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PubMed2002

Progression of vertebrate limb development through SHH-mediated counteraction of GLI3.

Distal limb development and specification of digit identities in tetrapods are under the control of a mesenchymal organizer called the polarizing region. Sonic Hedgehog (SHH) is the morphogenetic signal produced by the polarizing region in the posterior limb bud. Ectopic anterior SHH signaling induces digit duplications and has been suspected as a major cause underlying congenital malformations that result in digit polydactyly. Here, we report that the polydactyly of Gli3-deficient mice arises independently of SHH signaling. Disruption of one or both Gli3 alleles in mouse embryos lacking Shh progressively restores limb distal development and digit formation. Our genetic analysis indicates that SHH signaling counteracts GLI3-mediated repression of key regulator genes, cell survival, and distal progression of limb bud development.

Animals↗
PubMed1992

Dystrophin and a dystrophin-related protein in intrafusal muscle fibers, and neuromuscular and myotendinous junctions.

To determine whether or not and how dystrophin exists in neuromuscular junctions (NMJs) and myotendinous junctions (MTJs), we studied the mid-belly and peripheral portions of control and mdx muscles, immunohistochemically and immunoelectrophoretically, using six kinds of polyclonal antibodies, and an antibody against a dystrophin-related protein (DRP). In controls these regions and the polar region of intrafusal muscle fibers showed a rather clearer immunohistochemical dystrophin reaction than those of extrafusal muscle fibers with all antibodies used. In the muscles of mdx mice NMJs only showed a positive dystrophin reaction with the c-terminal antibody, that is, no reaction with the other five antibodies, and MTJs in mdx showed a positive reaction with the c-terminal antibody and a faint to negative reaction with the other five antibodies. In biopsied human muscles NMJs and MTJs also showed a clear reaction with all ten antibodies, i.e., six polyclonal and four monoclonal ones. Although an immunohistochemical DRP reaction was clearly seen at NMJs, only a faint or no reaction was seen on MTJs and on intrafusal muscle fibers in both mouse and human materials. Western blot analysis of control mouse muscle for dystrophin showed a clearer band for the peripheral portion, which contains many MTJs, than for the mid-belly portion. These data suggest that dystrophin really exists on MTJs, and that dystrophin and DRP exist on NMJs in mouse and human muscles.

Animals↗
PubMed2001

Localization of bilirubin in phospholipid bilayers by parallax analysis of fluorescence quenching.

It has been proposed that the neurotoxicity observed in severely jaundiced infants results from the binding of unconjugated bilirubin to nerve cell membranes. However, despite potentially important clinical ramifications, there remains significant controversy regarding the physical nature of bilirubin-membrane interactions. We used the technique of parallax analysis of fluorescence quenching (Chattopadhyay, A., and E. London. 1987. Biochemistry. 26: 39;-45) to measure the depth of penetration of bilirubin in model phospholipid bilayers. The localization of unconjugated bilirubin and ditaurobilirubin within small unilamellar vesicles composed of dioleoylphosphatidylcholine was determined through an analysis of the quenching of bilirubin fluorescence by spin-labeled phospholipids, and by bilirubin-mediated quenching of a series of anthroyloxy fatty acid probes at various depths within the membrane bilayer. Findings were further verified with potassium iodide as an aqueous quencher. Our results indicate that, at pH 10, unconjugated bilirubin localizes approximately 20 A from the bilayer center, in the region of the polar head groups. Further analyses suggest a modest influence of pH, membrane cholesterol content, and vesicle diameter on the bilirubin penetration depth. Taken together, these data support that, under physiologic conditions, bilirubin localizes to the polar region of phospholipid bilayers, near the membrane-water interface.

Bilirubin↗
PubMed1980

The time required for positional signalling in the chick wing bud.

An estimate of the time required for positional signalling in the chick wing bud was obtained by grafting irradiated quail polarizing regions to host wing buds and removing them at various times. Such polarizing regions must be present for at least 15 h to induce additional structures in the host wings. This results is discussed in terms of a diffusible morphogen model.

Animals↗
PubMed1989

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles.

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.

