Differential regulation of polygalacturonase and pectin methylesterase gene expression during and after heat stress in ripening tomato (Lycopersicon esculentum Mill.) fruits

The effects of extended heat stress on polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) gene expression at mRNA, protein and activity levels in ripening tomato fruits were investigated. Steady state levels of PG mRNA declined at temperatures of 27°C and above, and a marked reduction in PG protein and activity was observed at temperatures of 32°C and above. Exogenous ethylene treatment did not reverse heat stress-induced inhibition of PG gene expression. Transfer of heat-stressed fruits to 20°C partly restored PG mRNA accumulation, but the rate of PG mRNA accumulation declined exponentially with duration of heat stress. Heat stress-induced inhibition of PME mRNA accumulation was recoverable even after 14 days of heat stress. In fruits held at 34°C, both PG and PME protein and activity continued to accumulate for about 4 days, but thereafter PG protein and activity declined while little change was observed in PME protein and activity. In spite of increases in mRNA levels of both PG and PME during the recovery of heat-stressed fruit at 20°C, levels of PG protein and activity declined in fruits heat-stressed for four or more days while PME protein and activity levels remained unchanged. Collectively, these data suggest that PG gene expression is being gradually and irreversibly shut off during heat stress, while PME gene expression is much less sensitive to heat stress.     

Tropolone as a substrate for horseradish peroxidase

Tropolone (2,4,6-cycloheptatrien-1-one), in the presence of hydrogen peroxide but not in its absence, can serve as a donor for the horseradish peroxidase catalysed reaction. The product formed is yellow and is characterized by a new peak at 418 nm. The relationship between the rate of oxidation of tropolone (ΔA at 418 nm/min) and various concentrations of horseradish peroxidase, tropolone and hydrogen peroxide is described. The yellow product obtained by the oxidation of tropolone by horseradish peroxidase in the presence of hydrogen peroxide was purified by chromatography on Sephadex G-10 and its spectral properties at different pHs are presented. The M, of the yellow product was estimated to be ca 500, suggesting that tropolone, in the presence of horseradish peroxidase and hydrogen peroxide is converted to a tetratropolone.     

Diversity of β-Globin Mutations in Israeli Ethnic Groups Reflects Recent Historic Events

We characterized nearly 500 β-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. We found 28 different mutations in the β-globin gene, including three mutations (β^S, β^C, and β^O-Arab) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates—Druze and Samaritans—had a single mutation each. Fifteen of the β-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele—nonsense codon 37—appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of β-globin mutations can be largely explained by migration events that occurred in the past millennium.     

Oligomerization of the mitochondrial protein voltage-dependent anion channel is coupled to the induction of apoptosis

Accumulating evidence implicates that the voltage-dependent anion channel (VDAC) functions in mitochondrion-mediated apoptosis and as a critical player in the release of apoptogenic proteins, such as cytochrome c, triggering caspase activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c-conducting channel remain unknown. Here the relationship between VDAC oligomerization and the induction of apoptosis was examined. We demonstrated that apoptosis induction by various stimuli was accompanied by highly increased VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using bioluminescence resonance energy transfer technology. VDAC oligomerization was induced in all cell types and with all apoptosis inducers used, including staurosporine, curcumin, As(2)O(3), etoposide, cisplatin, selenite, tumor necrosis factor alpha (TNF-α), H(2)O(2), and UV irradiation, all acting through different mechanisms yet all involving mitochondria. Moreover, correlation between the levels of VDAC oligomerization and apoptosis was observed. Furthermore, the apoptosis inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited VDAC oligomerization. Finally, a caspase inhibitor had no effect on VDAC oligomerization and cytochrome c release. We propose that VDAC oligomerization is involved in mitochondrion-mediated apoptosis and may represent a general mechanism common to numerous apoptogens acting via different initiating cascades. Thus, targeting the oligomeric status of VDAC, and hence apoptosis, offers a therapeutic strategy for combating cancers and neurodegenerative diseases.     

