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C B Caputo

Publications and source records attributed to C B Caputo.

At least 19 recordsLinked to original sources
PubMed1992

Role of tau in the polymerization of peptides from beta-amyloid precursor protein.

The composition of paired helical filaments (PHFs), the intracellular amyloid fibrils that accumulate in the brains of Alzheimer patients, is not completely known. We investigated whether synthetic peptides from beta-amyloid precursor protein (APP) can form PHF-like fibrils. Two peptides formed fibrils morphologically similar to PHFs. The presence of tau protein, a known PHF component, greatly enhanced the numbers of fibrils formed from one peptide, from the C-terminus of APP, and became associated with the fibrils. A tau fragment corresponding to the tubulin-binding region was sufficient to induce fibril formation. Tau did not alter fibril formation by the other peptide, which was from the beta/A4 region of APP. These results raise the possibility that a C-terminal fragment of APP, along with tau, may be involved in PHF formation. Thus the proteolytic processing of APP may generate fragments that contribute to both amyloids and both histopathologic lesions of Alzheimer's disease.

Alzheimer Disease↗
PubMed1992

Association of the carboxy-terminus of beta-amyloid protein precursor with Alzheimer paired helical filaments.

We investigated whether a peptide fragment from the C-terminus of beta-amyloid protein precursor is associated with Alzheimer paired helical filaments (PHFs). Antiserum BR188, to the last 20 amino acids of the precursor, did not cross-react with tau protein, known to be in PHFs. It did react with all five pronase-treated PHF preparations assayed by ELISA and immunogold-labelled the same PHF fibrils that a PHF-specific tau antibody labelled. Neither antibody labelled beta/A4 fibrils. These results suggest that a fragment from the C-terminus of beta-amyloid precursor protein copurifies with pronase-treated PHFs and may play a role in their molecular pathogenesis.

Actin Cytoskeleton↗
PubMed1992

Tau protein induces bundling of microtubules in vitro: comparison of different tau isoforms and a tau protein fragment.

Expression of tau protein in non-neuronal cells can result in a redistribution of the microtubule cytoskeleton into thick bundles of tau-containing microtubules (Lewis et al.: Nature 342:498-505, 1989; Kanai et al.: J Cell Biol 109:1173-1184, 1989). We reconstituted microtubule bundles using purified tubulin and tau in order to study the assembly of these structures. Taxol-stabilized tubulin polymers were incubated with various concentrations of recombinant human tau and examined by electron microscopy. With increasing concentrations of tau 3 (tau isoform containing three microtubule binding domains) or tau 4 (isoform containing four microtubule binding domains) the microtubules changed orientation from a random distribution to loosely and tightly packed parallel arrays and then to thick cables. In contrast, tau 4L, the tau isoform containing four microtubule binding domains plus a 58-amino acid insert near the N-terminus, showed minimal bundling activity. tau 4-induced bundling could be inhibited by the addition of 0.5 M NaCl or 0.4 mM estramustine phosphate, conditions which are known to inhibit tau binding to microtubules. A tau construct that contained only the microtubule binding domains plus 19 amino acids to the C-terminus was fully capable of bundling microtubules. Phosphorylation of tau 3 with cAMP-dependent protein kinase had no effect on its ability to induce microtubule bundling. These results indicate that tau protein is directly capable of bundling microtubules in vitro, and suggests that different tau isoforms differ in their ability to bundle microtubule filaments.

Cytoskeleton↗
PubMed1992

Amyloid-like properties of a synthetic peptide corresponding to the carboxy terminus of beta-amyloid protein precursor.

A synthetic peptide whose sequence corresponds to the 20 carboxy-terminal amino acids of beta-amyloid protein precursor (APP) was found to form fibrils in vitro. These fibrils showed birefringence in polarized light when stained with Congo red, fluoresced when bound with thioflavin S, were resistant to proteases, and had a cross-beta conformation. By contrast, peptides with other sequences from the intracellular domain of APP and a peptide corresponding to this entire domain did not exhibit the full range of beta-amyloid properties. These results suggest that a fragment from the C-terminus of the beta-amyloid protein precursor could bind to intraneuronal paired helical filaments and account for some of its amyloid-like properties.

