Control of protein translation by phosphorylation of the mRNA 5'-cap-binding complex.

Initiation of mRNA translation is a key regulatory step in the control of gene expression. Microarray analysis indicates that total mRNA levels do not always reflect protein levels, since mRNA association with polyribosomes is necessary for protein synthesis. Phosphorylation of translation initiation factors offers a cost-effective and rapid way to adapt to physiological and environmental changes, and there is increasing evidence that many of these factors are subject to multiple regulatory phosphorylation events. The present article focuses on the nature of reversible phosphorylation and the function of the 5'-cap-binding complex in plants.

Developmental regulation of pectic epitopes during potato tuberisation.

We show, by immunogold labelling, that potato (Solanum tuberosum L. cv Karnico) pectic epitopes are developmentally regulated within regions of the stolon, in addition to showing tissue-specific differences in abundance and localisation. The (1-->4)-beta-D-galactan and (1-->5)-alpha-arabinan epitopes demarcate two distinct zones within stolons; galactans are enriched in primary walls of elongating cells proximal to the stolon hook, whilst arabinans predominate in younger cells distal to the hook. Low-methoxyl homogalacturonan epitopes are concentrated in the middle lamella and show a proximo-distal gradient in stolons similar to that of galactans, whilst high-methoxyl homogalacturonan is uniformly abundant. Calcium pectate is restricted to the middle lamella at cell corners and pit fields. Calcium-binding sites are uniformly present in stolon cell walls, but their total density is reduced and they become localised to a few cell corners in mature tubers, as determined by image-electron energy loss spectroscopy. During the transition from elongation growth to isodiametric expansion during tuberisation of the stolon hook, there were no detectable changes in pectic epitope abundance or localisation. As tubers matured, all epitopes increased in abundance in parenchymal cell walls, except for calcium pectate. We conclude that potentially significant changes in pectic composition occur as young cells distal to the stolon hook move into the zone of cell elongation proximal to the hook.

Monoclonal antibody 2G13, a new axonal growth cone marker.

Growth cones are specialized sensorimotor structures at the tips of neurites implicated in pathfinding decisions and axonal outgrowth during neuronal development. We generated a mouse monoclonal antibody (mAb 2G13) against chick tectum and found that the antibody exclusively labelled axonal growth cones, particularly their filopodia and lamellipodia, in developing rat CNS and in embryonic neurons in culture. The high fidelity of the staining of growth cones by mAb 2G13 means that the antibody will be a useful marker for identifying growth cones. In growth cones of cultured neurons, mAb 2G13 labelling is intracellular and mainly associated with the filamentous actin cytoskeleton. Experiments with cytochalasins, which depolymerise filamentous actin, showed that 2G13p (the protein recognised by mAb 2G13) is physically associated with filamentous actin in growth cones. These properties of 2G13p suggest a role in growth cone motility.

Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in regenerating axons of the sciatic nerve.

Monoclonal antibodies SMI-31 and 150 recognize phosphorylation epitopes on microtubule-associated protein 1B that have been shown to be developmentally down-regulated in the nervous system. We have used these antibodies to establish changes in the pattern of expression of their epitopes on microtubule-associated protein 1B in regenerating axons of the sciatic nerves in the adult mouse and rat. Immunohistochemical studies showed that, in the sciatic nerve, regenerating axons in both adult mice and rats were labelled with monoclonal antibody 150 in a proximodistal gradient which was highest at the growth cone. This is the first report of expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in regenerating axons. Immunoblotting showed that the expression of the isoform recognized by monoclonal antibody 150 is present in normal adult mouse sciatic nerve and in regenerating axons following crush or cut lesions, but was not detectable in the normal or regenerating adult rat peripheral nervous system. Regenerating axons were also labelled by monoclonal antibody SMI-31, but the labelling, unlike antibody 150 labelling, was uniform along the entire length of the axon and immunoblotting showed that it was due to recognition of neurofilament protein. We conclude that the phosphorylated isoforms of microtubule-associated protein 1B recognized by monoclonal antibody 150 that are developmentally down-regulated in the adult rat central and peripheral nervous systems and adult mouse cerebellum are maintained in the normal peripheral nervous system of the adult mouse. When peripheral axons regenerate in the adult mouse, the regenerating axons also contain these isoforms. Adult rat regenerating axons are stained by antibody 150 only in tissue sections, not in immunoblots. The maintenance of immature isoforms of microtubule-associated protein 1B in mouse peripheral axons may relate to a continual capacity for growth and remodelling. The immunohistochemical localization of the antibody 150 epitope in growth cone-like structures and sprouts in injured nerves shows that phosphorylation of microtubule-associated protein 1B is likely to be an integral part of the regenerative response. These results also show that the phosphorylation epitopes on microtubule-associated protein 1B recognized by monoclonal antibodies 150 and SMI-31 are different and that only expression of the former correlates with axonal regeneration.

