The influence of passive stretch and NF-κB inhibitors on the morphology of dystrophic muscle fibers.

The triangularis sterni (TS) is an expiratory muscle that is passively stretched during inspiration. The magnitude of passive stretch depends upon the location of individual fibers within the TS muscle, with fibers located more caudally being stretched ∼ 5% to 10% more than fibers in the cephalad region. In the mdx mouse model for muscular dystrophy, the TS exhibits severe pathological alterations that are ameliorated by treatment with inhibitors of the NF-κB pathway. The purpose of this study was to assess the influence of passive stretch in vivo on fiber morphology in nondystrophic and mdx TS muscles, and the morphological benefits of treating mdx mice with two distinct NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC), and ursodeoxycholic acid (UDCA). Transmission electron microscopy revealed Z-line streaming, hypercontraction, and disassociation of the plasma membrane from the basal lamina in mdx fibers. In both nondystrophic and mdx TS muscles, fiber density was larger in more caudal regions. In comparison with nondystrophic TS, fibers in the mdx TS exhibited substantial reductions in diameter throughout all regions. In vivo treatment with either PDTC or UDCA tended to increase fiber diameter in the middle and decrease fiber diameter in the caudal TS, while reducing centronucleation in the middle region. These results suggest that passive stretch induces hypercontraction and plasma membrane abnormalities in dystrophic muscle, and that differences in the magnitude of passive stretch may influence fiber morphology and the actions of NF-κB inhibitors on dystrophic morphology.

Fourier transform infrared observation of the nu1(sigma) mode of linear CoC3 trapped in solid Ar.

Fourier transform infrared (FTIR) and density functional theory (DFT) isotopic studies on cobalt-carbon species have resulted in the detection of linear CoC3. Dual laser ablation of carbon and cobalt rods, followed by trapping the products in solid Ar at approximately 10 K, produced the CoC3 chain. FTIR measurements of 13C isotopic shifts are in good agreement with the predictions of DFT calculations using the B3LYP and BPW91 functionals and the 6-311+G(3df) basis set, confirming the assignment of the nu1(sigma) fundamental of linear CoC3 at 1918.2 cm(-1).

Tamm-Horsfall glycoprotein binds IgG with high affinity.

Tamm-Horsfall protein (THP), a monomeric glycoprotein (M(r) 80 to 100 kDa), is produced by the mammalian kidney's thick ascending limb of Henle cells and excreted into the urine. The function of THP is uncertain. Here we report that a high molecular weight contaminant in sheep THP (sTHP) preparations was identified as sheep IgG by its positive reaction with donkey anti-sheep IgG antibody and with protein G. To answer the question of whether sTHP and sheep IgG co-purified because of a physical interaction between the two proteins, an enzyme-linked immunosorbent assay (ELISA) using immobilized sTHP and soluble sheep IgG was performed. Analysis of the ELISA data identified the presence of two sets of binding sites: a high affinity site (Kd 10(-8) to 10(-9) M) and a lower affinity site (Kd 10(-6) to 10(-7) M) [corrected]. The ELISA detected a similar high affinity interaction between human THP (hTHP) and human IgG. The binding of sheep IgG to immobilized sTHP was inhibited by soluble sTHP. These observations suggest an additional factor to be considered in studies addressing THP's potential immunoregulatory function.

Cation-induced aggregation of cat Tamm-Horsfall glycoprotein and its possible role in feline urolithiasis.

The in vitro cation-induced aggregation properties of cat Tamm-Horsfall protein (cTHP), a urinary glycoprotein, were examined and related to the potential role of cTHP in feline urolithiasis. The aggregation assay involved adding either CaCl2, MgCl2, or NaCl to solutions containing purified cTHP, and then separating the aggregated cTHP by centrifugation. The concentration of cTHP remaining in the supernatant was quantified using a previously developed enzyme-linked immunosorbent assay (ELISA). The effect that buffer pH, cTHP concentration, and urea concentration had on cTHP aggregation also were examined. Of the three salts, CaCl2 consistently was most efficient at precipitating cTHP, while MgCl2 was slightly less efficient. At least ten times more NaCl than CaCl2 or MgCl2 was required for comparable cTHP aggregation. As the pH decreased, increasing concentrations of the salts were required to aggregate cTHP. Increased amounts of CaCl2 and MgCl2 also were required to aggregate cTHP when the urea concentration was increased. As cTHP concentration increased within the physiological range, lower concentrations of CaCl2 and MgCl2 were required to precipitate 50% of the cTHP. Several aspects of the in vitro aggregation properties of cTHP correlate closely with previously identified risk factors for feline urolithiasis, strengthening the theory that cTHP aggregation may be important in this disease.

