentration before or after plasma exchange. Treatment of five patients with intravenous desferrioxamine increased the plasma aluminium concentration and urinary output of aluminium in those with evidence of aluminium retention. These studies show that patients with poor renal function receiving treatment with albumin contaminated with aluminium retain the metal and deposit it in bone, where it may eventually cause aluminium bone disease. Plasma exchange should be used with caution in patients with renal impairment.     

Mitochondrial DNA in the Bark Weevils: Size, Structure and Heteroplasmy

Mitochondrial DNA of higher animals has been described as an example of extreme efficiency in genome structure and function. Where exceptionally large size molecules have been found (greater than 20 kb), most have occurred as rare variants within a species, suggesting that these variants arise infrequently and do not persist for long periods in evolutionary time. In contrast, all individuals of at least three species of bark weevil (Curculionidae: Pissodes) possess a mitochondrial genome of unusually large size (30-36 kb). The molecule owes its large size to a dramatically enlarged A + T-rich region (9-13 kb). Gene content and order outside of this region appear to be identical to that found in Drosophila. A series of 0.8-2.0-kb repeated sequences occur adjacent to the large A + T rich region and have perhaps played a role in the generation of the large size as well as an unprecedented frequency of size variant heteroplasmy. Every weevil sampled in all three species (n = 219) exhibits anywhere from two to five distinct size classes of mtDNA. The persistence of this large amount of size polymorphism through two speciation events combined with the abundant size variation within individuals suggests that these molecules may not be subject to strong selection for small overall size and efficiency of replication. This pattern of variation contrasts strongly with the conservation of gene content and arrangement in the coding region of the molecule.     

Heterologous cross-linking of Lyt-2 (CD8) to the alpha beta-T cell receptor is more effective in T cell activation than homologous alpha beta-T cell receptor cross-linking

Ag recognition of Lyt-2 (CD8)-positive T lymphocytes requires the presentation by APC of a suitably processed Ag in association with MHC class I molecules. In previous studies we have obtained evidence that, for optimal activation, both the alpha beta-TCR and Lyt-2 have to participate in this recognition process. In the current study we investigate the functional consequences of limited cross-linking of these cell surface molecules by using soluble, dimeric hetero- and homoconjugates of mAb to Lyt-2 and to the TCR beta-chain (F23.1). Heterologous cross-linking of Lyt-2 to the TCR induced a vigorous, selective Lyt-2+ T cell proliferative response. Functionally active cytotoxic cells were generated, and a high frequency of responding cells was observed in limiting dilution analyses. In contrast, homologous TCR cross-linking initiated a less pronounced proliferation with a relatively low frequency of response, whereas Lyt-2 cross-linking resulted in no cellular proliferation. Significant T cell activation occurred with exposure to anti-Lyt-2: F23.1 mAb dimers at concentrations an order of magnitude lower than those required for stimulation by F23.1:F23.1 mAb dimers. The induction of proliferation by mAb dimers occurred in the absence of Fc components and in rigorously APC depleted, purified T cell preparations. Effective stimulation of resting T cells could be induced also by heterodimers of monovalent Fab fragments. Heterologous cross-linking of Lyt-2 to the TCR was superior to homologous TCR cross-linking primarily with respect to proliferation in IL-2 containing media and to IL-2R expression, whereas proliferation in response to other lymphokines and the production of IL-2 itself were similar under both cross-linking regimens. Thus, when linked to the TCR, Lyt-2 contributed a strong, positive signal toward IL-2-dependent growth of resting T cells. We assume that in the case of Ag-driven T cell activation, the class I MHC molecule acts as the physiologic cross-linking ligand for Lyt-2 and the TCR.     

