Inhomogeneous plastic flow investigated by X-ray absorption microtomography of an aluminium alloy containing marker particles.

By combining a synchrotron X-ray source and the microtomography technique, the displacement gradient tensor components can be measured directly throughout the volume of a metal sample containing highly absorbing marker particles to detect material flow. The present article describes application of these techniques to compression of a rectangular Al-W specimen with a central hole. The results show that the externally imposed axial displacement gradient is accommodated mainly in two 45 degrees shear bands passing through the hole. A simple deformation analysis provides insight into shear band formation.

Surfactant protein (SP) B associations and interactions with SP-A in white and black subjects with respiratory distress syndrome.

BACKGROUND: The etiology of respiratory distress syndrome (RDS) is multifactorial and/or multigenic. Surfactant protein A (SP-A) and/or SP-B genetic variants have been identified as risk or protection factors for RDS. METHODS: We genotyped subjects with and without RDS for the SP-B intron 4 size variants (invariant (inv), deletion (del), insertion (ins) and for four (-18 (A/C), 1013 (A/C), 1580 (C/T), 9306 (A/G)) SP-B single nucleotide polymorphisms (SNP), to study case-control associations in black and white subjects. We also determined whether specific SP-B variants interact with RDS susceptibility or protective SP-A variants to enhance or reduce risk for RDS. RESULTS: Based on odds ratio: (1) the SP-B intron 4 del variant in white subjects is more of an RDS risk factor for males and for subjects of 28 weeks

Cytosine methylation represses glutathione S-transferase P1 (GSTP1) gene expression in human prostate cancer cells.

Methylation of the glutathione S-transferase P1 (GSTP1) gene has been described as a highly specific and sensitive biomarker for prostate cancer. However, at present, it is not known whether methylation represses GSTP1 gene expression in human prostate cancer. We found the GSTP1 gene promoter to be completely methylated in the LNCaP prostate cancer cell line, where this gene is transcriptionally inactive. In contrast, Du145 and PC3 prostate cancer cells express the GSTP1 gene and exhibit methylated and unmethylated GSTP1 alleles. In a transient transfection assay using LNCaP cells, methylation of the GSTP1 promoter-driven luciferase reporter vector (GSTP1-pGL3) resulted in a >20-fold inhibition of transcription, and this repression was not relieved by the presence of a histone deacetylase inhibitor, trichostatin A (TSA). Treatment of LNCaP cells with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine, resulted in demethylation and activation of the GSTP1 gene. In contrast, TSA treatment failed to demethylate or activate the GSTP1 gene. Fully methylated but not unmethylated GSTP1 promoter fragment was shown to bind to a complex similar to methyl cytosine-binding protein complex 1 that contains methyl-CpG-binding domain 2 protein (MBD2) in electrophoretic mobility shift assays using LNCaP cell nuclear extracts. These data demonstrate that cytosine methylation can repress GSTP1 gene expression in LNCaP prostate cancer cells and that this effect is possibly mediated by a methyl cytosine-binding protein complex 1-like complex. Furthermore, these data also support the notion of the dominance of methylation over TSA-sensitive histone deacetylation in silencing genes with a high CpG density in the promoter region.

Expression and cDNA sequence of helix destabilizing protein (HDP) in rat lung.

The current study was undertaken to investigate genes that show alteration in expression during late fetal rat lung development. Using the differential display (DD) method, an initially unknown transcript was found to be abundant on day 18 compared to day 22 of gestation. This finding was confirmed by RNA blot analysis. Subsequent screening of a rat lung cDNA library with the unknown DD cDNA as a probe revealed a clone that encodes helix destabilizing protein (HDP), similar to that reported in rat brain (J Biol Chem. 1986, 261:3536-3543). Although there were several differences between lung and brain cDNAs at the 3' untranslated region (UTR), the 5' end was well conserved. There was a gradual decrease in HDP mRNA content beyond day 18 of gestation, as lung maturation increased. Similarly, a lower content of HDP mRNA was observed in maternal heart and brain tissues than in fetal heart and brain tissues from day 18 of gestation. Maternal dexamethasone treatment that promotes lung maturation did not have an impact on HDP expression. Because of the much higher abundance of HDP mRNA during fetal organ development compared to adult tissues, we speculate that HDP plays an important role during lung development. A portion of this work was previously presented (FASEB J. 1996;10:A26).

Regulation of transcription of the testis-specific histone H1t gene by multiple promoter elements.

