Cellular localization of thiol-proteinase inhibitor in the epidermis of the newborn rat

Summary In cichlid, poecilid and centrarchid fishes luteinizing hormone releasing hormone (LHRH)-immunoreactive neurons are found in a cell group (nucleus olfactoretinalis) located at the transition between the ventral telencephalon and olfactory bulb. Processes of these neurons project to the contralateral retina, traveling along the border between the internal plexiform and internal nuclear layer, and probably terminating on amacrine or bipolar cells. Horseradish peroxidase (HRP) injected into the eye or optic nerve is transported retrogradely in the optic nerve to the contralateral nucleus olfactoretinalis where neuronal perikarya are labeled. Labeled processes leave this nucleus in a rostral direction and terminate in the olfactory bulb. The nucleus olfactoretinalis is present only in fishes, such as cichlids, poecilids and centrarchids, in which the olfactory bulbs border directly the telencephalic hemispheres. In cyprinid, silurid and notopterid fishes, in which the olfactory bulbs lie beneath the olfactory epithelium and are connected to the telencephalon via olfactory stalks, the nucleus olfactoretinalis or a comparable arrangement of LHRH-immunoreactive neurons is lacking. After retrograde transport of HRP in the optic nerve of these fishes no labeling of neurons in the telencephalon occurred. It is proposed that the nucleus olfactoretinalis anatomically and functionally interconnects and integrates parts of the olfactory and optic systems.     

D-loop cycle. A circular reaction sequence which comprises formation and dissociation of D-loops and inactivation and reactivation of superhelical closed circular DNA promoted by recA protein of Escherichia coli

Excess recA protein, a protein essential to general genetic recombination in Escherichia coli, promotes a sequence of formation and dissociation of D-loops from negative superhelical closed circular double-stranded DNA (form I DNA) and homologous single-stranded fragments in the presence of excess ATP, resulting in inactivation of the form I DNA without apparent damage to the DNA. The dissociation of D-loops is accompanied by hydrolysis of ATP to ADP that apparently depends on homologous DNA molecules (homology-dependent ATP hydrolysis). However, at a lower concentrations of ATP, we observed anomalous kinetics in the formation and dissociation of D-loops; as the concentration of ATP was decreased, there was a progressively smaller dissociation of D-loops and a faster resynthesis in the second phase, without changing the rate of the first formation of D-loops. This anomaly might suggest that, as the increase in the amount of ADP relative to that of ATP, dissociation form I DNA is stimulated before formation of D-loops is inhibited. We found that addition of ADP inhibited competitively both formation and dissociation of D-loops and that the latter process was more sensitive to the inhibition than was the former process. Addition of a sufficient amount of ADP to inhibit both formation and dissociation of D-loops, cessation of homology-dependent hydrolysis of ATP, or incubation at low temperature resulted in reactivation of form I DNA that had been inactivated by the sequence. In the presence of an ATP-regenerating system, we confirmed our previous result that limiting the amount of recA protein also causes anomalous kinetics in the formation and dissociation of D-loops. These observations indicate that the formation and dissociation of D-loops and the inactivation and reactivation of form I DNA make a circular reaction sequence.     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene.

Inactivation of the retinoblastoma gene appears to have a fundamental role in the genesis of retinoblastoma, osteosarcoma, and other malignant tumors. The gene is generally inactivated because of loss-of-function mutations, although epigenetic phenomena, such as hypermethylation of the promoter region, could possibly have the same effect. We investigated the methylation pattern at the 5' end of the retinoblastoma gene, including its promoter region and exon 1, in DNA purified from 56 primary retinoblastomas. We found five tumors with evidence for hypermethylation, all from unilateral, simplex patients. No methylation abnormalities were detected in DNA purified from the leukocytes from these patients. It is interesting that in one of these tumors the hypermethylation was confined to one allele. There were no mutations in a 1,306-bp sequence including the hypermethylated region that might account for the allele-specific hypermethylation. We believe that the hypermethylation of the retinoblastoma gene that we found in these tumors corresponds to the allelic inactivation of the gene, and we speculate that erroneous hypermethylation without alteration of nucleotide sequence occasionally plays a role in the genesis of this cancer. If this is true, then retinoblastomas with hypermethylation might be treatable with chemotherapeutic agents that interfere with methylation of DNA.     

