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M Tytell

Publications and source records attributed to M Tytell.

At least 55 records · Page 3Linked to original sources
PubMed1986

Protein modification by RNA-dependent posttranslational aminoacylation in synaptoplasm.

A soluble enzyme system that posttranslationally adds [3H]arginine to proteins in a ribosome-free preparation of guinea pig synaptoplasm is described. The reaction in synaptoplasm is inhibited by the addition of ribonuclease-A and puromycin, indicating tRNA dependence. A limited number of proteins in synaptoplasm (molecular weights of 20, 37, and 50 kilodaltons) were found to accept arginine. We suggest that RNA-dependent posttranslational amino acylation is used by the mammalian neuron for protein processing at the synaptic terminal.

Acylation↗
PubMed1984

Glial polypeptides transferred into the squid giant axon.

The proteins synthesized by the glial sheath of an isolated segment of squid giant axon and by the cell bodies of the giant axon in the isolated stellate ganglion were labeled by incubation in the presence of [3H]leucine. The axoplasm, which contained labeled proteins transferred from the glial sheath, was separated from the sheath by mechanical extrusion. The labeled proteins in the axoplasm, the empty sheath and the stellate ganglion were analyzed and compared by one- and two-dimensional polyacrylamide gel electrophoresis. Over 80 glial polypeptides were found to be selectively transferred into the axoplasm and many of these were distinct from stellate ganglion polypeptides which presumably could be supplied to the axon via axonal transport. Three of the more highly labeled transferred glial polypeptides (TGPs) were actin, a fodrin-like polypeptide and a polypeptide we have named traversin. Our observations, considered in the context of other reports, suggest that the squid axon receives a large number of polypeptides from its surrounding glia either by phagocytozing glial cell process that project into it or via cytoplasmic channels between adaxonal glia and the axon. These TGPs may help the axon survive unfavorable conditions.

Animals↗
PubMed1984

Axonal transport of a subclass of tau proteins: evidence for the regional differentiation of microtubules in neurons.

Tubulin, the major constituent of microtubules, is anterogradely transported within the axon as part of slow component a (SCa; 0.2-1.0 mm/day). This raises the possibility that the microtubule-associated proteins (MAPs) may be transported at the same rate. To examine this question, the high molecular weight and tau MAPs obtained from whole brain preparations of microtubules were compared with the proteins of SCa in guinea pig retinal ganglion cell axons by using phosphocellulose chromatography and one- and two-dimensional polyacrylamide gel electrophoresis. Only two of the tau proteins were found to be cotransported with axonal tubulin, although four tau and two high molecular weight MAPs were synthesized in the retina. This result suggests either that the retinal ganglion cell synthesizes only those two tau proteins or that it synthesizes several of the MAPs, but commits to axonal transport just two of the tau proteins. In either case, these observations are consistent with the transport of intact microtubules and demonstrate that axonal microtubules represent a distinct subset of brain microtubules. Such a distinction may be related to unique properties of the axonal cytoskeleton.

Animals↗
PubMed1984

Axonal tubulin and axonal microtubules: biochemical evidence for cold stability.

Nerve extracts containing tubulin labeled by axonal transport were analyzed by electrophoresis and differential extraction. We found that a substantial fraction of the tubulin in the axons of the retinal ganglion cell of guinea pigs is not solubilized by conventional methods for preparation of microtubules from whole brain. In two-dimensional polyacrylamide gel electrophoresis this cold-insoluble tubulin was biochemically distinct from tubulin obtained from whole brain microtubules prepared by cold cycling. Cleveland peptide maps also indicated some differences between the cold-extractable and cold-insoluble tubulins. The demonstration of cold-insoluble tubulin that is specifically axonal in origin permits consideration of the physiological role of cold-insoluble tubulin in a specific cellular structure. It appears likely that much of this material is in the form of cold-stable microtubules. We propose that the physiological role of cold-insoluble tubulin in the axon may be associated with the regulation of the axonal microtubule complexes in neurons.

Animals↗
PubMed1983

Phospholipid synthesis in the squid giant axon: incorporation of lipid precursors.