Amino Acid Sequence↗
PubMed1992

Immunoreactivity of an antibody to a beta/A4 sequence with Alzheimer paired helical filaments and tau protein.

beta/A4, a peptide that forms the extracellular amyloid fibrils of Alzheimer senile plaques, has also been proposed to be a component of Alzheimer paired helical filaments (PHFs). We compared BR88, an antiserum to amino acids 1-12 of beta/A4, with BR126, an antiserum to the sequence SEKLDFKDRVQS in tau protein, since tau protein is the only confirmed component of PHFs. In enzyme-linked immunosorbent assays (ELISAs), both antibodies reacted with pronase-treated PHFs better after PHFs were treated with guanidine. tau protein shares no sequence homology with beta/A4. Nevertheless, BR88 cross-reacted with human recombinant tau isoforms by ELISA and Western blot analysis with potencies comparable to those for anti-tau antibodies. BR88 reacted with a beta/A4 peptide as well on a molar basis as with tau protein and showed some reactivity to the tubulin-binding region of tau protein. In conclusion, the beta/A4 antiserum BR88 cross-reacts with tau protein, possibly explaining its reactivity with PHFs.

Aged↗
PubMed1991

Differences in the abilities of human tau isoforms to promote microtubule assembly.

Three isoforms of human tau protein were compared for their abilities to induce microtubule assembly. The three isoforms, tau 3 (tau containing three microtubule-binding domains), tau 4 (tau containing four microtubule-binding domains) and tau 4L (tau containing four microtubule binding domains plus a 58-amino-acid insert near the N-terminus) were expressed in E. coli and purified using ammonium sulfate precipitation, ion exchange, and size exclusion chromatography. All three isoforms induced microtubule assembly at micromolar concentrations and showed similar critical concentrations for assembly of 0.4-0.45 microM. However, tau 4 induced microtubule formation at a rate five- to tenfold faster than either tau 3 or tau 4L. The rate of microtubule elongation seen with tau 4 was twofold greater than with tau 3 or tau 4L, suggesting that the faster rate of microtubule assembly seen with tau 4 was due, at least in part, to faster elongation. Tau 4 induced a greater number of microtubules to form at steady state than did tau 3 or tau 4L. The microtubules generated with each tau isoform had similar steady-state length distributions and were equally susceptible to cold-induced disassembly. These results indicate that the additional microtubule-binding domain in tau 4 enhances microtubule assembly, while the 58-amino-acid insert negates the stimulatory effect of the fourth microtubule-binding domain.

Chromatography, Gel↗
PubMed1989

Properties of a microtubule-associated cofactor-independent protein kinase from pig brain.

A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures.

Amino Acid Sequence↗
PubMed1989

Properties of several protein kinases that copurify with rat spinal cord neurofilaments.

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.

Affinity Labels↗
PubMed1988

Protease inhibitors decrease rabbit cartilage degradation after meniscectomy.

In vitro proteoglycan (PG) synthesis and release were measured on cartilage removed from rabbit knees within 1 week of meniscectomy. Three days following partial lateral meniscectomy, 72% of the femurs and 82% of the tibias had visible ulcers. Cartilage from the weight-bearing areas incorporated 2.0-2.9 times more 35S-sulfate in vitro than cartilage from the opposite, unoperated knees. 3H-thymidine incorporation was 2.5-3.4 times higher for surgical than control groups. 35S-sulfate incorporation by the surgical group was inhibited by 22% in the presence of 10(-4) M U24522, an inhibitor of rabbit chondrocyte metalloprotease (CMP). 3H-thymidine incorporation by the surgical group was inhibited by 28% by 10(-4) M U24522. In vitro PG release from cartilage removed 2 days after surgery was 1.6-3.7 times higher for the surgical than the control group. PG release by the surgical group after 22 h of incubation was reduced to the control level by three CMP inhibitors, U24278, U24279, and U24522. PG release by cartilage from the nonsurgical group was also reduced by these compounds at 22 h. These results suggest that both the anabolic and catabolic processes that are stimulated by surgery can be isolated in vitro and that CMP may be involved in the catabolic process.

