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M Lindenbaum

Publications and source records attributed to M Lindenbaum.

13 recordsLinked to original sources
PubMed2002

Retrofitting of a satellite repeat DNA-based murine artificial chromosome (ACes) to contain loxP recombination sites.

A satellite DNA-based mammalian artificial chromosome (ACes) was generated and subsequently modified by targeting of a loxP-red fluorescent protein (RFP) expression cassette via homologous recombination into a ribosomal DNA (rDNA)-containing locus. Clones containing correctly targeted ACes were identified by PCR from populations of RFP-expressing cells enriched by FACS sorting and were further characterized by fluorescent in situ hybridization. The targeted ACes maintained its ability to be purified to near homogeneity. Studies are currently underway to further characterize the functionality, carrying capacity, stability and transfectability of this modified ACes.

Animals↗
PubMed2001

Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes.

The introduction of mammalian artificial chromosomes (ACs) into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. Bovine oocytes aspirated from 2-5 mm size follicles were matured in vitro for 22 hr. Mature oocytes were fertilized in vitro with frozen- thawed bull spermatozoa. Artificial chromosome carrying either beta-galactosidase (Lac-Z) gene or green fluorescence protein (GFP) gene were isolated by flow cytometry. A single chromosome was microinjected into one of the two pronuclei of bovine zygotes. Sham injected zygotes served as controls. Injected zygotes were cultured in G 1.2 medium for 7 days. Hatched blastocysts were cultured on blocked STO cell feeder layer for attachment and outgrowth of ICM and trophectoderm cells. The results showed a high zygote survival rate following LacZ-ACs microinjection (74%). However, the blastocyst development rate after 7 days of culture was significantly lower than that of sham injected zygotes (7.5 vs. 22%). Embryonic cells positive for Lac-Z gene were detected by PCR in three of nine outgrowth colonies. In addition, GFP gene expression was observed in 15 out of 85 (18%) embryos at the arrested 2-cell stage to blastocyst stage. Six blastocysts successfully outgrew, three outgrowths were GFP positive for up to 3 weeks in culture. We conclude that the methodology for artificial chromosome delivery into bovine zygotes could lead to viable blastocyst development, and reporter gene expression could be sustained during pre-implantation development.

Animals↗
PubMed2000

Neural regulation of alpha-dystroglycan biosynthesis and glycosylation in skeletal muscle.

Alpha-dystroglycan (alpha-DG) is part of a complex of cell surface proteins linked to dystrophin or utrophin, which is distributed over the myofiber surface and is concentrated at neuromuscular junctions. In laminin overlays of muscle extracts from developing chick hindlimb muscle, alpha-DG first appears at embryonic day (E) 10 with an apparent molecular mass of 120 kDa. By E15 it is replaced by smaller (approximately 100 kDa) and larger (approximately 150 kDa) isoforms. The larger form increases in amount and in molecular mass (>200 kDa) as the muscle is innervated and the postsynaptic membrane differentiates (E10-E20), and then decreases dramatically in amount after hatching. In myoblasts differentiating in culture the molecular mass of alpha-DG is not significantly increased by their replication, fusion, or differentiation into myotubes. Monoclonal antibody IIH6, which recognizes a carbohydrate epitope on alpha-DG, preferentially binds to the larger forms, suggesting that the core protein is differentially glycosylated beginning at E16. Consistent with prior observations implicating the IIH6 epitope in laminin binding, the smaller forms of alpha-DG bind more weakly to laminin affinity columns than the larger ones. In blots of adult rat skeletal muscle probed with radiolabeled laminin or monoclonal antibody IIH6, alpha-DG appears as a >200-kDa band that decreases in molecular mass but increases in intensity following denervation. Northern blots reveal a single mRNA transcript, indicating that the reduction in molecular mass of alpha-DG after denervation is not obviously a result of alternative splicing but is likely due to posttranslational modification of newly synthesized molecules. The regulation of alpha-DG by the nerve and its increased affinity for laminin suggest that glycosylation of this protein may be important in myofiber-basement membrane interactions during development and after denervation.