The squid giant axon and extruded axoplasm from the giant axon were used to study the capacity of axoplasm for phospholipid synthesis. Extruded axoplasm, suspended in chemically defined media, catalyzed the synthesis of phospholipids from all of the precursors tested. 32P-Labeled inorganic phosphate and gamma-labeled ATP were actively incorporated into phosphatidylinositol phosphate, while [2-3H]myo-inositol and L-[3H(G)]serine were actively incorporated into phosphatidylinositol and phosphatidylserine, respectively. Though less well utilized. [2-3H]glycerol was incorporated into phosphatidic acid, phosphatidylinositol, and triglyceride, and methyl-3H]choline and [1-3H]ethanolamine were incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Isolated squid giant axons were incubated in artificial seawater containing the above precursors. The axoplasm was extruded following the incubations. Although most of the product lipids were recovered in the sheath (composed of cortical axoplasm, axolemma, and surrounding satellite cells), significant amounts (4-20%) were present in the extruded axoplasm. With tritiated choline and myo-inositol, the major labeled phospholipids found in both the extruded axoplasm and the sheath were phosphatidylcholine and phosphatidylinositol, respectively. With both glycerol and phosphate, phosphatidylethanolamine was a major labeled lipid in both axoplasm and sheath. These findings demonstrate that all classes of phospholipids are formed by endogenous synthetic enzymes in axoplasm. In addition, we feel that the different patterns of incorporation by intact axons and extruded axoplasm indicate that surrounding sheath cells contribute lipids to axoplasm. A comprehensive picture of axonal lipid metabolism should include axoplasmic synthesis and glial-axon transfer as pathways complementing the axonal transport of perikaryally formed lipids.

Animals↗
PubMed1981

Axonal transport: each major rate component reflects the movement of distinct macromolecular complexes.

The proteins of the three major rate components of axonal transport in guinea pig retinal ganglion cells were analyzed by one- and two-dimensional gel electrophoresis. Each rate component consisted of a different set of proteins that remained associated with each other during transport. This suggests that each rate component represents a distinct macromolecular complex and that these complexes may be definable organelles such as microtubules, microfilaments, and smooth endoplasmic reticulum. Thus, the transport of radiolabeled proteins in the axon reflects the movement of complete subcellular rather than the movement of individual proteins.

Animals↗
PubMed1981

Axonal transport of calmodulin: a physiologic approach to identification of long-term associations between proteins.

Calmodulin is a soluble, heat-stable protein which has been shown to modulate both membrane-bound and soluble enzymes, but relatively little has been known about the in vivo associations of calmodulin. A 17,000-dalton heat-stable protein was found to move in axonal transport in the guinea pig visual system with the proteins of slow component b (SCb; 2 mm/d) along with actin and the bulk of the soluble proteins of the axon. Co-electrophoresis of purified calmodulin and radioactively labeled SCb proteins in two dimensional polyacrylamide gel electrophoresis (PAGE) demonstrated the identity of the heat-stable SCb protein and calmodulin on the basis of pI, molecular weight, and anomalous migration in the presence of Ca2+-chelating agents. No proteins co-migrating with calmodulin in two-dimensional PAGE could be detected among the proteins of slow component a (SCa; 0.3 mm/d, microtubules and neurofilaments) or fast component (FC; 250 mm/d, membrane-associated proteins). We conclude that calmodulin is transported solely as part of the SCb complex of proteins, the axoplasmic matrix. Calmodulin moves in axonal transport independent of the movements of microtubules (SCa) and membranes (FC), which suggests that the interactions of calmodulin with these structures may represent a transient interaction between groups of proteins moving in axonal transport at different rates. Axonal transport has been shown to be an effective tool for the demonstration of long-term in vivo protein associations.

Actins↗
PubMed1980

Properties of LHRH release from a hypothalamic synaptosomal fraction of estrogen-primed ovariectomized rats.

The release of luteinizing hormone-releasing hormone (LHRH) from mitochondrial-synaptosomal fractions (P2) of basomedial hypothalamus was examined under various conditions. Less than 3% of the LHRH in P2 suspensions was released under control conditions while the addition of 60 mM KCl or NaCl effected an 8-fold increase in LHRH as measured by radioimmunoassay. Equiosmolar sucrose effected only a 1.8-fold increase in LHRH release. The stimulatory effects of both Na+ and K+ were significantly inhibited by Mn2+ or La3+. Two forms of released LHRH were observed, one soluble and the other particulate. Soluble LHRH release was effected by hypertonic sucrose or 60 mM KCl and was not inhibited by Ca2+ antagonists. The release of particulate LHRH was unaffected by hpertonic sucrose, was stimulated 10-fold by 60 mM KCl, and was abolished with Ca2+ antagonists. These results suggest that the released soluble LHRH results from nonspecific leakage while the release of particulate LHRH reflects a Ca2+-dependent secretory process.