Urinary Tamm-Horsfall glycoprotein concentrations in normal and urolithiasis-affected male cats determined by an ELISA.

A precise and reproducible enzyme-linked immunosorbent assay (ELISA) which measures urinary cat Tamm-Horsfall glycoprotein (cTHP) was developed in order to investigate the possible role of cTHP in the pathogenesis of feline urolithiasis. Reproducible quantification required that the cTHP be disaggregated with 2M urea and 0.05% Tween 20. It was necessary to standardize rigidly the handling of the samples prior to analysis, since the apparent cTHP concentration varied depending on the preanalysis protocols. Using the sample handling protocol of freezing urine at -70 degrees C before dialysis, urinary cTHP was quantified in male cats with no history of urolithiasis ("normal" cats) and in male cats with a history of urolith formation ("urolithiasis" cats). The mean cTHP concentration in adult, male "normal" cats of 49.2 +/- 35.5 micrograms/ml (N = 23) was significantly lower than the mean cTHP concentration of 95.4 +/- 34.1 micrograms/ml (N = 9) in "urolithiasis" cats (p < 0.01, Students' T-test). These findings indicate a correlation between urolithiasis and high urine cTHP concentrations in male cats which warrants further investigation.

Isolation and characterization of a glycosylated form of beta nerve growth factor in mouse submandibular glands.

In the course of characterizing polyclonal antibodies to beta nerve growth factor (NGF) on immunoblot replicas of sodium dodecyl sulfate gels, we observed a protein (designated C protein) migrating as two bands (14.0 and 13.5 kDa) that copurifies with NGF and reacts strongly with its antibodies. The molecule is detectable in the 7 S, beta, and 2.5 S forms of NGF, accounting in the latter two for approximately 2% of total protein. The C protein can be separated from the A and B chains of beta-NGF on acetic acid-urea gels and on two-dimensional gels but not by isoelectric focusing alone. The molecule has been isolated to near purity on reversed-phase high performance liquid chromatography. Amino acid analyses and sequencing through 49 Edman cycles revealed that the protein preparation is composed of the intact and desoctapeptide (des-(1-8] polypeptide chains and suggested a glycosylation site at Asn-45. Following digestion with N-glycanase, the chains migrated on sodium dodecyl sulfate gels identically with the A and B chains of beta-NGF. Although this was accompanied by some degree of proteolytic degradation, the presence of glucosamine (approximately 4 mol/mol of single chain) was confirmed in acid hydrolysates on the amino acid analyzer. No amino sugars were detected in hydrolysates of the A chain nor was galactosamine recovered in either preparation. Glycosylated NGF promotes neuronal growth and survival in a manner indistinguishable from native 2.5 S NGF when tested in the chick sensory ganglion assay and with rat postnatal sympathetic neurons in a dissociated culture cell survival assay or in a compartmentalized culture growth assay. These studies reveal that NGF can be modified by glycosylation in a manner that does not reduce its biological activity.

Epidermal growth factor-induced precocious incisor eruption is associated with decreased tooth size.

Epidermal growth factor (EGF) causes precocious eruption of incisors in vivo and is mitogenic for tooth-derived cells in vitro. These two observations lead to the hypothesis that the EGF-induced precocious eruption is the result of an increase in the size of the incisor. To test this hypothesis, neonatal mice were injected daily with various doses of EGF and, seven days after birth, were perfused with fixative. EGF causes a retardation of overall growth (as measured by body weight) and a dose-dependent thickening of the epidermis. The incisors were examined in midsagittal histological sections and in X-ray microradiographs. Contrary to our expectations, EGF causes a dose-dependent decrease in the size of the incisors. This result suggests that the stimulation of the growth of odontogenic cells seen in tissue culture is not part of the physiological response to EGF in vivo and that EGF-induced precocious eruption of incisors is not due to an increase in the growth rate of the tooth.

Transforming growth factor alpha inhibits secretion of gastric acid.

Transforming growth factor alpha (TGF-alpha), a protein secreted by transformed cells and related to epidermal growth factor (EGF), was tested for its effects on gastric acid secretion. Guinea pig gastric mucosae were mounted in Ussing chambers and the rate of acid release was monitored by the pH-stat method. When administered prior to the secretagogue, TGF-alpha prevented the histamine-induced increase in the rate of acid secretion. Similarly, TGF-alpha caused a decrease in the rate of acid release in tissues that had already been stimulated with histamine. These data show that TGF-alpha inhibits gastric acid secretion in a manner similar to EGF and that the two growth factors share at least one physiological action unrelated to their mitogenic properties.

