Involvement of Yeast HSP90 Isoforms in Response to Stress and Cell Death Induced by Acetic Acid

Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress.     

NMDA receptors in afferent synapses of frog semicircular canals

The effects of competitive (2-amino-phosphonovaleric acid) and noncompetitive (Mg²⁺, ketamine, kynurenic acid) antagonists of N-methyl-D-aspartate (NMDA) receptors on synaptic transmission were studied in afferent synapses of the frog semicircular canals. All of these antagonists reduced the rate of background activity in the nerve of posterior semicircular canal by 30–50%, which confirms the presence of glutamate NMDA receptors in the hair cell synapses in the frog semicircular canals.Neurofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 168–169, May–June, 1993.     

Cryopreservation of bovine hemopoietic progenitor cells in liquid nitrogen

Bovine marrow granulocyte/macrophage and erythroid progenitor cells maintained viability after storage in liquid nitrogen for 2 to 4 weeks. The granulocyte/macrophage progenitor cells maintained 100% viability for 4 weeks, while the erythroid progenitor cells maintained 100% viability for at least 2 weeks. The optimum concentration of either DMSO or glycerol was found to be 5–10%. DMSO was superior to glycerol as a cryopreservative of bovine granulocyte/ macrophage progenitor cells. Glycerol was found to be unable to cryopreserve bovine erythroid progenitor cells.     

Unilateral microfibrillar abnormalities in a case of asymmetric Marfan syndrome.

The Marfan syndrome is a dominantly inherited connective-tissue disorder characterized by ocular, cardiovascular, and musculoskeletal abnormalities. Although the underlying biochemical and molecular defect(s) of this pleiotropic disease is currently unknown, we have consistently observed apparent diminished content of elastin-associated microfibrillar fibers accumulating in skin, or produced by cultured fibroblasts, from patients with the Marfan syndrome and have documented the cosegregation of these immunofluorescent abnormalities of microfibrillar fibers with the Marfan syndrome phenotype in family studies. Recently, an unusual patient has been described with unilateral phenotypic features of the Marfan syndrome, providing an unique opportunity to compare microfibrillar fibers and other connective-tissue components between the affected and nonaffected sides. In the present report, we demonstrate striking differences in apparent content of microfibrillar fibers, as determined by indirect immunofluorescence of skin and fibroblast cultures, that are revealed when multiple homologous samples derived from different sides of the patient's body are compared. In contrast, no differences in apparent content of type III collagen or in the biosynthesis and apparent structure of types I and III (pro)collagens were found. HLA types and chromosome heteromorphisms were identical in fibroblasts from both sides of the body, eliminating the formal possibility of chimerism and suggesting that a postzygotic mutation accounts for the asymmetric manifestation of the Marfan syndrome in this patient. The observation of striking decreases in microfibrillar fibers on the affected side of the body provides further evidence that abnormalities of this component of the elastic fiber system may be central to the pathogenesis and possibly the etiology of the Marfan syndrome.     

Resonance Raman spectroscopic characterization of compound III of lignin peroxidase

Resonance Raman (RR) spectra of several compounds III of lignin peroxidase (LiP) have been measured at 90 K with Soret and visible excitation wavelengths. The samples include LiPIIIa (or oxyLiP) prepared by oxygenation of the ferrous enzyme, LiPIIIb generated by reaction of the native ferric enzyme with superoxide, LiPIIIc prepared from native LiP plus H2O2 followed by removal of excess peroxide with catalase, and LiPIII* made by addition of excess H2O2 to the native enzyme. The RR spectra of these four products appear to be similar and, thus, indicate that the environments of these hexacoordinate, low-spin ferriheme species must also be very similar. Nonetheless, the Soret absorption band of LiPIII* is red-shifted by 5 nm from the 414-nm maximum common to LiPIIIa, -b, and -c [Wariishi, H., & Gold, M.H. (1990) J. Biol. Chem. 265, 2070-2077]. Analysis of the iron-porphyrin vibrational frequencies indicates that the electronic structures for the various compounds III are consistent with an FeIIIO2.-formulation. The spectral changes observed between the oxygenated complex and the ferrous heme of lignin peroxidase are similar to those between oxymyoglobin and deoxymyoglobin. The contraction in the core sizes in compound III relative to the native peroxidase is analyzed and compared with that of other heme systems. EPR spectra confirm that the high-spin ferric form of the native enzyme, with an apparent g = 5.83, is converted into the EPR-silent LiPIII* upon addition of excess H2O2. Its magnetic behavior may be explained by anti-ferromagnetic coupling between the low-spin FeIII and the superoxide ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: role of veratryl alcohol in lignin biodegradation

