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Biomedical subjects

D A Kirschner

Publications and source records attributed to D A Kirschner.

At least 91 records · Page 5Linked to original sources
PubMed1983

Cytologic and molecular analysis of 46,XXq- cells to identify a DNA segment that might serve as a probe for a putative human X chromosome inactivation center.

Cloned human X chromosome-specific DNA segments, derived from a recombinant phage library enriched for the human X and previously localized to different regions of the X, were used as probes in Southern blots to confirm the nature of a deletion of the long arm of the X chromosome as del(X)(q13) in a patient with some features of Turner's syndrome and suspected from cytologic studies to have a 46,XXq- karyotype. Two dimensional scanning densitometry of autoradiograms of the Southern blots was used to quantitate hybridization of the 32P-labeled probes, reinforcing visual analysis and permitting distinction between sequences present at one or two copies per diploid genome. Once thus characterized, DNA from the patient's cells was used in quantitatively analyzed Southern blots to refine the location of an additional DNA segment, previously mapped to somewhere in the proximal part of the long arm of the X chromosome, to the juxtacentromeric region of Xq, which has been hypothesized to be critical for X-inactivation. Cloned DNA probes such as that localized to the juxtacentromeric region of Xq should be useful for evaluating this hypothesis.

Absorptiometry, Photon↗
PubMed1983

Ganglioside localization on myelinated nerve fibres by cholera toxin binding.

GM1 ganglioside has been localized on the surfaces of myelinated, peripheral nerve fibres by using immunofluorescence to detect cholera toxin receptors. Unfixed, mouse sciatic nerves were teased into individual, intact fibres in order to expose their extracellular surfaces. Cholera toxin binding sites were abundant at all nodes of Ranvier; they were scarce on the internodal fibre surfaces. The nodal receptors were resistant to various degradative enzymes, including trypsin and proteinase K. Proteases did not unmask receptors on the internodal surfaces. Exogenous GM1 successfully competed for the toxin binding sites on the fibres. From this evidence and the specificity of cholera toxin binding, we conclude that GM1 ganglioside is abundantly present on the membrane surfaces of peripheral nodes of Ranvier and is not present on the internodal Schwann cell surfaces in an appreciable amount. The patterns of fluorescence within the node suggest that the axon and Schwann cell structures are sites where GM1 is localized. Treatment of the teased fibres with Vibrio cholerae neuraminidase, which is known to reduce polysialogangliosides to the monosialoganglioside GM1, induced cholera toxin binding on the internodal Schwann cell surfaces. The induced receptors, as well as their precursors, were resistant to trypsin and proteinase K. We conclude that the internodal Schwann cell surface is rich in an unidentified polysialoganglioside(s) that can be converted to GM1 by neuraminidase.

Animals↗
PubMed1983

Myelin membrane from adrenoleukodystrophy brain white matter--biochemical properties.

Adrenoleukodystrophy (ALD) is an X-linked progressive neurological disorder characterized by the accumulation of saturated very-long-chain fatty acids (C24 to C30) in lipids, especially cholesterol esters of the brain white matter and adrenal cortex. In the present study we have investigated the localization of accumulated cholesterol esters in brain white matter. During isolation of purified myelin membrane from regions of active demyelination, significant enrichment in cholesterol ester was found in two fractions, mainly in a low-density floating fraction and to a lesser degree in the purified myelin preparation. The fatty acid composition of cholesterol esters from both the ALD floating and myelin fractions was enriched approximately 10-fold in saturated very-long-chain fatty acids (greater than or equal to C24) compared with control preparations.

Adrenoleukodystrophy↗
PubMed1982

Triethyl tin-induced myelin oedema: an intermediate swelling state detected by X-ray diffraction.

X-ray diffraction was used to probe the effects of triethyl tin (TET) on the periodicity and amount of membrane disorientation in the lamellar myelin from respiring optic and sciatic nerves in vitro as well as from nerves of rats treated in vivo through their drinking water. The diffraction patterns show that in vitro TET at concentrations of 4-100 microM affects C.N.S. but not P.N.S. myelin structure. A planar, concentric membrane array with a 200 A period is detected in the C.N.S.; this ordered, swollen myelin contrasts with the vacuolar and vesicular structure seen in thin-sections in TET-induced oedema. No effects of short-term in vivo treatment with TET are observed in either the C.N.S. or P.N.S. The finding that carbonic anhydrase (CA) inhibitors have no effect on the TET-induced structural changes indicates that the swelling we observe is not related to a CA-dependent process. In comparison, the TET effect is prevented by replacing the mobile ions with isotonic sucrose. We conclude that TET-induced swelling in C.N.S. myelin arises from an increase in ion transport followed by obligatory fluid movement. Further, the ordered, swollen structure we detect may be an intermediate state that exists transiently in vivo in TET intoxication and that precedes the gross swelling and vacuolization usually observed.