Animals↗
PubMed2003

The photochemical characteristics of aromatic enediyne compounds substituted with electron donating and electron withdrawing groups.

trans- and cis-1-(4-Dimethylaminophenyl)-6-(4-nitrophenyl)hex-3-ene-1,5-diynes (trans- and cis-DANE) were synthesized and their photochemical properties were studied. The absorption spectra of trans-DANE red-shifted compared with the parent compound bisphenylethynylethene (BEE) due to intramolecular charge transfer. The fluorescence spectra, Stokes shift, fluorescence lifetime, fluorescence quantum yield, and quantum yield of trans-to-cis photoisomerization of trans-DANE showed strong dependence upon the solvent polarity in the less-polar region. No fluorescence emission from trans-DANE was observed in medium-polar and polar solvents. The quantum yield of cis-to-trans isomerization was almost solvent independent. The donor-acceptor substituents shifted the equilibrium between the trans perpendicular triplet state and the trans planar triplet state to the trans triplet state, and resulted in an increase in the triplet lifetime. Comparison of the photochemical properties of trans-DANE with trans-4-dimethylamino-4'-nitrostilbene (DANS) suggests that trans-DANE is a possible fluorescent probe in the non-polar region.

Journal Article↗
PubMed1989

Contours and distribution of sites that react with antiacetylcholinesterase in chicken intrafusal fibers.

Serial cross and longitudinal sections of intrafusal fibers from the intracapsular portions of chicken tibialis anterior muscle spindles were incubated with a monoclonal antibody specific for chicken acetylcholinesterase (AchE) and examined by immunofluorescence for the presence of the enzyme on presynaptic and postsynaptic membranes of neuromuscular junctions. The midequatorial sensory region which lacks organized sarcomeres was negative, but immediately distal to it faintly staining regions of AchE localization were observed on intrafusal fibers. In cross sections at the juxtaequator, the outlines of areas that were positive for AchE were either thin and crescentlike or thick and compact. The distribution of both types of localization continued into the polar region. Toward the more distal polar region, the intensity of sites on the postsynaptic membrane that reacted with the anti-AchE progressively increased. In longitudinal sections, AchE localization was largely limited to two configurations. One was elongate, while the other was more round or oval and often also smaller. Both types might occur on the same, or on different, intrafusal fibers. Examination of silver-impregnated sections revealed the presence of platelike and of traillike axon terminals. The variety of shapes observed on presynaptic and postsynaptic membranes warrants further study to determine whether chicken muscle spindles are innervated by more than one type of motor neuron.

Acetylcholinesterase↗
PubMed1981

The thickness of monoolein lipid bilayers as determined from reflectance measurements.

Measurements of the reflectance of monoolein/n-alkane and monoolein/squalene lipid bilayers have been made. The total thickness of the bilayer was calculated from the dependence of reflectance on the refractive index of the aqueous salt or sucrose solution surrounding the bilayer. The total thickness was then compared to the thickness of the hydrocarbon chain region as determined from capacitance measurements. From this comparison, we found that the thickness of each polar region of the bilayers in salt solutions was 0.5 +/- 0.1 nm, independent of the hydrocarbon solvent used. When the aqueous solutions contained sucrose, each polar region was approx. 0.9 nm thick. When n-tetradecane and n-hexadecane were used as solvents, microlenses of solvent trapped in the monoolein bilayer increased the reflectance. After about one hour, the coalescence of microlenses into larger lenses allowed the reflectance of the bilayer alone to be measured. The use of reflectance to measure the thickness of monoolein bilayers appears to be consistent with other methods and to give useful information about the structure of lipid bilayers.

Chemical Phenomena↗
PubMed1979

The blood supply of muscle spindles in some mammals and the pigeon.

The blood supply of muscle spindles was studied in serial cross sections in macaque, cat, rabbit, guinea pig, mouse and pigeon muscles which had been incubated in a medium containing 3,3' diaminobenzidine. Lumina of blood vessels were recognized by the reaction product that was localized within erythrocytes. The outer capsule was well vascularized, but few or no capillaries were seen in the periaxial space. The inner spindle capsule, which closely invests the axial bundle, was rarely contacted by periaxial capillaries at the equator and juxtequator. Capillaries occurred more frequently adjacent to intrafusal fibers at the polar region and beyond the end of the outer capsule. Shorter diffusion distances and, usually, higher capillary densities were found at the polar region than at the spindle midsection. This suggests that transcapillary exchange at the polar segment is nearer to conditions prevalent in extrafusal muscle than elsewhere in the spindle, provided the inner and outer capsules are not less permeable at the poles than at the midsection. Differences in blood supply among mammalian species appear to be related to receptor size.

Animals↗
PubMed2005

Effect of pressure on the Prodan fluorescence in bilayer membranes of phospholipids with varying acyl chain lengths.