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H2O2-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H2O2 concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H2O2 to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H2O2 and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H2O2. AA competitively inhibited the oxidation of VA by LiP; the Ki for AA inhibition was 32 microM. Addition of VA to LiPIII* resulted in its conversion to the native enzyme. In contrast, AA did not convert LiPIII* to the native enzyme; instead, LiPIII* was bleached in the presence of AA. Thus, AA does not protect LiP from inactivation by H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of 2,7-dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions, the white-rot basidiomycete Phanerochaete chrysosporium degraded 2,7-dichlorodibenzo-p-dioxin (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell-free extracts. The multistep pathway involves the degradation of I and subsequent intermediates by oxidation, reduction, and methylation reactions to yield the key intermediate 1,2,4-trihydroxybenzene (III). In the first step, the oxidative cleavage of the dioxin ring of I, catalyzed by LiP, generates 4-chloro-1,2-benzoquinone (V), 2-hydroxy-1,4-benzoquinone (VIII), and chloride. The intermediate V is then reduced to 1-chloro-3,4-dihydroxybenzene (II), and the latter is methylated to form 1-chloro-3,4-dimethoxybenzene (VI). VI in turn is oxidized by LiP to generate chloride and 2-methoxy-1,4-benzoquinone (VII), which is reduced to 2-methoxy-1,4-dihydroxybenzene (IV). IV is oxidized by either LiP or MnP to generate 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (III). The other aromatic product generated by the initial LiP-catalyzed cleavage of I is 2-hydroxy-1,4-benzoquinone (VIII). This intermediate is also generated during the LiP- or MnP-catalyzed oxidation of the intermediate chlorocatechol (II). VIII is also reduced to 1,2,4-trihydroxybenzene (III). The key intermediate III is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial oxidative cleavage of both C-O-C bonds in I by LiP generates two quinone products, 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). The former is recycled by reduction and methylation reactions to generate an intermediate which is also a substrate for peroxidase-catalyzed oxidation, leading to the removal of a second chlorine atom. This unique pathway results in the removal of both aromatic chlorines before aromatic ring cleavage takes place.     

A base substitution at IVS-19 3′-end splice junction causes exon 20 skipping in proα2(I) collagen mRNA and produces mild osteogenesis imperfecta

Molecular investigations on a young patient and her family were undertaken to identify the molecular defect responsible for a mild form of osteogenesis imperfecta (OI) with blue sclerae, dentinogenesis imperfecta and joint laxity. Analysis of collagenous proteins from the proband's fibroblasts showed the presence of two populations of 2(I) chains, one normal and one migrating faster on SDS gels, thereby suggesting deletion of amino acid sequences. The faster migrating chains were retained mainly in the cell layer and not found in the extracellular matrix deposited by cultured fibroblasts. Chemical cleavage of mismatch (CCM) analysis on the patient's pro2(I) mRNA:normal cDNA heteroduplexes localized the molecular defect. cDNA sequencing revealed a deletion of exon 20 (54 bp) in about half of the molecules. Genomic DNA sequencing revealed heterozygosity for a G-to-C transversion of the last nucleotide of intron 19, which changed the 3 consensus splicing site. As a consequence pro2(I)mRNA was abnormally spliced from the last codon of exon 19 to the first codon of exon 21. To our knowledge, this is the first acceptor site mutation so far described in an OI patient. Restriction analysis indicated that the mutation was present also in three other affected family members. The full sequence of COL1A2 introns 19 and 20 are reported.