Animals↗
PubMed1999

alpha-Dystroglycan is a laminin receptor involved in extracellular matrix assembly on myotubes and muscle cell viability.

alpha-Dystroglycan (alpha-DG) is a laminin-binding protein and member of a glycoprotein complex associated with dystrophin that has been implicated in the etiology of several muscular dystrophies. To study the function of DG, C2 myoblasts were transfected stably with an antisense DG expression construct. Myotubes from two resulting clones (11F and 11E) had at least a 40-50% and 80-90% reduction, respectively, in alpha-DG but normal or near normal levels of alpha-sarcoglycan, integrin beta1 subunit, acetylcholine receptors (AChRs), and muscle-specific kinase (MuSK) when compared with parental C2 cells or three clones (11A, 9B, and 10C) which went through the same transfection and selection procedures but expressed normal levels of alpha-DG. Antisense DG-expressing myoblasts proliferate at the same rate as parental C2 cells and differentiate into myotubes, however, a gradual loss of cells was observed in these cultures. This loss correlates with increased apoptosis as indicated by greater numbers of nuclei with condensed chromatin and more nuclei labeled by the TUNEL method. Moreover, there was no sign of increased membrane permeability to Trypan blue as would be expected with necrosis. Unlike parental C2 myotubes, 11F and 11E myotubes had very little laminin (LN) on their surfaces; LN instead tended to accumulate on the substratum between myotubes. Exogenous LN bound to C2 myotubes and was redistributed into plaques along with alpha-DG on their surfaces but far fewer LN/alpha-DG plaques were seen after LN addition to 11F or 11E myotubes. These results suggest that alpha-DG is a functional LN receptor in situ which is required for deposition of LN on the cell and, further, implicate alpha-DG in the maintenance of myotube viability.

Animals↗
PubMed1999

Chimaeric mice deficient in dystroglycans develop muscular dystrophy and have disrupted myoneural synapses.

Mutations in the dystrophin gene (DMD) and in genes encoding several dystrophin-associated proteins result in Duchenne and other forms of muscular dystrophy. alpha-Dystroglycan (Dg) binds to laminins in the basement membrane surrounding each myofibre and docks with beta-Dg, a transmembrane protein, which in turn interacts with dystrophin or utrophin in the subplasmalemmal cytoskeleton. alpha- and beta-Dgs are thought to form the functional core of a larger complex of proteins extending from the basement membrane to the intracellular cytoskeleton, which serves as a superstructure necessary for sarcolemmal integrity. Dgs have also been implicated in the formation of synaptic densities of acetylcholine receptors (AChRs) on skeletal muscle. Here we report that chimaeric mice generated with ES cells targeted for both Dg alleles have skeletal muscles essentially devoid of Dgs and develop a progressive muscle pathology with changes emblematic of muscular dystrophies in humans. In addition, many neuromuscular junctions are disrupted in these mice. The ultrastructure of basement membranes and the deposition of laminin within them, however, appears unaffected in Dg-deficient muscles. We conclude that Dgs are necessary for myofibre survival and synapse differentiation or stability, but not for the formation of the muscle basement membrane, and that Dgs may have more than a purely structural function in maintaining muscle integrity.

Animals↗
PubMed1998

Disruption of the NF-H gene increases axonal microtubule content and velocity of neurofilament transport: relief of axonopathy resulting from the toxin beta,beta'-iminodipropionitrile.

To investigate the role of the neurofilament heavy (NF-H) subunit in neuronal function, we generated mice bearing a targeted disruption of the gene coding for the NF-H subunit. Surprisingly, the lack of NF-H subunits had little effect on axonal calibers and electron microscopy revealed no significant changes in the number and packing density of neurofilaments made up of only the neurofilament light (NF-L) and neurofilament medium (NF-M) subunits. However, our analysis of NF-H knockout mice revealed an approximately 2.4-fold increase of microtubule density in their large ventral root axons. This finding was further corroborated by a corresponding increase in the ratio of assembled tubulin to NF-L protein in insoluble cytoskeletal preparations from the sciatic nerve. Axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed an increased transport velocity of newly synthesized NF-L and NF-M proteins in motor axons of NF-H knockout mice. When treated with beta,beta'-iminodipropionitrile (IDPN), a neurotoxin that segregates microtubules and retards neurofilament transport, mice heterozygous or homozygous for the NF-H null mutation did not develop neurofilamentous swellings in motor neurons, unlike normal mouse littermates. These results indicate that the NF-H subunit is a key mediator of IDPN-induced axonopathy.

