Proteolytic cleavage of the six known insulinlike growth factor binding proteins (IGFBPs) is a powerful means of rapid structure and function modification of these important growth-regulatory proteins. Intact IGFBP-4 is a potent inhibitor of IGF action in vitro, and cleavage of IG-FBP-4 has been shown to abolish its ability to inhibit IGF stimulatory effects in a variety of systems, suggesting that IGFBP-4 proteolysis acts as a positive regulator of IGF bioavailability. Here we report the isolation of an IGFdependent IGFBP-4-specific protease from human fibroblastconditioned media and its identification by mass spectrometry microsequencing as pregnancy-associated plasma protein-A (PAPP-A), a protein of unknown function found in high concentrations in the maternal circulation during pregnancy. Antibodies raised against PAPP-A both inhibited and immunodepleted IGFBP-4 protease activity in human fibroblastconditioned media. Moreover, PAPP-A purified from pregnancy sera had IGF-dependent IGFBP-4 protease activity. PAPP-A mRNA was expressed by the human fibroblasts and osteoblasts, and PAPP-A protein was secreted into the culture medium. In conclusion, we have identified an IGF-dependent IGFBP protease and at the same time assigned a function to PAPP-A. This represents an unanticipated union of two areas of research that were not linked in any way before this report.

Soluble ␤-amyloid has been shown to regulate presynaptic Ca 2ϩ and synaptic plasticity. In particular, picomolar ␤-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within ␤-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of ␣-and ␤-secretases, and resident carboxypeptidase. The N-terminal ␤-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length ␤-amyloid, the N-terminal ␤-amyloid fragment is monomeric and nontoxic. In Ca 2ϩ imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal ␤-amyloid fragment proved to be highly potent and more effective than full-length ␤-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal ␤-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length ␤-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal ␤-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal ␤-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal ␤-amyloid fragment may serve as a potent and effective endogenous neuromodulator.

PAPP-A/proMBP, the complex of pregnancy-associated plasma protein-A (PAPP-A) and the proform of eosinophil major basic protein (proMBP), circulates at increasing levels during pregnancy. The major site of synthesis is the placenta, in which PAPP-A mRNA has been localized to the syncytiotrophoblast and the placental X cells, whereas proMBP mRNA has been localized to the placental X cells only. The function of PAPP-A/proMBP and its components has remained speculative for years. Recently, however, it has been shown that PAPP-A specifically cleaves insulin-like growth factor (IGF) binding protein-4 in an IGF-dependent manner. Female reproductive and nonreproductive tissues have previously been reported to contain PAPP-A immunoreactivity, based on studies using preparations of anti(PAPP-A/proMBP), now known to recognize both PAPP-A and proMBP, and other irrelevant antigens. To analyze for the presence of PAPP-A and proMBP mRNA, a sensitive semiquantitative reverse transcription (RT) polymerase chain reaction (PCR) method was developed. Reverse-transcribed poly(A)(+) RNA was used as a template in a competitive PCR. PAPP-A and proMBP mRNA levels were normalized against the level of beta-actin mRNA. Both mRNA species were significantly more abundant in term placenta than in other tissues analyzed. All analyzed tissues, including endometrium, myometrium, colon, and kidney, contained both PAPP-A and proMBP mRNA.

Plasma membranes of rat liver isolated by aqueous two-phase partition exhibited basal levels of NADH oxidase activity that were increased approx. 2-fold by addition of hormones and growth factors to which liver cells were known to respond. In contrast, hepatoma plasma membranes demonstrated an intrinsically increased level of NADH oxidase, which was not stimulated further by addition of growth factors. The results suggest that the NADH oxidase of the hepatoma plasma membrane is no longer correctly coupled to hormone and growth-factor receptors. This biochemical defect may parallel the loss of growth control that is characteristic of neoplastic transformation in hepatocarcinogenesis and other transformation systems. INTRODUCTIONAn NADH oxidase activity (transfer of electrons from NADH to oxygen) of plasma membranes purified from rat liver was described that was stimulated by the growth factor transferrin [1]. The stimulated activity was not inhibited by cyanide and was not seen in plasma membranes prepared from hyperplastic nodules from livers of animals given the hepatocarcinogen 2-acetylaminofluorene, nor was it due to reduction of iron associated with diferric transferrin [1]. In the present study, these findings were extended to a comparison of the effects of several growth factors and hormones to which liver cells are known to respond for both liver plasma membranes and plasma membranes prepared from rat hepatomas carried in syngeneic recipients. The findings show that the cyanide-resistant NADH oxidase was stimulated by several other growth factors in addition to diferric transferrin. Additionally, the ability of the growth factors and hormones to stimulate the NADH oxidase activity was lost in the plasma membranes of hepatomas. The latter exhibited intrinsically higher levels of activity, comparable with the hormonestimulated activity levels for liver plasma membranes. The findings point to a fundamental alteration of the hepatoma plasma membrane involving regulation of the activity of the NADH oxidase, which may be important to the unregulated growth that is characteristic of hepatomas in situ.