Electrophoretic study of enzymes from cereal aphid populations : 4. Detection of hidden genetic variation within populations of the grain aphid Sitobion avenae (F.) (Hemiptera: Aphididae).

A study of variation in three peptidases (PEP-3 to -5) in a parthenogenetic S. avenae field population at Rothamsted using serial one-dimensional polyacrylamide gel electrophoresis (involving changes of gel concentration and electrophoretic run-time) increased the overall number of "allozymes" (mobility variants) detected from 10 under standard conditions (6% gels, 2 h run-time) to 22, as well as revealing putative heterozygous banding patterns under some test conditions. However, an examination of another enzyme, 6-phosphogluconate dehydrogenase (6-PGD) in a sample collected at Rothamsted the following year failed, using a combination of serial methods (changes of gel concentration) and isoelectric focusing, to increase the total number of 6-PGD bands separated (seven, none of which appeared to be allelic in origin). Nevertheless, some major bands were split into several bands, whilst other infrequent bands were either gained or lost. The findings are briefly discussed.

Slow internalization of human chorionic gonadotropin by cultured granulosa cells.

Kinetic studies were performed on two-day cultures of rat ovarian granulosa cells to follow the fate of surface-bound 125I-labeled human chorionic gonadotropin (125I-hCG). Low pH was used to release hCG from its surface receptor, allowing us to distinguish between surface-bound and internalized hormone. Because our results indicated that hormone is lost from the cell surface by dissociation as well as internalization, equations were derived to determine independent rate constants for each process. We calculate that if hormone binding were irreversible, the t 1/2 for internalization would be 8.5 hour. Morphometric studies on the uptake of horseradish peroxidase indicate that the t 1/2 for internalization of bulk membrane in granulosa cells is 55 to 77 minutes. Thus, the rate of uptake of surface-bound hCG appears to be seven to nine times slower than the rate of uptake of bulk plasma membrane, which suggests that the LH/hCG receptor may be selectively excluded from the endocytic vesicles of granulosa cells.

The contractile basis of amoeboid movement III. Structure and dynamics of motile extracts and membrane fragments from Dictyostelium discoideum and Amoeba proteus.

Motile extracts from D, discoideum and A. proteus have been characterized in order to compare the structural dynamics and chemical regulation of movement in 2 different types of amoeboid cells. The structural dynamics of both extracts involve the formation of a nonmotile cytoskeleton followed by the contraction of actin and myosin to generate both direct contractile force and cytoplasmic streaming. The contractions are regulated by calcium ions and a threshold of ca. 1.0 X 10(-6) M calcium induces a transformation of actin to the free F-actin state which is capable of interacting with myosin. Furthermore, 3 low molecular weight proteins are concentrated along with actin and myosin during contraction and might play a regulatory role in movement. Several common characteristics of amoeba cytoplasm have been exhibited by these two types of amoeboid cells. The major contractile and "associated" proteins are similar, actin and associated proteins are structurally dynamic, and movement is regulated by calcium. The different modes of movement observed in different types of amoeboid cells could result from the site, rate, and extent of actin transformation followed in some regions by contractions.

The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts.

The role of calcium and magnesium-ATP on the structure and contractility in motile extracts of Amoeba proteus and plasmalemma-ectoplasm "ghosts" of Chaos carolinensis has been investigated by correlating light and electron microscope observations with turbidity and birefringence measurements. The extract is nonmotile and contains very few F-actin filaments and myosin aggregates when prepared in the presence of both low calcium ion and ATP concentrations at an ionic strength of I = 0.05, pH 6.8. The addition of 1.0 mM magnesium chloride, 1.0 mM ATP, in the presence of a low calcium ion concentration (relaxation solution) induced the formation of some fibrous bundles of actin without contracting, whereas the addition of a micromolar concentration of calcium in addition to 1.0 mM magnesium-ATP (contraction solution) (Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen. 1973. J. Cell Biol. 59:378-394) initiated the formation of large arrays of F-actin filaments followed by contractions. Furthermore, plasmalemma-ectoplasm ghosts prepared in the relaxation solution exhibited very few straight F-actin filaments and myosin aggregates. In contrast, plasmalemmaectoplasm ghosts treated with the contraction solution contained many straight F-actin filaments and myosin aggregates. The increase in the structure of ameba cytoplasm at the endoplasm-ectoplasm interface can be explained by a combination of the transformation of actin from a less filamentous to a more structured filamentous state possibly involving the cross-linking of actin to form fibrillar arrays (see above-mentioned reference) followed by contractions of the actin and myosin along an undetermined distance of the endoplasm and/or ectoplasm.