Bio-fortification and shelf-life extension of idli batter using curry leaves (Murraya koenigii).

Among several traditional foods of India, idli is one of the most popular and commonly consumed steamed products. A new method of adding Murraya koenigii (curry leaves) to idli batter as a vehicle for fortification and extension of shelf-life has been developed. Dried curry leaves powder was incorporated with other ingredients like rice and dehusked black gram in different proportions to optimize the most palatable formulation. Rate of fermentation and microbial changes in the batter; nutritional qualities, texture and sensory properties of the prepared product were assessed. Incorporation of curry leaves powder (5 %) in idli batter increased the shelf-life and also increased the flavour, texture and appearance of the idli. The calcium content of the prepared idli was 10 times more than that of the control idli, while dietary fiber content increased by 18.6 %. Anti-microbial activity of the curry leaves in idli batter extended the shelf-life from 2 to 5 days when stored at 30 °C.

Properties of exogenously added GPI-anchored proteins following their incorporation into cells.

Isolated glycosylphosphatidylinositol (GPI)-anchored proteins, when added to cells in vitro, incorporate into their surface membranes and, once incorporated, exert their native functions. Virtually any protein of interest, if expressed as a GPI-reanchored derivative, can be modified to acquire this capacity. Such transfer of proteins directly to cells, termed "protein engineering" or "painting" constitutes an alternative to conventional gene transfer for manipulating cell surface composition that has many potential applications. Previous studies with incorporated GPI-anchored proteins have focused almost entirely on their extracellular functions. In this study, biotinylated human erythrocyte (E(hu)) decay accelerating factor, E(hu) acetylcholinesterase, and GPI-reanchored murine B7-1 and B7-2 were used as GPI-anchored reporters to characterize their plasma membrane organization and cell signalling properties following addition to Hela or Chinese hamster ovary cells. For each reporter, three types of cell-association were documented; (1) nonphysiological attachment and/or incomplete insertion, (2) uncomplexed membrane integration, and (3) organization into TX-100-resistant microdomains. Transit from the first two compartments into the third, i.e., microdomains, progressed slowly, continuing even after 24 to 36 h and was associated with the acquisition of cell signalling capacity. All four reporters, incorporated in two different detergents, behaved similarly. When organized in microdomains, caveolin and other GPI proteins co-isolated with the incorporated reporter. These results have implications for protein engineering of cells in general, and in particular, for cells such as modified tumor cell immunogens administered to patients for therapeutic purposes.

Intracellular pathways linking hypoxia to activation of c-fos and AP-1.

Organisms respond to hypoxia through detection of blood oxygen levels by sensors at peripheral chemoreceptors and by receptors in certain key cells of the body. The pathways over which peripheral chemoreceptor signals are transmitted to respiratory muscles are well established. However, the intracellular pathways that transmit hypoxic stimulus to gene activation are just being identified. Using anti-sense c-fos strategy, we have shown that c-fos is essential for the activation of activator protein-1 transcription factor complex (AP-1) and subsequent stimulation of downstream genes such as tyrosine hydroxylase (TH; Mishra et al. 1998). The purpose of the present study was to identify intracellular pathways that link hypoxia to activation of c-fos. The results of the present study show that hypoxia causes Ca2+ influx through L-type voltage gated Ca2+ channels and that hypoxia-induced c-fos gene expression is Ca2+/calmodulin dependent. We also demonstrate that hypoxia activates the extracellular-regulated kinase (ERK) and p38, but not JNK. Further, phosphorylation of ERK is essential for c-fos activation via SRE cis-element. Further characterization of nuclear signalling pathways provides evidence for the involvement of Src, a non receptor protein tyrosine kinase, and Ras, a small G protein, in the hypoxia-induced c-fos gene expression. These results suggest a possible role for non-receptor protein tyrosine kinases in propagating signals from G-protein coupled receptors to the activation of immediate early genes such as c-fos during hypoxia.