Mutant p53 Gain-of-Function in Cancer

In its wild-type form, p53 is a major tumor suppressor whose function is critical for protection against cancer. Many human tumors carry missense mutations in the TP53 gene, encoding p53. Typically, the affected tumor cells accumulate excessive amounts of the mutant p53 protein. Various lines of evidence indicate that, in addition to abrogating the tumor suppressor functions of wild-type p53, the common types of cancer-associated p53 mutations also endow the mutant protein with new activities that can contribute actively to various stages of tumor progression and to increased resistance to anticancer treatments. Collectively, these activities are referred to as mutant p53 gain-of-function. This article addresses the biological manifestations of mutant p53 gain-of-function, the underlying molecular mechanisms, and their possible clinical implications.Mutations in the TP53 gene, encoding the p53 tumor suppressor, are arguably the most frequent type of gene-specific alterations in human cancer. This attests to the centrality of p53 as a major mainstay in the body’s built-in anticancer defense mechanisms. Not surprisingly, this pivotal role of the wild-type p53 (wtp53) protein in tumor suppression has attracted many researchers to study it in detail, resulting in an avalanche of information and publications. One might expect that, similar to other tumor suppressor genes, the sole outcome of mutations in the TP53 gene will be loss of wtp53 function, characteristically manifested as total lack of p53 expression or production of unstable or truncated mutant proteins. Yet, quite strikingly, the vast majority of cancer-associated p53 mutations actually lead to production of full length protein, typically with only a single amino acid substitution, which tends to accumulate in the tumor cells and reach steady-state levels that greatly exceed those of wtp53 in noncancerous cells (Rotter 1983). This remarkable feature has suggested early on in p53 research that cancer-associated mutant p53 (mutp53) isoforms may be more than just relics of wtp53 inactivation, and may instead play distinctive roles in the tumor cells.In principle, emergence of a p53 mutation within a cell might have three, not mutually exclusive, types of outcome (Michalovitz et al. 1991; Sigal and Rotter 2000; Weisz et al. 2007b). First, such mutation is expected to abrogate the tumor suppressor function of the affected TP53 allele, reducing the overall capacity of the cell to mount a proper p53 response; if both alleles eventually become mutated, or if the remaining allele is lost, such cells will be totally deprived of anticancer protection by p53. Second, many common mutp53 isoforms can exert dominant–negative effects over coexpressed wtp53, largely by forming mixed tetramers that are incapable of DNA binding and transactivation. Hence, even if one wt allele is retained, the cell may be rendered practically devoid of wtp53 function through such mechanism, particularly if the mutant protein is expressed in excess over its wt counterpart. Third, and most relevant for this article, the emergent mutp53 protein might possess activities of its own, often not present in the original wtp53 protein, which can actively contribute to various aspects of tumor progression. Such activities, commonly described as mutp53 gain-of-function (GOF), are the subject of this article. Several recent reviews address in detail the various aspects of mutp53 GOF (Brosh and Rotter 2009; Donzelli et al. 2008; Lozano 2007; Olivier et al. 2009; Peart and Prives 2006; Petitjean et al. 2007; Song and Xu 2007; Strano et al. 2007; Weisz et al. 2007b). Therefore, we focus here mainly on general principles as well as on some of the more recent findings.     

Mycorrhized Ri-transformed roots facilitate in vitro inoculation of Cistus incanus with Tuber melanosporum

Progress was made towards a reliable in vitro system for mycorrhizing Cistus incanus seedlings with Tuber melanosporum. A rich growth medium favored extensive growth of mycorrhized Ri-transformed roots (MTR) but inhibited mycelial outgrowth into the medium. A minimal medium, on the other hand, inhibited MTR growth but supported considerable mycelial outgrowth into the medium. While the presence of a C.␣incanus propagule clearly enhanced mycelial growth into the minimal medium, a highly significant factor appeared to be the use of MTR inoculant, which supported mycorrhizal development to the Hartig net stage. The advantages of MTR for in vitro mycorrhization of host plant seedlings are discussed.     

Nucleotide-binding sites in the voltage-dependent anion channel: characterization and localization

In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC. [alpha-(32)P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5'-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [alpha-(32)P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of approximately 17- and approximately 14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotide-binding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.