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Biomedical subjects

R B Nelson

Publications and source records attributed to R B Nelson.

18 recordsLinked to original sources
PubMed1991

Contrasting patterns of protein phosphorylation in human normal and Alzheimer brain: focus on protein kinase C and protein F1/GAP-43.

We introduce a new procedure to study kinase substrates in postmortem human brain. By adding purified exogenous protein kinase C (PKC) and the phospholipid phosphatidylserine to brain homogenates in vitro we are able to analyze PKC substrates. A human 53-kDa phosphoprotein is described that appears to be homologous to rat and monkey protein F1 (GAP-43). This identity is based on molecular weight, isoelectric point, phosphorylation by exogenous protein kinase C, enhancement of its phosphorylation by three activators (phospholipids, calcium and phorbol esters), phosphopeptide maps, and cross-reactivity with an antibody raised against rat protein F1. Protein F1 is a PKC substrate associated with synaptic plasticity and nerve growth. Its phosphorylation in rat brain has been correlated with long-term potentiation, an electrophysiological model of memory. In the present study of normal brain, human protein F1 shows an occipitotemporal in vitro phosphorylation gradient. This is consistent with previous observations in nonhuman primates. This gradient is less pronounced in Alzheimer's disease (AD). Changes in the in vitro phosphorylation pattern of three other non-PKC substrates in Alzheimer's disease, including one with characteristics similar to microtubule-associated protein tau, are also reported. These results suggest that protein phosphorylation can be studied in postmortem human brain and that PKC-mediated phosphorylation of protein F1, already linked to synaptic plasticity and memory, may be altered in AD.

Alzheimer Disease↗
PubMed1990

Thrombin and its inhibitors regulate morphological and biochemical differentiation of astrocytes in vitro.

Flat, amorphous astroblasts in culture differentiate into rounded process-bearing cells after removal of serum from the media or following addition of dibutyryl cyclic-AMP (dbcAMP). We report here that addition of thrombin (10 nM) to rat primary astroglial cultures reversed both the spontaneous morphological differentiation of astroblasts caused by serum removal, and the more extensive morphological differentiation caused by pre-treatment with dbcAMP. The astroblasts retained the ability to differentiate upon removal of thrombin from the medium. Proteolytic activity of thrombin was required for the reversal of differentiation. Moreover, addition of serine protease inhibitors active against thrombin elicited a prolonged morphological differentiation rivaling that induced by dbcAMP, suggesting that inactivation of cell-associated thrombin might be sufficient for morphological differentiation to occur. Two other serine proteases with a cleavage specificity similar to thrombin were ineffective in reversing differentiation. Both the induction of morphological differentiation by dbcAMP and its reversal by thrombin were rapid, being essentially complete by 1 h. With more prolonged treatments, thrombin also reduced the dbcAMP-mediated increase in glutamine synthetase, a biochemical marker for astroglial differentiation. Thrombin also inhibited morphological differentiation in C6 glioma and altered the morphology of microglial cells; however, thrombin did not prevent neurite outgrowth in primary central neuronal cultures in contrast to its previously reported effects on the neuroblastoma 2a cell line. These findings indicate that a proteolytic mechanism mediated by thrombin and its inhibitors may underlie the regulation of astroglial differentiation.

Animals↗
PubMed1990

Clipsin, a chymotrypsin-like protease in rat brain which is irreversibly inhibited by alpha-1-antichymotrypsin.

The protease inhibitor alpha-1-antichymotrypsin, which binds to chymotrypsin-like enzymes in a sodium dodecyl sulfate-resistant manner, has been shown recently to be both a normal constituent of brain and an integral component of the neuritic plaques that form in Down's syndrome and Alzheimer's disease. We have now identified in rat brain a Mr 25,000 alpha-1-antichymotrypsin-binding protein classified as a chymotrypsin-like protease by its inhibitor profile and substrate specificity. Release of 125I-labeled breakdown products from bands containing the protease in substrate-linked polyacrylamide gels was examined in parallel with hydrolysis of tetrapeptide chromogenic substrates in vitro to establish conditions under which the Mr 25,000 protease was the only activity being measured in vitro. The protease was completely membrane associated but was extractable using 1 M MgCl2; prior extraction of detergent- and low ionic strength-soluble proteins from membranes was used to increase its specific activity. The formation of sodium dodecyl sulfate-resistant bonds between human alpha-1-antichymotrypsin and the protease (kassoc = 2.9 X 10(6) M-1 s-1) was used to titrate the concentration of free protease solubilized from membranes. The protease cleaved both succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and methoxy-succinyl-Ala-Ala-Pro-Met-p-nitroanilide, the latter being of interest because cleavage after a methionine residue is predicted to generate the amino terminus of the neuritic plaque component beta-amyloid from its precursor protein. In fact, the solubilized protease degraded 90% of membrane-associated beta-amyloid precursor protein detected by Western blot analysis. The protease was kinetically distinct from both chymotrypsin and cathepsin G in direct comparisons and did not match kinetic values published for the rat mast cell proteases against comparable substrates; we therefore refer to the protease with the descriptive acronym clipsin (for chymotrypsin-like protease). Proteases similar to and potentially identical to clipsin were detected by enzymography in other organs from rat (most notably spleen and adult lung). The enzyme in brain was distinguished by a narrow window of elevated activity surrounding postnatal day 5, which was 12-14-fold higher than levels in day 1 or adult brain. Because independent lines of evidence suggest that a brain chymotrypsin-like protease may be involved in the etiology of Down's syndrome and Alzheimer's disease, clipsin is discussed as a candidate for such a role.

