Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5.

Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3beta. Here we show that tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus/hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases.

Deep vein thrombosis in lumbar spinal fusion: a prospective study of antiembolic and pneumatic compression stockings.

We prospectively studied the incidence of deep vein thrombosis (DVT) of the thigh in 117 patients having posterior lumbar spinal fusion with instrumentation and bone grafting for degenerative disk disease or spondylolisthesis. Patients with neoplasm, infection, trauma, or history of DVT were excluded. Patients were randomized into two groups. In the operating room, group 1 patients were placed in thigh-high antiembolic compression stockings (TED hose), and group 2 patients were placed in antiembolic stockings and pneumatic compression stockings. In both groups, the stockings were used until discharge. Postoperatively, patients in both groups received 600 mg buffered aspirin twice daily. Comparative analysis of the two groups showed no difference in operative time, blood loss, number of levels of lumbar vertebrae fused, time to mobilization, weight, age, or sex. All patients had duplex scanning of the thigh postoperatively. No patient in the series was observed to have acute DVT by clinical examination or by ultrasonography.

Duration of vecuronium-induced neuromuscular block as a predictor of liver allograft dysfunction.

The major causes of liver graft failure are acute rejection, technical failure, and primary nonfunction (PNF). This study was undertaken to determine whether delayed return of neuromuscular function correlates with allograft primary dysfunction in humans given vecuronium. Twenty-two adult patients undergoing orthotopic liver transplantation were given an initial dose of vecuronium, 0.1 mg/kg intravenously (i.v.). All patients recovered from vecuronium-induced neuromuscular block prior to explantation. No additional neuromuscular blocker was given until the liver graft was implanted and reperfused. Fifteen minutes after reperfusion another 0.1 mg/kg vecuronium was given IV and recovery time from attaining complete neuromuscular block to return of the fourth twitch of a train-of-four was recorded. Patients were divided into three groups according to postoperative liver function. Group I consisted of 17 patients with immediate normal liver graft function. Group II consisted of four patients with primary dysfunction (PDF) [peak aspartate aminotransferase (AST) and alanine aminotransferase (ALT) > 2000 U/L, prothrombin time > 16 s, and poor quality and quantity of bile within 3 days postoperatively] which eventually recovered normal function. Group III consisted of one patient with PNF (uncorrectable coagulopathy, severe metabolic acidosis, rising AST and ALT, and minimal or no bile output), whose graft never recovered. Recovery time in Groups II and III was prolonged compared to Group I (P < 0.05). Recovery time in Group III was prolonged compared to Group II (P < 0.05). A test based on these results using a recovery time of > 135 min as a predictor of PDF has a sensitivity and specificity of 80% and 76%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Applications of capillary electrophoresis in the eye-care pharmaceutical industry.

The speed and resolution of capillary electrophoresis (CE), combined with its low cost and low volume requirements, make it a promising method of separating eye-care pharmaceuticals and analyzing the ionic composition of tears. Capillary zone electrophoresis (CZE) offers linearity, precision, and recovery comparable to high-performance liquid chromatography (HPLC) at a fraction of its cost. In addition, CZE separates and quantitates cocoamphocarboxyglycinate in the presence of a non-ionic fatty acid amide surfactant seven times faster than HPLC. CE easily detects such cations as sodium and potassium in human tears.

Identification of a chymotrypsin-like mast cell protease in rat brain capable of generating the N-terminus of the Alzheimer amyloid beta-protein.

Cleavage after Met596 of the beta-amyloid precursor protein to generate the N-terminus of beta-protein indicates the activity of a protease having chymotrypsin-like specificity. A chymotrypsin-like protease is further implicated in Alzheimer's disease by the increased synthesis of the protease inhibitor alpha 1-antichymotrypsin in pathologically affected brain regions and by the presence in the amyloid deposits of inactivated forms of alpha 1-antichymotrypsin (indicating irreversible binding to a target chymotrypsin-like protease). In the present report, we have purified from rat brain a chymotrypsin-like protease that (a) binds with high affinity to human alpha 1-antichymotrypsin, (b) proteolytically generates a beta-protein-containing C-terminal fragment from full-length recombinant human beta-amyloid precursor protein, and (c) selectively cleaves methoxysucinyl-Glu-Val-Lys-Met- p-nitroanilide (a substrate modeling the protease recognition domain for the beta-protein N-terminal cleavage site). Amino acid sequences of tryptic fragments of the purified rat brain chymotrypsin-like protease indicate an identity with rat mast cell protease I. Moreover, the ontogeny and compartmentalization of rat brain chymotrypsin-like protease are consistent with those of connective tissue-type mast cells in the meningeal and intracortical perivasculature. Because these areas in human brain form extensive beta-amyloid deposits in Alzheimer's disease, Down's syndrome, and hereditary cerebral hemorrhage with amyloidosis of Dutch origin, the present findings suggest that a brain mast cell chymotrypsin-like protease may participate in generating perivascular beta-protein, which ultimately aggregates into beta-amyloid deposits.

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.

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.

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.

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.

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.