Dual influence of nitric oxide on gene regulation during hypoxia.

It is being increasingly recognized that nitric oxide (NO) is associated with many physiological processes, including regulation of gene expression. NO shares certain similarities with molecular oxygen (O2). Previous studies have shown that hypoxia up-regulates c-fos, an immediate early gene, and tyrosine hydroxylase (TH), a late response gene that encodes rate limiting enzyme in catecholamine synthesis. Given the similarities between NO and O2, we hypothesized that NO inhibits hypoxia-induced up-regulation of c-fos and TH. Experiments were performed on rat pheochromocytoma (PC12) cells. c-fos and TH mRNA's were analysed by Northern blot and promoter activities by reporter gene assays, respectively. Hypoxia (1% O2 for 6 h) up-regulated c-fos and TH mRNA and increased c-fos promoter activity. Hypoxia-induced c-fos mRNA expression, and promoter activities were significantly potentiated in presence of spermine nitric oxide (SNO), a NO donor. By contrast, SNO significantly inhibited TH mRNA expression and TH promoter activity during hypoxia. Electrophoretic mobility shift-assay showed increased binding of AP-1 and HIF-1 transcription factors to the TH promoter in cells exposed to hypoxia. SNO abolished the binding of AP-1 and HIF-1 to the TH promoter during hypoxia, suggesting that inhibition of hypoxia-induced TH transcription by NO are due to reduced binding of AP-1 and HIF-1 transcription factors. These result demonstrate that NO has both positive and negative influence on gene regulation by hypoxia and suggest that although NO resembles O2 does not always inhibit gene expression during low oxygen.

L-type Ca(2+) channel activation regulates induction of c-fos transcription by hypoxia.

In the present study we examined the intracellular pathways that link hypoxia to activation of c-fos gene expression. Experiments were performed on rat pheocromocytoma-12 (PC-12) cells. c-fos mRNA and promoter activities were analyzed by RT-PCR and reporter gene assays, respectively. BAPTA, a Ca(2+) chelator, inhibited c-fos mRNA and promoter activation by hypoxia. Nitrendipine, an L-type Ca(2+)-channel blocker, abolished, whereas BAY K 8644, an L-type channel agonist, enhanced c-fos activation by hypoxia. Ca(2+) currents were augmented reversibly by hypoxia, suggesting that Ca(2+) influx mediated by L-type Ca(2+) channels is essential for c-fos activation by hypoxia. We next determined downstream pathways activated by intracellular Ca(2+) concentration. Immunoblot analysis revealed Ca(2+)/calmodulin-dependent kinase II (CaMKII) protein in PC-12 cells and revealed that hypoxia increased the enzyme activity. KN-93, a CaMK inhibitor, blocked CaMKII activation and c-fos promoter stimulation by hypoxia. Ectopic expression of an active mutant of CaMKII (pCaMKII290) stimulated c-fos promoter activity under normoxia. Hypoxia increased phosphorylation of CREB at the serine residue 133 (Ser-133), and KN-93 attenuated this effect. Point mutations at the Ca(2+)/cAMP-responsive cis-element (Ca/CRE) attenuated, whereas point mutations in the serum-responsive cis-element (SRE) abolished transcriptional activation of c-fos by hypoxia. These results demonstrate that c-fos activation by hypoxia involves CaMK activation and CREB phosphorylation at Ser-133 and requires Ca/CRE and SRE. These observations demonstrate that Ca(2+)-dependent signaling pathways play a crucial role in induction of c-fos gene expression, which may underlie long-term adaptive responses to hypoxia.

Blunted respiratory responses to hypoxia in mutant mice deficient in nitric oxide synthase-3.