Animals↗
PubMed1980

Effects of estrogen and progesterone on LHRH release from a hypothalamic synaptosomal fraction of ovariectomized rats.

Mitochondrial-synaptosomal fractions (P2) from the basomedial hypothalamus of adult ovariectomized rats were employed to study the effects of estradiol benzoate (EB) and progesterone (P) on the release of luteinizing hormone-releasing hormone (LHRH). Treatment of ovariectomized rats with 5 or 50 micrograms of EB significantly reduced the total LHRH released from P2 under both control and K+-stimulated conditions. Furthermore, rats given 50 microgram EB demonstrated cyclic variations in the magnitude of inhibition of LHRH release. Comparison of LHRH release from P2 of rats sacrificed at 0900 hr with that from those sacrificed at 1500 hr revealed a small but persistent facilitation of LHRH release each afternoon. This facilitation, associated with an increase in the soluble component of LHRH release, was absent when rats also received 5 mg of P. No effects on LHRH release were observed when 17 beta-estradiol alone or when P was applied to P2 in vitro. The data show that the regulatory effects of estrogen and progesterone given in vivo on LHRH secretion can be observed in a subcellular fraction of the hypothalamus containing neurosecretory cell terminals.

Animals↗
PubMed1980

Estrogen and the subcellular distribution of luteinizing hormone releasing hormone: rate sedimentation studies.

Rate sedimentation of the 900 X G supernatants (S1) of hypothalamic homogenates from untreated male rats or ovariectomized rats with or without 5 micrograms estradiol benzoate (EB) revealed two populations of LHRH particles: a minor, slowly sedimenting one (peak 1) and a major, more rapidly sedimenting one (peak 2). Some LHRH-containing material also sedimented to the bottom of the gradient. The ovariectomized rats displayed more heterogeneity of particulate LHRH than did the male rats. Furthermore, the administration of EB to ovariectomized rats altered the relative sedimentation pattern of LHRH. In ovariectomized rats, hypotonic shock of S1 prior to rate sedimentation eliminated peak 2 and post-peak 2 LHRH and increased free LHRH at the top of the gradient. Peak 1 LHRH was still present and was elevated after EB treatment. Also, EB treatment lowered the free LHRH at the top of the gradient. These data demonstrate that the administration of EB to an ovariectomized rat alters the subcellular distribution of LHRH.

Animals↗
PubMed1980

Fast axonal transport in auditory neurons of the guinea pig: a rapidly turned-over glycoprotein.

Proteins of the fast component of axonal transport were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis in the guinea pig spiral ganglion, which has its cell bodies in the cochlea and its axons in the eighth cranial nerve projecting to the ipsilateral cochlear nucleus. We found that we could easily identify the proteins of the fast component even though these axons are only about 3 mm long because the cochlea minimized diffusion of labeled precursor into the cochlear nucleus. The composition of the fast component of the spiral ganglion cells was similar, but not identical, to the fast component of guinea pig retinal ganglion cells. One difference was the predominance in the spiral ganglion cell fast component of a rapidly turned-over glycoprotein (RTGP) with a molecular weight of 110,000-140,000 and an isoelectric point of 5.0 RTGP accumulated in the cochlear nucleus for just the first 3 hr after the application of the labeled precursor and then rapidly disappeared, whereas the other major fast component polypeptides continued to accumulate for 12-24 hr. RTGP was also tentatively identified in the fast component of retinal ganglion cells, but was not as prominently labeled relative to the other fast-component proteins in those cells. The rapid disappearance of RTGP from spiral ganglion cell terminals in the cochlear nucleus may be a result of secretion, perhaps as part of a synaptic vesicle, or retrograde transport as a feedback signal. The difference in the relative amounts of RTGP found in spiral ganglion and retinal ganglion cell terminals may reflect differences in the fundamental properties of the two groups of neurons.

Animals↗
PubMed1980

Effects of estrogen on luteinizing hormone releasing hormone in a hypothalamic synaptosomal fraction from the ovariectomized rat.