Amino Acid Sequence↗
PubMed1989

Phosphoproteins localized to presynaptic terminal linked to persistence of long-term potentiation (LTP): quantitative analysis of two-dimensional gels.

Previous findings suggest: (1) that altering protein kinase C (PKC) activity alters the persistence of long-term potentiation (LTP) in the intact hippocampal formation; and (2) that PKC activity is directly correlated with persistence of LTP in vivo as measured by the in vitro phosphorylation of two major PKC substrates in adult hippocampus, protein F1 and 80k. Using quantitative analysis of two-dimensional gels, we report here two additional phosphoproteins of 72 and 55 kDa which were directly correlated to persistence of LTP induced in the intact dorsal hippocampal formation. The phosphorylation of both proteins in response to addition of different kinase stimulators was distinct from that of protein F1 and 80k. Moreover, neither protein was a substrate for exogenous PKC. The physicochemical properties of these phosphoproteins suggest they are identical to the previously described synaptic vesicle proteins IIIa and IIIb, and as such are immunologically indistinguishable. Because proteins IIIa and IIIb are known to be phosphorylated by a Ca2+/calmodulin (CaM)-stimulated kinase, and protein F1 is known to be a plasma membrane-associated protein (P-57) which releases bound CaM in response to phosphorylation by PKC, the present findings suggest a potential mechanism in which PKC-mediated changes in plasma membrane proteins produce CaM kinase-mediated changes in synaptic vesicle proteins through a phosphorylation cascade. These membrane/vesicle alterations are postulated to underlie the increased synaptic efficacy which marks persistent LTP.

Adaptation, Physiological↗
PubMed1989

Expression of beta-amyloid precursor protein in reactive astrocytes following neuronal damage.

Although the beta-amyloid peptide is an established core component of neuritic plaques that accumulate in Alzheimer's disease, the mechanisms responsible for its deposition are not well understood. We now report that lesions of rat hippocampal neurons cause a time-dependent, long-lasting elevation of immunoreactivity for the beta-amyloid precursor protein (APP) in neighboring astrocytes, a cell type not normally containing the protein. The increase represents astroglial expression of the protein rather than a scavenging of APP released by damaged neurons. Immunoelectron microscopy confirmed that APP-containing cells are reactive astroglia, both surrounding capillaries and within the neuropil. These results demonstrate that neuronal damage stimulates APP expression in adult brain and suggest that reactive astrocytes may be a source of the beta-amyloid that forms neuropathological plaques in Alzheimer's disease.

Amyloid↗
PubMed1989

Identification and characterization of calcium-dependent metalloproteases in rat brain.

We have begun to identify and characterize brain protease activities separated by and assayed in substrate-containing polyacrylamide gels. In the present report, we focus on four proteolytic activities identified from rat brain that are dependent on micromolar and millimolar Ca2+ concentrations for activity. In contrast to the previously described Ca2+-dependent neutral cysteine proteases (calpains), all four activities appear to be metalloproteases based on their inhibition by EDTA, EGTA, and 1,10-o-phenanthroline, but not by blockers of serine, cysteine, or aspartic proteases. In the presence of excess Ca2+ and the Zn2+-chelating inhibitor 1,10-o-phenanthroline, activity of the enzymes was reconstituted by addition of lower concentrations of Zn2+, and inhibited by higher Zn2+ concentrations. The four metalloproteases were designated MP-112, MP-92, MP-70, and MP-65 on the basis of their apparent molecular masses in kilodaltons. MP-70, the major activity detected, had an apparent kact for Ca2+ greater than 100 microM versus 10-25 microM for MP-65 and 50-100 microM for MP-92. MP-112 was a minor activity for which Ca2+ activation levels were not determined. MP-112, MP-70, and MP-65 were similar in being most active in the soluble fraction of 7-day neonate forebrain. In contrast, MP-92 activity was highest in the particulate fraction of adult forebrain. About half of the MP-92 activity and lower levels of the other three activities were still detectable in particulate fractions after detergent extraction of membrane, suggesting an association with cytoskeletal or other structural proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

The two major phosphoproteins in growth cones are probably identical to two protein kinase C substrates correlated with persistence of long-term potentiation.

