Evidence that the rab5 effector APPL1 mediates APP-ßCTF-induced dysfunction of endosomes in Down syndrome and Alzheimer's disease.

ß-Amyloid precursor protein (APP) and its cleaved products are strongly implicated in Alzheimer's disease (AD). Endosomes are highly active APP processing sites, and endosome anomalies associated with upregulated expression of early endosomal regulator, rab5, are the earliest known disease-specific neuronal response in AD. Here, we show that the rab5 effector APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif) mediates rab5 overactivation in Down syndrome (DS) and AD, which is caused by elevated levels of the ß-cleaved carboxy-terminal fragment of APP (ßCTF). ßCTF recruits APPL1 to rab5 endosomes, where it stabilizes active GTP-rab5, leading to pathologically accelerated endocytosis, endosome swelling and selectively impaired axonal transport of rab5 endosomes. In DS fibroblasts, APPL1 knockdown corrects these endosomal anomalies. ßCTF levels are also elevated in AD brain, which is accompanied by abnormally high recruitment of APPL1 to rab5 endosomes as seen in DS fibroblasts. These studies indicate that persistent rab5 overactivation through ßCTF-APPL1 interactions constitutes a novel APP-dependent pathogenic pathway in AD.

Neurofilament subunits are integral components of synapses and modulate neurotransmission and behavior in vivo.

Synaptic roles for neurofilament (NF) proteins have rarely been considered. Here, we establish all four NF subunits as integral resident proteins of synapses. Compared with the population in axons, NF subunits isolated from synapses have distinctive stoichiometry and phosphorylation state, and respond differently to perturbations in vivo. Completely eliminating NF proteins from brain by genetically deleting three subunits (α-internexin, NFH and NFL) markedly depresses hippocampal long-term potentiation induction without detectably altering synapse morphology. Deletion of NFM in mice, but not the deletion of any other NF subunit, amplifies dopamine D1-receptor-mediated motor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to plasma membrane, consistent with a specific modulatory role of NFM in D1-receptor recycling. These results identify a distinct pool of synaptic NF subunits and establish their key role in neurotransmission in vivo, suggesting potential novel influences of NF proteins in psychiatric as well as neurological states.

(2E)-2-Benzyl-idene-9-phenyl-3,4-di-hydro-acridin-1(2H)-one.

In the title compound, C26H19NO, the plane of the aromatic heterocycle makes a dihedral angle of 75.22 (4)° with that of the attached phenyl ring. In the crystal, mol-ecules are connected by C-H⋯O inter-actions, generating R 2 (2)(12) dimers. These dimers are further connected by C-H⋯π inter-actions, linking the mol-ecules into chains running along the a-axis direction.

(1R*,3'S*,4'R*)-4'-(4-Chloro-phen-yl)-3'-[(4-hy-droxy-2-oxo-1,2-dihydro-quinolin-3-yl)carbon-yl]-1'-methyl-spiro-[ace-naphthyl-ene-1,2'-pyrrolidin]-2-one.

The title compound, C(32)H(23)ClN(2)O(4), has a quinoline, a chloro-phenyl and an acenaphthalene ring system attached to a central pyrrolidine ring, which has three stereogenic centers. Nevertheless, the compound crystallizes as a racemate with two mol-ecules of identical chirality in the asymmetric unit. They differ in the conformation of the five-membered pyrrolidine ring; in one molecule it has an envelope conformation, while in the other molecule it has a twisted conformation. In each molecule there is an intra-molecular O-H⋯O hydrogen bond making an S(6) ring motif. In the crystal, pairs of N-H⋯O hydrogen bonds produce inversion dimers with R(2) (2)(8) motifs. There are also C-H⋯O interactions present. The crystal structure contains voids (60 Å(3)) within which there is no evidence of solvent mol-ecules.

4-Hydr-oxy-3-(1'-methyl-2-oxo-4'-phenyl-spiro-[indoline-3,2'-pyrrolidine]-3'-yl-carbon-yl)quinolin-2(1H)-one.