Previous investigations have shown that hypothalamic luteinizing hormone releasing hormone (LHRH) is localized within a subcellular fraction eqivalent or similar to the synaptosome or nerve ending particle. In the present study, the ovariectomized adult rat was employed to investigate the effects of estrogen on the distribution of LHRH among various subcellular fractions of the hypothalamus. In addition, the liberation of LHRH from a synaptosomal fraction by repeated freeze-thaw cycles was examined before and after in vivo estrogen treatment. Following a single injection of 5 micrograms of estradiol benzoate (EB), the LHRH concentration of the synaptosomal fraction rose as much as 100% in 27 h. Increasing the dose of EB to 50 micrograms shortened the latency of the rise and prolonged its duration, but did not alter its magnitude compared to the lower dose. Furthermore, the LHRH in that synaptosomal fraction was significantly more resistant to solubilization by repetitive freezing and thawing than the LHRH in a synaptosomal fraction from rats which had not received EB. These observations led us to conclude that estrogens can alter the physical properties of the hypothalamic LHRH-containing particles or the storage form of the peptide within those particles so that it is less easily released. This phenomenon may represent one aspect of the regulatory effects of estrogens on hypothalamic LHRH secretion.

Animals↗
PubMed1972

Fever: reciprocal shift in brain sodium to calcium ratio as the set-point temperature rises.

A bacterial pyrogen acts on the brain by disturbing the natural balance between two essential cations in the cerebral region involved in thermoregulation. After typhoid vaccine is administered to the unanesthetized cat, (45)Ca(2+) efflux into the third cerebral ventricle increases while (22)Na(+) is retained in hypothalamic tissue at the same time that the set-point temperature begins to rise. The subsequent rates of (22)Na(+) and (45)Ca(2+) efflux parallel the course of the bacterial fever but in a reciprocal fashion. This supports the theory that a change in the set-point temperature is determined by an alteration in the inherent ratio of Na(+) to Ca(2+) ions in the hypothalamus.

Animals↗
PubMedDate not supplied

Immunohistochemical assessment of constitutive and inducible heat-shock protein 70 and ubiquitin in human cerebellum and caudate nucleus.

The distributions of constitutive and inducible 70-kDa heatshock proteins (Hsc70 and Hsp70, respectively) and ubiquitin (Ub) were investigated in autopsy specimens from 24 adult human brains. The objectives were to verify that the milder fixation and celloidin embedding applied to those specimens preserved protein immunoreactivity in the tissue sections, even with extended intervals between death and fixation, and to determine the typical pattern of distribution of the proteins in aged human cerebellum and caudate nucleus. To achieve these objectives, the patterns of immunoreactivity in human specimens were compared with those in normal rat brain after three methods of immersion fixation: 1. 1% Formalin; 2. 10% Formalin; 3. Methacarn (a modification of Carnoy's solution). Additionally, some rats were left refrigerated, but unfixed for up to 24 h to mimic the postmortem interval that commonly occurs prior to fixation of human autopsy material. Tissues were embedded in celloidin, sectioned at 100 microns, and the celloidin dissolved to permit immunostaining. Immunoreactivity for all antigens was greatly diminished in the rat brain by fixation in 10% formalin compared to 1% formalin or methacarn. Rat and human brain tissues fixed in the latter two solutions showed similar patterns of low levels of Hsp70 immunostaining in gray matter and other areas where neuronal somata were concentrated, whereas Hsc70 immunostaining was much greater in those same areas. Little Hsc70 or Hsp70 immunoreactivity was detected in the white matter from either source, but immunoblots of human gray and white matter suggested that white matter contained more Hsc70 and Hsp70 than apparent by tissue section immunoreactivity. Ubiquitin immunostaining in rat and human brain showed the same high levels as Hsc70 in gray matter, but unlike Hsc70, was also visible in white matter. These patterns remained the same in rat brains even if fixation was delayed for 24 h. In three human brain specimens, elevated Hsc70 staining, but not Hsp70 or Ub, was found in a ring pattern similar to that described as the ischemic penumbra in experimentally induced brain ischemia. These results indicated that dilute formalin preserved Hsc/Hsp70 and Ub antigenicity well, and that the proteins had similar distributions in human and rat brains, despite the extended postmortem delay in fixation of the former. They also suggested that evidence of premortem, localized cellular metabolic stress may be preserved in the postmortem human brain by an alteration in the typical distribution of Hsc70.

Adult↗