Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.

We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.     

A model for sieving of macromolecules by the glomerular membrane of the kidney.

The transport of water and of macromolecules across the glomerular membrane of the kidney depends on the membrane parameters (radius, length and number of pores) as well as on the hydrostatic and oncotic pressures on either side of the membrane. The filtration pressure decreases along the capillary loops from afferent to efferent end. Water and solute flows are thus given by a system of two differential equations. The sieving coefficient of the macromolecules is the ratio of solute to water flow. In the program described the differential equations are solved by the Runge-Kutta method (fourth order). Rosenbrock's method of minimization is used to adjust the theoretical to the experimental sieving coefficients. The pore radius, total pore area per unit of path length and conductance of the membrane, as well as the intracapillary hydrostatic pressure and its gradient can thus be determined.     

Permeability of artificial membranes to a pluridisperse solution of 125I-polyvinylpyrrolidone.

The validity of the transport equation for uncharged macromolecules across a porous membrane developed by Verniory et al. (1973, J. Gen. Physiol. 62:489) has been tested on several types of artificial membranes (Amicon PM-30, XM-50, and XM-100 Diaflo ultrafilters) using a pluridisperse solution of polyvinylpyrrolidone as filtrand. The influence of the filtration pressure on the shape of the sieving curve predicted by the theory has been verified. A mean pore radius and the width of the pore demonstrated that the method used previously to determine the effective filtration pressure in the glomerulus from sieving data is valid. The transport equation previously proposed by Renkin (1954, J. Gen. Physiol. 38:225) gives results that are less consistent than those obtained with the new transport equation.     

Inbreeding in recessive diseases

Summary The consanguinity of parents (born in France) of individuals who have a recessive disease has been studied. The frequency of first cousin marriages is less than 0.2% in the general French population. Among the parents of affected individuals the following frequencies of first cousin matings were observed:cystic fibrosis: 1.4%cystinosis: 7.1%nephronophtisis: 5.6%spinal muscular atrophy: 4.5%albinsism: 5.0%achromatopsia: 12.5%(Albinism and spinal muscular atrophy are heterogeneous conditions). The increase in the frequency of first cousin marriages relative to that of the general population is much greater, as expected, in cystinosis, which is a rare disease, than in cystic fibrosis, which is the most frequent recessive disorder in France.Inbreeding in cystinosis and cystic fibrosis was also studied by computing the distance between parental birth places. This distance is smaller in cystinosis than in cystic fibrosis.     

Immunoglobulin subclasses of antibodies to human T-cell leukemia/lymphoma virus I-associated antigens in acquired immune deficiency syndrome and lymphadenopathy syndrome

Immunoglobulin G (IgG) and IgM antibodies to human T-cell leukemia/lymphoma virus-I (HTLV-I)-associated membrane antigens (HTLV-I-MA) were assayed by indirect cytospin immunofluorescence, and IgG and IgM antibodies to purified HTLV-I were assayed by enzyme-linked immunosorbent assay in sera from 119 immunologically well-characterized promiscuous male homosexuals in The Netherlands, of whom 9 suffered from acquired immune deficiency syndrome (AIDS), 18 suffered from lymphadenopathy syndrome (LAS), and 5 suffered from gay bowel syndrome. Antibodies to HTLV-I-MA were present in four of nine AIDS patients, including one patient with antibodies to purified HTLV-I. Antibodies to HTLV-I-MA were present in 6 of 18 LAS patients, including 3 patients with antibodies to purified HTLV-I. Of five patients with gay bowel syndrome, one had IgG and IgM antibodies to HTLV-I-MA. Of the four HTLV-I seropositive AIDS patients, two had IgG and IgM antibodies to HTLV-I or HTLV-I-MA, one had only IgG antibodies, and one had only IgM antibodies. Of the six HTLV-I seropositive LAS patients, four had IgG and IgM antibodies to HTLV-I or HTLV-I-MA, and two had only IgM antibodies. In the sera from 27 healthy homosexuals with and 60 without T-cell subset imbalances, no antibodies to HTLV-I or HTLV-I-MA were detected.     

Molecular cloning, sequence and expression of Aa-polB, a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5′ region) and a gene for tRNA^Asn (3′ region), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5′ region) and 3864 (3′ region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons). The domains involved in the 3′→5′ exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe. As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol?γ in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol?γ from yeast to human.     

Effect of glycosidases and proteinases on cobalamin binding and physicochemical properties of purified saturated haptocorrin and intrinsic factor

The effect of exoglycosidase, N-glycanase, trypsin and chymotrypsin was studied on the binding capacity and physicochemical properties of intrinsic factor and of haptocorrin using Superose 6 gel filtration. Intrinsic factor was purified as recently described by us. Haptocorrin was purified 6000-fold from human saliva using thermolabile affinity chromatography and high-performance cationic exchange chromatography with a specific activity of 20.6 nmol of cobalamin (Cbl) per mg protein and a yield of 44.7%. Exoglycosidases provoked a decrease of 54.3 and 78.2% of the Cbl binding capacity of haptocorrin and intrinsic factor, respectively. The sequential incubation of haptocorrin and intrinsic factor wit exoglycosidases and proteinases provoked a decrease of, respectively, 100 and 92.7% of their Cbl binding capacity, whereas the incubation with proteinase decreased the Cbl binding capacity of, respectively, 67.9 and 7.9%. The result of the incubation of [3H]intrinsic factor or [3H]haptocorrin with chymotrypsin and trypsin gave, respectively, no change in the elution position and a shift corresponding to a decrease of 50% of the estimated molecular mass. The estimated molecular mass of Cbl-intrinsic factor and of Cbl-haptocorrin decreased, respectively, to 57.1 kDa and to 88.1 kDa after incubation with exoglycosidases. It was concluded that (1) the carbohydrate core of intrinsic factor protects the whole protein whereas the carbohydrate core of haptocorrin protects only half part of the protein and (2) the carbohydrates are implicated in the formation of the cobalamin binding site of haptocorrin and intrinsic factor.     

Nucleosome occupancy landscape and dynamics at mouse recombination hotspots

During meiosis, paternal and maternal homologous chromosomes recombine at specific recombination sites named hotspots. What renders 2% of the mammalian genomes permissive to meiotic recombination by allowing Spo11 endonuclease to initiate double‐strand breaks is largely unknown. Work in yeast has shown that chromatin accessibility seems to be important for this activity. Here, we define nucleosome profiles and dynamics at four mouse recombination hotspots by purifying highly enriched fractions of meiotic cells. We found that nucleosome occupancy is generally stable during meiosis progression. Interestingly, the cores of recombination hotspots have largely open chromatin structure, and the localization of the few nucleosomes present in these cores correlates precisely with the crossover‐free zones in recombinogenic domains. Collectively, these high‐resolution studies suggest that nucleosome occupancy seems to direct, at least in part, how meiotic recombination events are processed.