Animals↗
PubMed1988

Degradation of rat chondrosarcoma proteoglycans by a neutral metalloprotease from rabbit chondrocytes.

Rat chondrosarcoma proteoglycan aggregate with radiolabeled core protein was digested with a chondrocyte metalloprotease (CMP) or clostripain (CP) at neutral pH. The rates of product formation and the sizes and antigenicities of the products were studied using column chromatography and monoclonal antibodies. Sixteen percent of [35S]methionine label and 17-18% of [3H]serine label in core protein were freed from glycosaminoglycan bound peptides by 50 U/ml (760 micrograms/ml) of CP or 10 micrograms/ml (estimated) of CMP in 180 min. The CP reaction was almost complete at 5 minutes while the CMP reaction proceeded slowly from 5 to 180 min. The chondroitin-sulfate rich fragments were smaller after CP than CMP treatment. The 180 min CMP digest contained protein that migrated in 2 peaks on Sepharose CL6B. These two peaks corresponded to the peaks where hyaluronic acid binding region produced by CP and link protein migrate. Metalloenzyme inhibitors inhibited CMP with IC50s of 5 x 10(-5)M, 1 x 10(-3)M, and 80 micrograms/ml for phenanthroline, EDTA, and alpha 2-macroglobulin, respectively.

Animals↗
PubMed1987

Proteoglycan degradation by a chondrocyte metalloprotease. Effects of synthetic protease inhibitors.

Synthetic inhibitors of a chondrocyte metalloprotease (CMP) were assessed for potency. Proteoglycan core protein was used as substrate. The IC50 values were between 2 X 10(-6) and 7 X 10(-6) M for two types of inhibitors, thiol tripeptides and N-carboxyalkyl peptides. Hydroxamic acid peptides were more potent, with IC50 values of 3.2 X 10(-8) to 6.0 X 10(-8) M. These results confirm inhibitory concentrations reported using a proteoglycan-polyacrylamide bead assay. The slopes of the dose-response curves for the thiol compounds were steeper than the slopes for the other two types of compounds. All of the culture media tested inhibited CMP to some extent. Some media also interfered with inhibitor activity. In Ham's F10 nutrient medium, minimum CMP inhibition occurred, and all four hydroxamic acid peptides retained their activity for 1-2 days at 37 degrees. One thiol peptide compound assayed lost activity in 1 hr in thiocyanate-treated serum. All four hydroxamic acid peptides assayed retained activity in thiocyanate-treated serum after 3 days at 37 degrees. The hydroxamic acid peptides may provide a way to block endogenous CMP activity in vivo and to assess the role of CMP in normal and experimentally altered cartilage. They are more potent than other known CMP inhibitors. They retain activity in culture media and serum conditions used for in vivo and in vitro tests of CMP activity and toxicity.

Animals↗
PubMed1987

Effect of synthetic metalloprotease inhibitors on cartilage autolysis in vitro.

The role of chondrocyte metalloprotease (CMP) in mediating cartilage autolysis was studied. Proteoglycan (PG) release and synthesis by rabbit articular cartilage explants were measured. After a 1-day preculture in control medium, 3.3 X 10(-6) M retinoic acid (RET) treatment for 1 day stimulated PG release several fold. RET also caused a large decrease in PG synthesis that returned to the control level after a 3-day recovery period. The effect on PG synthesis was observed at serum levels of 5 and 0.05%. The effect of RET on PG release required protein synthesis, inasmuch as it was lost in cultures maintained in media without amino acids or in a low volume of media. Interleukin-1 (IL-1) and lipopolysaccharide (LPS) treatment for 2 days also stimulated PG release. More PG was released after RET than after IL-1 or LPS, and only RET produced an effect that was evident by day 1. The amount of CMP that produced the same size effect on PG release as these stimulators was below the detection level of PG protease assays. Three potent CMP inhibitors reduced RET-, IL-1- and LPS-stimulated PG release to control levels. These inhibitors did not block another action of RET on chondrocytes, namely the inhibition of PG synthesis by RET immediately after treatment. The inhibitors did not act by reducing cell viability, because recovery of the rate of PG synthesis 3 days post-treatment occurred in inhibitor-treated cultures. These studies suggest that CMP is involved in cartilage autolysis that is stimulated by RET and IL-1.