Actin Cytoskeleton↗
PubMed1998

alpha-Dystroglycan functions in acetylcholine receptor aggregation but is not a coreceptor for agrin-MuSK signaling.

alpha-dystroglycan (alpha-DG) is an agrin-binding protein that has been implicated in acetylcholine receptor (AChR) clustering, but it is unclear whether it acts as a coreceptor involved in initial agrin signaling or as a component involved in later events. To investigate its role, we have generated antisense derivatives of the C2 mouse muscle cell line, which have reduced alpha-DG expression. When compared with wild-type cells, the alpha-DG-deficient myotubes have a dramatic reduction in the number of spontaneous and agrin-induced AChR clusters. Several findings suggest that this decrease in AChR clustering is likely not because of a defect in agrin signaling through the MuSK receptor tyrosine kinase. Compared with wild-type cells, the alpha-DG-deficient cell lines showed only a transient reduction in the level of agrin-induced MuSK tyrosine phosphorylation and no reduction in AChR beta-subunit tyrosine phosphorylation. Additionally, agrin-induced phosphorylation of MuSK in wild-type myotubes was not decreased using agrin fragments that lack the domain primarily responsible for binding to alpha-DG. Finally, neural agrin-induced phosphorylation of MuSK was unaffected by treatments such as excess muscle agrin or anti-alpha-DG antibodies, both of which block agrin-alpha-DG binding. Together, these results suggest that alpha-DG is not required for agrin-MuSK signaling but rather that it may play a role elsewhere in the clustering pathway, such as in the downstream consolidation or maintenance of AChR clusters.

Agrin↗
PubMed1995

Dystroglycan expression in the wild type and mdx mouse neural retina: synaptic colocalization with dystrophin, dystrophin-related protein but not laminin.

Alpha- and beta-dystroglycan (alpha- and beta-DG) are members of a dystrophin-associated glycoprotein complex (DGC) in skeletal muscle which binds to agrin and laminin, and has been postulated to be involved in myoneural snyapse formation. The absence of functional dystrophin in Duchenne muscular dystrophy (DMD) and in one of its animal models, the mdx mouse, leads to a reduction of alpha- and beta-DG in muscle, and is often associated with mental retardation and abnormal retinal synaptic transmission in DMD. Using immunohistochemistry, we find that alpha- and beta-DG are expressed in the outer plexiform layer of both wild type and mdx retina, where both dystrophin and dystrophin-related protein (DRP), but not laminin are present. In situ hybridization identifies two neuronal populations, photoreceptors and retinal ganglion cells, that express DG mRNA. Alpha- and beta-DG are also expressed in the inner limiting membrane and around blood vessels where they colocalize with laminin and DRP. Western blot analysis revealed the expression of several dystrophin isoforms in wild type and mdx retina, possibly explaining the unaltered expression of alpha- and beta-dystroglycan in the mdx central nervous system (CNS). Our results support the hypothesis that alpha- and beta-DG can interact with dystrophin and DRP in the CNS and perform functions analogous to those of the DGC in muscle.

Animals↗
PubMed1995

The basement membrane at the neuromuscular junction: a synaptic mediatrix.

The basement membrane at the neuromuscular junction directs formation of pre- and postsynaptic elements at this synapse. Efforts to understand the molecular basis for development of the postsynaptic specialization have brought new insights into extracellular matrix proteins and their cell-surface receptors. Recent evidence for an agrin receptor and mice null for the s-laminin gene have reinforced the function of the basement membrane in both orthograde and retrograde signalling across the synapse.

Agrin↗
PubMed1993

Functional analysis of the human neurofilament light chain gene promoter.

We have carried out a structural and functional analysis on the human NF-L (H-NF-L) gene. It contains a methylation-free island, spanning the 5' flanking sequences and the first exon and a number of neuronal-specific DNase I hypersensitive sites have been identified in the upstream region as well as within the body of the gene. Analysis in cell lines and transgenic mice using a combination of these sites has revealed the presence of a conserved element(s) between -300bp and -190bp which is required for neuronal-specific expression.

Animals↗
PubMed1990

Response to hepatitis B vaccine in Alaska natives with chronic alcoholism compared with non-alcoholic control subjects.

