Bibliothek

Notizen: Abstract: We measured the activity of cathepsin D, the major cerebral protease, in 50 separate areas of the central nervous system of adult and aged humans, using hemoglobin as the substrate. The activity showed significant regional heterogeneity, with average differences of 50–100% between the lower and higher level areas, and a more than threefold difference between the lowest and highest levels. The forebrain, midbrain, and hindbrain each had areas of high and low activity; cerebellum and cord areas were among those with low activity. Cathepsin levels tended to increase with age in about half of the areas analyzed, and the increases were significant in 14. Statistically significant decreases with aging were observed in two areas. The increases varied between 30 and 60%, and the decreases were 20%. Enzyme activity in thalamus, hypothalamus, pons, medulla, and cerebellum increased with age. In the ventrolateral medulla, which contains the major portion of the cerebral noradrenergic cells, the cathepsin D levels increased with age; in the dorsal raphe area, which contains the major portion of the cerebral serotonergic cells, the enzyme levels decreased. The change with age in human brain seems to be less than what we observed in rat brain, where activity more than doubled in most areas. The changes in enzyme levels need to be tested at more ages to establish a pattern of changes in activity throughout life.

Notizen: Abstract Fish optic nerves, unlike mammalian optic nerves, are endowed with a high capacity to regenerate. Injury to fish optic nerves causes pronounced changes in the composition of pulse-labeled substances derived from the surrounding non-neuronal cells. The most prominent of these injury-induced changes is in a 28-kilodalton (kDa) polypeptide whose level increases after injury, as revealed by one-dimensional gel electrophoresis and autoradiography. The present study identified as apolipoprotein A-I (apo-A-I) a polypeptide of 28 kDa in media conditioned by regenerating fish optic nerves. The level of this polypeptide increased after injury by approximately 35%. Apo-A-I was isolated by gel-permeation chromatography from delipidated high-density lipoproteins (HDL) that had been obtained from carp plasma by sequential ultracentrifugation. Further identification of the purified protein as apo-A-I was based on its molecular mass (28 kDa) as determined by gel electrophoresis, amino acid composition, and microheterogeneity studies. The isolated protein was further analyzed by immunoblots of two-dimensional gels and was found to contain six isoforms. Western blot analysis using antibodies directed against the isolated plasma protein showed that the 28-kDa polypeptide in the preparation of soluble substances derived from the fish optic nerves (conditioned media, CM) cross-reacted immunologically with the isolated fish plasma apo-A-I. Immunoblots of two-dimensional gels revealed the presence of three apo-A-I isoforms in the CM of regenerating fish optic nerves (pIs: 6.49, 6.64, and 6.73). At least some of the apo-A-I found in the CM is derived from the nerve, as was shown by pulse labeling with [35S]methionine, followed by immunoprecipitation. The apo-A-I immunoactive polypeptides in the CM of the fish optic nerve were found in high molecular-weight, putative HDL-like particles. Immunocytochemical staining revealed that apo-A-I immunoreactive sites were present in the fish optic nerves. Higher labeling was found in injured nerves (between the site of injury and the brain) than in non-injured nerves. The accumulation of apo-A-I in nerves that are capable of regenerating may be similar to that of apo-E in sciatic nerves of mammals (a regenerative system); in contrast, although its synthesis is increased, apo-A-I does not accumulate in avian optic nerves nor does apo-E in rat optic nerves (two nonregenerative systems).

Notizen: Abstract: Translation products of mRNA from retinas of goldfish optic nerve (representing a regenerative CNS) and adult rabbit optic nerve (representing a nonregenerative CNS which can be induced to express regenerative characteristics) were examined by one- and two-dimensional gel electrophoresis. Translation products from retinas of the regenerating goldfish optic nerve included polypeptides barely detectable in the translation products of mRNA derived from retinas of uninjured controls. Some of these polypeptides, of apparent molecular weights 24–28, 43–49, 60, and 65 kilodaltons can be considered as growth-associated polypeptides described in other regenerative and developing systems. The induction of regeneration-associated characteristics in the injured adult rabbit optic nerve, “implanted” with diffusible substances from nonneuronal cells of regenerative or growing nerve, is reflected by changes in the mRNA translation products of the retina. Among such translation products are those of the following molecular weights: 16–18, 28, 32–35, 43–47, and 56–60 kilodaltons, and some higher-molecular-weight species.

Notizen: Abstract: This study provides evidence that apolipoprotein-A-I (apo-A-I), derived from fish plasma and nerve, has heparin binding activity. We have shown previously that injury in a regenerative CNS, such as that of fish optic nerves, leads to increased levels of apo-A-I in media conditioned by these nerves, as compared with media conditioned by noninjured nerves. In the present study, we have purified and characterized apo-A-I from both fish plasma and optic nerves. Sequence analysis of the 15 N-terminal amino acids revealed that at least 14 amino acids are identical in these two purified apo-A-I samples. The purified apo-A-I derived from both fish plasma and optic nerves binds to heparin. Binding measurements using [3H]heparin followed by Scatchard analysis revealed that apo-A-I binds to heparin with relatively low affinity (KD= 2.8 × 10−6M). Results are discussed with respect to the possibility that accumulation of apo-A-I in the extracellular matrix of fish optic nerves is made possible via heparin binding, like that to apolipoprotein-E in mammals.