In the present study, the role of nitric oxide (NO) generated by endothelial nitric oxide synthase (NOS-3) in the control of respiration during hypoxia and hypercapnia was assessed using mutant mice deficient in NOS-3. Experiments were performed on awake and anesthetized mutant and wild-type (WT) control mice. Respiratory responses to 100, 21, and 12% O(2) and 3 and 5% CO(2)-balance O(2) were analyzed. In awake animals, respiration was monitored by body plethysmography along with O(2) consumption (VO(2)) and CO(2) production (VCO(2)). In anesthetized, spontaneously breathing mice, integrated efferent phrenic nerve activity was monitored as an index of neural respiration along with arterial blood pressure and blood gases. Under both experimental conditions, WT mice responded with greater increases in respiration during 12% O(2) than mutant mice. Respiratory responses to hyperoxic hypercapnia were comparable between both groups of mice. Arterial blood gases, changes in blood pressure, VO(2), and VCO(2) during hypoxia were comparable between both groups of mice. Respiratory responses to cyanide and brief hyperoxia were attenuated in mutant compared with WT mice, indicating reduced peripheral chemoreceptor sensitivity. cGMP levels in the brain stem during 12% O(2), taken as an index of NO production, were greater in mutant compared with WT mice. These observations demonstrate that NOS-3 mutant mice exhibit selective blunting of the respiratory responses to hypoxia but not to hypercapnia, which in part is due to reduced peripheral chemosensitivity. These results support the idea that NO generated by NOS-3 is an important physiological modulator of respiration during hypoxia.

Vascular endothelial growth factor in Alzheimer's disease and experimental cerebral ischemia.

Several growth factors have been implicated in the pathogenesis of Alzheimer's disease (AD). We considered whether the vascular endothelial growth factor (VEGF) is involved in the vascular pathology associated with most cases of AD. We observed enhanced VEGF immunoreactivity in clusters of reactive astrocytes in the neocortex of subjects with AD compared to elderly controls. VEGF reactivity was also noted in walls of many large intraparenchymal vessels and diffuse perivascular deposits. In addition, we established that astrocytic and perivascular VEGF reactivity was enhanced in cerebral cortex of rats subjected to cerebral ischemia and to chronic hypoxia; experimental conditions known to be associated with astrogliosis and angiogenesis. We suggest the increased VEGF reactivity, also observed in infarcted human brain tissue, implicates compensatory mechanisms to counter insufficient vascularity or reduced perfusion (oligemia) apparent in AD.

Identification and expression of the Na+/H+ exchanger in mammalian cerebrovascular and choroidal tissues: characterization by amiloride-sensitive [3H]MIA binding and RT-PCR analysis.

We report the initial characterization of [3H]5-(N-methyl-N-isobutyl)amiloride (MIA) binding to the Na+/H+ exchanger (NHE) and expression of its gene in mammalian cerebrovascular, choroidal and neocortical tissues. [3H]MIA bound reversibly to particulate fractions of rat, pig and human cerebral microvessels, choroid plexus and cerebral cortex. Scatchard analyses revealed binding to a single amiloride-sensitive site with dissociation constants (Kd) ranging from 20 to 90 nM for the various tissue preparations. The maximal binding capacities (Bmax) were between 2 to 17 pmol/mg protein and were several-fold greater in cerebral microvessels compared to the cerebral cortex. Amiloride, MIA, 5-(N, N-hexamethylene)amiloride (HMA), 5-(N, N-dimethyl)amiloride (DMA) and 5-(N-methyl-N-isopropyl)amiloride (IPA) variably displaced [3H]MIA binding to the microvessels in the following rank order: MIA>HMA>/=IPA>DMA>amiloride. Benzamil, a potent ligand of the Na+/Ca+ transporter was the least sensitive. These binding results were most compatible with the existence of the amiloride-sensitive NHE type 1 in the brain vascular and choroidal tissues. To substantiate this, we utilized reverse transcription polymerase chain reaction (RT-PCR) techniques to search for NHE-1 mRNA. Using primers corresponding to conserved sequences of the human growth factor-activatable NHE gene, RT-PCR revealed strong expression of NHE-1 mRNA in cerebral microvessels, choroid plexus, pial vessels and vascular smooth muscle cells relative to neocortical tissues from several species including rat, pig, cow, monkey and human subjects. Further confirmation of NHE-1 isoform mRNA expression in the cerebrovascular tissues was obtained by HpaII restriction digestion analysis and by subcloning and sequencing of the PCR amplified products. Our study suggests that mammalian cerebrovascular and choroidal tissues contain high amounts of the ubiquitous amiloride-sensitive [3H]MIA binding proteins consistent with the expression of NHE type 1 mRNA.

