The Cdk5-p35 kinase associates with the Golgi apparatus and regulates membrane traffic.

We show here that an active Cdk5-p35 kinase is present in Golgi membranes, where it associates with a detergent-insoluble fraction containing actin. In addition, Cdk5-p35-dependent phosphorylation of alpha-PAK immunoreactive protein species was detected in Golgi membranes, as well as an interaction with the small GTPase, Cdc42. Moreover, antisense oligonucleotide suppression of Cdk5 or p35 in young cultured neurons, as well as inhibition of Cdk5 activity with olomoucine, blocks the formation of membrane vesicles from the Golgi apparatus. Taken together, these results show a novel subcellular localization of this kinase and suggest a role for Cdk5-p35 in membrane traffic during neuronal process outgrowth.

Long-term rat survival after malignant brain tumor regression by retroviral gene therapy.

Total regression of malignant brain tumors was observed in Wistar rats after retrovirus-mediated gene therapy. Tumors were induced by inoculation of C6 rat glioblastoma cells to a specific location in the rat brain and the tumors that developed were visualized by magnetic resonance imaging (MR). Retroviral vectors were constructed from a defective murine retrovirus to which the thymidine kinase (tk 1) gene from herpes simplex was added (HSV1tk). The vectors produced therapeutic viruses upon their introduction into retrovirus packaging cells. Delivery of the producer cells to the tumor mass and subsequent antiherpetic treatment eradicated the tumors completely, as observed using MRI. Some of the treated animals have been followed for over 8 months and show no signs of recurrence.

Role of phosphorylated MAPlB in neuritogenesis.

The distribution of microtubule-associated protein lB (MAPlB) phosphorylated by either proline-directed protein kinase (PDPK) or casein kinase II (CK II) in neuroblastoma cells and hippocampal neurons has been studied by immunofluorescence using specific antibodies to distinct phosphorylation-sensitive epitopes. A proximo-distal gradient of increasing PDPK-catalyzed phosphorylation of MAPlB is superimposed on a proximo distal gradient of decreasing CK II-catalyzed MAPlB phosphorylation within growing axon-like neurites. Additionally, CK II-phosphorylated MAPlB is present in cell bodies and dendrites where no PDPK-phosphorylated MAPlB is observed. These results suggest distinct roles for both types of modifications of MAPlB in developing neurons.

Effect of bilirubin on the membrane potential of rat brain synaptosomes.

The effect of the neurotoxic pigment bilirubin on the membrane potential of rat brain synaptosomes was studied by using the tetraphenylphosphonium ion (TTP+) technique. Bilirubin induces a rapid depolarization of synaptosomes, as reflected by an efflux of previously accumulated [3H]TTP+. This phenomenon persisted when the membrane potential across either the plasma membrane of the synaptosome or the inner membrane of the entrapped mitochondria was selectively depressed, thus indicating that both components of the synaptosomal membrane potential were affected by bilirubin. Bovine serum albumin, used at a albumin/bilirubin molar ratio of 1:1, had the capacity to completely prevent and reverse the effect of bilirubin. This fact demonstrates that the bilirubin-induced TPP+ release from synaptosomes is a reversible process that requires the presence of bilirubin interacting with the synaptosomal membranes. These results, together with the inhibition by bilirubin of [3H]TPP+ and [2-14C]acetate uptake by synaptosomal plasma membrane vesicles isolated from rat brain, suggest that bilirubin depresses the membrane potential across the synaptosomal plasma membrane by a mechanism involving alterations in ion permeability. This effect could be of relevance in the pathogenesis of bilirubin encephalopathy.

Bovine chromaffin granules: immunological studies with antisera against neuropeptide Y, [Met]enkephalin and bombesin.

