Cell type-specific association between two types of spectrin and two types of intermediate filaments

We have demonstrated a differential association between two types of spectrin, from erythrocytes and brain, with two types of intermediate filaments, vimentin filaments and neurofilaments. Electron microscopy showed that erythrocyte spectrin promoted the binding of vimentin filaments to red cell inside-out vesicles via lateral associations with the filaments. In vitro binding studies showed that the association of spectrin with vimentin filaments was apparently saturable, increased with temperature, and could be prevented by heat denaturation of the spectrin. Comparisons were made between erythrocyte and brain spectrin binding to both vimentin filaments and neurofilaments. We found that vimentin filaments bound more erythrocyte spectrin than brain spectrin, while neurofilaments bound more brain spectrin than erythrocyte spectrin. Our results show that both erythroid and nonerythroid spectrins are capable of binding to intermediate filaments and that such associations may be characterized by differential affinities of the various types of spectrin with the several classes of intermediate filaments present in cells. Our results also suggest a role for both erythroid and nonerythroid spectrins in mediating the association of intermediate filaments with plasma membranes or other cytoskeletal elements.     

Kinetic measurement of bacterial adherence to eukaryotic cells

We developed a kinetic assay using a monolayer of differentiated respiratory epithelium in culture to assess bacterial adherence. Mean residence time of bacteria in the tissue culture chamber was estimated from a model-independent (moment) analysis of the rate of bacterial washout from perfused Rose chambers. Results with this method compared favorably with visual assessment of adherence and double radiolabel method with H. influenzae. Adherence was assessed with low inoculae of H. influenzae, P. cepacia and P. aeruginosa avoiding cytotoxic effects seen when large inoculae are added to eukaryotic cells. This method will provide a means of assessing adherence of pathogenic respiratory bacteria to their cellular target at low inoculae.     

The ``hitchhiking Effect'''' Revisited

The number of selectively neutral polymorphic sites in a random sample of genes can be affected by ancestral selectively favored substitutions at linked loci. The degree to which this happens depends on when in the history of the sample the selected substitutions happen, the strength of selection and the amount of crossing over between the sampled locus and the loci at which the selected substitutions occur. This phenomenon is commonly called hitchhiking. Using the coalescent process for a random sample of genes from a selectively neutral locus that is linked to a locus at which selection is taking place, a stochastic, finite population model is developed that describes the steady state effect of hitchhiking on the distribution of the number of selectively neutral polymorphic sites in a random sample. A prediction of the model is that, in regions of low crossing over, strongly selected substitutions in the history of the sample can substantially reduce the number of polymorphic sites in a random sample of genes from that expected under a neutral model.     

Restriction map and alpha-amylase activity variation among Drosophila mutation accumulation lines

The specific activities of alpha-amylase were measured for two sets of mutation accumulation lines, each set having originated from a different lethal-carrying second chromosome and SM1(Cy) chromosome and having been maintained by a balanced lethal system for about 300 generations. Significant variation was found to have accumulated among lines of both sets. Because of dysgenic crosses in the early generations of mutation accumulation, insertions or deletions of transposable elements in the Amy gene region were suspected of being the cause of this variation. In order to test this possibility, the structural changes in the 14 kb region of these chromosomes that includes the structural genes for alpha-amylase were investigated by restriction map analysis. We found that most part of the activity variation is due to replacements of a chromosomal region of SM1(Cy), including the structural genes for alpha-amylase, by the corresponding regions of the lethal chromosomes. One line also contained an insertion in this region but this line has an intermediate activity value. Thus, insertions of transposable elements into the Amy gene region were not found to be responsible for the new variation observed in alpha-amylase activity. If we remove those lines with structural changes from the analysis, the genetic variance of alpha-amylase specific activity among lines becomes non-significant in both sets of chromosomes.     

