Syntheses of p-aminophenyl α-d-talopyranoside and 1-thio α-d-talopyranoside as analogs of glycosidase substrates

p-Nitrophenyl and p-aminophenyl α-d-talopyranoside and 1-thio-α-d-talopyranosides were prepared for studies on specificity of glycosidases. Reaction of α-d-talopyranose pentaacetate with p-nitrophenol gave exclusively p-nitrophenyl 2,3,4,6-tetra-O-acetyl-α-d-talopyranoside (2) in 63% yield. A similar reaction with p-nitrobenzenethiol afforded the 1-thio analog (3) of 2 in 41.8% yield; the p-nitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-d-talopyranoside (6) was also obtained in low yield (6.7%). The two α-d-talosides 2 and 3 were catalytically deacetylated in near-quantitative yields by methanolic sodium methoxide. The p-nitrophenyl α-d-talopyranoside (4) and 1-thio-α-d-talopyranoside (5) were reduced with palladium on barium sulfate catalyst to the corresponding p-aminophenyl talosides. The acetylated p-nitrophenyl d-talosides 2, 3, and 6 were determined, from their 250-MHz n.m.r. spectra, to exist in the ⁴C₁ (d) conformation in chloroform solution.     

Induction of α1-antitrypsin mRNA and cloning of its cDNA

Local inflammation was inflicted in a baboon by turpentine administration in order to induce the plasma level of α1-antitrypsin, an acute phase protein synthesized in the liver. Comparison of the α1-antitrypsin mRNA activity in the induced and non-induced baboon liver indicated that the “acute phase” response to chemical-inflicted inflammation is mediated through an increase in the steady-state level of cellular mRNA. Alpha-1-antitrypsin was then enriched from the induced baboon liver to a purity of greater than 90% by specific immunoprecipitation of polysomes. Double-stranded DNA was synthesized from the enriched mRNA and inserted into the $\text{Pst}$ I site of pBR322. Recombinant clones containing α1-antitrypsin cDNA sequences were identified by hybridselected translation and confirmed by DNA sequence analysis.     

The N-terminal globular domain of the laminin α1 chain binds to α1β1 and α2β1 integrins and to the heparan sulfate-containing domains of perlecan

The N-terminal domains VI plus V (62 kDa) and V alone (43 kDa) of the laminin α1 chain were obtained as recombinant products and shown to be folded into a native form by electron microscopy and immunological assays. Domain VI alone, which corresponds to an LN module, did not represent an autonomously folding unit in mammalian cells, however. Fragment α1VI/V, but not fragment α1V, bound to purified α1β1 and α2β1 integrins, to heparin, and to heparan sulfate-substituted domains I and V of perlecan. This localized the binding activities to the LN module, which contains two basic sequences suitable for heparin interactions.     

Immunolocalization of α1A-adrenoceptors in rat and human epididymis

Immunohistochemistry was conducted to analyze the cellular localization of alpha(1A)-adrenoceptors along rat and human epididymis. ADR-A, a polyclonal antibody that recognizes the specific C-terminal region of alpha(1A)-adrenoceptors, immunostained this adrenoceptor subtype in smooth muscle cells surrounding the epididymal tubules and interstitial blood vessels and in subpopulations of epithelial cells from adult rat and human caput and cauda epididymidis. The same cell types from rat epididymidis were immunostained by ADR-1, a polyclonal antibody that recognizes a common region of the three alpha(1)-adrenoceptor subtypes, alpha(1A), alpha(1B), and alpha(1D). Immunostaining with both antibodies was also conducted in adult rat and human vas deferens and seminal vesicle used as positive controls because of the abundance of alpha(1A)-adrenoceptors in these tissues. ADR-A- and ADR-1-positive immunostaining was differentially distributed depending on the antibody, method of tissue fixation (Bouin-fixed and fresh frozen tissues), species (rat and human), tissue (caput and cauda epididymidis), and age (immature and adult rats) analyzed. This is the first report immunolocalizing alpha(1A)-adrenoceptor along rat and human epididymis. The presence of this adrenoceptor subtype in epididymal smooth muscle and epithelial cells indicates their contribution to smooth muscle contractile responses and a possible role in the absorptive and/or secretory activities of the epithelium lining the epididymal duct. Taken together, our results should contribute to a better understanding of the physiological role of alpha(1)-adrenoceptors in the epididymidis and the importance of the sympathetic nervous system for male (in)fertility.     