In the title compound, C(28)H(23)N(3)O(4), the dihedral angle between the quinoline and indole ring systems is 29.30 (5)°. The pyrrolidine ring adopts a twist conformation. An intra-molecular O-H⋯O hydrogen bond generates an S(6) ring motif. A weak intra-molecular C3-H3⋯O3 inter-action is also observed. In the crystal, mol-ecules are linked by two sets of N-H⋯O hydrogen bonds, forming centrosymmetric dimers containing two R(2) (2)(8) ring motifs. The dimers are linked via C-H⋯π inter-actions.

2-Meth-oxy-quinoline-3-carbaldehyde.

In the title compound, C(11)H(9)NO(2), the quinoline ring system is essentially planar (r.m.s. deviation = 0.005 Å) and the meth-oxy and aldehyde groups are almost coplanar with it [N-C-O-C = 6.24 (19) and O-C-C-C = 0.3 (2)°]. In the crystal, mol-ecules are linked by pairs of C-H⋯O hydrogen bonds, forming centrosymmetric R(2) (2)(10) dimers. The dimers are linked via π-π inter-actions involving the pyridine and benzene rings [centroid-centroid distance = 3.639 (1) Å].

8-Meth-oxy-3,3,5-trimethyl-3,11-dihydro-pyrano[3,2-a]carbazole.

In the title compound, C(19)H(19)NO(2), commonly called koenimbine, the pyran ring adopts a sofa conformation. The carbazole ring system is planar [r.m.s. deviation = 0.063 (1) Å]. A C(10) zigzag chain running along the b axis is formed through inter-molecular C-H⋯O hydrogen bonds. The chains are linked via weak C-H⋯π and N-H⋯π inter-actions.

Association of Multifaceted Mobile Technology-Enabled Primary Care Intervention With Cardiovascular Disease Risk Management in Rural Indonesia.

Importance: Cardiovascular diseases (CVDs) are the leading cause of disease burden in Indonesia. Implementation of effective interventions for CVD prevention is limited. Objective: To evaluate whether a mobile technology-supported primary health care intervention, compared with usual care, would improve the use of preventive drug treatment among people in rural Indonesia with a high risk of CVD. Design, Setting, and Participants: A quasi-experimental study involving 6579 high-risk individuals in 4 intervention and 4 control villages in Malang district, Indonesia, was conducted between August 16, 2016, and March 31, 2018. Median duration of follow-up was 12.2 months. Residents 40 years or older were invited to participate. Those with high estimated 10-year risk of CVD risk (previously diagnosed CVD, systolic blood pressure [BP] >160 mm Hg or diastolic BP >100 mm Hg, 10-year estimated CVD risk of 30% or more, or 10-year estimated CVD risk of 20%-29% and a systolic BP >140 mm Hg) were followed up. Interventions: A multifaceted mobile technology-supported intervention facilitating community-based CVD risk screening with referral, tailored clinical decision support for drug prescription, and patient follow-up. Main Outcomes and Measures: The primary outcome was the proportion of individuals taking appropriate preventive CVD medications, defined as at least 1 BP-lowering drug and a statin for all high-risk individuals, and an antiplatelet drug for those with prior diagnosed CVD. Secondary outcomes included mean change in BP from baseline. Results: Among 22 635 adults, 3494 of 11 647 in the intervention villages (30.0%; 2166 women and 1328 men; mean [SD] age, 58.3 [10.9] years) and 3085 of 10 988 in the control villages (28.1%; 1838 women and 1247 men; mean [SD] age, 59.0 [11.5] years) had high estimated risk of CVD. Of these, follow-up was completed in 2632 individuals (75.3%) from intervention villages and 2429 individuals (78.7%) from control villages. At follow-up, 409 high-risk individuals in intervention villages (15.5%) were taking appropriate preventive CVD medications, compared with 25 (1.0%) in control villages (adjusted risk difference, 14.1%; 95% CI, 12.7%-15.6%). This difference was driven by higher use of BP-lowering medication in those in the intervention villages (1495 [56.8%] vs 382 [15.7%]; adjusted risk difference, 39.4%; 95% CI, 37.0%-41.7%). The adjusted mean difference in change in systolic BP from baseline was -8.3 mm Hg (95% CI, -10.1 to -6.6 mm Hg). Conclusions and Relevance: This study found that a multifaceted mobile technology-supported primary health care intervention was associated with greater use of preventive CVD medication and lower BP levels among high-risk individuals in a rural Indonesian population.