Carbohydrate Metabolism in Taproots of Medicago sativa L. during Winter Adaptation and Spring Regrowth

Our objective was to identify amylases that may participate in starch degradation in alfalfa (Medicago sativa L.) taproots during winter hardening and subsequent spring regrowth. Taproots from field-grown plants were sampled at intervals throughout fall, winter, and early spring. In experiment 1, taproots were separated into bark and wood tissues. Concentrations of soluble sugars, starch, and buffer-soluble proteins and activities of endo- and exoamylase were determined. Starch concentrations declined in late fall, whereas concentrations of sucrose increased. Total amylolytic activity (primarily exoamylase) was not consistently associated with starch degradation but followed trends in soluble protein concentration of taproots. This was especially evident in spring when both declined as starch degradation increased and shoot growth resumed. Activity of endoamylase increased during periods of starch degradation, especially in bark tissues. In experiment 2, a low starch line had higher specific activity of taproot amylases. This line depleted its taproot starch by late winter, after which taproot sugar concentrations declined. As in experiment 1, total amylolytic activity declined in spring in both lines, whereas that of endoamylase increased in both lines even though little starch remained in taproots of the low starch line. Several isoforms of both amylases were distinguished using native polyacrylamide electrophoresis, with isoforms being similar in bark and wood tissues. The slowest migrating isoform of endoamylase was most prominent at each sampling. Activity of all endoamylase isoforms increased during winter adaptation and in spring when shoot growth resumed. Endoamylase activity consistently increased at times of starch utilization in alfalfa taproots (hardening, spring regrowth, after defoliation), indicating that it may serve an important role in starch degradation.     

A Computer Simulation Study of Vntr Population Genetics: Constrained Recombination Rules Out the Infinite Alleles Model

Extensive allelic diversity in variable numbers of tandem repeats (VNTRs) has been discovered in the human genome. For population genetic studies of VNTRs, such as forensic applications, it is important to know whether a neutral mutation-drift balance of VNTR polymorphism can be represented by the infinite alleles model. The assumption of the infinite alleles model that each new mutant is unique is very likely to be violated by unequal sister chromatid exchange (USCE), the primary process believed to generate VNTR mutants. We show that increasing both mutation rates and misalignment constraint for intrachromosomal recombination in a computer simulation model reduces simulated VNTR diversity below the expectations of the infinite alleles model. Maximal constraint, represented as slippage of single repeats, reduces simulated VNTR diversity to levels expected from the stepwise mutation model. Although misalignment rule is the more important variable, mutation rate also has an effect. At moderate rates of USCE, simulated VNTR diversity fluctuates around infinite alleles expectation. However, if rates of USCE are high, as for hypervariable VNTRs, simulated VNTR diversity is consistently lower than predicted by the infinite alleles model. This has been observed for many VNTRs and accounted for by technical problems in distinguishing alleles of neighboring size classes. We use sampling theory to confirm the intrinsically poor fit to the infinite alleles model of both simulated VNTR diversity and observed VNTR polymorphisms sampled from two Papua New Guinean populations.     

Why are some genetic diseases common?

Various processes (selection, mutation, migration and genetic dirft) are known to determine the frequency of genetic disease in human populations, but so far it has proved almost impossible to decide to what extent each is responsible for the presence of a particular genetic disease. The techniques of gene and haplotype analysis offer new hope in addressing this issue, and we review relevant studies of three haemoglobinopathies: sickle cell anaemia, and and thalassaemia. We show how for each disease it is possible to recognize a pattern of regionally specific mutations, found in association with one or a few haplotypes, that is best explained as the result of selection; other patterns are due to population migration and genetic drift. However, we caution that such conclusions can be drawn in special circumstances only. In the case of the haemoglobinopathies it is possible because a selective agent (malaria) was already suspected, and the investigations could be carried out in relatively genetically homogenous populations whose migratory histories are known. Moreover, some data reviewed here suggest that gene conversion and the haplotype composition of a population may affect the frequency of a mutation, making interpretation of gene frequencies difficult on the basis of standard population genetics theory. Hence attempts to use the same approaches with other genetic diseases are likely to be frustrated by a lack of suitably untrammelled populations and by difficulties accounting for poorly understood genetic processes. We conclude that although this combination of molecular and population genetics is successful when applied to the study of haemoglobinopathies, it may not be so easy to apply it to the study of other genetic diseases.