Mechanism of the cyanide-catalyzed oxidation of α-ketoaldehydes and α-ketoalcohols

Cyanide catalyzes the reduction of dioxygen or of ferricytochrome c by dihydroxyacetone phosphate. The rapid initial phase of these reactions, but not the subsequent slow phase, was augmented by incubating the triose phosphate aerobically or anaerobically at pH 9.0 prior to adding the cyanide. The aerobic incubation, which was most effective, was associated with a decline in enediol, whereas the less effective anaerobic incubation was accompanied by an increase in enediol content. This suggested that the α-ketoaldehyde product of autoxidation of the enediol, rather than the enediol itself, was responsible for the rapid phase reaction which followed addition of cyanide. This was confirmed by exploring the cyanide-catalyzed oxidation of the α-ketoaldehyde, phenylglyoxal. The inhibitory effect of the manganese-containing superoxide dismutase indicated that O₂⁻ was a kinetically important intermediate of the rapid phase reaction. A reaction mechanism is proposed which is consistent with the results presented.     

Physiological and Pathological Roles of α3β1 Integrin

α3β1 integrin has been considered to be a mysterious adhesion molecule due to the pleiotropy in its ligand-binding specificity. However, recent studies have identified laminin isoforms as high-affinity ligands for this integrin, and demonstrated that α3β1 integrin plays a number of essential roles in development and differentiation, mainly by mediating the establishment and maintenance of epithelial tissues. Furthermore, α3β1 integrin is also implicated in many other biological phenomena, including cell growth and apoptosis, angiogenesis and neural functions. This integrin receptor forms complexes with various other membrane proteins, such as the transmembrane-4 superfamily proteins (tetraspanins), cytoskeletal proteins and signaling molecules. Recently, lines of evidence have been reported showing that complex formation regulates integrin functions in cell adhesion and migration, signal transduction across cell membranes, and cytoskeletal organization. In addition to these roles in physiological processes, α3β1 integrin performs crucial functions in various pathological processes, especially in wound healing, tumor invasion and metastasis, and infection by pathogenic microorganisms.This revised version was published online in June 2005 with a corrected cover date.     

Kinetics of the inhibition of free and elastin-bound human pancreatic elastase by α1-proteinase inhibitor and α2-macroglobulin

At pH 8.0 and 25°C α₁-proteinase inhibitor and α₂-macroglobulin bind human pancreatic elastase with rate constants of 4.7·10⁵ M⁻¹·s⁻¹ and 6.4·10⁶ M⁻¹·s⁻¹, respectively. The corresponding delay times of elastase inhibition in plasma are 0.4 s and 0.2 s, respectively, indicating that both inhibitors may act as physiological antielastases. Elastin impairs the elastase inhibitory capacity of α₁-proteinase inhibitor and α₂-macroglobulin. In presence of human elastin, the former behaves like a slow-binding elastase inhibitor, with a rate constant of about 260 M⁻¹·s⁻¹. In contrast, α₂-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin.     

Locus assignment of human α-globin structural mutants by selective enzymatic amplification of α1 and α2-globin cDNAs

Summary We have used the powerful methodology of DNA enzymatic amplification in order to assign human -globin structural mutants to one of the two highly homologous -globin genes. Selectively amplified 1 and 2-globin cDNAs were dot-blotted and further hybridized to synthetic oligonucleotides encompassing either the normal or the mutated sequences. The generated signals corresponded specifically to one of the two -globin genes. Using this approach the -globin structural mutants J-Buda and G-Pest were found to be encoded by the 2 and the 1-globin genes, respectively. Furthermore, the exact nucleotide changes were determined. We propose this technique to serve as a simple and definitive method for assigning -globin structural mutants.     