Synthesis, antimicrobial activities and cytogenetic studies of newer diazepino quinoline derivatives via Vilsmeier-Haack reaction.

The study of the Vilsmeier-Haack reagent on 4-hydroxyquinaldines resulted in a new versatile intermediate 4-chloro-3-formyl-2-(2-hydroxy-ethene-1-yl)quinolines, which on further treatment with hydrazine hydrate yielded the desired diazepino quinoline derivatives. All the synthesized diazepino quinoline derivatives are screened for their antibacterial and antifungal activities. Cytogenetic analysis of the samples is also reported.

Substitutional impact on biological activity of new water soluble Ni(II) complexes: Preparation, spectral characterization, X-ray crystallography, DNA/protein binding, antibacterial activity and in vitro cytotoxicity.

A series of new water soluble nickel(II) complexes containing triphenylphosphine and 4-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones were synthesized and characterized. Crystallographic investigations confirmed the structure of the complexes (1-4) having the general structure [Ni(4-Msal-Rtsc)(PPh3)] (Where R=H (1); CH3 (2); C2H5 (3); C6H5 (4)) which showed that thiosemicarbazone ligands coordinated to nickel(II) ion as ONS tridentate bibasic donor. DNA/BSA protein binding ability of the ligands and their new complexes were studied by taking calf-thymus DNA (CT-DNA) and Bovine serum albumin (BSA) through absorption and emission titrations. Ethidium bromide (EB) displacement study showed the intercalative binding trend of the complexes to DNA. From the albumin binding studies, the mechanism of quenching was found as static and the alterations in the secondary structure of BSA by the compounds were confirmed with synchronous spectral studies. The binding affinity of the complexes to CT-DNA and BSA has the order of [Ni(4-Msal-etsc)(PPh3)] (3) >[Ni(4-Msal-mtsc)(PPh3)] (2) >[Ni(4-Msal-tsc)(PPh3)] (1) >[Ni(4-Msal-ptsc)(PPh3)] (4). In vitro cytotoxicity of the complexes was tested on human lung cancer cells (A549), human cervical cancer cells (HeLa), human liver carcinoma cells (Hep G2). All the complexes exhibited significant activity against three cancer cells. Among them, complex 4 exhibited almost 2.5 fold activity than cisplatin in A549 and HepG2 cell lines. In HeLa cell line, the complexes exhibited significant activity which is less than cisplatin. While comparing the activity of the complexes in A549 and HepG2 cell lines it falls in the order 4>1>2>3>cisplatin. The results obtained from DNA, protein binding and cytotoxicity studies, it is concluded that the cytotoxicity of the complexes as determined by MTT assay were not unduly influenced by the complexes having different binding efficiency with DNA and protein. The complexes exhibited good spectrum of antibacterial activity against four pathogenic bacteria such as E. faecalis (gram +ve), S. aureus (gram +ve), E. coli (gram -ve) and P. aeruginosa (gram -ve).

Occurrence of type VI collagen in extracellular matrix of renal glomeruli and its increase in diabetes.

Human and bovine glomerular basement membrane (GBM) preparations, representing the extracellular matrix of the renal filtration units, were found to contain type VI collagen. This protein was solubilized by guanidine and guanidine-dithiothreitol extractions and characterized after polyacrylamide gel electrophoretic resolution by immunoblotting with an antiserum directed against the alpha 1(VI) and alpha 2(VI) polypeptide chains and by its insensitivity to collagenase digestion in the nonreduced state. In contrast to GBM, which is the product of three distinct cells, type VI collagen could not be detected in extracts from calf lens capsule, an epithelial cell-derived basement membrane. Quantitation by radioimmunoassay of the type VI collagen content of GBM from 17 diabetic and 15 nondiabetic human subjects indicated a 2.8-fold higher level (P less than 0.001) in the diabetic preparations. Because in the glomerulus type VI collagen is considered on the basis of immunohistochemistry to be localized to the mesangium, we believe that measurement of this protein in GBM preparations can provide a valuable index of mesangial expansion in diabetic and other glomerulopathies.

