PROTEIN KINASE C AND TRANSMITTER RELEASE

1. Protein kinase C (PKC) is an important second messenger-activated enzyme. In noradrenergic nerves it appears to be tonically activated by diacylglycerol (DAG) to facilitate transmitter release and the steps in this involve activation of phospholipase C, generation of DAG and activation of PKC. It is suggested that the subsequent facilitation of transmitter release is due to the phosphorylation of proteins involved in the release process distal to Ca²⁺entry, presumably those involved in vesicle dynamics. 2. There are differences between central noradrenergic neurons and sympathetic nerves. In central neurons PKC appears to be tonically active and its inhibition results in a decrease in noradrenaline release under most, if not all, conditions. 3. In sympathetic nerves PKC inhibitors only decrease transmitter release during high-frequency stimulation and not during low-frequency stimulation. At high frequency there is a gradual increase in the effect of PKC inhibitors on transmitter release during the first 15 s of a stimulation train. It is suggested that this is due to a progressive rise in intracellular Ca²⁺ and a consequent activation of PKC. 4. Activation of PKC by phorbol esters produces a large enhancement in action potential-evoked noradrenaline release in both the central nervous system and in peripheral tissues. The structural requirements of the phorbol esters for maximal effect suggest that the phorbol esters must access the interior of the nerve terminal to activate PKC and the neural membrane acts as a barrier for highly lipophilic phorbol esters, thereby reducing their activity. Activation of PKC represents one of the most powerful ways to enhance transmitter release and may have therapeutic potential.     

Adherence of human polymorphonuclear leukocytes to endothelial monolayers: effects of temperature, divalent cations, and chemotactic factors on the strength of adherence measured with a new centrifugation assay

Polymorphonuclear leukocytes (PMNs) adhere to endothelial cells at sites of acute inflammation. To examine this phenomenon in vitro, we have developed a new assay to measure adherence of PMNs to cultured endothelial cells. Human PMNs were labeled with 111indium-oxine and incubated in microtiter wells with monolayers of either human umbilical vein or bovine aortic endothelial cells. Following incubation, the wells were sealed, inverted, and centrifuged at varying speeds. Results are expressed as the percentage of PMNs added initially that remained attached to the monolayers after being subjected to dislodgment forces (ie, relative centrifugal forces) ranging from 1 to 1,200 g. Adherence of PMNs to endothelial monolayers was temperature dependent, dependent on the concentration of extracellular Mg2+ (but not Ca2+), and enhanced significantly by the chemotactic peptides, N-formyl-methionyl-leucyl- phenylalanine (fMLP) and human C5a. It was found that fMLP and C5a not only increased the number of PMNs that adhered to endothelial cells, but also increased the strength of adherence.     

Deficient activity of alanyl‐tRNA synthetase underlies an autosomal recessive syndrome of progressive microcephaly,hypomyelination, and epileptic encephalopathy

Aminoacyl‐transfer RNA (tRNA) synthetases ligate amino acids to specific tRNAs and are essential for protein synthesis. Although alanyl‐tRNA synthetase (AARS) is a synthetase implicated in a wide range of neurological disorders from Charcot‐Marie‐Tooth disease to infantile epileptic encephalopathy, there have been limited data on their pathogenesis. Here, we report loss‐of‐function mutations in AARS in two siblings with progressive microcephaly with hypomyelination, intractable epilepsy, and spasticity. Whole‐exome sequencing identified that the affected individuals were compound heterozygous for mutations in AARS gene, c.2067dupC (p.Tyr690Leufs*3) and c.2738G>A (p.Gly913Asp). A lymphoblastoid cell line developed from one of the affected individuals showed a strong reduction in AARS abundance. The mutations decrease aminoacylation efficiency by 70%–90%. The p.Tyr690Leufs*3 mutation also abolished editing activity required for hydrolyzing misacylated tRNAs, thereby increasing errors during aminoacylation. Our study has extended potential mechanisms underlying AARS‐related disorders to include destabilization of the protein, aminoacylation dysfunction, and defective editing activity.     

Changes in cartilage and bone metabolism identified by serum markers in early osteoarthritis of the knee joint

Serum concentrations of two extracellular matrix molecules were determined over a 3 yr period in individuals with chronic knee pain to investigate whether sequential serum measurements of cartilage- and bone-derived molecular fragments reflect early stages of osteoarthritis (OA) of the knee joints. Thirty-eight individuals with chronic knee pain (> 3 months at inclusion) with or without radiographic evidence of knee joint OA at the 3 yr follow-up radiographic examination were studied. Serum concentrations of cartilage oligomeric matrix protein (COMP) and bone sialoprotein (BSP) increased significantly (P < 0.001) in the 23 individuals with radiographic OA at follow-up, while remaining unchanged in the 15 individuals with normal radiographs at follow-up. The baseline concentrations of the two variables did not differ between the groups. These findings suggest that pathological processes in cartilage and subchondral bone coincide in OA, and appear to be reflected by circulating levels of macromolecules released from cartilage and bone. Changes in serum levels of COMP and BSP are potential tools in studies of knee joint OA in subjects with chronic knee pain.      

Osteopontin: a new role for a familiar actor

Our understanding of the events that occur in cancer progression has been enhanced by the use of cell lines in vitro. Changes in gene expression, induction of signalling, and cell motility can all be investigated in this setting. However, other aspects of progression can be revealed only in vivo, especially the interactions of tumour cells with host cells and organ systems. In one such in vivo model, described by McAllister and colleagues, it proved possible to establish a novel function of an already well-characterised protein, osteopontin, adding to its attractiveness as a target in cancer therapy.     

Felbamate-Induced Headache

We prospectively investigated drug-induced headaches (HA) among 60 epileptic patients receiving felbamate (FBM). Twenty patients (33%) experienced HA. HA was pounding in 11 (55%), steady in 9 (45%), moderate or severe in 19 (95%), occurred at least once a week in all patients, and was relieved by nonnarcotic analgesics in 14 (70%). Mean duration on FBM before HA onset was 19 days. HA occurred with higher FBM doses and was relieved in 8 of 13 patients (62%) with FBM dose reduction. FBM was discontinued in most cases because of risks of anemia or hepatitis; not because of HA. Other side effects included insomnia (25%), gastrointestinal symptoms (27%), and agitation or restlessness (23%). HA is a common dose-related complication of FBM, occurs early after initiation of FBM treatment, and is relieved by dose reduction.     

Retractable-barb needle for breast lesion localization: use in 60 cases

A needle system with a retractable barb was developed for localization of nonpalpable breast lesions. The system can be repositioned without major adjustments and strongly anchors to breast tissue, eliminating failure due to dislodgment or transection of the localizing wire. Its use may necessitate fewer confirmatory radiographs, resulting in decreased radiation exposure. In addition, the needle can be retracted during surgery, to ease extraction of the specimen.