An analysis of an axonal gradient of phosphorylated MAP 1B in cultured rat sensory neurons.

The present study investigated the cellular distribution of a developmentally regulated phosphorylated form of MAP 1B recognized by monoclonal antibody (mAb) 150 in cultures of dorsal root ganglia. The cell soma and the whole axon, when it first appears, are labelled, but longer axons label with a proximodistal gradient, such that the cell soma and proximal axon become unlabelled, whilst the distal axon and growth cone label strongly. Double-labelling experiments with mAb 150 and a polyclonal antibody (N1-15) that recognizes all forms of MAP 1B demonstrated that MAP 1B is distributed along the entire length of axons with gradients, so the gradient of phosphorylated MAP 1B is not due to a loss or absence of MAP 1B from the proximal axon. The proportion of axons from 20 h cultures that were labelled with a mAb 150 gradient was at least 80% and this proportion was independent of the nerve growth factor concentration of the culture medium. Analysis of axons ranging in length from 100 to 700 microm and labelled with a gradient showed that the unlabelled proximal portions of axons increased in length more slowly than the labelled distal axon. Axons labelled along their entire length accounted for no more than 19% of th axonal population and analysis of these showed them to be frequently < 400 microm long. After simultaneously fixing and detergent-extracting cultures this proportion rose significantly to 93%, suggesting that in the proximal axon the mAb 150 epitope is masked by some factor(s) that is removed by detergent extraction. The possibility that mAb 150 could not access the epitope in the proximal axon was discounted because another IgM, mAb 125, which recognizes a different phosphorylation epitope on MAP 1B, labelled the proximal axon of conventionally fixed cultures. In growth cones of fixed and extracted neurons examined by immunofluorescence, the mAb 150 labelling strongly colocalized to bundled microtubules in the distal axon shaft and the C-domain. In the P-domain, mAb 150 staining was weaker and more widely distributed than the microtubules. Immunogold electron microscopy confirmed that antibody N1-15 and mAb 150 strongly labelled the bundled microtubules in the C-domain and also showed that individual microtubules in the P-domain, some of which lie alongside actin filament bundles of filopodia, were labelled lightly and discontinuously with both antibodies. This suggests that the phosphorylated isoform of MAP 1B recognized by mAb 150 may be microtubules and actin filaments in the P-domain.

Distribution and expression of developmentally regulated phosphorylation epitopes on MAP 1B and neurofilament proteins in the developing rat spinal cord.

The distribution and expression of developmentally regulated phosphorylation epitopes on the microtubule-associated protein 1B and on neurofilament proteins recognized by monoclonal antibody (mAb) 150 and mAb SMI-31 was investigated in the developing rat spinal cord. In the embryonic day 11 spinal cord, mAb 150 stained the first axons to appear, whereas mAb SMI-31 staining did not appear until embryonic day 12. At the start of axonogenesis, mAb 150 stained neuronal cell bodies and axons whereas at later times only the distal axon was stained, this is the first demonstration in vivo of a mAb 150 axonal gradient similar to that seen previously in vitro (Mansfield et al., 1991). During the postnatal period, axonal staining by mAb 150 dramatically declined so that by the third postnatal week, only the corticospinal tract, which contains axons that are still growing, was labelled. There was no evidence of dendritic staining except of adult primary motoneurons. In contrast, mAb SMI-31 staining of axons was not present as a gradient. Instead, mAb SMI-31 staining increased progressively throughout this period, persisted into adulthood and was shown by immunoblotting to be related to the increased phosphorylation of the medium and heavy neurofilament proteins. Axonal staining by mAb 150 re-appears in a sub-population of the SMI-31-labelled myelinated axons in the adult spinal cord and PNS and in the perikarya and dendrites of primary motoneurons, where it probably recognizes a phosphorylation epitope on heavy neurofilament proteins. This late appearing epitope has some similarities to that recognized by mAb SMI-31 on neurofilaments, but it is not identical. These cross-reactivities of mAbs that recognize phosphorylation epitopes on otherwise unrelated proteins dictate caution in interpreting immunohistochemical data. It may now be necessary in some cases to re-appraise published studies using these two antibodies.