Amyloid-beta protein angiopathies masquerading as Alzheimer's disease?

Current evidence from genetic and epidemiological studies supports the view that Alzheimer's disease (AD) is a heterogeneous disorder. While the disease is pathologically defined by the presence of specified lesions in form of amyloid plaques and neurofibrillary tangles within the parenchyma, other features of pathology are often either neglected or considered coincidental. Our studies suggest that cerebrovascular pathology is inherently part of the disorder, which could be an important factor in a cause or effect manner. We have recently identified subjects having died with severe amyloid beta (A beta) protein cerebral amyloid angiopathy (CAA) in the absence of a profound Alzheimer pathology. These subjects, diagnosed with dementia had a late onset disease and were found at autopsy to exhibit severe CAA but paucity of typical AD changes. Immunocytochemical studies showed numerous microvascular abnormalities as well as characteristic degeneration of the vascular smooth muscle in both surface and intracortical vessels. The pathology was also characterized by occasional intracerebral hemorrhages and multiple infarcts. Further assessment of the abnormalities and amyloid infiltrated cerebral vessels with antibodies to the carboxyl terminus of A beta indicated that the longer, more pathogenic form of A beta(1-42) was found to be highly associated with intracerebral hemorrhages. Our observations suggest that these mild AD cases with a predominantly vascular pathology are variants of AD and bear resemblance to the familial Dutch and Flemish versions of cerebral amyloidosis. We propose that AD is a group of diseases with a variable pathology analogous to the prion diseases, in which a vascular variant also exists.

Evaluation of risk factors for Alzheimer's disease in elderly east Africans.

A number of biological risk factors have been implicated for Alzheimer's disease (AD). The investigation of prevalence rates of AD in crosscultural populations has much potential in validating these factors. We previously assessed brain amyloid beta (A beta) protein deposition and other lesions associated with AD as possible markers for preclinical AD in elderly nondemented East Africans. In further analysis, we demonstrate that 17-19% of elderly East African subjects without clinical neurological disease exhibited neocortical A beta deposits and minimal neurofibrillary changes at necropsy that was qualitatively and quantitatively similar to that in an age-matched elderly control sample from Cleveland, OH. A beta deposits varied from numerous diffuse to highly localized neuritic plaques and were predominantly reactive for the longer A beta 42 species. In parallel studies, we evaluated another recently implicated factor in AD, the apolipoprotein E genotype. We found relatively high frequencies of the apolipoprotein E-epsilon 4 allele in elderly nondemented East Africans. The frequencies were comparable to those in other African populations but higher than in subjects from developed countries. Our limited study suggests that elderly East Africans acquire cerebral lesions found in AD subjects but the apolipoprotein E-epsilon 4 allele may not be a highly specific factor for the disease among East Africans.

Apolipoprotein E polymorphism in elderly east Africans.

Current advances have shown the apolipoprotein E (APOE)-epsilon 4 allele to be highly associated with late-onset familial and sporadic Alzheimer's disease (AD) in Western populations. The association of APOE allele frequencies and dementia remain unknown in populations from developing countries. We recently initiated a project to examine APOE frequencies in non-demented and demented elderly East Africans. Blood DNA collected from two hospital-based populations showed that the APOE allele frequencies in a group of non-demented 67 Tanzanians over the age of 65 years were found to be 14% for epsilon 2, 61% for epsilon 3 and 25% for epsilon 4. By comparison, the frequency of APOE-epsilon 4 in an age-matched demented group was also 25%. Assessment of APOE genotypes in the group of elderly Kenyan subjects from Nairobi also revealed high frequencies of the epsilon 4 allele with no clear difference in frequency between demented and non-demented subjects. Our preliminary observations suggest that elderly East Africans with no apparent clinical AD possess relatively high APOE-epsilon 4 allele frequencies compared to normal ageing subjects from Western countries including African-Americans. These results appear similar to those reported in a recent study in Nigerian Africans where a lack of correlation between APOE-epsilon 4 allele frequency and Alzheimer type of dementia was noted, and imply that APOE-epsilon 4 allele may not necessarily be a risk factor in some populations of Africa.

