Arrangement of high molecular weight associated proteins on purified mammalian brain microtubules

The arrangement of the high molecular weight proteins associated with the walls of reconstituted mammalian brain microtubules has been investigated by electron microscopy of negatively stained preparations. The images are found to be consistent with an arrangement whereby the high molecular weight molecules are spaced 12 tubulin dimers apart, i.e., 960 A, along each protofilament of the microtubule, in agreement with the relative stoichiometry of tubulin and high molecular weight protein. Molecules on neighbouring protofilaments seem to be staggered so that they give rise to a helical superlattice, which can be superimposed on the underlying tubulin lattice. In micrographs of disintegrating tubules there is some indication of lateral interactions between neighbouring high molecular weight molecules. When the microtubules are depolymerized into a mixture of short spirals and rings, the high molecular weight proteins appear to remain attached to their respective protofilaments.     

Bile salts of the lungfishes Lepidosiren, Neoceratodus and Protopterus and those of the coelacanth Latimeria chalumnae Smith.

1. Bile salts of the coelacanth Latimeria chalumnae Smith (five specimens) and of the three living genera of lungfish (Dipnoi) were examined as completely as possible and compared. 2. The small 'bile acid' fractions include no more than traces of well-known C27 or C24 acids (free or conjugated) and the functioning bile salts must be regarded as alcohol sulphates. 3. Comparison of the alcohols suggest that (a) Latimeria stands biochemically outside the animal group which includes the Dipnoi, (b) Protopterus and Lepidosiren are more closely related to one another than either is to Neoceratodus, (c) all four primitive osteiychtheans have some amphibian affinities, (d) there are affinities between Latimeria and Dipnoi and ostariophysan families (especially Cyprinidae and Catostomidae) and (e) there are biochemical links between Dipnoi and lampreys.     

Repressor and int synthesis of bacteriophage lambda in the E. coli host mutant ER437.

Summary Analysis of phage infection of the host mutant ER437 by SDS polyacrylamide gel electrophoresis and autoradiography has revealed altered expression of repressor and integration function (Int). We show that in this host Int as well as repressor synthesis is not dependent upon the cIII gene product in the usual manner, nor is their synthesis turned off in the normal way.     

Antibodies to muscarinic acetylcholine receptors in myasthenia gravis

IgG obtained from patients with myasthenia gravis block the specific binding of the muscarinic antagonists (³H)-N-methyl-4-piperidyl benzilate (4NMPB) and (³H)-Quinuclidinyl benzilate to rat brain muscarinic acetylcholine receptors. IgG obtained from healthy controls have a much smaller effect. The inhibitory effect of the myasthenic IgG increases linearly with immunoglobulin concentration and has no effect on the affinity of the muscarinic receptors towards (³H)-4NMPB (K_D = 0.7 ± 0.1 nM). This implies that the inhibition is probably due to the blocking of some of the muscarinic receptors by the myasthenic IgG, and not due to alteration in affinity of all the receptors. it remains to be established whether the presence of antimuscarinic receptor activity in the serum of myasthenic patients is of importance in the pathophysiology and diagnosis of myasthenia gravis.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

Ultrastructural localization of bcl-2 protein.

Previous cell subfractionation studies have indicated that bcl-2 is an inner mitochondrial membrane protein. We have sought to determine the ultrastructural localization of bcl-2 protein in lymphoma and breast carcinoma cell lines and biopsy material known to overexpress bcl-2 using immunoelectron microscopy. To avoid the possibility of processing artifacts, samples were prepared by three different methods: progressive lowering of temperature, cryosectioning, and freeze-substitution. In all instances the labeling of bcl-2 protein was relatively weak but the distribution the same. In both lymphoma and breast carcinoma tissues, bcl-2 protein was detected on the periphery of mitochondria: little labeling of either the mitochondrial matrix or cristae could be detected. Labeling was also detected on the perinuclear membrane and throughout the cytoplasm, as also indicated by confocal microscopy. These data therefore indicate that bcl-2 protein can be detected at several intracellular sites and that at the likely functional destination, the mitochondria, there appears to be, contrary to expectations, a preferential association with the outer membrane.     

A multivariate method for detecting genetic linkage, with application to a pedigree with an adverse lipoprotein phenotype.

The robust or model-free method for detecting linkage developed by Haseman and Elston for data from sib pairs is extended to incorporate observations of multiple traits on each individual. A method is proposed that estimates the linear function that results in the strongest correlation between the squared pair differences in the trait measurements and identity by descent at a marker locus. The method is illustrated by the study of apolipoprotein and cholesterol levels in individuals from a large family that had many members diagnosed with coronary heart disease.     

Factors affecting the induction of secondary dormancy in lettuce

The relationship between the temperature at which germination of 50% of the seeds is inhibited in the light (GT₅₀ Light) and secondary dormancy was investigated in three cultivars of Lactuca sativa L. Seeds were incubated for varying periods under non-germinating conditions and subsequent germination in response to red light (R) was determined over a wide range of temperatures. Dark incubation at 32 C reduced the GT₅₀ Light of cv. New York but did not affect germination at temperatures below 24 C. Dark, 32 C incubation had no effect on the GT₅₀ Light of cv. Great Lakes. In cv. Grand Rapids, dark incubation at 15, 24, 32, or 35 C initially reduced the GT₅₀ Light. However, longer incubations induced a secondary dormancy, i.e., the seeds became unable to germinate at all temperatures in response to R given after the high temperature incubation. A single exposure to R at the beginning of a 32 C incubation slowed the induction of secondary dormancy. Repeated exposures to R prevented the induction of secondary dormancy, but did not prevent a decline in the GT₅₀ Light. GA₃ mimicked the effect of repeated R.     

HLA serum screening based on an heuristic solution of the set cover problem.

A computer program initially written by the Milwaukee Blood Bank has been modified to use a new algorithm for the assignment of HLA specificities to antisera. The assignment is based on the reactions of cells with known specificities. Specificities which are present only on cells which do not react are first ruled out. This step is followed by one or more steps in which the 'least reactive' specificities are ruled out. The rationale for the algorithm is discussed and an example is presented.