Human P301L-Mutant Tau Expression in Mouse Entorhinal-Hippocampal Network Causes Tau Aggregation and Presynaptic Pathology but No Cognitive Deficits

Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer’s disease (AD). It can occur before significant Aβ deposition and appears to “spread” into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here.     

The Amusic Brain: Lost in Music,but Not in Space

Congenital amusia is a neurogenetic disorder of music processing that is currently ascribed to a deficit in pitch processing. A recent study challenges this view and claims the disorder might arise as a consequence of a general spatial-processing deficit. Here, we assessed spatial processing abilities in two independent samples of individuals with congenital amusia by using line bisection tasks (Experiment 1) and a mental rotation task (Experiment 2). Both amusics and controls showed the classical spatial effects on bisection performance and on mental rotation performance, and amusics and controls did not differ from each other. These results indicate that the neurocognitive impairment of congenital amusia does not affect the processing of space.     

Involvement of phosphatidylinositol and insulin in the coordinate regulation of proteoheparan sulfate metabolism and hepatocyte growth

A rat hepatocyte cell line was cultured in Higuchi's medium with fetal calf serum and insulin and labeled with 35SO2/4-. The cells were treated with a number of ligands to displace the heparan 35SO4 proteoglycan (HSPG) from the pericellular matrix. Maximum release was obtained with D-mannose-6-PO4 (50 mM), D-glucose-6-PO4 (50 mM), myo-inositol-2-PO4 (2-5 mM), myo-inositol hexaphosphate (2-5 mM), and DL-myo-inositol-1-PO4 (1-2 mM). D-myo-Inositol-1,3,4-(PO4)3 (1 mM) and L-myo-inositol-1-PO4 (2 mM) were intermediate in their ability to release the cell surface HSPG, whereas heparin (2 mg/ml), yeast phosphomannan (4 mg/ml), D-xylose-1-PO4 (50 mM), D-glucose-6-SO4 (50 mM), and myo-inositol hexasulfate (5 mM) were ineffective. When 35SO2/4- was added to cell cultures, the total cell surface HSPG increased linearly, but the percentage of the total cell surface [35SO4]HSPG that was released by myo-inositol-PO4 increased with time during the labeling period, reaching a maximum of 65% after 5 h. When cells were labeled for 12 h without insulin in the medium, the maximum amount of cell surface HSPG that was released by myo-inositol-PO4 was reduced to 30%. However, when cells labeled in the absence of insulin were treated with phosphatidylinositol-specific phospholipase C and then myo-inositol-PO4, the release of the cell surface [35SO4]HSPG was increased to 73%. When the [35SO4]HSPG that was released from the cell surface by treatment with myo-inositol-PO4 was added to cultures of unlabeled hepatocytes, it was taken up very rapidly and a portion of the internalized HSPG was converted to free heparan SO4 chains which appeared in the nucleus. Uptake was Ca2+- and Mg2+-independent. The amount of [35SO4]HSPG taken up was markedly reduced when the myo-inositol-PO4-releasable [35SO4]HSPG was pretreated with trypsin, thermolysin, alkaline borohydride, or alkaline phosphatase. When the cells were grown in inositol-deficient medium or in the presence of myo-inositol-PO4, the amount of heparan SO4 found in the nucleus was markedly reduced, and the cells no longer exhibited contact inhibition. These effects of myo-inositol deficiency on the growth and nuclear heparan SO4 were accentuated by addition of LiCl to the cultures to prevent phosphatidylinositol synthesis from the endogenous myo-inositol-PO4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immobilization of cellulolytic and hemicellulolytic enzymes on inorganic supports

Commercial cellulase preparations from Trichoderma viride and Aspergillus niger were immobilized on porous silica glass and ceramics such as alumina and titania with titanium tetrachloride (TiCl(4)) and on their silanized derivatives with glutaraldehyde (GLUT). The amounts of the immobilized enzymes were in the range 10-50 mg/g carrier (dry) depending on the kind of carrier and immobilization method. Their activities toward carboxymethyl cellulose (CMC), xylan, aryl-beta-glucoside, and aryl-beta-xyloside were 3-53% of those of the native enzymes. The optimum pH of the enzymes shifted to the acidic side in most cases, whereas the optimum temperatures were nearly the same as those of native ones. The activity of immobilized enzyme preparations towards CMC did not change significantly during continuous operation over a periods of 60 days. Finally, xylan was hydrolyzed with the immobilized enzymes, and the sugars formed were investigated.     