High levels (μM) of beta amyloid (Aβ) oligomers are known to trigger neurotoxic effects, leading to synaptic impairment, behavioral deficits, and apoptotic cell death. The hydrophobic C-terminal domain of Aβ, together with sequences critical for oligomer formation, is essential for this neurotoxicity. However, Aβ at low levels (pM-nM) has been shown to function as a positive neuromodulator and this activity resides in the hydrophilic N-terminal domain of Aβ. An N-terminal Aβ fragment (1-15/16), found in cerebrospinal fluid, was also shown to be a highly active neuromodulator and to reverse Aβ-induced impairments of long-term potentiation. Here, we show the impact of this N-terminal Aβ fragment and a shorter hexapeptide core sequence in the Aβ fragment (Aβcore: 10-15) to protect or reverse Aβ-induced neuronal toxicity, fear memory deficits and apoptotic death. The neuroprotective effects of the N-terminal Aβ fragment and Aβcore on Aβ-induced changes in mitochondrial function, oxidative stress, and apoptotic neuronal death were demonstrated via mitochondrial membrane potential, live reactive oxygen species, DNA fragmentation and cell survival assays using a model neuroblastoma cell line (differentiated NG108-15) and mouse hippocampal neuron cultures. The protective action of the N-terminal Aβ fragment and Aβcore against spatial memory processing deficits in amyloid precursor protein/PSEN1 (5XFAD) mice was demonstrated in contextual fear conditioning. Stabilized derivatives of the N-terminal Aβcore were also shown to be fully protective against Aβ-triggered oxidative stress. Together, these findings indicate an endogenous neuroprotective role for the N-terminal Aβ fragment, while active stabilized N-terminal Aβcore derivatives offer the potential for therapeutic application.

No abstract

Conditioned culture media of HeLa S cells contain a soluble NADH oxidase activity inhibited by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N' -(4-chlorophenyl)urea (LY181984) similar to that associated with the outer surface of the plasma membrane. This activity was absent from media in which cells had not been grown and was present in conditioned culture media from which cells had been removed by centrifugation both for serum-containing and serum-free media. The Km with respect to NADH and response to thiol reagents were similar to those of the corresponding activity of the plasma membrane of HeLa cells. The conditioned HeLa culture media bound [3H]LY181984 with high affinity. Both antitumor sulfonylurea-inhibited and -resistant forms of the NADH oxidase were isolated by free-flow electrophoresis. The antitumor sulfonylurea-inhibited activity was purified to apparent homogeneity and was identified with a 33.5 kDa protein with an isoelectric point of about pH 4.5. The 33.5 kDa protein from conditioned HeLa culture medium both bound [3H]LY181984 and retained an LY181984-inhibited NADH oxidase activity. A polyclonal antisera was raised in rabbits to the purified 33.5 kDa constituent from conditioned HeLa culture medium. The antisera blocked the activity of the LY181984-inhibited NADH oxidase activity, immunoprecipitated the activity and reacted with a 33.5 kDa protein on Western blots while preimmune sera did not. Also inhibited and immunoprecipitated was NADH oxidase activity from HeLa plasma membranes. The findings are consistent with the 33.5 kDa drug-inhibited NADH oxidase activity of the culture media being a shed form of the corresponding native 34 kDa antitumor sulfonylurea-inhibited NADH oxidase activity of the HeLa cell plasma membrane.

Variation in pathogenicity of Plasmodiophora brassicae in Australia was studied using the European Clubroot Differential series of brassica hosts. From 41 collections of P. brassicae originating from important vegetable brassica production regions in Victoria, Western Australia, Tasmania, Queensland and New South Wales, 23 triplet codes were generated. These were more similar to populations of P. brassicae reported from the USA than those from Europe. The most common Australian pathotypes had triplet codes of 16/3/12 and 16/3/31 and were each assigned seven times to pathogen collections originating from three states of Australia. Other codes that occurred more than once were