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) were observed as a function of pressure for the bilayer membrane systems of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). The wavelength of the emission maximum, lambdamax, was found to be 480, 430, and 500 nm for the liquid crystalline (Lalpha), ripple gel (P'beta), and pressure-induced interdigitated gel (LbetaI) phase, respectively. Since the lambdamax reflects the solvent property around the probe molecules, we could speculate on the location of the Prodan molecules in the bilayer membranes; in the Lalpha phase of the lipid bilayer, the Prodan molecules distribute around the phosphate of the lipids (i.e. the polar region). The Lalpha/P'beta phase transition caused the Prodan molecules to move into the less polar region near the glycerol backbone. The fluorescence intensity of the Prodan in the P'beta phase was dependent on the chain length of the lipids and on pressure; the shorter the chain length of the lipid, the stronger the fluorescence intensity of the Prodan. Moreover, for the DLPC bilayer membrane system, the fluorescence intensity at 430 nm increased with increasing pressure, indicating that the partition of Prodan into the DLPC bilayer membrane is promoted by applying pressure. In the case of the DPPC and DSPC bilayers, as the pressure increased further, the pressure-induced interdigitation caused the Prodan molecules to squeeze out of the glycerol backbone region and to move the hydrophilic region near the bilayer surface. The ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430, showed a sharp change at the phase-transition pressure. In the case of the DPPC and DSPC bilayers, the values of F480/F430 showed an abrupt increase above a certain pressure higher than the Lalpha/P'beta transition pressure, which corresponds to the interdigitation from the P'beta to the LbetaI phase. The plot of F480/F430 versus pressure is available for recognition of the bilayer phase transitions, especially the bilayer interdigitation.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1976

Immunofluorescent polar tips of Rhizobium japonicum: possible site of attachment or lectin binding.

Rhizobium japonicum USDA 31 demonstrated marked polarity by binding homologous fluorescent antibody (FA) heavily on one end of the cell. FA prepared against R. japonicum strains 110 and 138, and against R. trifolii TA1 cross-reacted with strain 31 only in the polar tip region. No polar immunofluorescing tips could be seen with FA against two other strains of R. japonicum or with those against several unrelated microorganisms. Common antigens localized only in a polar region were seen in many rhizobia stained with R. japonicum 31 FA: 22 of 23 strains of R. japonicum, 10 of 17 strains of R. trifolii, 3 of 7 strains of R. melitolii, 3 of 6 strains of R. phaseoli, and 3 of 9 strains of R. leguminosarum had some cells with detectable polar tips. The proportion of R. japonicum 31 cells with polar tips was high throughout the growth cycle. Polar tip staining was not affected by drastic cell treatments. A function was proposed for the polar tip region as a site for attachment. R. japonicum 31 cells attached to each other in a tip-to-tip fashion and endwise to fungal hyphae with the polar tip in contact with the hyphal wall. Binding of fluorescein isothiocyanate-labeled soybean lectin to certain strains of R. japonicum gave additional evidence of polarity. Polar binding of both antibody and lectin may provide insights into relationships between rhizobia and roots of host legumes.

Antibodies, Bacterial↗
PubMed1960

The fine structure of muscle spindles in the lumbrical muscles of the rat.

Lumbrical muscles of young rats were fixed with OsO(4) and embedded in methacrylate for electron microscopy. The spindle capsule was found to be continuous with and similar in structure to the sheath of Henle surrounding the nerves supplying the spindle. The capsule consists of several closely applied concentric cytoplasmic sheets. Each sheet is about 1,000 A thick and has no fenestrations. Many caveolae and vesicles in the cytoplasm suggest active transport through the sheets. The periaxial space fluid contains much solid material. It is suggested that the capsule and periaxial space regulate internal chemical environment. The interfibrillar structures are less evident in the polar regions of intrafusal fibres than in extrafusal fibres. Simple motor end-plates occur on the polar regions of intrafusal fibres. In the myotube region of the intrafusal fibre a peripheral zone of myofibrils surrounds a cytoplasmic core containing nuclei, mitochondria, Golgi bodies, reticulum, and a few lipid-like granules. Naked sensory endings lie on the myotube "in parallel" with the underlying myofilaments. Naked processes of the primary sensory ending deeply indent the muscle plasma membrane and the underlying wisps of myofilament in the nuclear bag region. The plasma membranes of sensory nerve ending and intrafusal muscle fibre are about 200 A apart.

Animals↗
PubMedDate not supplied

[Immunopathology of leprosy: the complexity of the mechanisms of host immune response to Mycobacterium leprae].

