Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β-precursor protein. In our search for proteases that can generate the N-terminus of amyloid β protein (β-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid β protein, and generates amyloidogenic fragments from full-length recombinant amyloid β-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.     

Molecular Cloning, Expression Pattern, and Chromosomal Localization of the Human Na–Cl Thiazide-Sensitive Cotransporter (SLC12A3)

Electrolyte homeostasis is maintained by several ion transport systems. Na–(K)–Cl cotransporters promote the electrically silent movement of chloride across the membrane in absorptive and secretory epithelia. Two kidney-specific Na–(K)–Cl cotransporter isoforms are known, so far, according to their sensitivity to specific inhibitors. We have cloned the human cDNA coding for the renal Na–Cl cotransporter selectively inhibited by the thiazide class of diuretic agents. The predicted protein sequence of 1021 amino acids (112 kDa) shows a structure common to the other members of the Na–(K)–Cl cotransporter family: a central region harboring 12 transmembrane domains and the 2 intracellular hydrophilic amino and carboxyl termini. The ex- pression pattern of the human Na–Cl thiazide-sensitive cotransporter (hTSC, HGMW-approved symbol SLC12A3) confirms the kidney specificity. hTSC has been mapped to human chromosome 16q13 by fluorescencein situhybridization. The cloning and characterization of hTSC now render it possible to study the involvement of this cotransport system in the pathogenesis of tubulopathies such as Gitelman syndrome.     

Normal rat intestinal cells IEC-18: characterization and transfection with immortalizing oncogenes

IEC-18 cells, a cell line derived from the ileum of rat intestine, have the characteristics of normal cells since they have a contact inhibited cell growth, do not form colonies in soft agar and are not tumorigenic when injected in nude mice. IEC-18 cells were transfected with nuclear oncogenes, c-myc, v-myc and SV40 T antigen in order to obtain immortal cell lines. Independent clones were isolated and characterized for the growth properties. Expression of v-myc altered the morphology of the cells and shortened the doubling time. A slow growth together with a low cloning efficiency was associated with the expression of SV40 T antigen. No changes either in growth or in morphology were observed in c-myc-expressing IEC-18 cells. Expression of these nuclear oncogenes did not result in the neoplastic transformation of the IEC-18 cells, since none of the clones lost the anchorage dependence or were able to form tumors in vivo. The c-myc-containing IEC-18 cells were unable to secrete in the growth medium TGF and exposure to TGF inhibited the growth rate by 30%. All these observations are consistent with the conclusion that the expression of nuclear oncogenes does not lead to the neoplastic transformation of these cells.     

Pigment production from in vitro cultures of Alkanna tinctoria Tausch

Summary An in vitro culture of Alkanna tinctoria Tausch cells was set up in order to investigate the possibility of producing alkannin, a red naphthoquinone naturally present in the root bark of this plant. Furthermore, an in vitro culture of callusderived roots was established and the production of alkannin evaluated. In the different experimental conditions investigated, differences in the production of alkannin derivatives as well as in the type of pigments produced, were observed. The potential use of this technology is discussed.     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

Influence of a menhaden oil diet on cercarial penetration of Schistosoma mansoni.

The ability of a menhaden oil (MO) diet to influence cercarial penetration into mouse tail skin was evaluated. Male CD-1 mice 4-6 wk old (15.2 g average weight) were fed a 0, 10%, or 20% MO-supplemented diet for 2 wk. After this time mice were infected with either 65 +/- 3 or 145 +/- 3 [35S]methionine/cysteine-labeled cercariae for 1 hr by tail immersion. Twenty-four hours and 7 days later groups of mice were killed and their tail skin removed and autoradiographed. At 24 hr postinfection, mice fed a 20% MO diet had significantly higher cercarial penetration than controls and 10% MO diets (56% +/- 5.2 vs. 44% +/- 2.9, P = 0.02, 1-tailed t-test). After 7 days mice fed a 20% MO diet retained more radioactive foci than controls or 10% MO diets (21% +/- 2.0 vs. 15% +/- 1.3, P = 0.01, 1-tailed t-test).     

An elongation factor Tu (EF-Tu) resistant to the EF-Tu inhibitor GE2270 in the producing organism Planobispora rosea

SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner membrane, contains two ATP-binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high-affinity nucleotide-binding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross-linking with [α-³²P]-ATP. This analysis revealed that the nucleotide was cross-linked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP-binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low-affinity ATP-domain showed a normal distribution of cytosolic, peripheral and integral membrane forms. Interestingly, the SecA51(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high-affinity ATP-binding site. These results delimit more precisely the high-affinity ATP-binding domain of SecA, indicate the importance of the early amino-terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de-insertion from, the inner membrane by the activity of the high-affinity ATP-binding domain.     

Immunohistochemical distribution of S-100 protein and type IV collagen in human embryonic and fetal sympathetic neuroblasts

Summary The expression and distribution of S-100 protein and type IV collagen was studied immunohistochemically in sympathetic neuroblasts from the paravertebral region to the adrenal glands in human embryos and fetuses ranging from 7 to 12 weeks gestational age. Prom 7 weeks gestational age, S-100 protein was detected in round or oval cells mingling with sympathetic neuroblasts, and in spindle-shaped cells forming a continuous layer around them. The latter S-100 protein-positive cells were found in contact with the Schwann cells of nerve fibres entering the groups of sympathetic neuroblasts. Staining for type IV collagen showed that all groups of sympathetic neuroblasts were surrounded by a continuous basement membrane. By examining serial sections stained for type IV collagen and S-100 protein, a continuous basement membrane was found along the distribution pattern of the peripheral S-100 protein-positive spindle cells. The morphology of these cells, and their relationships with Schwann cells and with the basement membrane of the sympathetic neuroblasts, indicated that they were Schwann-like cells probably capable of synthesizing a continuous basement membrane separating the neuroblasts from the adjacent tissues. In contrast, the round or oval S-100 protein-positive cells, in contact with the sympathetic neuroblasts and not associated with nerve fibres, were considered as sustentacular or sustentacular precursor cells. At week 7 gestational age, the peri-adrenal sympathetic neuroblasts and their sustentacular and Schwann-like cells started to invade the adrenal glands and mingled with the adrenal cortical cells. These findings suggest the extra-adrenal origin of the sustentacular cells in embryonic and fetal adrenal glands.