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To improve Skindex, a dermatologic quality-of-life instrument.Cross-sectional and longitudinal questionnaire study.Dermatology clinic of a Veterans Affairs hospital and private dermatology practices.Patients waiting for dermatology appointments; 201 patients responded to the original version of Skindex and 692 additional patients to the revised version.Reproducibility, internal consistency reliability, and validity of the revised version of Skindex. The revised version was compared with the original in 3 ways: the amount of time patients need to complete it; discriminative capability, determined as the number of items to which patients chose the same response; and evaluative capability, determined as the number of scales that were responsive to patients' reports of clinical change.With the revised 29-item version of Skindex, scale scores were reproducible after 72 hours (r = 0.88-0.92) and were internally reliable (Cronbach alpha = 0.87-0.96). The instrument demonstrated both construct and content validity; patients with psoriasis and eczema responded with higher scores than those with isolated lesions; in an exploratory principal axes factor analysis with an oblique rotation, 97% of the common variance was explained by 3 factors that correlated with the a priori scales; and most patients' responses to an open-ended question about their skin disease were addressed by items in the instrument. The average time to complete the revised instrument was 5 minutes (compared with 15 minutes for the original version). For only 3 items (10%) did 70% or more of patients choose the same response (vs 17 [28%] of items in the original version). All scales changed significantly in the expected direction in patients who reported that their skin had changed after 3 months (vs only 3 of 8 scales originally).The 29-item version of Skindex remains reliable and valid, but has decreased respondent burden and improved discriminative and evaluative capability.

The squid giant axon is presented as a model for the study of macromolecular interaction between cells in the nervous system. When the isolated giant axon was incubated in sea water containing [ 3 H]leucine for 0.5-5 hr, newly synthesized proteins appeared in the sheath and axoplasm as demonstrated by: ( i ) radioautography, ( ii ) separation of the sheath and axoplasm by extrusion, and ( iii ) perfusion of electrically excitable axons. The absence of ribosomal RNA in the axoplasm [Lasek, R. J. et al. (1973) Nature 244, 162-165] coupled with other evidence indicates that the labeled proteins that are found in the axoplasm originate in the Schwann cells surrounding the axon. Approximately 50% of the newly synthesized Schwann cell proteins are transferred to the giant axon. These transferred proteins are soluble for the most part and range in molecular size from 12,000 to greater than 200,000 daltons. It is suggested that proteins transferred from the Schwann cell to the axon have a regulatory role in neuronal function.

Antibodies prepared in rabbits against Myxicola infundibulum neurofilaments have been employed to stain neurofilaments immunohistochemically in intact Myxicola infundibulum nervous tissue. Paraffin-embedded and frozen sections (5--6 mu) were examined at the light microscopic level with Sternberger's peroxidase-antiperoxidase method, and Vibratome (20--40 mu) sections were studied at the ultrastructural level with Nakane's conjugated peroxidase method. The neurofilament antibody stained only neurons and axons at the light microscopic level. The staining pattern at the electron microscopic level corresponded to the neurofilaments within axons and neurons. Glial cells, which surround the axons, contain large bundles of filaments that resemble astrocytic filaments in mammalian astrocytes. These filaments do not stain with the anti-neurofilament antibody. Neurons, neurofilaments, glial cells, glial filaments, and nonnervous tissue showed no peroxidase staining when specific antiserum absorbed with neurofilaments was used. These structures were also unstained when antiserum to the glial fibrillary acidic protein of mammalian central nervous system astrocytes was substituted for the neurofilament antiserum. Therefore, in Myxicola infundibulum, the antigenic determinants of the neurofilament protein, as recognized immunohistochemically by anti-neurofilament protein antibodies, are not shared with those of glial filaments.

We have examined the monomer-polymer equilibria which form the cytoskeletal polymers in squid axoplasm by extracting protein at low concentrations of monomer. The solution conditions inside the axon were matched as closely as possible by the extraction buffer (buffer P) to preserve the types of protein associations that occur in axoplasm. Upon extraction in buffer P, all of the neurofilament proteins in axoplasm remain polymerized as part of the stable neurofilament network. In contrast, most of the polymerized tubulin and actin in axoplasm is soluble although a fraction of these proteins also exists as a stable polymer. Thus, the axoplasmic cytoskeleton contains both stable polymers and soluble polymers. We propose that stable polymers, such as neurofilaments, conserve cytoskeletal organization because they tend to remain polymerized, whereas soluble polymers increase the plasticity of the cytoskeleton because they permit rapid and reversible changes in cytoskeletal organization.