Antisera against neuropeptide Y, [Met]enkephalin and bombesin were used for characterizing the immunoreactive material in subcellular fractions of bovine adrenal medulla. Neuropeptide Y was identified by high performance liquid chromatography and by immunoblotting. Subcellular fractionation established that neuropeptide Y is present in chromaffin granules. During stimulation of the adrenal it is released concomitantly with catecholamines. The soluble proteins of chromaffin granules contain 1.9 micrograms neuropeptide Y/mg protein which gives 429 copies of neuropeptide Y for a single granule. In two-dimensional immunoblots two peptides of the same molecular size, but with differing pI (6.4 and 7.3) react with the antiserum against neuropeptide Y. There was no evidence for the presence of a larger neuropeptide Y precursor in chromaffin granules. On the other hand, larger enkephalin-containing peptides could be detected by immunoblotting. The subcellular distribution of these enkephalin precursors differed. The larger peptides (23.3 and 18.2 kD) were more concentrated in lighter granules when compared to the smaller precursors (12.6 and 8.6 kD) which is consistent with proteolytic processing of these peptides during granule maturation. An antiserum against bombesin reacts in immunoblots with the chromogranin B family. This study further illustrates that chromaffin granules contain a complex mixture of neuropeptide-immunoreactive material. The combination of immunoblotting with subcellular fractionation appears as a useful tool to characterize these peptides and their precursors.

Developmental studies on the uptake of tyrosine by synaptosomes and plasma membrane vesicles derived from rat brain. Effect of thyroid hormones.

The uptake of L-tyrosine at various stages of development was examined in synaptosomes and in plasma membrane vesicles derived from rat brain. The total uptake has two components, Na+-dependent and Na+-independent, respectively. The Na+-dependent component of the transport system appears around the 5th postnatal day and increases with age. The affinity of the transport system for tyrosine does not vary substantially during development. The Vmax increases more than six-fold between day 15 and adulthood. Plasma membrane vesicles derived from T3-treated rats accumulate more tyrosine than those obtained from the control animals. The results support the view that thyroid hormones during development promote the establishment of the systems implicated in neurotransmission in the developing nervous system.

Ontogenetic studies on tryptophan transport into plasma membrane vesicles derived from rat brain synaptosomes: effect of thyroid hormones.

The uptake of tryptophan at various stages of development was examined in plasma membrane vesicles derived from rat brain. The total uptake has two components Na+-dependent and Na+-independent respectively. The Na+-dependent component of the transport system appears around the 5th postnatal day and increases with the age. The Km value of the system does not vary during development. The Vmax increases five-fold between 14 and 35 day of postnatal life. Plasma membrane vesicles derived from T3-treated rats are able to accumulate nearly three-fold more tryptophan than nontreated rats. The results support the idea that thyroid hormones at the earlier stages of life, promote the establishment of neurotransmission in the developing nervous system.

Effect of postnatal anoxia on bilirubin levels in rat brain.

The effects of a period of anoxia 18-24 h after birth on bilirubin levels in rat brain were investigated during anoxia, recovery, and development. Postnatal anoxia induces a significant, temporary increase (up to 200% with respect to control values) in newborn rat brain bilirubin levels during anoxia and short-term recovery. Pretreatment of the newborn rats with a single dose of the drug sulfixosazole markedly enhances bilirubin accumulation in the brain of the anoxic rats. A second rise in brain bilirubin concentration is detected in a group of the newborn rats 3-6 days after oxygen deprivation. Autoradiographic localization of radiolabeled bilirubin following in vivo experiments suggests that this substance is preferentially accumulated in some areas of the newborn rat brain as a consequence of postnatal anoxia, and indicates, together with the effect of sulfixosazole, that as a result of anoxia, a displacement of unbound bilirubin from blood to the nervous tissue occurs. Our results confirm the importance of anoxia as a risk factor for the development of bilirubin-induced encephalopathy. The possible relevance of intracerebral hemorrhages caused by perinatal asphyxia producing delayed bilirubin accumulation in the newborn rat brain is suggested.

Effect of thyroid hormones on the malic enzyme activity in rat brain during development.

Changes in the development pattern of cytoplasmic and mitochondrial malate dehydrogenase (decarboxylating; NADP+) activity in the brain of hypothyroid rats and the effect of triiodothyronine on the enzyme activity have been investigated. Hypothyroid rats showed lower malate dehydrogenase activity than controls during the suckling period. Results suggest that thyroid hormones promote the development of malic enzyme in the brain.

Effect of thyroid hormones on the 3-oxo acid CoA-transferase activity in rat brain during development.

The changes in the developmental pattern of 3-oxo acid CoA-transferase activity in the brain of hypothyroid rats and the effect of triiodothyronine on this enzyme activity have been investigated. Hypothyroid rats showed lower activity than controls during the suckling period. However, higher enzyme levels were found in treated rats after weaning in contrast to control animals. The results suggest that thyroid hormones promote the development of enzymes of ketone-body metabolism in the brain.