Genetic characterization of the mei-41 locus in Drosophila melanogaster

Summary The mutagen-sensitive mutant mus(1)104 ^D1 of Drosophila melanogaster maps to a position on the X chromosome very close to the meiotic mutant mei-41 ^D5 . Both mutants have been characterized as mutagen-sensitive and defective in post-replication repair. In the present report we show by complementation studies that mus(1)104 and mus(1)103 are allelic with mei-41. In addition, two reported alleles of mus(1)104 lie between the mei-41 alleles A10 and D5. The size of the mei-41 locus is estimated to be about 0.1 centimorgans (cM). Because several alleles of mei-41 have been shown to reduce recombination and increase meiotic chromosome loss and nondisjunction, mus(1)104 ^D1 females were examined for defects in meiosis. Although there was no evidence for reduced recombination on the second chromosome in homozygous mus(1)104 ^D1 females, heterozygous mus(1)104 ^D1 /mei-41 ^>D5 and mus(1)104 ^D1 /deficiency females showed reduced levels of recombination. However, there was no evidence of an increase in nondijunction in these females.We dedicate this article to the memory of Larry Sandler, who passed away suddenly on February 7, 1987     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Biochemical analysis of the activation of adherent neutrophils in vitro

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.     

Basic studies of cryochemotherapy in a murine tumor system

The combined effect of cryosurgery and anticancer drugs (cryochemotherapy) was studied in an experimental B16 melanoma/BDF1 tumor system. Vascular volume and vascular permeability after cryosurgery of normal skin and the tumor were measured by using 51Cr-labeled red blood cells and 125I-labeled serum albumin. The vascular volume and vascular permeability of both the normal vessels and the tumor vessels greatly increased immediately after cryosurgery, and their vascular volume decreased to less than the normal level within a few hours. However, the tumor vessels showed less dilatation and increase in permeability than the vessels of normal tissue. There was a difference in functional characteristics in response to cryoinjury between the normal vessels and the tumor vessels. The anticancer drugs, peplomycin and adriamycin, were administered intraperitoneally in combination with cryosurgery. When peplomycin was administered 5 min, 1 hr, and 3 hr after cryosurgery, the drug concentration in the frozen tumor was higher than that in the untreated tumor. But when administered 1 hr before cryosurgery, peplomycin was not trapped in the tumor. Trapping of adriamycin was not observed after the same treatment. In cryochemotherapy, it is necessary to administer the appropriate drug at the appropriate time. However, the trapping of the anticancer drug results in a high concentration and lasts for a long time, so that cryochemotherapy is expected to be a new mode of cancer therapy, particularly as a multidisciplinary treatment for cancer.     

The core molecule from type H proteoglycan. Release of mannose-containing oligosaccharides by digestion with N-oligosaccharide glycopeptidase.

Chick-embryo cartilage contains a unique set of proteoglycans. Type H proteoglycan (PG-H) is the most abundant, constituting over 90% of the total cartilage hexuronate. We previously showed that treatment of PG-H with chondroitinase ACII and keratanase yields a protein-enriched core molecule [PG(-CS,KS)] with enzymically modified linkage oligosaccharides of the chondroitin sulphate and keratan sulphate chains. We report here that further treatment of PG(-CS,KS) with pepsin and N-oligosaccharide glycopeptidase (almond glycopeptidase) released four distinct types of mannose-containing oligosaccharide. Two of them were shown to be: (Formula: see text). Of the mannose-containing glycopeptides formed by pepsin digestion, about 40% (as mannose) were resistant to N-oligosaccharide glycopeptidase. Since the resistant fraction was enriched in keratan sulphate remnants, it is suggest that the mannose-containing oligosaccharides in this fraction represent those located in a keratan sulphate-enriched region of PG-H.