Proteolysis of protein kinase C: mM and microM calcium-requiring calpains have different abilities to generate, and degrade the free catalytic subunit, protein kinase M.

Limited proteolysis of protein kinase C (PKC) by calpain under cell free conditions cleaves the regulatory and catalytic PKC subunits, generating a free, co-factor independent catalytic subunit, termed PKM. In the present study, we demonstrate distinct differences in the rate, nature, and lipid-sensitivity of PKC and PKM proteolysis by microM and mM calcium-requiring calpain isozymes (mu calpain or m calpain, respectively). PKC is a preferred substrate for m calpain; not even a 100-fold increase in mu calpain was capable of degrading PKC as fast as in calpain. PKM was generated by both m and mu calpains, but was itself rapidly degraded by m calpain and therefore was only transiently detectable. By contrast, PKM was formed but not degraded by mu calpain, and persisted in the presence of mu calpain long after all PKC had been degraded. Phosphatidyl serine (PS) inhibited PKC hydrolysis by m calpain yet enhanced PKC hydrolysis by mu calpain. The ability of either calpain isoenzyme to degrade [14C]azocasein was unaffected by PS, suggesting that the influence of PS was on PKC conformation. These findings point towards distinct roles for mu and m calpain in PKC regulation.

Up-regulation of the lysosomal system in experimental models of neuronal injury: implications for Alzheimer's disease.

Previous studies established that the populations of neurons that frequently degenerate in Alzheimer's disease exhibit robust up-regulation of the lysosomal system. In this study, we investigated alterations of the lysosomal system during different forms of experimental injury in rat hippocampal neurons in culture, utilizing a combination of immunocytochemical and biochemical methods. Using triple-label immnocytochemistry for activated caspase-3, fragmentation of DNA and the microtubule-associated protein-2, we characterized treatment paradigms as models of the apoptotic (staurosporine, camptothecin), the oncotic (high-dose menadione, glutamate), and the mixed apoptotic and oncotic (low-dose menadione) pathways of neuronal death. Slowly developing apoptotic or slowly developing mixed apoptotic and oncotic forms of neuronal injury were associated with substantial increases in the number and size of cathepsin D-positive vesicles (late endosomes and mature lysosomes) as determined by immunocytochemistry, and elevated levels of cathepsin D by western blotting. In agreement with our previous findings in Alzheimer's disease, where lysosomal system activation was not restricted to overtly degenerating neurons, up-regulation of this system was also detected quite early during the course of experimental neuronal injury, preceding the development of dystrophic neurites, nuclear segmentation or fragmentation of DNA. These findings implicate lysosomal system activation, both in Alzheimer's disease and in experimental models of neuronal injury, as an important event associated with early stages of neurodegeneration.

Limited proteolytic processing of the mature form of cathepsin D in human and mouse brain: postmortem stability of enzyme structure and activity.

The mature form of cathepsin D (Cat D), purified to homogeneity from postmortem human brain or mouse brain, behaved as a 42-kDa protein in its native state but revealed additional proteolytic processing under denaturing conditions. Human brain Cat D was composed of a 30-32 kDa heavy chain and a protein doublet consisting of 14 and 15 kDa light chains. Mouse Cat D, which closely resembled the human enzyme in amino acid composition, existed mainly as the uncleaved 42-kDa protein, but up to 40% existed as a complex of 30-32 kDa and 12-14 kDa chains. The 3:1 ratio of light to heavy (30-32 kDa) chains suggested processing of some 30-kDa chains. Cleavage of the 42-kDa chain could not be induced autolytically. Human brain Cat D had a 2-3-fold higher specific activity than the mouse enzyme but shared other properties, including similar biphasic pH optima (peaks at pH 3.30 and 4.2), Km values for methemoglobin and inhibitor profiles. Human Cat D displayed the same polypeptide chain composition when purified from brains differing in postmortem interval (3-28 h). Fresh SH-SY5Y human neuroblastoma cells analyzed on Western blots with anti-Cat D antibodies also displayed only cleaved forms of mature Cat D. Furthermore, brain Cat D isolated from mice stored after death for 5, 15 or 30 h at 25 degrees C contained the same molar ratios of cleaved and uncleaved enzyme found in fresh mouse brain . Cat D activity was stable in human brains with postmortem intervals up to 27 h and stored frozen for up to 3 years. Similarly, total Cat D activity was essentially unchanged in brains of mice subjected to stimulated postmortem conditions for 0.5-4.2 h, although 20% of the total soluble brain protein became insoluble during this postmortem interval. These results demonstrate a remarkable postmortem stability of Cat D and strongly suggest that limited proteolytic cleavage of mature brain Cat D is an in vivo event, the extent of which varies markedly in different species.

