TGF-beta 1-induced inhibition of mouse mammary ductal growth: developmental specificity and characterization

TGF-beta 1, implanted into growing mouse mammary glands, was previously shown to inhibit ductal growth in an apparently normal and fully reversible manner. In this report we extend these findings to show that TGF-beta 1 inhibition is highly specific. In pregnant or hormone-treated mice, doses of TGF-beta 1 that were capable of fully inhibiting ductal elongation had little effect on the proliferation of lobuloalveolar structures. Additionally, the inhibitory action of TGF-beta 1 on ducts is epithelium-specific, resulting in cessation of DNA synthesis in the rapidly proliferating epithelium of mammary end buds, but does not inhibit DNA synthesis in the stroma surrounding the end buds. At the cellular level, transplant studies showed that TGF-beta 1 inhibited the regeneration of mammary ductal cells when implanted into mammary gland-free fat pads by suppressing the formation of new end buds, without inhibiting maintenance DNA synthesis in ductal lumenal epithelium; this observation indicates the potential of TGF-beta 1 to maintain patterning by suppressing adventitious lateral branching. The time-course of TGF-beta 1 inhibition of end buds was rapid, with cessation of DNA synthesis by 12 hr, followed by loss of the stem cell (cap cell) layer. The question of glandular exposure to TGF-beta 1 administered in EVAc implants was also investigated. Incorporation of TGF-beta 1 into EVAc was found not to degrade the hormone, while the release kinetics of the ligand from implants, its retention in the gland, and the demonstrable zone of exposure were consistent with observed inhibitory effects. These results support the hypothesis that TGF-beta 1 is a natural regulator of mammary ductal growth.     

Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy

We show that Duchenne muscular dystrophy (DMD) selectively affects a subset of skeletal muscle fibers specialized for fast contraction. Muscle fiber types were characterized immunohistochemically with monoclonal antibodies that distinguish isoforms of fetal and adult-fast or adult-slow myosin heavy chain present in the same fiber. Fetal myosin expression increased with patient age and was not due to arrested development but rather to de novo synthesis, which served as a sensitive indicator of muscle regeneration. A subset of fast fibers were the first to degenerate (type IIb). Extensive fast fiber regeneration occurred before slow fibers were affected. These results suggest that the DMD gene product has a specific function in a subpopulation of muscle fibers specialized to respond to the highest frequency of neuronal stimulation with maximal rates of contraction.     

Quantitative determination of Coomassie R bound to proteins in polyacrylamide gel. A facilitated method for multi-spot analysis of electrograms

Dimethylsulfoxide (DMSO) was found to be an efficient solvent for extraction of Coomassie Blue R 250 (Coomassie R) from stained proteins on polyacrylamide gels. Kinetic measurements show that the extraction of the dye from a 2-D gel reached equilibrium in 48 h. Staining of E. coli ribosomal proteins by Coomassie R dissolved in trichloroacetic acid exhibited two types of dye-protein complexes, the majority of them yield a blue-purple colour, while the rest are stained with a light-blue tone and fluorescent appearance as well. The absorbance spectra of the complexes in the gel matrix differ significantly from each other. However, the DMSO-extracted Coomassie show identical absorbance profiles with lambda max at 602 nm, thus the amount of the bound dye can easily be measured spectrophotometrically.     

Developmental regulation of 16S acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line

We have studied the appearance, distribution and regulation of acetylcholinesterase (AChE) and acetylcholine receptors (AChRs) in a mouse skeletal muscle cell line (C2), that was originally isolated and described by Yaffe & Saxel [54]. In culture, cells from this line form spontaneously contracting myotubes, with overshooting action potentials that are TTX-sensitive. After fusion of myoblasts into myotubes, there was a dramatic increase in the amount of both AChE and AChR. Three forms of AChE, distinguished by their sedimentation on sucrose gradients, were synthesized: 4-6S, 10S, and 16S. The 4-6S and 10S forms appeared 1 day after the cells began to fuse, whereas the 16S form appeared only 2 days after fusion began. Maximal levels of the 16S AChE form (25-30% of the total) were obtained by reducing the concentration of horse serum in the fusion medium. Prevention of myoblast fusion by reducing the calcium levels in the medium decreased the total AChE by 70%, and only the 4-6S form was synthesized. Blocking spontaneous contractile activity of the myotubes by tetrodotoxin (TTX) led to a 50% reduction in all three esterase forms. Thus, the 16S, or endplate form of AChE is not specifically regulated by electrical or contractile activity in the C2 cell line. After fusion the number of AChRs increased rapidly for 3-4 days and then stabilized. Receptor clusters, ranging from 10-30 micron in length, appeared 1 day after myoblast fusion began. When cells were grown in medium containing reduced Ca2+, the total number of AChRs was decreased by 20-50%. Reduction of Ca2+ after myotubes and AChR clusters had formed resulted in dispersal of AChR clusters. Inhibition of muscle contractions with TTX did not affect the number of AChRs or their distribution.     