We have previously demonstrated that neurofilament protein (NFP) is endogenously phosphorylated in squid giant fiber axoplasm (1). Here the generality of this phenomenon is demonstrated, and optimal reaction conditions are described. Phosphorylation of Myxicola NFP is shown in vivo and in isolated axoplasm by 32P labeling from [32P]orthophosphoric acid and [gamma-32P]ATP, respectively. Phosphorylation of guinea pig NFP is shown in extracts of peripheral nerve and in enriched NFP preparations. Both Myxicola NFP subunits (150 and 160 kilodaltons) and all three guinea pig NFP subunits (200, 145, and 68 kilodaltons) are phosphorylated. Phosphorylation of 10-nm filament protein from cultured fibroblasts is also shown optimal reaction conditions with guinea pig enriched NFP are as follows. The reaction has high specificity for ATP as substrate. Divalent cations are required, Mn2+ being most effective. Monovalent cations decrease activity. The pH optimum is 8.0. Cyclic AMP and other low molecular weight factors have no significant modulatory effect.

Incubation of intracellulary perfused squid giant axons in [3H]leucine demonstrated that newly synthesized proteins appeared in the perfusate after a 45-min lag period. The transfer of labeled proteins was shown to occur steadily over 8 h of incubation, in the presence of an intact axonal plasma membrane as evidenced by the ability of the perfused axon to conduct propagated action potentials over this time-period. Intracellularly perfused RNase did not affect this transfer, whereas extracellularly applied puromycin, which blocked de novo protein synthesis in the glial sheath, prevented the appearance of labeled proteins in the perfusate. The uptake of exogenous 14C-labeled bovine serum albumin (BSA) into the axon had entirely different kinetics than the endogenous glial labeled protein transfer process. The data provide support for the glia-neuron protein transfer hypothesis.

The monomer-polymer equilibria for tubulin and actin were analyzed for the cytoskeleton of the squid giant axon. Two methods were evaluated for measuring the concentrations of monomer, soluble (equilibrium) polymer, and stable polymer in extruded axoplasm. One method, the Kinetic Equilibration Paradigm ( KEP ), employs the basic principles of diffusion to distinguish freely diffusible monomer from proteins that are present in the form of polymer. The other method is pharmacological and employs either taxol or phalloidin to stabilize the microtubules and microfilaments, respectively. The results of the two methods agree and demonstrate that 22-36% of the tubulin and 41-47% of the actin are monomeric. The in vivo concentration of monomeric actin and tubulin were two to three times higher than the concentration required to polymerize these proteins in vitro, suggesting that assembly of these proteins is regulated by additional mechanisms in the axon. A significant fraction of the polymerized actin and tubulin in the axoplasm was stable microtubules and microfilaments, which suggests that the dissociation reaction is blocked at both ends of these polymers. These results are discussed in relationship to the axonal transport of the cytoskeleton and with regard to the ability of axons to change their shape in response to environmental stimuli.

Recent studies of axonal transport indicate that cytoskeletal proteins are assembled into polymers in the neuron cell body and that these polymers move from the cell body toward the end of the axon. On the other hand, membranous elements appear to be inserted into the axonal plasma membrane preferentially at the end of the axon. These new observations are explored in relation to our current understanding of axonal elongation.

Axons do not contain polysomes and therefore are unable to synthesize proteins. On the other hand, the nerve cell body has a well-developed protein synthesizing capacity, and proteins are conveyed into the axon from the cell body by orthograde axonal transport. Studies on vertebrate neurones demonstrate that the proteins conveyed from the cell body constitute all of the major structures of the axon including : membranous vesicles, the endoplasmic reticulum, mitochondria, and the cytoskeleton which consists of microtubules, neurofilaments, and microfilaments. Although the nerve cell body is the major source of the macromolecules which provide the structural integrity of the axon, in some axons the glial cells surrounding the axon also supply proteins to the axon. The transfer of newly synthesized proteins from glial cells to the axon is documented most completely in the case of the squid giant axon. The transfer of proteins from glial cells to the neurone can be studied in the giant axon by incubating axons, which have been disconnected from their nerve cell bodies, in a solution which contains labelled amino acids. The glial cells transfer as much as 40 % of their newly synthesized proteins to the giant axon. These transferred proteins can be studied in axoplasm which is separated from the giant axon by extrusion. What are the comparative roles of proteins supplied to the axon by the glial cells and the nerve cell body? To begin to answer this question, we have compared the glial-transfer proteins with the proteins which constitute whole axoplasm. The proteins were analysed by two-dimensional polyacrylamide gel electrophoresis, and axoplasmic proteins were detected by staining the gels with Coomassie blue, whereas the labelled proteins were detected by fluorography of the same gels. The glial-transfer proteins differ substantially from the stained axoplasmic proteins. For example, neurofilament proteins and tubulin are major proteins of the axoplasm but are not represented among the labelled proteins which are transferred from the glial cells. In fact, these proteins are not synthesized to any significant degree by the glial cells. One of the glial-transfer proteins (molecular weight 70000) stands out because it is heavily labelled and is well resolved on the gels. This protein which we have named traversin is present among the stained proteins of whole axoplasm but is a relatively minor component of the axoplasm. In order to determine whether traversin is supplied to the axon by the nerve cell body we analyzed the proteins synthesized in the stellate ganglion which contains the nerve cell bodies of the giant axon. The pattern of proteins synthesized by the stellate ganglion was similar to that of whole axoplasm, but was very different from the constellation of proteins transferred from the glial cells. Traversin was not among the major labelled proteins synthesized by the ganglion and is at most a minor synthetic product of the ganglion. Because the ganglion contains glia, we can not ascertain whether the small amount of labelled traversin in the ganglion was synthesized in the glia or the nerve cell bodies. However, these results suggest that traversin is synthesized in much greater amounts by glial cells than by nerve cells. If, as our results suggest, traversin is supplied to the axon principally by the glial cells, then the proteins transferred from the glial cells are likely to be specialized in function and not simply an accessory source which supplements the proteins supplied by the nerve cell body. Although some of the proteins transferred from the glial cells are special and are probably not supplied to the axon by the nerve cell body, we have also found proteins which are supplied by both the nerve cell body and the glial cells. One of these proteins is actin. The presence of actin in the population of proteins which are transferred from the glial cells is particularly interesting because of the known contractile function of this protein. Actin could be involved in the mechanism of protein transfer from the glial cells. The transferred proteins apparently exist in the form of a complex in the axoplasm. This complex of proteins can be separated from axoplasm because of its chromatographic behaviour of Sephadex G-200. These results suggest that the glial-transfer proteins are conveyed from the glial cell to the axon in the form of a particle which includes actin in its structure.

