Ribosomes and polyribosomes are present in the squid giant axon: an immunocytochemical study.

Ribosomes and polyribosomes were detected by immuno-electron microscopy in the giant axon and small axons of the squid using a polyclonal antibody against rat brain ribosomes. The ribosomal fraction used as antigen was purified by ultracentrifugation on a sucrose density gradient and shown to contain ribosomal RNAs and native ribosomes. The polyclonal antibody raised in rabbits reacted with at least ten proteins on immunoblots of purified rat brain ribosomes as well as with a set of multiple ribosomal proteins prepared from the squid giant fiber lobe. Immunoreactions were performed on cryostat sections of the stellate nerve cut at a distance of more than 3 cm from the stellate ganglion, using pre-embedding techniques. Ribosomes and polyribosomes were identified within the giant axon and small axons using electron microscopic methods, following binding of peroxidase-conjugated anti-rabbit IgG secondary antibody. Polysomes were more frequently localized in peripheral axoplasm, including the cortical layer of the giant axon, and were generally associated with unidentified cytoskeletal filaments or with dense matrix material. The immunochemical demonstration of ribosomes and polyribosomes in the giant axon and small axons of the squid confirms similar observations in the squid and the goldfish obtained with the method of electron spectroscopic imaging, and strongly supports the view that a local system of protein synthesis is present in axons. The immunochemical method here described offers an alternative tool for the selective identification of ribosomes, and is likely to prove of value in the analyses of other axonal systems.

Local radiolabeling of the 68 kDa neurofilament protein in rat sciatic nerves.

Rat sciatic nerve segments, 4.5 cm distal to the dorsal root ganglion (DRG), were incubated in vivo with [35S]methionine. Fluorography of 2-D polyacrylamide gels of the nerve proteins demonstrated the labeling of the 68-kDa neurofilament protein, which was identified by immunoblotting. This experimental design excludes the dorsal root ganglion as the source of the radiolabeled neurofilament protein and suggests that this neuron-specific protein may be synthesized in axons.

Effects of gangliosides on the methionine uptake in crushed sciatic nerves of rats with alloxan diabetes.

Effect of gangliosides on the transmembrane transport of methionine in crushed sciatic nerves was studied in alloxan diabetic and control rats until the 4th day after nerve crushing. Methionine uptake rate decreases while time passes after injury in both control and diabetic rats, but the decrement is greater in diabetic rats than in controls. Ganglioside treatment causes an enhancement on the transmembrane transport in both diabetic and control animals, but the effect in time was greater in diabetic rats than in controls.

A high glucose environment improves survival of diabetic neurons in culture.

Dorsal root ganglia neurons from streptozotocin-induced diabetic and normal C57BL mice were cultured in serum-containing and serum-free media. The ratio of dead cells was higher in diabetic neurons than in controls in the early stages of culture. The effect of glucose concentration on survival in the culture medium was also measured for 1 week. Treatment with high glucose concentrations improved the survival of diabetic neurons, which was enhanced by duration of diabetes in the animal. These results indicate that exposure to hyperglycemia in vivo might adapt neurons to a high glucose environment in vitro.

An in vitro model to study diabetic neuropathy.

Adult dorsal root ganglion (DRG) neurons from diabetic and normal C57BL mice were cultured and compared for survival and neurite extension. Neurons from diabetic mice have improved their survival and neurite extension when the insulin concentration was increased but it was never as good as that of controls. The increase of glucose concentration in serum-free media to ten times higher than normal improved both survival and neurite extension of neurons from diabetic animals.

Nerve growth factor (NGF) restores depletions of calcitonin gene-related peptide and substance P in sensory neurons from diabetic mice in vitro.