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)

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)

Selective decline in protein F1 phosphorylation in hippocampus of senescent rats.

Certain forms of neuronal plasticity have been found to be expressed through alterations in brain protein phosphorylation, and its regulation by protein kinase activity. Of interest in this regard is the possibility that the decline in neuronal plasticity and cognitive function that occurs in advanced age may result in part from altered phosphorylation of specific proteins. As a first attempt to identify age-related changes in phosphoproteins, we assayed in vitro phosphorylation of proteins in hippocampus, cerebellum, entorhinal cortex, and frontal cortex from Fischer-344 rats of 5 months, 11 months, and 25 months of age. Compared to the middle-aged animals, the aged rats showed a selective 46% decline in phosphorylation of the 47 kDa protein (F1) in hippocampus, with no change in the phosphorylation of other proteins measured in this structure. Aged animals also showed decreased phosphorylation relative to young animals. No age-related change was observed in any protein band for the other brain areas examined. Since protein F1 is phosphorylated by protein kinase C (PKC), the cytosolic and membrane distribution of this enzyme was compared across age groups. The activity of PKC in hippocampus did not change across age. The explanation of this age-related decline in protein F1 phosphorylation is likely to be a decline in the substrate protein itself. The results are discussed in terms of protein F1's possible role in age-related decline of hippocampal synaptic plasticity.

Construct validity of the Kaufman Assessment Battery for Children with mildly mentally retarded students.

The construct validity of the Kaufman Assessment Battery for Children (K-ABC) with mildly mentally retarded children was examined. Subjects were given three intelligence tests: the K-ABC, WISC-R, and Stanford Binet, Form L-M. Analysis of variance procedures revealed differences between the K-ABC Mental Processing Composite and the WISC-R Full Scale IQ, whereas the Stanford-Binet IQ was not reliably different from either. Correlational analysis provided supportive evidence that the K-ABC is measuring general intelligence; however, discrepancy between Sequential/Simultaneous scales and intersubtest correlations may not support distinct processing styles for mildly mentally retarded students, although this sample represents a restricted range.

Gradients of protein kinase C substrate phosphorylation in primate visual system peak in visual memory storage areas.

Two protein kinase C (PKC) substrates of 50 and 81 kDa display topographical gradients in 32P-incorporation along the occipitotemporal visual processing pathway in rhesus monkey cerebral cortex. The 50 kDa protein appears to be homologous to protein F1 from rat (47 kDa) on the basis of isoelectric point, two-dimensional phosphopeptide maps, and kinase specificity, while the 81 kDa protein is probably the same as a previously described PKC substrate. The phosphorylation of protein F1 and 81 kDa was significantly higher in temporal regions of the occipitotemporal pathway, which have been implicated in the storage of visual representations, than in occipital regions, which appear to be less important for visual memory functions. These results suggest that the PKC phosphorylation system, which has been related previously to changes in neural plasticity, plays a progressively greater role in later stages of visual processing, and that this role may involve the storage of visual information in inferotemporal cortical areas.

Direct relation of long-term synaptic potentiation to phosphorylation of membrane protein F1, a substrate for membrane protein kinase C.

One hour after long-term potentiation (LTP) in the intact hippocampus, a selective increase in protein F1 in vitro phosphorylation was observed in homogenate prepared from dorsal hippocampus. Protein F1 phosphorylation was directly related to the magnitude and persistence of potentiation. No other phosphoprotein studied exhibited a relationship with synaptic enhancement. Low-frequency, non-potentiating stimulation did not increase protein F1 phosphorylation, and phosphorylation of F1 was not elevated when high-frequency stimulation did not produce potentiation. We also confirmed our earlier demonstration of a similar pattern of results 5 min after LTP. In related work we have previously observed: that protein F1 is a substrate for protein kinase C (PKC); that membrane PKC activity was increased by translocation from the cytosol following LTP; and that membrane PKC activity was directly related to the persistence of enhancement. We therefore predicted in the present study that protein F1 phosphorylation in a dorsal hippocampal membrane fraction would be related to LTP. Hippocampal membrane protein F1 was found to be directly related to both the magnitude and persistence of response enhancement. Thus the molecular events leading to prolonged potentiation may involve increased PKC/protein F1 association. Persistence of potentiation may be related to synaptic growth processes involving the growth-associated function of protein F1.

Effects of cognitive and psychomotor tasks on breath-holding span.

Bartlett (J. Appl. Physiol. 42: 717-721, 1977) demonstrated that a psychomotor task (hand dynamometer) extended breath holds at functional residual capacity (FRC) in a manner comparable to Valsalva and Mueller maneuvers. This led us to the hypothesis that distraction of a subject's attention from dyspneic sensations accounted for Bartlett's findings. This hypothesis was tested by comparing a hand dynamometer task (rubber-bulb squeeze) with a mental performance task (mental arithmetic). Results for tasks performed separately and concurrently in a within-subjects design showed comparable effects, without reaching a FRC breath-hold performance ceiling. Implications of results for effects of nonmechanical and nonchemical factors on respiratory drive are discussed.