Leprosy, whose etiologic agent Mycobacterium leprae, is an illness of ample clinical and immunopathological spectrum. Its clinical manifestations are correlated with distinct immunologic forms, varying from a vigorous immune response mediated by cells to M. leprae, with Th1 standard in the tuberculoid polar region, to an absence of specific cellular response to antigens of M. leprae in the lepromatous polar region, with predominance of Th2 response and exacerbation of humoral response. It is probable that different polymorphic genes determine susceptibility to M. leprae. Additional studies are necessary to clarify the complex interactions between cytokines and the role of the phenotypic diversity of cells network that contribute to the host defense. The comprehension of such mechanisms will provide new insights for the identification of agonists and/or antagonists for pro- or anti-inflammatory effects, and also will indicate possible situations for its appropriate use in immunologic and/or immunotherapeutic interventions.

Animals↗
PubMed1986

Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo.

The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizating organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self-reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubule-related processes.

Anaphase↗
PubMed1975

Neutron scattering study of human serum low density lipoprotein.

Regions of different proton density in the low density lipoprotein (LDL) particle from human serum have been determined by neutron scattering. From measurements in various H2O/D2O mixtures, the LDL particle appears to be quasi-spherical, with the centers of gravity of the hydrocarbon and polar regions coinciding. The average volume occupied by a particle was found to be 3.2 X 10-6 A-3, with the volume fraction occupied by the hydrocarbons being 60%. The ratius of gyration of the hydrocarbon region was 64 A, while that of the polar region was 100 A. consequently, the core of LDL is predominantly occupied by the hydrocarbon chains, while the outer shell is sparsely occupied by protein emerging from the lipid core.

Deuterium↗
PubMed1977

Localization of the membrane perturber adamantylsulfate in sarcoplasmic reticular vesicles.

The properties of a new membrane perturbing agent, adamantyl sulfate (A-1-S) are described. Lobster sarcoplasmic reticular vesicles (SRV) were used as a test membrane system and the activity of the Ca2+--Mg2+-dependent ATPase in conjunction with spin label data was used to determine the location of A-1-S in the membrane. The results suggest that A-1-S perturbs only the polar region of the SRV and that this causes loss of ATPase function. Thus, ATPase activity requires integrity of the membrane polar region.

Adamantane↗
PubMed1984

The sensory and motor innervation of muscle spindles in cat tail dorsolateral muscles.

The sensory and motor innervation of a sample of 202 muscle spindles from the dorsolateral tail muscles of eight cats were studied by silver and gold staining techniques. The range of diameters of Group Ia afferent nerve fibres to primary (P) sensory endings was 2.0-14.0 micron with a peak at 8.5 micron. The range of Group II fibres to secondary (S) sensory endings was 2.0-8.5 micron with a peak at 5.0 micron. In a sample of 234 spindle capsules, 171 (84.2%) were single capsule spindles and 31 (15.8%) were tandem spindles. With respect to the sensory endings of single capsule spindles, 19.7% showed only a P-ending, 32.5% had an additional S-ending and 15.8%, 3.0% and 2.1% had two, three and four S-endings, respectively. In the tandem spindle capsules, 0.8% of the larger proximal capsules showed only a P-ending, 5.1% had an additional S-ending and 4.7%, 2.6% and 0.4% had two, three and four S-endings, respectively. In the 31 tandem spindles, one of which had a triple capsule, there were 14 types of sensory ending combination in the proximal and distal capsules. The most common (44.4%) innervation was by a P-ending only, while 27.0% had an additional S-ending and 17.5%, 9.5% and 1.6% had two, three and four S-endings, respectively. Of the distal capsules, 92.9% showed only a P-ending. There were three types of motor nerve ending, namely the p1-plate, the p2-plate and the trail (tr) ending, in spindle polar regions. Spindle poles without a motor innervation also occurred. There were 16 types of motor ending combination in the two polar regions of muscle spindles. The most common type of combination was the trp2-ending (41.8%), receiving an average of 5.8 fusimotor fibre branches and the next common was the tr-ending only (38.7%), innervated by an average of 3.2 fibres. The least common combination was with the p1-plate ending (9.5%), receiving an average of 6.9 fibres. The mean number of fusimotor fibre branches per spindle pole was 4.2.

Animals↗
PubMed1990

Expression of myosin heavy chain isoforms in developing rat muscle spindles.

The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein. At 17-18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19-20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to anti-slow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