Non-specific resistance to Babesia divergens in the Mongolian gerbil (Meriones unguiculatus)

Inoculation of mature gerbils with BCG gave protection to subsequent infection with B. divergens when inoculated by the intracardiac and intraperitoneal routes, the latter showing a dose dependent relationship. BCG vaccination was most effective in immature gerbils (less than 4 weeks old), which are innately resistant to B. divergens. Vaccination of gerbils with killed Propionesbacterium acne and zymosan A failed to elicit a protective response, which contrasts conspicuously with rodent babesia studies. Incubation of B. divergens-infected gerbil blood with hydrogen peroxide produced parasite inhibition only at the highest concentration and treatment of parasitized gerbils with the oxidative radical inducer, alloxan monohydrate, gave equivocal results so it is evident that, unlike Plasmodium spp., B. divergens is not significantly susceptible to the action of reactive oxygen forms.     

Molybdoenzymes in Drosophila. IV. Further characterization of the cinnamon phenotype

Summary Mutations at the cin gene display drastically lowered levels of the molybdoenzymes, xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), and lack pyridoxal oxidase (PO) and sulfite oxidase (SO) activities. Certain mutations at cin also display varying degrees of female sterility, which is maternally affected. Here we characterize five new cin alleles with respect to the molybdoenzyme activities as well as the molybdenum cofactor, commonly required for molybdoenzyme activity. In complementing cin heterozygotes we find that, in addition to the previously reported unusually high levels of XDH and AO activities, there are unusually elevated levels of SO activity, as well as complementation for PO activity. The levels of immunologically crossreacting material in such heterozygotes indicate that the elevated levels of molybdoenzyme activities cannot be due to increases in the number of enzyme molecules. Measurements of the level of molybdenum cofactor activity normally present in XDH, AO, PO, and SO point to the possibility that a larger fraction of the enzyme molecules are active in these heterozygotes. The possible role of SO with respect to cinnamon's female sterility is also discussed.     

Densimetric determination of carbohydrate content in glycoproteins.

Carbohydrates play important roles in activity, stability and pharmacokinetics of glycoproteins and the degree of glycosylation varies with proteins. In this communication, a simple method of determining the carbohydrate content was developed, which consists of measuring the density increments of a glycoprotein and its non-glycosylated counterpart, and then dividing the difference between the two values by the density increment of carbohydrates. The density increment was relatively constant for various sugars except for sialic acid, and hence assumed to be 0.39. Thus, we obtained carbohydrate contents of 38, 28, 8 and 7% for Chinese hamster ovary cell-expressed erythropoietin (EPO), stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and platelet-derived growth factor (PDGF), respectively. These values are in close agreement with those determined by other methods.     

The Evolution of Restricted Recombination and the Accumulation of Repeated DNA Sequences

We suggest hypotheses to account for two major features of chromosomal organization in higher eukaryotes. The first of these is the general restriction of crossing over in the neighborhood of centromeres and telomeres. We propose that this is a consequence of selection for reduced rates of unequal exchange between repeated DNA sequences for which the copy number is subject to stabilizing selection: microtubule binding sites, in the case of centromeres, and the short repeated sequences needed for terminal replication of a linear DNA molecule, in the case of telomeres. An association between proximal crossing over and nondisjunction would also favor the restriction of crossing over near the centromere. The second feature is the association between highly repeated DNA sequences of no obvious functional significance and regions of restricted crossing over. We show that highly repeated sequences are likely to persist longest (over evolutionary time) when crossing over is infrequent. This is because unequal exchange among repeated sequences generates single copy sequences, and a population that becomes fixed for a single copy sequence by drift remains in this state indefinitely (in the absence of gene amplification processes). Increased rates of exchange thus speed up the process of stochastic loss of repeated sequences.     

Evolution and Extinction of Transposable Elements in Mendelian Populations

A model of the evolution of a transposable element family in a Mendelian host population is proposed that incorporates heritable phenotypic mutations in the elements. The temporal behavior of the numbers of mutant and wild-type elements is studied, and the expected extinction time of the transposable element family is examined. Our results indicate that, if the mutant can be transposed equally well in the presence of the wild type, then it can be expected to be found in preponderance, whereas elements, such as retroviruses, where the transposing genome and its phenotypic expression are coupled, may be characterized by a low mutant frequency.