Blood-nerve barrier: ultrastructural and endothelial surface charge alterations following nerve crush.

Nerve crush results in an enhanced vascular permeability of the endoneurial vessels distal to the lesion. Vascular permeability at the blood-nerve barrier (BNB) to serum proteins is influenced by many factors, including anionic surface charge, endothelial vesicular transcytosis and the presence or absence of fenestrated vessels. Using mice and rats, the present ultrastructural investigation examined the effect of nerve crush (axonotmesis) on: (1) the distribution of endothelial anionic sites and (2) the appearance of fenestrations in endoneurial vessels after 4 and 14 day intervals as demonstrated with cationic probes. Transient anionic fenestrations developed in a minority of mouse endoneurial vessels in 4-day crushed nerves, but were not found in 14-day crushed nerves of mice nor in crushed nerves of rats. The known increase in the permeability of endoneurial vessels in rats and mice was not associated with reduced luminal labelling with cationic ferritin at physiological pH. At pH 2.0 the labelling of glycocalyx moieties (such as sialic acid) with cationic colloidal gold was disrupted in some epi- and endoneurial vessels of 4-day rats, but in a greater proportion after 14 days. The enhanced permeability of the BNB during degeneration and regeneration is related to the formation of anionic fenestrations in endoneurial vessels of mice and to the reduced and uneven distribution of endothelial glycocalyx moieties that are anionic at pH 2.0 in rats.

Distribution of anionic sites on the perineurium.

The distribution of anionic sites on the perineurial basal lamina (BL) and plasmalemma of dorsal root ganglia and sciatic nerves was determined using cationic ferritin (CF) and cationic gold (CCG). The probes were applied to the tissue before and after resin embedding and visualised by electron microscopy. There were no apparent differences in charge distribution between the 2 tissues. At physiological pH a strong anionic charge was distributed evenly over the BL as demonstrated by pre-embedding labelling with CF; the plasmalemma was only moderately anionic. A similar application of CCG at pH 2.0 revealed a quasi-regular distribution of anionic sites (presumably due to acidic carbohydrate moieties) on the BL, whilst CCG-labelling of L. R. White sections indicated a differential distribution of these moieties on the BL of the inner and outer perineurial lamellae. Cationic ferritin (12 nm diameter) crossed the BL and entered perineurial caveolae, but CCG (effective diameter of 15 nm) did not, suggesting that the BL is a size-restrictive filter. These results are discussed with regard to the ultrastructure and function of the BL of other tissues and the possible role of perineurial BL charge as a determinant of perineurial permeability.

Blood-nerve barrier: distribution of anionic sites on the endothelial plasma membrane and basal lamina of dorsal root ganglia.

Previous investigations of the blood-nerve barrier have correlated the greater permeability of ganglionic endoneurial vessels, compared to those of nerve trunks, with the presence of fenestrations and open intercellular junctions. Recent studies have demonstrated reduced endothelial cell surface charge in blood vessels showing greater permeability. To determine the distribution of anionic sites on the plasma membranes and basal laminae of endothelial cells in dorsal root ganglia, cationic colloidal gold and cationic ferritin were used. Electron microscopy revealed the existence of endothelial microdomains with differing labelling densities. Labelling indicated that caveolar and fenestral diaphragms and basal laminae are highly anionic at physiological pH, luminal plasma membranes and endothelial processes are moderately charged and abluminal plasma membranes are weakly anionic. Tracers did not occur in caveolae or cytoplasmic vesicles. In vitro tracer experiments at pH values of 7.3, 5.0, 3.5 and 2.0 indicated that the anionic charge on the various endothelial domains was contributed by chemical groups with differing pKa values. In summary, the labelling of ganglionic and sciatic nerve vessels was similar except for the heavy labelling of diaphragms in a minority of endoneurial vessels in ganglia. This difference is likely to account in part for the greater permeability of ganglionic endoneurial vessels. The results are discussed with regard to the blood-nerve and -brain barriers and vascular permeability in other tissues and a comparison made between the ultrastructure and anionic microdomains of epi-, peri- and endoneurial vessels of dorsal root ganglia and sciatic nerves.