Adenosine Triphosphatases↗
PubMed1981

Compaction and particle segregation in myelin membrane arrays.

Compacted membrane arrays are formed in the nerve myelin sheath by lowering the water activity (through evaporation or immersion in hypertonic solutions of nonelectrolytes or monovalent salts) or by binding specific cations (Ca(++), La(+++), and tetracaine at concentrations above 5-10 mM). X-ray diffraction observations on intact, hydrated nerves treated to induce compaction provide a control to assess the significance of structural changes seen by freeze-fracture electron microscopy. Compaction inevitably leads to lateral segregation of particles away from the closely packed membrane arrays into contiguous normal, or slightly expanded, period arrays. In the particle-enriched layers, the E fracture face is more particle-dense than the P face, whereas no particles are found on either face in the compacted layers. Morphologically, compaction induced by the all-or-nothing, relatively irreversible action of specific cations cannot be distinguished from compaction to the same extent induced by the graded, reversible effects of nonelectrolytes. Compaction by sodium chloride resembles that by specific- cation binding in that the repeat period is independent of reagent concentration; but, like dehydration by nonelectrolytes, the extent of compaction is reversibly related to reagent concentration. Sodium chloride-compacted myelin can be distinguished morphologically by a lack of the elongated border particles at the boundary between smooth and particle-enriched membrane observed for other compacting treatments. Fracture faces in compacted arrays are not always smooth, but the unusual appearances can be duplicated in purified myelin lipid multilayers subjected to similar treatments, which indicates that the particle-free membrane fracture faces are uninterrupted lipid hydrocarbon layers. Correlation of x-ray diffraction and electron microscopy observations provides a direct basis for identifying the intramembrane particles with transmembrane protein. The transmembrane protein appears to play a significant role in maintaining the normal membrane separation; swelling of the particle-enriched arrays in myelin compacted by tetracaine at low ionic strength provides information about the charge distribution on the transmembrane protein. Swelling of the compacted arrays following irreversible particle segregation shows that the interaction properties of the particle-free membranes are similar to those of pure lipid multilayers. Compaction and the consequent particle segregation in lyelin results from conditions stabilizing close apposition of the lipid bilayers. Particle segregation in areas of close contact between other cell membranes may also be driven by interbilayer attractive forces.

Animals↗
PubMed1980

Collagen type II differs from type I in native molecular packing.

Native molecular packing of types I and II collagens were compared by low-angle X-ray diffraction. Fibers from human intervertebral disc that contained different proportions of types I and II collagens were studied by X-ray diffraction, and were then analyzed biochemically to measure the constituent collagen species. Other cartilages, containing exclusively type I or type II collagen, were also examined. The equatorial diffraction established that in wet, native type II collagen, the molecules are spaced farther apart laterally than in type I collagen under the same conditions. For the disc the average lateral spacing ranged from about 14 A for a fiber from the outer annulus fibrosus containing mostly type I collagen, to 16-17 A for nucleus pulposus that contained all type II collagen. No differences were evident among dried specimens. We have also found that the meridional diffraction pattern from dry fibers of type II collagen differed from that of type I collagen. The findings indicate that under physiological conditions type II collagen fibrils contain more water than type I fibrils. Calculations suggest 50-100% more water. We propose that this difference is an inherent property of type II collagen and that it may be significant for the function of type II collagen in tissues that dissipate compressive forces. The content of glycosylated hydroxylysine residues is the chemical variable likely to be modulating fibrillar hydration.

Adolescent↗
PubMed1980

Dynamics of myelin membrane contacts.

The crystal-like array of membranes in the nerve myelin sheath can be swollen or compacted by controlled changes in the environment. Swelling results when the ionic strength is reduced; compaction occurs when the water activity is lowered or when the divalent cation concentration is raised. X-ray diffraction patterns from frozen specimens show that dehydrating treatments, which induce compaction, are cryoprotective. Electron micrographs of freeze-fractured specimens show that the compacted domains are continuous with normal period arrays. Intramembrane particles, which are uniformly distributed in untreated membranes, are laterally displaced from the compacted membrane layers into particle-enriched domains. The more particle-dense of the alternating rough-surfaced membrane fracture faces is the E-face. The smooth fracture faces of compacted myelin membranes and of purified myelin lipids look the same. Comparison of the electron density profiles indicates that some protein remains associated with the lipid polar groups in compacted arrays. The dynamics of structural changes in myelin provides information about the intermembrane forces that stabilize the regular arrangement.