Quantitative effects of common genetic variations in the 3′UTR of the human LDL-receptor gene and their associations with plasma lipid levels in the Atherosclerosis Risk in Communities study

The low-density lipoprotein receptor (LDLR) plays a pivotal role in cholesterol homeostasis. However, the role of genetic variations in the 3′UTR of the LDLR in relation to plasma cholesterol has been largely understudied. Six SNPs, G44243A, G44332A, C44506G, G44695A, C44857T and A44964G, within the 5′ region of the 3′UTR fall into three common haplotypes, GGCGCA, AGCACG, and GGCGTA, occurring at frequencies of 0.45, 0.31 and 0.17, respectively, in Caucasians (n = 29) and 0.13, 0.13 and 0.38, respectively, in African Americans (n = 32), with three other haplotypes occurring at lesser frequencies. In a tissue culture based system, expression of a reporter gene carrying a 3′UTR that includes the 1 kb nucleotide sequences corresponding to the AGCACG or GGCGTA was 70 or 63%, respectively, of the same sequence with GGCGCA. Genotyping of two “haplotype tagging” SNPs, C44857T and A44964G, in the Atherosclerosis Risk in Communities (ARIC) study population showed that in Caucasians, but not in African Americans, the inferred TA haplotype had a significant LDL-cholesterol lowering effect. The adjusted LDL-cholesterol levels in the TA/TA diplotypes were lower by 6.10 mg/dl in men (P < 0.001) and by 4.63 mg/dl in women (P < 0.01) than in individuals with other diplotypes. Caucasian men homozygous for CA, in contrast, showed significantly higher LDL-cholesterol (P < 0.04), lower HDL-cholesterol (P < 0.02) and higher LDL/HDL ratios (P < 0.001). Thus our data shows that 3′UTR sequences that cause higher reporter gene expression in vitro are associated in Caucasians with plasma lipid profiles indicative of higher cardiovascular risk, suggesting that further studies of quantitative variants in the LDLR gene will be valuable. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy

We developed a method to measure the rupture forces between antibody and antigen by atomic force microscopy (AFM). Previous studies have reported that in the measurement of antibody–antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM.     

Induction of Bcl-xL Expression by Human T-Cell Leukemia Virus Type 1 Tax through NF-κB in Apoptosis-Resistant T-Cell Transfectants with Tax

Human T-cell leukemia virus type 1 (HTLV-1) Tax is thought to play a pivotal role in immortalization of T cells. We have recently shown that the expression of Tax protected the mouse T-cell line CTLL-2 against apoptosis induced by interleukin-2 (IL-2) deprivation and converted its growth from being IL-2 dependent to being IL-2 independent. In this study, we demonstrate that constitutive expression of bcl-xl but not bcl-2, bcl-xs, bak, bad, or bax was associated with apoptosis resistance after IL-2 deprivation in CTLL-2 cells that expressed Tax. Transient-transfection assays showed that bcl-x promoter was transactivated by wild-type Tax. Similar effects were observed in mutant Tax retaining transactivating ability through NF-kappaB. Deletion or substitution of a putative NF-kappaB binding site identified in the bcl-x promoter significantly decreased Tax-induced transactivation. This NF-kappaB-like element was able to form a complex with NF-kappaB family proteins in vitro. Furthermore, Tax-induced transactivation of the bcl-x promoter was also diminished by the mutant IkappaBalpha, which specifically inhibits NF-kappaB activity. Our findings suggest that constitutive expression of Bcl-x(L) induced by Tax through the NF-kappaB pathway contributes to the inhibition of apoptosis in CTLL-2 cells after IL-2 deprivation.     

In situ measurement of cell stiffness of Arabidopsis roots growing on a glass micropillar support by atomic force microscopy

Atomic force microscopy (AFM) can measure the mechanical properties of plant tissue at the cellular level, but for in situ observations, the sample must be held in place on a rigid support and it is difficult to obtain accurate data for living plants without inhibiting their growth. To investigate the dynamics of root cell stiffness during seedling growth, we circumvented these problems by using an array of glass micropillars as a support to hold an Arabidopsis thaliana root for AFM measurements without inhibiting root growth. The root elongated in the gaps between the pillars and was supported by the pillars. The AFM cantilever could contact the root for repeated measurements over the course of root growth. The elasticity of the root epidermal cells was used as an index of the stiffness. By contrast, we were not able to reliably observe roots on a smooth glass substrate because it was difficult to retain contact between the root and the cantilever without the support of the pillars. Using adhesive to fix the root on the smooth glass plane overcame this issue, but prevented root growth. The glass micropillar support allowed reproducible measurement of the spatial and temporal changes in root cell elasticity, making it possible to perform detailed AFM observations of the dynamics of root cell stiffness.