Calcium-activated neutral proteinase (calpain) system in aging and Alzheimer's disease.

Calpains (CANPs) are a family of calcium-dependent cysteine proteases under complex cellular regulation. By making selective limited proteolytic cleavages, they activate or alter the regulation of certain enzymes, including key protein kinases and phosphatases, and induce specific cytoskeletal rearrangements, accounting for their suspected involvement in intracellular signaling, vesicular trafficking, and structural stabilization. Calpain activity has been implicated in various aging phenomena, including cataract formation and erythrocyte senescence. Abnormal activation of the large stores of latent calpain in neurons induces cell injury and is believed to underlie neurodegeneration in excitotoxicity, Wallerian degeneration, and certain other neuropathologic states involving abnormal calcium influx. In Alzheimer's disease, we found the ratio of activated calpain I to its latent precursor isoform in neocortex to be threefold higher than that in normal individuals and those with Huntington's or Parkinson's disease. Immunoreactivity toward calpastatin, the endogenous inhibitor of calpain, was also markedly reduced in layers II-V of the neocortex in Alzheimer's disease. The excessive calpain system activation suggested by these findings represents a potential molecular basis for synaptic loss and neuronal cell death in the brain in Alzheimer's disease given the known destructive actions of calpain I and its preferential neuronal and synaptic localization. In surviving cells, persistent calpain activation may also contribute to neurofibrillary pathology and abnormal amyloid precursor protein trafficking/processing through its known actions on protein kinases and the membrane skeleton. The degree of abnormal calpain activation in the brain in Alzheimer's disease strongly correlated with the extent of decline in levels of secreted amyloid precursor protein in brain. Cytoskeletal proteins that are normally good calpain substrates become relatively calpain resistant when they are hyperphosphorylated, which may contribute to their accumulation in neurofibrillary tangles. As a major effector of calcium signals, calpain activity may mirror disturbances in calcium homeostasis and mediate important pathologic consequences of such disturbances.

Plasma somatomedin activity in vitamin A deficient children.

A role for vitamin A in the sulphation of mucopolysaccharides has been suggested. Somatomedin, a growth hormone dependent serum factor, has also been shown to stimulate the uptake of sulphate by cartilage. Therefore studies were undertaken on vitamin A deficient children to examine the possible interrelationship between vitamin A and somatomedin activity. Plasma somatomedin activity was markedly lowered in vitamin A deficient children and plasma HGH levels were in the normal range. The data suggest that vitamin A and somatomedin may be interrelated and also that plasma somatomedin activity may not always be determined by plasma growth hormone levels.

Antibacterial activity of Mappia foetida leaves and stem.

Successive petroleum ether, chloroform and methanol extracts of Mappia foetida leaves and stem were tested for their antibacterial activity. The methanol fractions were found to be most effective against all the tested organisms.

Placental pathology in pre-eclampsia eclampsia syndrome.

Quantitative analysis of placental pathology was carried out on 20 placentae from various grades of pre-eclampsia eclampsia syndrome and 20 placentae from control group. Placental weights were lower in the study group. The gross abnormalities noted were the placental infarcts, retroplacental haematoma and calcification. The striking villous lesions observed in the study group were cytotrophoblastic cell proliferation, thickening of villous basement membrane and paucity of vasculosyncytial membrane and these findings correlated well with the severity of maternal disease. These vascular villous lesions were considered secondary to uteroplacental ischaemia.