Blood-nerve barrier: distribution of anionic sites on the endothelial plasma membrane and basal lamina.

The distribution of anionic sites on the cell membranes and basal laminae of vascular endothelial cells in the rat sciatic nerve was investigated using cationic ferritin (CF) and cationic colloidal gold (CCG). Nerves fixed by perfusion followed by immersion were chopped into 400 microns thick slices and incubated in CF or embedded in LR White resin for staining with CCG. Using electron microscopy, the distribution of these tracers was investigated. The results indicated that microdomains of various charge densities exist. Diaphragms of caveolae and transendothelial channels, and luminal endothelial processes are highly anionic, the basal laminae of endothelial cells and pericytes and luminal membranes are medium and abluminal membranes least anionic. Inter-endothelial tight junctions were unlabelled and not penetrated by CF. These structures are thought to represent charge and size filters that control permeability of the vasa nervorum. The distribution of these charge-size filters is discussed in terms of the blood-nerve barrier, a physiological property present in the endo- but absent in the peri- and epineurial vessels.

A method for the identification of Streptococcus mutans in gingival margin plaque by immunofluorescence.

A method was developed to identify Streptococcus mutans in natural dental plaque by indirect immunofluorescence staining, using a high-titred polyclonal antiserum raised against a serotype c strain of S. mutans followed by an FITC conjugate. Specificity was determined by staining 45 representative strains of plaque organisms, which demonstrated minimal cross-reactions. In vitro incubation of S. mutans NCTC 10449 films with a human serum containing antibodies to S. mutans and the presence of extracellular polysaccharide did not inhibit staining. The staining method enabled 98% of the streptococci to be detected in mixtures of S. mutans NCTC 10449 and Lactobacillus casei NCTC 10302. S. mutans was detected at a ratio of 1:100,000 in mixtures of pure cultures. In plaque samples, S. mutans could be distinguished from other organisms, including an unidentified cross-reacting bacillus found in some gingival plaque samples. The results suggest that immunofluorescence is a fast, practical method for identifying specific bacteria in plaque and, therefore, could be of use in microbiological studies of caries.

The effect of bupropion, a new antidepressant drug, and alcohol and their interaction in man.

The effects of bupropion and ethanol were examined alone and in combination in a placebo controlled, double-blind, crossover study in 12 healthy volunteers. Results were subjected to analysis of variance and differences of p less than 0.05 taken as significant. In the main study using the Wilkinson auditory vigilance test, no active treatment or combination of treatments produced significant change compared with placebo. However, when compared with bupropion 100 mg, vigilance was significantly impaired by 32 ml alcohol alone though not when combined with bupropion. No significant changes in reaction time or short term memory occurred. Visual analogue scales indicated that the subjects were mentally slower after alcohol 32 ml than after placebo. Combination of bupropion 100 mg with alcohol 32 ml abolished this difference. A similar pattern occurred with group ratings indicating mental sedation. Subjects were clearly able to differentiate between the 16 ml and 32 ml doses of alcohol when assessing their degree of inebriation. Combination of bupropion with alcohol made no difference to the ratings of inebriation. The top dose of alcohol tended to increase energy in the low frequency EEG bands. Combination of the top alcohol dose with bupropion, however, produced a significant reversal with lowered energy in the 4-7.5 Hz band. Combination of bupropion with alcohol failed to change the blood alcohol concentration achieved.

The effects of chloridazepoxide on avoidance performance of mice subjected to undernutrition or handling stress in early life.

This experiment compared the effects of (u) "early undernutrition" by rearing in large litters and (ii) an early handlin g stress, on avoidance learning in Swiss white mice. The two treatments, the first leading to permanent physical stunting and the second not, had similar and additive detrimental effects on avoidance performance. Treatment with the minor tranquillizer Chlordiazepoxide improved performance in all groups but had a proportionately greater effect on previously undernourished, handled mice. Thus the poor avoidance performace of mice reared in large litters appears to be largely independent of the growth stunting effect and more closely related to an elevated stress response produced by stress in early life.