pH, EF-1alpha and the cytoskeleton

One of the unexpected cellular components found interacting with the cytoskeleton is elongation factor 1 alpha (EF-1alpha). How this interaction is regulated is not clear, but pH may be a potent regulator. Interestingly, pH also regulates the amount of protein translation occurring in many cell systems. In this paper, the authors suggest that sequestration of EF-1alpha in the cytoskeleton may play a key role in regulating the spatial distribution of macromolecular assembly in a way that is dependent on cytoplasmic pH.     

Opsonization with Antimyelin Antibody Increases the Uptake and Intracellular Metabolism of Myelin in Inflammatory Macrophages

In most demyelinating diseases, macrophages are believed to be active agents of myelin destruction. In experimental encephalomyelitis, these cells appear to strip off and ingest the myelin lamellae, and myelin debris has been observed within the cell body. We show here in vitro conditions in which rat peritoneal macrophages phagocytose and metabolize CNS myelin lipids. Purified rat myelin, prelabeled in vivo with [14C]acetate, was incubated with preimmune serum or rabbit antiserum to rat CNS myelin and added to macrophage monolayers. Myelin opsonized with antimyelin antibodies was more readily phagocytosed and metabolized by cultured macrophages than untreated myelin or that preincubated with preimmune serum. In the presence of macrophages, levels of myelin polar lipids and cholesterol decreased, whereas radioactive cholesterol ester and triglyceride accumulated. Up to five times as much radioactive cholesterol ester and about twice as much triglyceride accumulated in macrophage cultures containing antibody-treated myelin as in cultures fed preimmune serum-treated myelin or in those incubated with untreated myelin. Both the fatty acid and the cholesterol from cholesterol ester contained radioactive label; therefore, both were derived at least partly from the radioactive myelin lipid. Antiserum to myelin purified from peripheral nerve was almost as effective as that to CNS myelin in stimulating cholesterol metabolism, whereas antiserum to galactocerebroside was about 70% as active. Antiserum to basic protein had less effect, whereas antiserum to the myelin-associated glycoprotein and proteolipid protein was inactive. Of the polar lipids, ethanolamine phosphatide was most degraded in both the antiserum- and preimmune serum-treated myelin, with the diacyl form and plasmalogen form degraded about equally. These experiments indicate that myelin-specific antibodies in inflammatory CNS lesions may participate in and stimulate macrophage-mediated demyelination.     

The role of phospholipases from inflammatory macrophages in demyelination

Activated macrophages harvested from rat peritoneum were shown to contain phospholipase A₁, A₂ and lysophospholipase activities which were defined on a series of radiolabelled phospholipid substrates. During in vitro culture of these elicited macrophage populations, phospholipase enzymes were secreted into the culture medium. Radiolabelled myelin, prepared from young rats after intracerebral injection of¹⁴C acetate, was used as a substrate to analyze the susceptibility of central nervous system (CNS) myelin to attack by cell-associated and secreted macrophage enzymes. Homogenates of peritoneal macrophages degraded the myelin lipids at acid pH; phosphatidyl choline (PC) and ethanolamine phosphatide (EP) were both degraded with liberation of free fatty acid and small amounts of lysolipids. The ethanolamine lipids were most vulnerable; up to 20% of this fraction was degraded in six hours. Selected batches of macrophage culture supernatant similarly degraded the myelin EP at acid pH. These results suggest that phospholipase enzymes, released from activated macrophages in close proximity to the myelin sheath, may participate in primary demyelination in inflammatory CNS lesions.     

A Model of sodium channel-inactivation based upon the modulated blocker

An attempt is made to model sodium channel inactivation based upon real physical processes. The principle involved, which is supported by calculation and by direct appeal to experimental results, is that the gating dipole reversal or gating charge transfer that occurs when the channel is activated, markedly modulates the electrical properties of charged groups at the channel ends. Four examples of possible mechanisms that lead to channel inactivation are described. The simple four-state model that results is able to predict: (a) the steep voltage dependence of the equilibrium inactivation characteristic without the presence of any appreciable displacement current associated with inactivation; (b) the negative shift in membrane voltage of the equilibrium inactivation characteristic relative to the activation characteristic; (c) the bell-shaped dependence of inactivation time constant on membrane voltage; (d) the similarity of the membrane voltage dependence of the time constant of recovery from inactivation, to that of inactivation itself. A brief discussion of a model for sodium channel activation based upon the same physical principle is included.     

Sesquiterpenes in leaf pocket resins of Copaifera species

Four sesquiterpene leaf resin components were isolated and identified from Copaifera leaf resin. Additional GC and mass spectrometric evidence support the close similarity of Copaifera leaf pocket resin composition with that of the related genus, Hymenaea.     