Animals↗
PubMed1979

X-ray diffraction study of the kinetics of myelin lattice swelling. Effect of divalent cations.

The time-course of myelin lattice swelling and its reversal in dissected peripheral nerves was determined by small-angle x-ray diffraction using a position-sensitive proportional detector. The process of swelling can take place either in several hours or in less than 1 h depending on pretreatment of the nerves. The reversal of swelling was always completed within 1 h. The rapid structural transitions involved the disordering of membrane pairs as indicated by the transient appearance of a continuous intensity distribution similar to the membrane pair transform for myelin. The slow transitions involved the gradual replacement of the discrete reflections from the native structure by the reflections from the swollen lattice. Myelin membrane arrays reformed in normal Ringer's solution were much more stable to subsequent swelling than arrays reformed in Ca+2 and Mg+2-free Ringer's. These results suggest that these ions participate in stabilizing the interactions between the external surfaces of adjacent membrane pairs.

Animals↗
PubMed1979

Structural states of myelin observed by x-ray diffraction and freeze-fracture electron microscopy.

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.

Animals↗
PubMed1976

X-ray diffraction study of myelin structure in immature and mutant mice.

X-ray diffraction patterns were obtained from freshly dissected central and peripheral nerves of quaking, myelin synthesis deficiency (msd), and trembler mutants, as well as immature and adult normal mice. The patterns were compared with respect to strength of myelin diffraction, background scatter level, repeat period, and intensity and linewidth of Bragg reflections. The deficiency of myelin in optic nerves was found to be (in decreasing severity): quaking greater immature greater trembler approximately normal adult; and in sciatic nerves: trembler greater immature greater quaking greater msd approximately normal adult. Repeat periods about 3 A less than that for normal adult sciatic myelin were detected in corresponding nerves from immature, quaking, and trembler mice. In some trembler sciatic nerves a second phase having a 190-200 A period and accounting for about 60% of the total ordered myelin was also evident. Comparison of electron density profiles of membrane units calculated from the repeat periods and diffracted intensities for sciatic myelins indicate structural differences at the molecular level. The main findings are: (1) quaking myelin shows a significant elevation of density in the external protein-water layer between membrane bilayers; (2) the membrane bilayer of immature myelin is approximately equal to 2 A thinner than that for normal adult; (3) the membrane bilayer of the more compact phase in trembler myelin is approximately equal to 5 A thinner than for normal; and (4) the difference in repeat periods for the two phases present in some of the trembler nerves can be accounted for predominantly by distinct membrane bilayer separations at the external boundary.

Aging↗
PubMed1976

Morphological evidence of alteration in myelinstructure with maturation.

The periodicity of the myelin sheath in glutaraldehyde carbohydrazide (GACH) embedded tissues from three 10-day-old and two 43-day-old mice was investigated with the electron microscope. The mean repeat period and number of repeats per fiber was measured in 31-89 fibers in the trapezoid bodies, olfactory tract and sciatic nerves. The mean period of the myelin sheaths in both the peripheral and the central nervous systems of the 10-day-old mice (15.1 +/- 0.07 nm (S.E.M.) and 13.4 +/- 0.09 nm respectively) are smaller (rho less than 0.001) than those of the adult mice (15.9 +/- 0.08 nm and 14.7 +/- 0.06 nm respectively). These differences may be related to the known differences in chemical composition of immature and mature myelin.

Age Factors↗
PubMed1975

Myelin structure transformed by dimethylsulfoxide.

X-ray diffraction patterns from nerves bathed for about one-half hour in Ringer's solution containing dimethylsulfoxide at concentrations of 10% or more show reflections from a new, highly ordered structure with a repeat period about two-thirds that of native myelin. The proportion of myelin transformed is greater at higher concentrations, and above 40% the native pattern is no longer observed. Replacing the dimethylsulfoxide with Ringer's solution leads to the rapid reappearance of the native diffraction pattern. The effect of dimethylsulfoxide can be accounted for by the loss of water from the spaces between the membrane units without significant modification of the bilayer structure.

Animals↗