Dorsal root ganglion neurons from streptozotocin (STZ)-induced diabetic, genetic diabetic and normal mice were cultured in serum-containing media with or without nerve growth factor (NGF). The immunocytochemical analysis carried out after 1 week in culture revealed that the ratios of neurons immunoreactive to calcitonin gene-related peptide (CGRP) in NGF-free medium in the STZ-diabetic mice (average 23.2%) were significantly lower than those in the normal mice (45.1%). The ratios of neurons immunoreactive to CGRP and substance P (SP) in the NGF-free medium were also lower in the genetic diabetic mice (23.6% and 21.8%) than those in the normal ones (40.7% and 34.2%). However, treatment with NGF restored these reduced immunoreactivities in the diabetic groups in a dose-dependent manner. These results show that NGF can be effective for the diabetes-induced depletion of CGRP and SP in sensory neurons, and suggest its possible role in the prevention and improvement of diabetic sensory neuropathy.

Removal of intact follicles from immature mice ovaries.

A method was devised with the purpose of preparing significant amounts of isolated intact follicles suitable for "in vitro" studies on the protein synthesis machinery of mammalian oocytes. Ovaries of immature mice(8-14-days old), free of annexes and surrounding fat are squashed gently on a wire screen placed over the rim of a centrifuge tube. This step is followed by washing the screen in a saline-gelatin solution which is centrifuged twice at low speed to separate intact follicles from smaller ovarian components. At the mentioned ages about 170-190 follicles of 80 mu in diameter are found in the ovary. 20% of this population comes out intact from the screen which means that 30 ovaries may yield about 1,000 free follicles. The samples were examined under the light microscope to check their homogeneity and in the electron microscope to see whether squashing introduced changes in the cell cytoplasm. No significant changes were observed. This method is therefore considered useful as a previous step to studying, at the molecular level, the metabolic phenomena related to oocyte growth and further differentiation.

Large scale isolation of zonae pellucidae from ovarian oocytes of mice.

A large scale method for the isolation of zonae pellucidae (ZP) from mouse ovaries is described. It involves the squashing of the ovaries on a screen (70 X 70 M mesh) and sedimentation of the material in a discontinuous gradient. It is possible to obtain free ZP in useful quantities in about 1 h's work.

Protein-synthesizing machinery in the growing oocyte of the cyclic mouse. A quantitative electron microscopic study.

A quantitative ultrastructural evaluation of the oocyte ribosomal population was carried out during the oocyte growth, bearing in mind that this period of the mouse oogenesis displays the greatest activity of ribosomal RNA synthesis. At the onset of growth almost 3/4 of the oocyte ribosomes exist as singles, these become polysomal ribosomes as growth progresses. At the same time the number of ribosomes increases. Once the major growth period has elapsed, the number of ribosomes starts to decrease just when lattice-like structures exhibiting a periodic organization begin to accumulate in the oocyte cytoplasmic matrix. Evidence, like the particulate organization of these lattices, the size of their particles, its digestion by RNase, and the time of the lattice appearance, together with data reported by several authors, allows one to suggest that near the end of the oocyte growth a great part of the ribosomes are stored in the lattices to be used during early development.

Calcium efflux from platelet vesicles of the dense tubular system. Analysis of the possible contribution of the Ca2+ pump.

The ATP dependent Ca2+ uptake of platelet vesicles was inhibited by the two hydrophobic drugs trifluoperazine (TFP) and propranolol (PROP). Inhibition was significantly lowered when Pi was used instead of oxalate as a precipitant agent. When the ATPase ligands substrate (Mg2+ and Pi) were absent of the efflux medium, a slow release of Ca2+ which did not couple with ATP synthesis (passive Ca2+ efflux) was observed. Both, TFP and PROP enhanced the passive Ca2+ efflux. This enhanced efflux was partially inhibited only when Mg2+ and Pi were added together to the efflux reaction media, but it was not affected by spermidine, ruthenium red or thapsigargin (TG). The Ca2+ ionophores A23187 and ionomycin, also enhanced passive Ca2+ efflux. However, in this case, Ca2+ efflux was inhibited just by inclusion of Mg2+ to the medium. Ca2+ efflux promoted by Triton X-100 was not affected by either Mg2+ or Pi, included together or separately into the efflux medium. The ATP <==> Pi measured in the presence of Triton X-100 and millimolar Ca2+ concentrations was inhibited by both TFP and PROP, but not by Ca2+ ionophores up to 4 microM. The data suggest that the observed enhancement of passive Ca2+ efflux promoted by TFP and PROP could be attributed to a direct effect of these drugs over the platelet Ca2+ pump isoforms (Sarco Endoplasmic Reticulum Calcium ATPase, SERCA2b and SERCA3) themselves, as it was reported for the sarcoplasmic reticulum Ca2+ ATPase (SERCA1).