Animals↗
PubMed1987

Regrowth of the rabbit meniscus after lateral and medial meniscectomies.

The rate of regrowth of the meniscus was compared after medial and lateral meniscectomies. Articular cartilage ulcers were observed 21 days after lateral meniscectomies and femoral pitting was observed after either procedure. Histologically, lesions were observed only after the lateral procedure. Regrowth of the meniscus was evident within 1 week of surgery as increased 3H-thymidine uptake and increased collagen synthesis by meniscal tissue from both surgical groups. These parameters returned to normal sooner after the medial than the lateral procedure. The excised meniscus was completely replaced by new tissue within 3 weeks of either procedure. In conclusion, the meniscus heals quickly following surgery and the small differences in the rate of regrowth between the two procedures do not seem to be adequate to explain the significant differences in femoral and tibial lesions.

Animals↗
PubMed1986

Lack of response of rat chondrosarcoma chondrocytes to interleukin-1 to produce proteoglycan- and collagen-degrading enzymes.

Chondrocytes obtained from rat articular cartilage and Swarm rat chondrosarcoma were maintained in culture. Human interleukin-1 (IL-1) was added at confluency, and three days later the spent media was evaluated for proteoglycan- and collagen-degrading enzymes. Unlike chondrocytes from articular cartilage from several species, chondrocytes from Swarm rat chondrosarcoma did not respond to IL-1 stimulation. This lack of sensitivity to IL-1 may be a useful biochemical marker to classify various types of chondrosarcomas. However, it appears that the use of chondrosarcoma chondrocyte cultures are not useful as a source for metalloproteinases.

Animals↗
PubMed1976

Failure of estrogens and androgens to inhibit bone resorption in tissue culture.

The effects of estrogens, androgens, and glucocorticoids on bone resorption were compared in organ culture. At a concentration of 10(-5) M, corticosterone, testosterone, dihydrotestosterone, dehydroepiandosterone, estradiol, ethinyl estradiol and estrone did not inhibit the release of 45Ca from 19-day fetal rat long bone shafts in control or para-thyroid hormone (PTH)-treated cultures after 2 or 5 days. Cortisol inhibited both control and PTH-stimulated resorption at 10(-5) M and 10(-6) M. 17beta-Estradiol was inhibitory at 3 X 10(4) M. However, it was less effective than the estrogenically inactive epimer 17alpha-estradiol, in 8 day control cultures or in cultures containing low concentrations of PTH. At 10(-5) m, neither 17beta-estradiol nor 17alpha-estradiol inhibited resorption stimulated by prostaglandin E2 or by osteoclast activating factor.

Androgens↗
PubMedDate not supplied

The amyloid proteins of Alzheimer's disease as potential targets for drug therapy.

Two amyloid proteins accumulate in Alzheimer's disease. These proteins, beta amyloid protein and paired helical filament protein, are present in the hallmark lesions of Alzheimer's disease, neuritic plaques and neurofibrillary tangles. Although the amino acid sequences of these two proteins are likely to be different, they nevertheless share certain physical characteristics which define each as belonging to a common class of proteins, amyloid proteins. Since these proteins are probably important in the pathology of Alzheimer's disease, drugs that prevent their accumulation should have therapeutic utility. Based on the amyloidoses associated with other diseases, three mechanisms for amyloid formation have emerged. These mechanisms form a framework for studying Alzheimer amyloids and designing interventions. One mechanism involves posttranslational events which render a normal protein amyloidogenic. Proteolysis, phosphorylation, glycosylation, and transglutamination may be relevant posttranslational events in Alzheimer's disease. If more conclusive evidence can be generated suggesting that these events are involved in the abnormal formation of amyloid in Alzheimer's disease, then these events will become viable targets for drug therapy. Another mechanism for amyloid formation results from expression of an abnormal gene which, in the case of familial Alzheimer's disease, may be an important etiological component. A third mechanism involves the accumulation of a normal protein to a threshold concentration that spontaneously forms amyloid. An effective therapeutic approach for these last two mechanisms could likely include pharmacological manipulation of gene expression.

Alzheimer Disease↗