This is a review of mechanisms that contribute to testis-specific transcription of the histone H1t gene. The mammalian testis-specific histone H1t gene is transcribed only in primary spermatocytes during spermatogenesis. Linker histones bind to DNA and contribute to chromatin condensation by formation of the 30 nm chromatin fiber. Furthermore, linker histones contribute to regulation of transcription of specific genes. Histone H1t, which binds more weakly to DNA than the other six known linker histones, is expressed in cells that are involved in the essential processes of crossing over and mismatch repair of DNA and in cells that undergo a dramatic alteration in gene expression. However, contributions of this linker histone to these processes are unknown. Subtle differences are found in the H1t promoter compared to the other H1 promoters. Nevertheless, several lines of evidence support the hypothesis that a sequence element designated TE that is located within the H1t promoter is essential for enhanced testis-specific transcription of this gene. Transgenic mice bearing a rat H1t transgene which contains a replacement of the TE element with stuffer DNA fail to express rat H1t mRNA. In addition, an upstream sequence appears to function as a silencer element that leads to transcriptional repression of the H1t gene in nongerminal cells. Thus, multiple promoter elements appear to contribute to regulation of transcription of the histone H1t gene.

Expression of the testis-specific histone H1t gene: evidence for involvement of multiple cis-acting promoter elements.

The histone H1t gene is expressed exclusively in testis primary spermatocytes. Previous studies indicate that accumulation of H1t mRNA occurs only in primary spermatocytes in normal rats and in transgenic mice bearing the rat H1t transgene. In this study, DNA sequences of human, monkey, mouse, and rat H1t genes were compared and found to be almost identical in the proximal promoter region extending from the H1/AC box through the TATAA box. In addition to conserved elements common to replication-dependent H1 promoters, the H1t promoter contains a unique TE element, and sequences within this element may contribute to enhanced expression of the gene in primary spermatocytes. Two imperfect inverted repeat sequences designated TE1 and TE2, that are located within the larger TE element, overlap a central GC-rich region and bind specifically to nuclear proteins derived from primary spermatocytes. Protein interactions characterized by methylation interference and UV cross-linking experiments indicate that a complex of proteins with a molecular mass of approximately 180 kDa binds TE1. The GC-rich region in H1t and in some replication dependent histone H1 promoters contains an Sp1 consensus sequence. Although the H1t/TE element that contains the GC-rich region binds nuclear proteins, it does not appear to bind Sp1 obtained from cell populations enriched in primary spermatocytes as determined by electrophoretic mobility supershift assays using polyclonal anti-Sp1 antibodies.

Leucine-specific binding of photoreactive Leu7-MAP to a high molecular weight protein on the plasma membrane of the isolated rat hepatocyte.

Leu8-MAP (Multiple Antigen Peptide) is an effective inhibitor of macroautophagy and proteolysis in the isolated rat hepatocyte, having an apparent Km (0.1 mM) equaling leucine. Since it is not transported into the cytosolic compartment, it very likely mediates its effect through a plasma membrane site. In an attempt to identify the site we photoreacted intact cells with a biologically active, iodinatable azide derivative of Leu7-MAP. A approximately 340,000 M(r) protein whose labeling was protected 83% with 20 mM Leu was found in plasma membrane fractions when electrophoresed in 7.5-20% gradient gels under nonreducing conditions; addition of 20 mM dithiothreitol generated smaller m.w. products, possibly subunits, of consistent size. No specific labeling was observed with photoreactive derivatives of Ile7-MAP or Val7-MAP.

4-Amino-6-methylhept-2-enoic acid: a leucine analogue and potential probe for localizing sites of proteolytic control in the hepatocyte.

A recent analysis of leucine analogues has suggested that the carboxyl group is not required for mediating low concentration proteolytic inhibition in liver cells. In designing a probe to localize the regulatory site(s), we tested this hypothesis by synthesizing an analogue with a 2-carbon insert between the carboxyl and alpha-carbon. The Wittig product, a trans olefin, was fully active. Surprisingly, low concentration activity was lost when the double bond was eliminated by hydrogenation although some inhibitory effectiveness at high concentrations was evident. Since the double bond extends the carboxyl group away from the alpha-carbon, the results support the above hypothesis as well as the feasibility of adding functional groups to the carboxyl end of leucine.

Synthesis and secretion of proteins by postpartum human oviductal tissue in culture.