Hydroxylysine in the N-terminal regions of the α1- and α2-chains of various collagens

The degree of hydroxylation of the lysine residue located in both alpha(1)- and alpha(2)-chains of collagen in the N-terminal, non-helical telopeptide region of the molecule has been determined in collagen from various sources after isolation of the peptides (alpha(1)- and alpha(2)-CB1) that contain the lysine residue in question and are obtained by cyanogen bromide cleavage of collagen alpha(1)- and alpha(2)-chains respectively. As with collagen from chick tibia, bone collagens from rat tibia and femur and embryonic chick frontal bone, have a high degree of hydroxylation (approx. 50% or more) of the lysine residue in both alpha(1)- and alpha(2)-CB1 peptides. This is in contrast with the lack of hydroxylation of this residue in both alpha(1)- and alpha(2)-chains of all skin collagens so far examined. The presence of hydroxylysine in alpha(1)- and alpha(2)-CB1 peptides from tendon collagen is also indicated. In rat tail tendon collagen the amount of hydroxylation is only slight but in the much less soluble tendon collagen from embryonic chick leg tendons, approximately one-third of the lysine is hydroxylated.     

Comparative analysis of the sequences of the three collagen chains α1(I), α2 and α1(III): Functional and genetic aspects

The amino acid sequences of type I collagen containing α1(I) and α2 chains at a ratio of 2:1, and of type III collagen consisting of α1 (III) chains are known. A statistical analysis of the sequences of these α chains is presented. The inter-chain comparison showed a high level of homology between the three α chains. The interactive amino acids, such as the polar charged and part of the hydrophobic residues responsible for the assembly of the molecules, are strongly conserved. The intra-chain analysis revealed that the α chains are divided into four related D units, each with a length of 234 residues. Between the D units within a chain the polar residues show a higher variability than the hydrophobic amino acids.Besides the D units, other periodicities such as $\text{D}\text{3}$ (78 residues), $\text{D}\text{6}$ (39 residues), $\text{solD}\text{11}$ (21 residues) and $\text{solD}\text{13}$ (18 residues) were observed, particularly in α1 (I) and α1 (III). The D unit is a functional repeat that is formed by the interactive polar charged and hydrophobic residues and which determines the aggregation of the molecules. The $\text{solD}\text{3}$ unit is mainly pronounced by the non-interactive residues such as proline and alanine and appears to be a reminiscence of a primordial gene. The smaller periodic repeating units may be considered as additional genetic units or as structural units, which determine the triplehelical pitch and thus the lateral aggregation of the molecules.In contrast to α1 (I) and α1 (III), the α2 chain shows less regularity in its internal structure.     

The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein

α_S-Casein, the major milk protein, comprises α_S1- and α_S2-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α_S-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α_S-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α_S2-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α_S1-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α_S1-, α- and α_S-casein were all significantly less effective. We conclude that α_S1- and α-casein stabilise each other to facilitate optimal chaperone activity of α_S-casein. This work highlights the interdependency of casein proteins for their structural stability.     

Fluorescence Polarization of αs1, κ-Caseins and αs1-κ-Casein Complex

Conjugates of α^s1-,κ-caseins and α^s1-,κ-casein complex were prepared with dimethylaminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesulfonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec.

The rotational relaxation time of pyrenebutyrate α^s1-,κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of α^s1- and κ-casein polymers led to dissociation of the κ-casein polymer.

Changes of the rotational relaxation time as a function of weight ratio of α^s1- and κ-casein polymers (α^s1/κ) showed the specific variation and it was suggested that 4 moles of α^s1-κ-casein complex were formed from one mole of κ-casein polymer.     

The development of the antigenic structures of human α1-antichymotrypsin and C 1 q

Summary The cross-reactions of human ₁-antichymotrypsin and C 1 q with their homologues in the plasmas of the chimpanzee, several Old World monkeys and nine non-primate eutheria were investigated by standard procedures. The results show that cross-reactions are limited to the first species mentioned. Comparative Ouchterlony tests and absorption controls revealed the presence of two (human) determinants on both human and chimpanzee molecules, while the cercopithecoids analyzed carried only one of them on their homologue. The results are discussed briefly with reference to earlier findings from this laboratory.