PURPOSE: This study was designed to determine if (1) alcoholics have a higher prevalence of hepatitis B virus (HBV) serologic markers than do non-alcoholic controls and (2) if they respond to hepatitis B vaccination in a manner similar to that of non-alcoholic controls. PATIENTS AND METHODS: The study was designed as a case-control study, and 129 Alaska Natives were recruited. Alcoholics were recruited from inpatient wards, outpatient clinics, a soup kitchen serving the homeless, and several alcohol rehabilitation centers; control subjects were recruited primarily from among Alaska Native Hospital employees. A standardized questionnaire, the Alcohol Dependency Scale (ADS), was administered to all participants. Each participant was screened for hepatitis B serologic markers, had liver function studies performed, and was examined for evidence of liver disease. Participants seronegative for HBV markers received three doses of hepatitis B vaccine. Linear regression analysis was performed to compare the amount of alcohol intake and variables associated with liver disease with the response to hepatitis B vaccination and antibody levels achieved. Using an ADS score of greater than 13, 64 participants were classified as chronic alcoholics, and 60 were classified as controls. RESULTS: HBV seropositivity was found in 22 alcoholics (34.4%) and seven controls (11.7%). After adjusting for age and sex, this difference was significant (chi 2 MH = 6.57, df = 1; p = 0.012). Abnormal levels of liver transaminase occurred significantly more often in alcoholic participants than in control subjects (chi 2 MH = 4.91, df = 1; p = 0.026). Of 95 seronegative persons, 72 received three doses of hepatitis B plasma-derived vaccine. Alcoholic subjects and control subjects did not differ significantly in their response to vaccination. Only four alcoholics and two controls did not develop antibody to hepatitis B surface antigen (anti-HBs) after hepatitis B vaccination, and two alcoholics and three controls had anti-HBs levels less than 10 SRU by radioimmunoassay. Mean anti-HBs levels measured in milli-international units (mIU) for the 62 responders showed a decrease in the anti-HBs level with increasing age (p less than 0.001). There was no difference in the mean anti-HBs log10 mIU between alcoholics and controls younger than 45 years of age, but in persons greater than 45 years of age, alcoholics had a lower mean anti-HBs log10 mIU level than did controls; this difference, however, was not significant (p greater than 0.10). CONCLUSION: Chronic alcoholics have a higher prevalence of HBV seromarkers than do age-matched controls. Seronegative alcoholics, especially those under age 45, respond well to hepatitis B vaccination, and such vaccination should be considered in all chronic alcoholic persons.

Adult↗
PubMed1981

Interaction of steroids with dexamethasone-binding receptor and corticosteroid-binding globulin in the mammary gland of the mouse in relation to lactation.

Dexamethasone (9 alpha-fluoro-11 beta, 17, 21-trihydroxy-16 alpha-methyl-pregna-1,4-diene-3,20-dione) binding by cytosolic components of mammary glands from pregnant and lactating mice was characterized and quantified. The unfilled receptor for mice was characterized and quantified. The unfilled receptor for dexamethasone lost binding activity rapidly by a process that was largely, but not completely, reversed by the addition of sulfhydryl reagents. The quantity of dexamethasone receptors increased 4-fold during the last 8 days of pregnancy. The apparent binding affinity (Ka) calculated from Scatchard plots was maximal at parturition (1.5 x 10(8)M-1), but declined gradually as lactation progressed. A mixture of cytosols from pregnancy and lactation gave a linear Scatchard plot, with a Ka that was the mean of the Ka values obtained in the separate cytosols. The decreased Ka in lactating mice was not due to a different class of receptors, but may be attributed to the increased levels of endogenous free steroids in the cytosol. Some differences, however, were noted between the sedimentation properties of the dexamethasone-receptor complexes in cytosols of pregnant and lactating mice. In addition to the dexamethasone-binding activity, a binding substance identical to corticosteroid-binding globulin (CBG) was identified in the cytosol fraction of mammary glands. CBG in glands of lactating mice was saturated with endogenous steroids, but that in pregnant mice was only 8% saturated. The presence of high concentrations of unsaturated CBG in the vicinity of the parenchymal cells would decrease the availability of corticosteroids to the receptors in pregnancy and may be an important mechanism for the control of the initiation of milk secretion.

Animals↗