— RNA and DNA were determined in the axoplasm and sheath of squid giant axons. If the RNA was related to axonal length, equivalent amounts of RNA were found in the axoplasm and sheath. However, when it was expressed as a function of dry wt. or residue protein, the concentration of RNA in the sheath was four times greater than in the axoplasm. A ratio of 1·1 was found for sheath RNA/sheath DNA. Less DNA was present in the axoplasm than the amount which could be accurately determined with the methods employed.

We have previously demonstrated that neurofilament protein (NFP) is endogenously phosphorylated in squid giant fiber axoplasm (1). Here the generality of this phenomenon is demonstrated, and optimal reaction conditions are described. Phosphorylation of Myxicola NFP is shown in vivo and in isolated axoplasm by 32P labeling from [32P]orthophosphoric acid and [gamma-32P]ATP, respectively. Phosphorylation of guinea pig NFP is shown in extracts of peripheral nerve and in enriched NFP preparations. Both Myxicola NFP subunits (150 and 160 kilodaltons) and all three guinea pig NFP subunits (200, 145, and 68 kilodaltons) are phosphorylated. Phosphorylation of 10-nm filament protein from cultured fibroblasts is also shown optimal reaction conditions with guinea pig enriched NFP are as follows. The reaction has high specificity for ATP as substrate. Divalent cations are required, Mn2+ being most effective. Monovalent cations decrease activity. The pH optimum is 8.0. Cyclic AMP and other low molecular weight factors have no significant modulatory effect.

During axonal transport, membranes travel down axons at a rapid rate, whereas the cytoskeletal elements travel in either of two slow components, SCa (with tubulin and neurofilament protein) and SCb (with actin). Clathrin, the highly ordered, structural coat protein of coated vesicles, has recently been shown to be able to interact in vitro with cytoskeletal proteins in addition to membranes. The present study examines whether clathrin travels preferentially with the membrane elements or the cytoskeletal elements when it is axonally transported. Guinea pig visual system was labeled with tritiated amino acids. Radioactive SDS-polyacrylamide gel electrophoresis profiles from the major components of transport were coelectrophoresed with clathrin. Only SCb had a band comigrating with clathrin. In addition, radioactive clathrin was purified from guinea pig brain containing only radioactive SCb polypeptides. Kinetic analysis of the putative clathrin band in SCb revealed that it travels entirely within the SCb wave. Thus we conclude that clathrin travels preferentially with the cytoskeletal proteins making up SCb, rather than with the membranes and membrane-associated proteins in the fast component.

Cytoskeletal protein transport and metabolism are studied in the somatic motor and parasympathetic axons of the chicken oculomotor system. Kinetic analyses of pulse-labeled proteins indicate that the neurofilaments are transported 2–3 times more rapidly in the somatic motor axons than in the parasympathetic axons. By contrast, the transport rates of the slow component b (SCb) proteins are very similar in these axons. The parasympathetic axons terminate in the ciliary ganglion, and radiolabeling curves from the ciliary ganglion can be used to study the kinetics of cytoskeletal protein removal from the terminals. The rate of removal directly determines the residence time of the cytoskeletal proteins in the ciliary ganglion, and the residence time directly affects the shape and amplitude of the transport curves of the ganglion. A computer model was used to analyze these transport curves and to determine the half-residence time of the cytoskeletal proteins in the terminal regions. From the computer experiments, we estimate that the half-residence times of the neurofilament proteins actin and tubulin are 2, 3.5, and 7 d, respectively. The differences between the half-residence times of the cytoskeletal proteins indicate that the rate of protein removal from the terminals differs for these proteins. On the basis of these results, we propose that the removal mechanisms critically control the composition of the cytoskeleton in the terminal regions. Through their effects on its composition, the removal mechanisms have a crucial role in converting the cytoskeleton of the axon proper into the specialized cytoskeleton of the axon terminal.