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Zusammenfassung Die Kreuzreaktionen des menschlichen ₁-Antichymotrypsin und des C 1 q mit seinen Homologen im Plasma des Schimpansen, einiger Altweltaffen und demjenigen von 9 Nichtprimaten (Eutheria) wurden mit Standardmethoden untersucht. Die Ergebnisse zeigen, daß Kreuzreaktionen auf die zuerst genannten Species beschränkt sind. Vergleichende Ouchterlony-Tests und Absorptionskontrollen ließen die Anwesenheit zweier (menschlicher) Determinaten auf den Molekülen des Menschen und des Schimpansen erkennbar werden, während die untersuchten Cercopithecoidea nur eine dieser Determinanten besitzen. Die Ergebnisse werden kurz im Zusammenhang mit früheren Befunden aus unserem Laboratorium diskutiert.

     

Identification and biology of α-secretase

Our knowledge of the etiology of Alzheimer's disease (AD) has advanced tremendously since the discovery of amyloid beta (Aβ) aggregation in diseased brains. Accumulating evidence suggests that Aβ plays a causative role in AD. The β-secretase enzyme, beta-site APP cleaving enzyme-1 (BACE1), is also implicated in AD pathogenesis, given that BACE1 cleavage of amyloid precursor protein is the initiating step in the formation of Aβ. As a result, BACE1 inhibition has been branded as a potential AD therapy. In this study, we review the identification and basic characteristics of BACE1, as well as the progress in our understanding of BACE1 cell biology, substrates, and phenotypes of BACE1 knockout mice that are informative about the physiological functions of BACE1 beyond amyloid precursor protein cleavage. These data are crucial for predicting potential mechanism-based toxicity that would arise from inhibiting BACE1 for the treatment or prevention of AD.     

Specificity and stereospecificity of α-chymotrypsin

1. The optically pure p-nitrophenyl esters of the d and l enantiomers of N-acetyl-tryptophan, N-acetylphenylalanine and N-acetyl-leucine, and the p-nitrophenyl ester of N-acetylglycine, have been prepared. 2. These materials are all substrates of α-chymotrypsin, and the rates of deacylation of the corresponding acyl-α-chymotrypsins have been determined. 3. As the size of the amino acid side chain increases, the l series deacylate progressively faster than the N-acetylglycyl-enzyme, and the d series progressively more slowly. 4. The results are interpreted in terms of a three-locus model of the enzyme's active site, which accounts for the interrelationship between substrate specificity and stereospecificity observed. 5. The concepts of negative specificity and of specificity saturation are introduced.     

Expression of core-binding factor α1 and osteocalcin in fluoride-treated fibroblasts and osteoblasts

To study the effects and importance of fluoride on FBs in the development of extraperiosteal calcification and the ossification of skeletal fluorosis, the presence of the osteogenic phenotype, which is indicated by the expression of core-binding factor α1 (Cbfa1) and osteocalcin (OCN), in an FB cell line (L929) and in osteoblasts (OBs) exposed to fluoride was determined. Fibroblasts and osteoblasts were exposed to different concentrations of fluoride (0, 0.0001, 0.001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻). By using RT-PCR and ELISA, the mRNA levels of Cbfa1 and OCN were measured at 48 h, and the protein levels of Cbfa1 and OCN were measured at 2, 4, 24, 48 and 72 h. The data demonstrated the following: (1) The Cbfa1 protein level in fluoride-treated fibroblasts clearly increased at 48 h in the groups treated with 0.0001, 0.001, 0.1, 1.0 and 20.0 mg/L F⁻. The Cbfa1 protein level of the group treated with 10 mg/L F⁻ at 72 h was higher than that of the control group. The level of Cbfa1 mRNA in the fibroblasts was much higher at 48 h in the group treated with 10.0 mg/L F⁻ than in the control group. (2) The OCN protein level in fluoride-treated fibroblasts was significantly higher than that of the control group in the 0.0001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻ groups at 2 h, and in the 0.001 and 0.1 F⁻ groups at 4 h. A slightly higher level of OCN mRNA in fluoride-treated fibroblasts was also found in the 1.0 and 20.0 mg/L F⁻ groups compared to the control group. (3) The expressions of Cbfa1 and OCN in osteoblasts treated with the same experimental conditions as the fibroblasts were up-regulated by fluoride following the same trend as in the fibroblasts. Our results showed an increase in the expression of Cbfa1 and OCN in fibroblasts and osteoblasts exposed to fluoride and suggested that the osteogenic function of fibroblasts induced by fluoride could play an important role in the development of extraperiosteal ossification during skeletal fluorosis.