Regulation of neural protein kinase C (PKC) activity appears to directly affect the persistence of long-term potentiation (LTP; Akers and Routtenberg, 1985; Lovinger et al., 1985, 1986, 1987; Routtenberg et al., 1985, 1986; Akers et al., 1986; Linden et al., 1987), a model of neural plasticity (Bliss and Lomo, 1973). In addition, the in vitro phosphorylation of a brain-specific PKC substrate, protein F1 (Mr 47 kDa, pl 4.5), has been directly correlated with persistence of LTP (Lovinger et al., 1986). Because PKC has been implicated in neurite outgrowth and is present at high levels in growth cone-rich areas of fetal brain, we investigated and characterized PKC substrates in a preparation of isolated nerve growth cone fragments from fetal rat brain and compared them with PKC substrates found in adult rat hippocampus. Four major proteins in the growth cone preparation showed endogenous phosphorylation levels at least 10-fold greater than any other phosphoproteins. Three of these 4 phosphoproteins, termed pp40, pp46, and pp80 (Katz et al., 1985), were phosphorylated by exogenous PKC in a dose-dependent manner, indicating that PKC activity might be of particular importance relative to other kinases in growth cone function. The 2 most highly labeled PKC substrates, pp46 and pp80, comigrated on 2-dimensional gels with the adult hippocampal proteins F1 and "80k" (Mr 78-80 kDa, pl 4.0), respectively. In addition, similarities in charge heterogeneity, 2-dimensional phosphopeptide maps, and increased phosphorylation in the presence of exogenous PKC or PKC stimulators suggest that protein F1 and 80k are highly homologous to, if not identical to, pp46 and pp80, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

A selective increase in phosporylation of protein F1, a protein kinase C substrate, directly related to three day growth of long term synaptic enhancement.

Increased in vitro phosphorylation of the 47 kdalton, 4.5 pI protein F1 was observed in dorsal hippocampal tissue from animals exhibiting long term enhancement (LTE) three days after high frequency stimulation of the perforant pathway, as compared to tissue from low frequency stimulated controls or from unoperated animals. The increase in protein F1 phosphorylation was related to LTE rather than simple activation of perforant path-dentate gyrus synapses. This is the first report of a change in brain protein phosphorylation accompanying synaptic enhancement lasting days. The extent of growth of LTE over the three days following stimulation was directly related (r = +0.66, P less than 0.05) to protein F1 phosphorylation. Among the phosphoproteins studied this relationship between LTE and phosphorylation was selective for protein F1. This suggests that protein F1 may regulate growth of synaptic plasticity for at least a three day period. The mechanism for the LTE-related increase in protein F1 phosphorylation has not been established. However, recent evidence from this laboratory indicates: that protein F1 is phosphorylated by the calcium/phospholipid-dependent protein kinase C; and that kinase C is activated 1 h after LTE. Therefore, the increase in protein F1 phosphorylation following LTE may result from long term activation of protein C kinase.

Animals↗
PubMed1979

Clinical implications of sickle-cell trait and glucose-6-phosphate dehydrogenase deficiency in hospitalized black male patients.

To determine whether sickle-cell trait and glucose-6-phosphate dehydrogenase deficiency influence the course and fatality rates of certain diseases requiring hospitalization, especially those associated with thrombotic phenomena, we conducted a co-operative study of 65,154 consecutively admitted, black male patients in 13 Veterans Administration hospitals. The overall frequency of sickle-cell trait was 7.8 per cent and of glucose-6-phosphate dehydrogenase dificiency 11.2 per cent. Both conditions were present in 0.9 per cent of those examined. There were regional, but no age-dependent, differences in the frequency of sickle-cell trait. Sickle-cell trait had no effect on average age at hospitalization or death, overall mortality, length of hospitalization on medical and surgical wards and frequency of any diagnosis, except essential hematuria and pulmonary embolism. Although statistically significant (P less than 0.001), the differences for the latter were small (1.5 per cent of all patients with normal hemoglobin and 2.2 per cent of patients with sickle-cell trait). Glucose-6-phosphate dehydrogenase deficiency had no adverse effect.

Adult↗
PubMed1977

The role of the pharmacist in drug information for the agricultural sector.

Discussion with veterinarians practicing in southeastern Wyoming suggests that increased pharmaceutical services are needed for the agricultural sector. Such services should include more control of veterinary drugs, informational services, and a forum in which to report untoward responses to drugs. Certain educational and organizational requirements for implementing these services are discussed. Recommendations are advanced that: 1)Some veterinary drugs should be subject to sale by prescription only and be more closely controlled by the pharmacist, 2)A specially in veterinary pharmacy should be established, 3)This specialty should be included as an option on state board examinations and required as a qualification for dispensing animal health drugs, 4)Veterinarians should be appointed to state boards of pharmacy, and 5)Programs in continuing education should be developed to facilitate the entry of existing pharmacists into veterinary pharmacy.

Agriculture↗