Cellular aspects of the inflammatory response in Alzheimer's disease.

Cerebral amyloid beta protein deposition in Alzheimer's disease is associated with a predominantly local acute phase response that kindles release of various inflammatory and immune system mediators. The molecular events are accompanied by a profound cellular response which is largely orchestrated by microglia. Current evidence suggests microglia are primarily involved in phagocytic activity and may be responsible for inducing further neuronal damage by generating reactive oxygen species and proteolytic enzymes. Antiinflammatory measures that target complement activation as well as microglial-mediated oxidative damage would provide rational therapeutic strategies.

Activation of nitric oxide synthase gene expression by hypoxia in central and peripheral neurons.

In the present study we examined the effects of hypobaric hypoxia on neuronal (n) and endothelial (e) nitric oxide synthase (NOS) gene expression in the central and peripheral nervous system. Adult rats were exposed either to normoxia (room air) on to hypobaric hypoxia (0.4 atm) for 4, 12 or 24 h and cerebellum and nodose ganglion representing the central and peripheral neurons, respectively, were removed. Messenger RNAs encoding n- and eNOS as well as beta-actin were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) technique. Hypoxia increased nNOS mRNA expression with maximal changes occurring after 12 h wherein mRNA levels were increased by 10.4 +/- 1.3 and 2 +/- 0.4 fold in nodose ganglion and cerebellum, respectively. Hypoxia, on the other hand, had no significant effect on eNOS and beta-actin mRNA levels. Analysis of nNOS protein and enzyme activity showed near doubling of these variables in both tissues after 24 h of hypoxia, indicating that nNOS protein levels are increased and that the protein is functionally active. These observations demonstrate that 12-24 h of hypobaric hypoxia selectively activates nNOS gene expression, which is reflected in an increase in nNOS protein in central and peripheral neurons. It is suggested that up-regulation of nNOS leads to increased generation of nitric oxide, which in turn may contribute to the readjustments of cardio-respiratory systems during the early stages of chronic hypoxia.

Molecular aspects of inflammatory and immune responses in Alzheimer's disease.

Recent advances indicate numerous molecular and cellular elements of the immune system are involved in the pathogenesis of Alzheimer's disease. Amyloid beta protein deposition induces many molecules associated with a predominantly local inflammatory response within the brain parenchyma. These responses also provoke the release of immune system mediators including cytokines, which all seem largely to be produced by reactive cells such as astrocytes and microglia. Classical acute phase proteins of the pentraxin and serine protease inhibitor (serpin) families as well as a host of complement proteins and some coagulation factor seem the most intrinsically involved. These secreted molecules display variable binding with the amyloidotic lesions. Although our understanding of the molecular specificity and significance of the interaction of these proteins within the lesions is not replete, the development of unique inhibitors of the inflammatory reactions could provide therapeutic strategies to impede the pathogenetic process. Currently, this appears a more viable option than to inhibit amyloid beta production or modify amyloid beta precursor protein processing, an approach which seems more complex.

Apolipoprotein E-epsilon4 alleles in cerebral amyloid angiopathy and cerebrovascular pathology associated with Alzheimer's disease.