An explant culture system that used labelled leucine and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with fluorography was used to identify specific de novo synthesized and released polypeptides by the human postpartum oviduct. Both ampulla and isthmus tissue in culture exhibited de novo synthesis and release of a large number of polypeptide subunits. Immunoglobulins A and G appear to be the major proteins produced in the ampulla. In addition, two complexes of acidic (pI less than 5) polypeptide subunits are found primarily in ampulla culture medium. Two families of proteins (Mr 51,000 and 60,000) are released by the isthmus but appear to be minor in the ampulla cultures.

Modulation of the amino acid control of hepatic protein degradation by caloric deprivation. Two modes of alanine co-regulation.

Intracellular protein degradation in perfused livers of fed rats has been shown to be directly regulated by 7 amino acids (Leu, Tyr, Gln, Pro, Met, His, and Trp) and co-regulated by alanine. Responses to graded increases of regulatory amino acids (individually or combined) are multiphasic and include (a) an initial inhibition at 0.5 times normal plasma concentrations, (b) a localized, zonal loss of inhibition at normal levels, and (c) suppression to basal rates at 4 times normal concentrations or greater; the zonal loss of inhibition is prevented by 0.5 mM (normal) alanine. In further perfusion studies carried out at the usual time (1100 h), we have occasionally observed a sharp decrease in proteolytic responsiveness at normal amino acid concentrations. The decrease, which occurred spontaneously in normal fed rats, was attributed to a nearly 90% loss in the sensitivity of alanine co-regulation. In all instances, alanine sensitivity was restored after 4 to 24 h of starvation. The cause of the insensitivity and the mechanism of its reversal by caloric deprivation are not presently known. Starvation for 24 h also appeared to alter the individual inhibitory effectiveness of Leu, Tyr, and Gln. On the other hand, inhibition by the full regulatory group at 4 times normal plasma levels was unchanged when compared with the complete plasma mixture except for a concentration shift in the peak zonal loss of proteolytic inhibition from 1.25 to 0.6 times plasma levels. Since the shift paralleled known changes in portal vein regulatory amino acids, it may have been adaptive in nature. As with fed animals, the zonal loss in starvation was abolished by 0.5 mM alanine, but not with high levels of lactate and pyruvate (10 mM), a finding consistent with the view that co-regulation is mediated by the recognition of alanine per se rather than its metabolism.

Uptake and intracellular fate of [14C]sucrose-insulin in perfused rat livers.

Insulin was covalently linked to [14C]sucrose by means of cyanuric chloride to provide a label that would remain entrapped within the vacuolar system. The uptake of the conjugate by the perfused rat liver was rapid (half-life = 2.9 min), competitively inhibited by native insulin, and abolished by alkali denaturation. As assessed by its distribution on self-generating gradients of colloidal silica-povidone, label in lysosome-enriched samples of liver taken at different times after the addition of the conjugate moved progressively during 15 min from the plasma membrane into an intermediate peak and then to dense lysosomal fractions. After 30-60 min, the label had equilibrated throughout the lysosomal-vacuolar system. The initial movement from the plasma membrane to the intermediate peak occurred between 2 and 5 min. Because label in the peak could be physically separated from the lysosomal marker, beta-acetylglucosaminidase, by dispersing the sample through the gradient mixture before centrifugation rather than layering it, we concluded that the intermediate particles in question were not lysosomal in nature. On gel-filtration chromatography, label extracted from the intermediate peak did not move with insulin but rather as a broad band of lower molecular weight products, suggesting that insulin is subject to early proteolytic attack within a nonlysosomal compartment.

Multiphasic control of hepatic protein degradation by regulatory amino acids. General features and hormonal modulation.

Previous studies with livers from fed rats perfused in the single-pass mode have shown that regulatory amino acids (Leu, Tyr, Gln, Pro, Met, His, and Trp) as a group as well as leucine alone inhibit deprivation-induced protein degradation optimally at 0.5 and 4 times (X) normal plasma amino acid concentrations. However, they lose inhibitory effectiveness almost completely within a narrow zone centered at normal (1 X) levels (Pösö, A. R., Wert, J. J., Jr., and Mortimore, G.E. (1982) J. Biol. Chem. 257, 12114-12120; Pösö, A. R., and Mortimore, G. E. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4270-4274). We now report similar effects for tyrosine and glutamine and suggest that this multiphasic dose response is a general feature of the regulatory group. Insulin (2.4 micrograms h-1) selectively modulated the response by abolishing the zonal loss, whereas glucagon (10 micrograms h-1) blocked the initial inhibition (0.5 X); proteolytic suppression was restored at 4 X normal plasma levels. Although the zonal loss of inhibition at 1 X was associated with a near maximal increase in the volume density of macroautophagy, the vacuoles differed from those induced by stringent amino acid deprivation in containing 4.5-fold more smooth than rough endoplasmic reticulum and thus represented a separate population. Surprisingly, the leucine analog, L-alpha-hydroxyisocaproate, elicited multiphasic responses identical to those of L-leucine, including inhibition at 0.1 mM (equivalent to 0.5 X Leu). Inasmuch as alpha-ketoisocaproate is not effective at this concentration, the initial suppression of protein degradation could be mediated from a site that recognizes structural features common to leucine and its hydroxyl analog.