A new method for stoichiometric analysis of proteins in complex mixture--reevaluation of the stoichiometry of E. coli ribosomal proteins

A novel way is presented for determination of the stoichiometry of ribosomal proteins in the ribosome. The 70S E. coli r-proteins, completely separated on a two-dimensional gel system, were used throughout our experiments. The method is based on our previous observation that the amount of Coomassie R bound to a protein molecule is directly proportional to the number of positive charges on that protein. By plotting the amount of bound Coomassie as a function of the number of positive charges of each r-protein, and relating the experimental amount of the dye bound to each r-protein to the value obtained from the linear regression line based on all (a total of some 50 proteins), one can obtain the molar concentration of every protein in the ribosome. A parallel experiment can be carried out, which relates the radioactivity contributed by 3H-labeled amino acid in each r-protein to its amino acid content in that molecule. The two sets of data, which are completely independent of each other, are well correlated. Further verification of the validity of our procedure is provided by the fact that we found the known proportions of four copies of L7/L12 and one copy of S6 per ribosome. The rationale behind the present study was our finding that recalculation of Hardy's data (Hardy, S.J.S. (1975) Mol. Gen. Genet. 140, 253-274), with the accurate molecular weight value of the r-proteins provided by Giri et al. (Adv. Protein Chem. (1984) 36, 1-78), raises some doubt with regard to his experimental results, although we agree with his final conclusion that E. coli ribosome is homogeneous with respect to its proteins.     

Primary products of break-induced recombination by Escherichia coli RecE pathway.

Alternative models for break-induced recombination predict different distributions of primary products. The double-stranded break-repair model predicts a noncrossover product and equimolar amounts of two crossover products. The one-end pairing model predicts two crossover products, but not necessarily in equimolar amounts, and the single-stranded annealing model predicts deletion of the fragment between the pairing sequences. Depending on the structure of the recombining substrate(s) and the nature of the resectioning step that precedes strand annealing, the single-stranded annealing mechanism would yield only one or both crossover products. We tested these predictions for the RecE recombination pathway of Escherichia coli. Nonreplicating intramolecular recombination substrates with a double-stranded break (DSB) within one copy of a direct repeat were released from chimera lambda phage by in vivo restriction, and the distribution of primary circular recombination products was determined. Noncrossover products were barely detectable, and the molar ratio of the two crossover products was proportional to the length ratio of the homologous ends flanking the DSB. These results suggest an independent pairing of each end with the intact homolog and argue against the double-stranded break-repair model. However, the results do not distinguish alternative pairing mechanisms (strand invasion and strand annealing). The kinetics of heteroduplex formation and heteroduplex strand polarity were investigated. Immediately following the DSB induction, heteroduplex formation was done by pairing the strands ending 3' at the break. A slow accumulation of the complementary heteroduplex made by the pairing of the strands ending 5' at the break (5' heteroduplexes) was observed at a larger stage. The observed bias in heteroduplex strand polarity depended on DSB induction at a specific site. The 5' heteroduplexes may have been generated by reciprocal strand exchange, pairing that is not strand specific, or strand-specific pairing induced at random breaks.     

Structural basis of a conserved idiotope expressed by an autoreactive human B cell lymphoma. Evidence that a VH CDR3 mutation alters idiotypy and specificity

Our laboratory has previously investigated the relationship of autoimmune disease and B cell neoplasia in a patient with a diffuse, well differentiated splenic B cell lymphoma and associated autoimmune hemolysis due to an anti-Pr2 antibody. EBV-immortalized B cell clones, established from this lymphoma, were shown to secrete the same pathologic anti-Pr2 antibody. The antiidiotypic mAb, RI.1, defined a private Id (IdRI.1) of the anti-Pr2 antibody that was related to the Ag-binding site and was expressed by both the lymphoma and derived cell lines. This unique Id was expressed by the majority of splenic tumor B cells and also was conserved over a period of 4 yr. In this report, the structural basis of IdRI.1 expression was investigated by analysis of Id- variants isolated by flow microfluorimetry using RI.1. Six Id- cell lines that secrete IgM kappa but lack Pr2 specificity were generated from an Id+ cell line, LS2. These lines were shown to be related to LS2 and the lymphoma by karyotype and by restriction fragment analysis of Ig gene rearrangements. Shared and unshared nucleotide substitutions in the VH and VL regions of the six independent clones were used to construct a genealogic tree relating the Id- clonal members to a common Id+ precursor. The tree illustrates that the base changes occurred sequentially, suggesting that they were introduced by somatic point mutation. Only one VH CDR3 bp difference from the LS2 nucleic acid sequence is common to all Id- sequences, resulting in an amino acid substitution of cysteine 108 to tyrosine. Taken together, these findings suggest that both the expression of IdRI.1 and Ag binding are affected by a single mutation localized to the D region of the anti-Pr2 antibody.     

How do ionic channel properties depend on the structure of polyene antibiotic molecules?

A study has been made of the properties of ionic channels formed in phospholipid-cholesterol bilayers by polyene antibiotics of various molecular structures. Properties of channels created by natural antibiotics with different structures of the lactone ring (amphotericin B-nystatin-mycoheptin) as well as by some derivatives of amphotericin B modified with respect to the amino and carboxyl groups are compared. Neutralization of one or both charges of the amphotericin B molecule (both by chemical modification and by pH shift) increases the probability of the channel to be in a nonconducting state. An increase of cholesterol concentration in the membrane produces an opposite effect. It is assumed that the electrostatic interaction of the amino group of an antibiotic molecule with the carboxyl group of an adjacent one stabilized the channel. Conductance and selectivity of an open channel are not influenced by changes in the charged groups. These properties strongly depend on the structure of the polar chain of the lactone ring. For example, the appearance of one more carbonyl group in the mycoheptin molecule results in a sharply decreasing anion permeability of channels. An antibiotic concentration which is necessary to observe single channels depends on the polyene chain structure: this is about 10(-7) M for tetraene nystatin and 2.10(-8) M for heptaene amphotericin B an mycoheptin.