The presence of apolipoprotein E-epsilon4 (APOE-epsilon4) allele has been implicated as a risk factor for Alzheimer's disease (AD). We examined the frequencies of APOE-epsilon4 alleles in age-matched controls and subgroups of 190 AD subjects exhibited cerebral amyloid angiopathy (CAA) and other frequently associated lesions. CAA was evident in 96% of the AD subjects, which were divided into two groups, one bearing mild or no apparent CAA and the other with moderate to severe CAA. APOE-epsilon4 allele frequency (48%) in the latter advanced CAA group was six times higher than in those who exhibited mild CAA. In the advanced CAA subjects, the occurrence of an epsilon4 allele was increased by a factor of 17 (95% confidence interval, 7.56 to 38.9). This was despite the fact that neocortical amyloid-beta plaque densities in the two groups were similar and that all of the AD subjects had met the accepted neuropathological criteria. We also observed that the degree of CAA severity was greatest in the group of subjects with the epsilon4/epsilon4 genotype. The association between CAA and APOE-epsilon4 was further implicated in two non-AD subjects among neurological controls with severe CAA. These two subjects, both homozygous for the APOE-epsilon4 allele, were primarily diagnosed as having Creutzfeldt-Jakob disease and Pick's disease in the absence of significant neocortical amyloid deposition. Allele frequency comparisons between neurological control subjects with CAA and those without likewise accorded a strong relationship between the APOE-epsilon4 allele and the presence of CAA. More remarkably, the epsilon4 allele frequency was highly associated with AD subjects exhibiting lobar or intracerebral hemorrhage, all of whom had advanced CAA. We observed that 36% of the AD subjects had concomitant cerebrovascular pathology resulting from single infarcts, multiple microinfarcts, ischemic white matter lesions, or petechial hemorrhages. Although the difference in APOE genotype distribution between subjects with and without cerebrovascular lesions did not reach statistical significance, we did note that the frequency of the epsilon4 allele was significantly higher in subjects with such pathology as compared with those without. However, we found no evidence to suggest that the acquisition of an APOE-epsilon4 allele or one of the alleles, epsilon2 or epsilon3, was a factor in the occurrence of atherosclerosis localized in the basal surface arteries. Analyses of our sample also confirm that there was a lower frequency of the APOE-epsilon2 allele in AD subjects and that the frequency of the epsilon4 allele in AD subjects with concomitant diffuse Lewy body disease was intermediate between controls and AD subjects. Our results suggest that the APOE-epsilon4 allele is a significant factor in the development of CAA in AD and reveal the possibility that APOE is an independent factor in CAA and other vascular abnormalities associated with AD.

The nef gene from a long-term HIV type 1 nonprogressor.

We examined the nef gene of HIV-1 in a long-term nonprogressor to look for evidence suggesting an attenuated virus. The nef gene was previously shown to be required for induction of AIDS. Simian immunodeficiency virus (SIV) deleted in nef, while infectious, fails to sustain the high viral loads necessary for the induction of AIDS in infected adult rhesus monkeys. The human subject of this report was found to harbor virus (HIV-1 Sur25) encoding open-nef reading frames. However, the nef genes of this subject bore a signature point mutation: a cysteine at amino acid 138. The sequence at this position was identical in all clones examined over a 3-year period. When this sequence was compared to the sequence database for AIDS and human retroviruses at Los Alamos, New Mexico, several isolates from other asymptomatic individuals were also found to encode nef genes with a cysteine at position 138. Furthermore, Cys-138 was found in chimpanzee immunodeficiency virus (CIV), a lentivirus that is similar to HIV but does not cause AIDS in chimpanzees. Multiple cysteines are also found in the nef gene of African green monkey virus, SVIagm, including cysteine at the position analogous to Cys-138. While seroprevalence of SIVagm is high in the wild, there is no known disease associated with this virus. The pathogenic virus isolated from Asian macaques, SIVmac, encodes a Nef protein that has few cysteines. Although the virus HIVSur25 encodes a completely open-nef gene, the virus from this individual is similar to attenuated SIVmac (SIVmac239/nef-deletion) as well as HIV deleted in nef in its growth properties in H9 cells. Nef containing a cysteine at position 138 was shown to be responsible for determining the ability to grow in H9.

