New frontiers in the study of memory mechanisms

We review recent work on three major lines of memory research: a) the possible role of the protein kinase M-zeta (PKMzeta) in memory persistence; b) the processes of “synaptic tagging and capture” in memory formation; c) the modulation of extinction learning, widely used in the psychotherapy of fear memories under the name of “exposure therapy”. PKMzeta is a form of protein kinase C (PKC) that apparently remains stimulated for months after the consolidation of a given memory. Synaptic tagging is a mechanism whereby the weak activation of one synapse can tag it with a protein so other synapses in the same cell can reactivate it by producing other proteins that bind to the tag. Extinction, once mistakenly labeled as a form of forgetting, is by itself a form of learning; through it animals can learn to inhibit a response. We now know it can be modulated by neurotransmitters or by synaptic tagging, which should enable better control of its clinical use.

Estresse oxidativo: revisão da sinalização metabólica no diabetes tipo 1 / Oxidative stress: a review on metabolic signaling in type 1 diabetes

O diabetes melito e suas complicações apresentam origem multifatorial. Mecanismos bioquímicos e patológicos estão associados com hiperglicemia crônica no diabetes e o aumento do estresse oxidativo tem sido postulado com papel central nestas desordens. Evidências sugerem que a lesão celular oxidativa causada pelos radicais livres contribuem para o desenvolvimento das complicações no diabetes tipo 1 (DM1) e a diminuição das defesas antioxidantes (enzimáticas e não-enzimáticas) parecem correlacionar-se com a gravidade das alterações patológicas no DM1. Nesta revisão, relata-se como o estresse oxidativo pode exercer efeitos deletérios no diabetes e são apresentadas as opções terapêuticas em estudo para modulação da injúria vascular.

Diabetic complications appear to be multifactorial process. The biochemical and pathological mechanisms are associated with chronic hyperglycemia of diabetes and the increased oxidative stress which has been postulated to play a central role in these disorders. Accumulating evidence suggests that oxidative cell injury caused by free radicals contributes to the development of type 1 diabetes (DM1) complications and decreased efficiency of antioxidant defenses (both enzymatic and nonenzymatic) seems to correlate with the severity of pathological tissue changes in DM1. In this review, we report as oxidative stress may exert deleterious effects in diabetes, as well as address current strategies in study to down-regulating vascular injury.

Efeitos da fosforilação de proteínas em Leishmania L. amazonensis / Effects of protein phosphorylation in Leshmania L. amazonensis

(...) Com o objetivo de abordar tais vias parasito, estudamos bioquimicamente e citoquimicamente a atividade fosfatase ácida. Parasitos tratados com os três inibidores po 1h e 24h apresetaram atividade fosfatase ácida secretada significativametne dimunuída. com a finalidade de estudar as vias de sinalização do parasito na interação com a célula hospedeira, promastigotas pré-tratados com os antagonistas foram incubados com macrófagos peritoneais. Observamos que estaurosporina 1μM inibiu, de forma significativa, a internalização e a sobrevivência intracelular dos parasitos. Nossos dados sugerem que inibidores de proteína cinases podem exercer efeitos na morfologia, infectividade e proliferação de Leishmania, bloqueando o ciclo celular desses parasitos.

Effect of cigarette smoke extract on the role of protein kinase C in the proliferation of passively sensitized human airway smooth muscle cells / 华中科技大学学报（医学）（英德文版）

To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A1, A2 and A3 and the group B was sub-divided into the group of B1, B2, B3, B4 and B5. No other agents were added to the group A1 and B1. The cells of group A2 and B2 were stimulated with 5% CSE for 24 h. HASMCs from group A3 and B3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5%) for 24 h. PKC inhibitor Ro-31-8220 (5 micromol/L) was added to the HASMCs of group B4 for 24 h. The cells from group B5 were stimulated with Ro-31-8220 (5 micromol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-a in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-alpha mRNA and the A value of PKC-alpha protein in HASMCs from group B1, B2 and B3 were significantly increased compared to those of group A1, A2 and A3 correspondingly and respectively (P< 0.01). The proliferation of HASMCs of group A2 and B2 stimulated with CSE and group A3 and B3 stimulated with CSE and PMA were also significantly enhanced when group A1, A2 and A3 and group B1, B2 and B3 compared to each other (P<0.05, P<0.01, respectively). The percentage of S phase, absorbency (A) value, 3H-TdR incorporation rate, the ratios of A value of PKC-alpha mRNA and the A value of PKC-alpha protein in HASMCs from group B4 treated with Ro-31-8220 and group B5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B1 and B2 correspondingly and respectively (P<0.05, P<0.01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.

Calcium- and zinc-binding proteins in intracellular transport

The complex mechanism of intracellular transport is regulated by free calcium in different manners. Calcium binding proteins regulate several aspects of the vesicle fusion mechanism mediated by NSF (N-ethylmaleimide sensitive fusion factor). At least in some regulated exocytosis, calcium-binding proteins are the trigger for fusion downstream of NSF, Still, calcium-binding proteins, such as annexins, may be part of a different fusion mechanism mediating some specific transport steps or working in parallel to the NSF-dependent fusion process. Calcium is not the only ion necessary for the function of factors involved in vesicular transport. A zinc requirement has been also proposed. One of the zinc-dependent factors is probably a protein with a cysteine-rich region that coordinates zinc and binds phorbol esters. Although protein kinase C is the more prominent family of proteins carrying this domain, the factor necessary for transport does not appear to function as a kinase.