Metabolic alterations and distribution of rat liver lysosomes in colloidal silica.

Lysosome-enriched samples from normal fed rat livers have been shown to separate into dense and buoyant bands when layered on self-generating gradients of colloidal silica. The hypothesis that the buoyant peak is comprised largely of autophagic vacuoles that are selectively restrained by sedimenting mitochondria was tested in livers subjected to conditions known to accelerate autophagy or suppress it. In all cases, a sharp peak of beta-N-acetyl-D-glucosaminidase activity was evident when autophagy was present but was undetectable when it was eliminated or virtually eliminated. Moreover, the sensitivity to osmotic shock of buoyant peak particles, but not those of the dense band, was increased after autophagic stimulation. The total amount of enzyme that moved to buoyant fractions with the induction of autophagy generally correlated with the degree of autophagic acceleration. However, with maximal autophagy, the buoyant peak was broad, and only 70% was capable of being dislodged and returned to the dense peak by dispersing samples in the gradient medium and/or reducing the sample size. On the other hand, at roughly half-maximal stimulation and lower, the buoyant distribution was sharply defined and completely reversible. These findings support the above hypothesis and suggest that the buoyant peak could be used as a measure of autophagy in the physiological range of regulation.

Role of particle interaction on distribution of liver lysosomes in colloidal silica.

Rat liver mitochondrial-lysosomal fractions were separated on gradients of colloidal silica. Lysosomal enzymes were distributed bimodally. The dense peak (1.117 g/ml) was nearly free from contaminants; beta-N-acetyl-D-glucosaminidase was enriched nearly 60-fold. By contrast, the buoyant peak (1.085 g/ml) co-sedimented with mitochondria, microsomes, peroxisomes, and Golgi particles. Decreasing the amount of protein layered on the gradient medium or dispersing a full sample through it shifted lysosomal marker from the buoyant to the dense peak. Thus the majority of lysosomes in the two peaks appeared to have equivalent densities. Electron microscopic examination of particles separated from gradients with layered samples showed that the dense peak contained most of the dense bodies, whereas the buoyant peak was relatively enriched in autophagic vacuoles. Dispersion, however, shifted autophagic vacuoles from the buoyant to the dense peak without affecting the distribution of dense bodies. We conclude that the bulk of buoyant particles act as a sieve to retard the density equilibration of autophagic vacuoles without specifically affecting other lysosomal enzyme-containing components.

Multifunctional control of amino acids of deprivation-induced proteolysis in liver. Role of leucine.

The control of intracellular proteolysis by amino acids was investigated in rat livers perfused in the single pass mode with various mixtures at multiples of normal plasma concentrations. Twelve amino acids together exhibited no suppressive activity while the remainder (Leu, Phe, Tyr, Gln, Pro, His, Trp, and Met) were inhibitory. Because (a) the effects of phenylalanine and tyrosine were equal and nonadditive, and (b) the former is rapidly converted to the latter, tyrosine rather than phenylalanine was considered to be an active regulator. The inhibitory group, minus phenylalanine, duplicated responses of the complete mixture between 0 and 4 times (4x) normal plasma levels. When tested individually, leucine was the strongest inhibitor, suppressing deprivation-accelerated proteolysis by 63% at 4x and 10x. However, at lower concentrations its effects differed dramatically from those of the regulatory mixture in showing a sharp modal loss of responsiveness at approximately 1x, a finding which suggests that proteolytic regulation at normal levels is achieved largely by the remaining 6 amino acids. Leucine responses from 1x to 4x (0.2 to 0.8 mM) were mimicked by equimolar additions of alpha-ketoisocaproate. The fact that 0.8 mM alpha-ketoisocaproate increased intracellular leucine by only 7.8% of the value obtained with equimolar (4x) leucine supports observations of others that transamination limits interconversion between leucine and its alpha-ketoacid to 4-10% of total alpha-ketoisocaproate utilization at this level. We conclude that the effects of leucine and alpha-ketoisocaproate on hepatic proteolysis are mediated by separate pathways.