Apolipoprotein E alleles and brain vascular pathology in Alzheimer's disease.

The presence of apolipoprotein E (ApoE)-E4 allele has been implicated as a risk factor for Alzheimer's disease (AD). We examined the occurrence of ApoE 4 alleles in AD associated with cerebral amyloid angiopathy and other vascular lesions. We found significantly high frequency of the ApoE 4 allele in AD with moderate to severe CAA. The frequency of the allele was also high in AD cases with other vascular lesions such as multiple infarcts and lacunes. As previously reported, we confirm a greater frequency of the ApoE 4 allele in the diffuse Lewy body variant of AD. Our results suggest ApoE 4 allele to be a significant factor in the development of CAA in AD. While this may be related to increased brain amyloid load as a consequence of ApoE genotype, the possibility exists that ApoE may be a specific factor in vascular abnormalities associated with AD.

Altered expression of amyloid beta precursor mRNAs in cerebral vessels, meninges, and choroid plexus in Alzheimer's disease.

Altered tissue-specific processing or production of the amyloid precursor protein (APP) is thought to be central to amyloid deposition in cerebrovascular and the neocortical tissues in Alzheimer's disease (AD). We demonstrate that A beta peptide(s) is readily detectable and increased in cerebral vessels, meninges and choroid plexus obtained at autopsy from AD subjects compared to age-matched controls. Using the reverse transcription (RT)-polymerase chain reaction (PCR), we further found that A beta transcripts encoding the A beta sequence in all forms of APP containing exons 16 and 17 (of APP770) were significantly increased in vessel samples in AD subjects. This was also evident in the neocortical samples and not related to pre-mortem factors or postmortem interval. It is possible that the increased A beta mRNAs reflect enhanced expression of the L-APP isoform (APP770 without exon 15) expressed in leukocytes and glia alike. We also found evidence for changed proportions of APP 770, 756 and 695 mRNAs in cerebral vessel samples from AD subjects compared to controls. Whereas APP770 and APP751, the predominant forms, were significantly decreased, APP695 transcript was increased in vessel samples from AD subjects. Such changes were not evident in neocortical samples from the same subjects. These observations suggest tissue-specific changes in expression of APP isoforms implicating one of the mechanisms for increased accumulation of A beta in cerebrovascular tissues in AD.

Production and increased detection of amyloid beta protein and amyloidogenic fragments in brain microvessels, meningeal vessels and choroid plexus in Alzheimer's disease.

Recent advances indicate soluble amyloid beta (A beta) protein is produced constitutively during normal metabolism of the amyloid precursor protein (APP). This has not been directly examined in human brain vascular tissues. Using a panel of well-characterized antibodies, here we show that increased amounts of soluble A beta were found in isolated vascular tissues from AD subjects compared to age-matched controls without significant Alzheimer pathology. Immunocytochemical analyses of isolated vessel preparations showed characteristic transverse patterns of A beta deposits in large vessels with smooth muscle, however, fine A beta deposits were apparent even in capillaries. A proportion of such A beta protein and potentially amyloidogenic carboxyl terminal fragments were released by solubilization and disruption of the vascular basement membrane by collagenase treatments. We further demonstrated by in vitro metabolic labelling that soluble A beta or an A beta-like peptide is associated and produced by cerebral microvessels, meningeal vessels and the choroid plexus isolated postmortem from human as well as rat brain. Compared to those from young rats, cerebral microvessels from aging rats showed increased release of carboxyl terminal fragments of APP and A beta-like peptide. Our observations provide the first direct demonstration that human vascular tissues produce soluble A beta, a product of the secretory pathway in APP processing. Our findings also suggest that aging associated alterations in the basement membranes are a factor in A beta accumulation that results in vascular amyloid deposition, the principal feature of cerebral amyloid angiopathy.