STUDIES ON THE MECHANISM OF DEMYELINATION: TRIETHYL TIN-INDUCED DEMYELINATION

The metabolism of myelin undergoing breakdown as a result of edema induced by chronic administration of triethyl tin (TET) dissolved in the drinking water (10 mg/l.) was examined. The spinal cord showed more edema and loss of myelin than the brain. Uptake in vitro of [1-¹⁴C]acetate into myelin lipids of slices of brain or spinal cord from TET-treated rats was depressed until 4–5 weeks after the beginning of the regime, then rose to above normal levels. The uptake of [l-¹⁴C]leucine into myelin protein rose within several weeks of TET treatment to levels averaging over 300 per cent of normal and remained high even after the TET was removed. The high levels of [l-¹⁴C]leucine incorporation were inhibited by cycloheximide and were not explained by an increase in the size of the free amino acid pool. The three classes of myelin proteins, basic, proteolipid protein, and Wolfgram protein shared in the increased incorporation. Spinal cord myelin showed the greatest metabolic response, brain stem myelin less, and myelin from the forebrain was minimally affected by the TET treatment. Myelin prelabelled by intracisternal injection of [l-¹⁴C]acetate and [l-¹⁴C]leucine before the onset of TET administration showed faster turnover in myelin proteins in relation to the myelin lipids than the control in the most severely affected animals, but not in others less affected. A ‘floating fraction’ was observed floating on 10.5% (w/v) sucrose during the myelin purification. This fraction showed metabolic characteristics typical of myelin, and myelin-labelling studies at various stages of the animal's development showed it to be derived from recently synthesized myelin. The floating fraction from the brain contained less cerebroside and more lecithin than myelin, while the spinal cord floating fraction composition was much like that of myelin. The floating fractions contained less protein typical of myelin (basic and proteolipid protein) and more highmolecular-weight protein which may have been derived from contaminating microsomes. The floating fraction was presumed to be partially deproteinated myelin. The use of TET-treatment as model for demyelination as a result of edema and proceeding in the absence of macrophages is discussed.     

DNA Changes in Spinal Cords of Rats with Experimental Allergic Encephalomyelitis

DNA levels were measured in the spinal cords of Lewis rats during the development of and recovery from experimental allergic encephalomyelitis (EAE). Spinal cord DNA was first increased 11 days after immunizing the rats with guinea pig myelin and rose to levels four times that of the Freund's adjuvant controls at day 14, then subsided after day 22. Spinal cord DNA was still 150% of control levels 60 days after immunization. These DNA changes were compared with fluctuations in spinal cord acid proteinase in the same animals. Acid proteinase activity in EAE spinal cord increased later than the rise in DNA and attained a level of 170% of control at days 15-17, then subsided. Spinal cord DNA was higher in rats immunized with whole myelin than in those administered equivalent amounts of purified myelin basic protein. Furthermore DNA was higher in spinal cords of rats immunized with a larger dose of myelin (1.0 mg) than with a lower amount (0.5 mg). Various protease inhibitors including pepstatin, nitrophenyl p-guanidino benzoate, polylysine, and dipropionyl rhein, previously shown to protect Lewis rats against EAE, suppressed the increase of DNA in the spinal cord. Measurement of DNA increases in the spinal cord of EAE animals provides a convenient reproducible measurement of the severity of inflammation in the CNS and provides an objective criterion for assessment of the efficacy of various agents screened as possible therapeutic treatment for multiple sclerosis.     

Structure determination of a new fluorescent tricyclic nucleoside from archaebacterial tRNA.

A highly fluorescent nucleoside was detected in enzymatic digests of the extremely thermophilic archaebacterium Sulfolobus solfataricus by combined liquid chromatography-mass spectrometry (LC/MS). Following isolation, the structure was determined primarily by mass spectrometry, to be 3-(beta-D-ribofuranosyl)-4,9-dihydro-4,6,7-trimethyl-9-oxoimidazo[ 1, 2-a]purine (mimG), a new derivative of the Y (wye) nucleoside. The structural assignment was verified by comparison of the base released by acid hydrolysis with the corresponding synthetic base, using mass spectrometry, chromatography, and UV absorption and fluorescence properties. Nucleoside mimG was also detected by LC/MS in hydrolysates of the thermophiles Thermoproteus neutrophilus and Pyrodictium occultum. These results constitute the first finding of a member of the hypermodified Y family of nucleosides in archaebacteria.     

A comparison of sodium channel kinetics in the squid axon,the frog node and the frog node with BTX using the “silent gate” model

In this paper it is shown that the very different kinetics measured for the rise of the sodium current which follows a depolarization of the membrane in the squid giant axon, the frog node and the frog node treated with Batrachotoxin may be accurately predicted using only the measured equilibrium and static characteristics for the three preparations and the kinetics measured for the gating charge transfer.The kinetic predictions follow the use of the silent gate model for ion channel gating. The model is electrostatic and its chief assumptions are that the channel gate, called here the N-system, has fast kinetics and responds to the gating charge that transfers but not directly to the trans-membrane voltage applied. Because channel gating, corresponding here to the motion of the N-system, does not change its energy in the trans-membrane applied electric field the gating is electrically silent as far as gating charge transfer measurement is concerned. However the probability of gating rises with the quantity of gating charge that transfers due to the electrostatic interaction between the N-system and the gating charge, redistributed under the influence of the applied trans-membrane electric field. With these assumptions the kinetics of sodium channel gating are predictable using only the static and equilibrium characteristics of gating charge and channel activation measured as a function of membrane voltage, and the kinetics of the gating charge transfer. Because of the fast kinetics assumed for the N-system the predicted kinetics are the same for channels with any number of equivalent and independent N-systems or gates acting in parallel.The model predictions for sodium permeability kinetics are compared in detail with those recently measured for the frog node treated with Batrachotoxin and excellent agreement is obtained.     

National survey of spinal injuries in hockey players.

There has been an alarming increase in the number of spinal injuries in hockey players. Between 1976 and 1983, 42 were reported to the Committee on Prevention of Spinal Injuries due to Hockey. The median age of the injured players was 17 years. Of the 42 players 28 had spinal cord injuries, and 17 of them had complete paralysis below the vertebral level of the injury. Strikes from behind and collisions with the boards were common mechanisms of injury. Many of the players had suffered a burst fracture of the cervical spine following a blow to the top of the helmet when the neck was slightly flexed. The committee studied a number of possible etiologic factors and made several recommendations regarding prevention. League officials, coaches, players and equipment manufacturers can all play a role in prevention.