Mechanism of formation of post Golgi vesicles from TGN membranes: Arf-dependent coat assembly and PKC-regulated vesicle scission

We have developed an experimental system that utilizes purified Golgi fractions obtained from virus infected infected MDCK cells to reproduce in vitro the process of vesicle generation in the trans Golgi network, an important site for the sorting of proteins addressed to the plasma membrane, secretory vesicles, or lysosomes. Using an integrated biochemical and electron microscopic approach, we have shown that the formation of post Golgi vesicles carrying proteins destined to both plasma membrane domains of epithelial cells requires the activation of an ArF-like GTP-binding protein that serves to promote the assembly of the protein coat necessary to deform the donor membrane and generate a vesicle. The formation of the post Golgi vesicles also requires the participation of a Golgi membrane-associated Protein Kinase C, but not its phosphorylating activity. Other authors have shown that this is also the case for the PKC activation of the enzyme phospholipase D, which generates phosphatidic acid from phosphatidyl choline and may be involved in remodeling of membranes. We have been able to dissect the process of post Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and a subsequent one of vesicle scission. The first stage can occur at 20 degrees C and requires the activation of the Arf protein necessary for coat assembly. The second stage does not require nucleotides or an energy supply, but requires cytosolic proteins, and in particular, an NEM sensitive membrane scission promoting activity that operates only at a higher temperature of incubation. Because various PKC inhibitors blocked vesicle scission without preventing bud formation, we propose that the PKC is required for the activation of a PLD in the TGN, which leads to remodeling of the donor membrane and the severing of connections between the emerging vesicles and the membranes.

Slow cluster formation of purified human or rhesus T cells requires protein kinasse C and LFA-1

Ab: Homotropic T cell adhesion, as generally studied, consists of a rapid, transient binding process that is measured over a 15-120 min. period. Here we report a slow type of adhesion process occurring with human or rhesus T cells, purified from peripheral blood, that manifests itself by the formation of rounded, multi-layer clusters which may contain hundreds of cells. The maximal number and size of the clusters peak 1-2 days after the addition of phorbol ester, an absolute requirement. The number of clusters formed is proportional to phorbol ester concentration up to 1.25 ng/mL. Phorbol esters such as phorbol myristate acetate (PMA), phorbol dibutyrate (PDB), and 7-octylindolactam (OIL) induced optimal cluster formation at 1-13 ng/mL, levels slightly higher than that required to induce mitogenesis of purified T cells. Phorbol itself and the alpha-form of the ester were inactive. Both cluster formation and mitogenesis (stimulated by Con A or anti-CD3) are completely inhibited by staurosporin at 12.5 ng/mL. Even at 2.5 ng/mL, 74 percent of cluster formation was inhibited, which strongly implies a crucial role for protein kinase C. In the presence of accessory cells, T cell clusters were suppressed. Monoclonal Ab such as anti-CD3, mouse anti-CD3 followed by anti-mouse IgG, anti-CD4, anti-CD4A, anti-CD2, anti-CD8, and anti-CD45 did not induce cluster formation. None were inhibitory or stimulatory in the presence of PMA, except for anti-CD3 which enhanced cluster formation by 26 percent. However, anti-LFA-1 beta-chain (mouse monoclonal) completely blocked cluster formation over the range studied (63-1000 ng/mL) for both human and rhesus cells; rat anti-LFA-1 only blocked human cell adhesion. Anti LFA-1 only partially inhibited T cell mitogenesis. These results show that slow cluster formation shares the LFA-1 and phorbol ester requirements of the rapid adhesion of T cells requiring LFA-1 and ICAM-1. However, cluster occurs at a very low phorbol ester concentration, appears more sensitive to staurosporin inhibition, and is not stimulated via the TCR receptor like the rapid adhesion process. We hypothesize that certain neuronal processes, induced by phorbol ester, and which also show a similar protein kinase C activation time course, may share mechanisms in common with cluster formation

Phosphorylation of ribosomal protein S6 and its regulation during differentiation of human leukemic cells

We attempted to study the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the cascade of phosphorylation of ribosomal protein S6 during differentiation of leukemic cells (HL-60, THP-1, and RWLeu-4). Neither activation nor inhibition of colony stimulating factor-1 (CSF-1) receptor's PTK activity with CSF-1 or genistein respectively affected the phosphorylation of S6. However, vanadate which is a protein tyrosine phosphatase (PTP) inhibitor showed enhancement of S6 phosphorylation. Dimethylsulfoxide which does not affect either PTK or PKC demonstrated no change in S6 phosphorylation. PKC activation by acute 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment induced monocytic differentiation and S6 phosphorylation. Surprisingly, the more prominent phosphorylation of S6 protein was observed in PKC-depleted cells by prolonged TPA treatment. Our results suggest that PTK/PTP play a lesser role in S6 phosphorylation of HL-60 cells than PKC does. In addition, two different mechanisms seem to be involved in TPA-induced S6 phosphorylation during HL-60 differentiation: PKC activation by acute TPA treatment and PKC depletion which may lead to the synthesis of some endogenous protein responsible for the differentiation by chronic TPA treatment.