Development of the innervation of the urogenital complex in the fetal mouse.

The developing urogenital complex of the fetal mouse was studied by means of silver impregnation and electron microscopy. These studies showed that: 1) the mesonephric field is innervated during prenatal stages (Wolffian nerve); 2) nerve penetration precedes the differentiation of the gonads and related ducts; and 3) the Wolffian nerve arises during the earliest stages from the first pair of abdominal rami communicantes. The identity between the fetal Wolffian nerve and the nerve of the suspensory ligament (higher pathway) of the adult is discussed.

Ca(2+) dynamics in synaptosomes isolated from the squid optic lobe.

Synaptosomes from the optic lobes of squid (Loligo forbesi) were prepared by homogenization and allowed to settle onto glass coverslips. Synaptosomes were loaded with Ca(2+) sensitive dyes (Fura-2 AM, Calcium Green-1 AM and Calcium Green-5N AM), visualized by light microscopy and Ca(2+) sensitive fluorescence signals recorded and analyzed. With Fura-2, resting Ca(2+) was found to be 80 nM (n = 10, SEM 5.7). Addition of K(+) (30 mM), caffeine (3 mM) and thapsigargin (10 microM) evoked transient increases in cytoplasmic Ca(2+). Addition of BAPTA-AM (20 microM) decreased intrasynaptosomal free Ca(2+). Similar results were obtained with Calcium Green-1 AM but not with Calcium Green-5N AM. We conclude that synaptosomes from the squid optic lobe posses intact membranes and mechanisms to regulate intrasynaptosomal free [Ca(2+)], as well as caffeine sensitive Ca(2+) stores. The results of this study are discussed with respect to the role of Ca(2+) in presynaptic protein synthesis.

Neurofilament mRNAs are present and translated in the normal and severed sciatic nerve.

Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT-PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT-PCR demonstrated the presence of NF-L, NF-M and NF-H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury. In situ hybridization showed that NF-L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon.

Myosin Va is locally synthesized following nerve injury.

The presence of Myosin Va (an actin-based molecular motor) in the peripheral nervous system was examined and its subcellular distribution within the axons of the sciatic nerve was demonstrated via immunocytochemistry. Myosin Va (M-Va) in the nerve was detected by using SDS-PAGE and Western blot techniques with a polyclonal antibody specifically raised against the M-Va globular tail domain. In addition, purification of M-Va from the rat sciatic nerve prior to immunoblotting yielded a M-Va standard band. Likewise, optical immunocytochemical procedures revealed the presence of M-Va, particularly in the cortical axoplasmic territory, but also in the Schwann cell soma. The above experiments were carried out both on intact as well as on severed sciatic nerves with similar results. The proximal stumps of severed sciatic nerves (from 0 to 72 h after injury) were labelled in vivo with (35)S-methionine. SDS-PAGE autoradiography of the immunoabsorbed M-Va from the radiolabelled homogenized nerve tissue showed a significant increment of the radioactive intensity of M-Va heavy chain band through time. Moreover, a significant increment of transcripts coding for M-Va heavy chain was detected through time using RT-PCR after nerve injury and compared to intact nerves. This data suggest that M-Va is up-regulated in a time-dependent manner. The latter suggests a possible involvement of M-Va in nerve regeneration processes.