Evaluation of Postharvest-Processed Oysters by Using PCR-Based Most-Probable-Number Enumeration of Vibrio vulnificus Bacteria

Postharvest processing (PHP) is used to reduce levels of Vibrio vulnificus in oysters, but process validation is labor-intensive and expensive. Therefore, quantitative PCR was evaluated as a rapid confirmation method for most-probable-number enumeration (QPCR-MPN) of V. vulnificus bacteria in PHP oysters. QPCR-MPN showed excellent correlation (R² = 0.97) with standard MPN and increased assay sensitivity and efficiency.     

Tissue kallikrein proteolytic cascade pathways in normal physiology and cancer

Human tissue kallikreins (KLKs or kallikrein-related peptidases) are a subgroup of extracellular serine proteases that act on a wide variety of physiological substrates, while they display aberrant expression patterns in certain types of cancer. Differential expression patterns lead to the exploitation of these proteins as new cancer biomarkers for hormone-dependent malignancies, in particular. The prostate-specific antigen or kallikrein-related peptidase 3 (PSA/KLK3) is an established tumor marker for the diagnosis and monitoring of prostate cancer. It is well documented that specific KLK genes are co-expressed in tissues and in various pathologies suggesting their participation in complex proteolytic cascades. Here, we review the currently established knowledge on the involvement of KLK proteolytic cascades in the regulation of physiological and pathological processes in prostate tissue and in skin. It is well established that the activity of KLKs is often regulated by auto-activation and subsequent autolytic internal cleavage leading to enzymatic inactivation, as well as by inhibitory serpins or by allosteric inhibition by zinc ions. Redistribution of zinc ions and alterations in their concentration due to physiological or pathological reasons activates specific KLKs initiating the kallikrein cascade(s). Recent studies on kallikrein substrate specificity allowed for the construction of a kallikrein interaction network involved in semen liquefaction and prostate cancer, as well as in skin pathologies, such as skin desquamation, psoriasis and cancer. Furthermore, we discuss the crosstalks between known proteolytic pathways and the kallikrein cascades, with emphasis on the activation of plasmin and its implications in prostate cancer. These findings may have clinical implications for the underlying molecular mechanism and management of cancer and other disorders in which KLK activity is elevated.     

Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria.

The import of metals, iron in particular, into mitochondria is poorly understood. Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins. We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria. By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import. Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP. Only reduced iron is functional in generating heme. Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane. Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining why in vivo these ions are not incorporated into porphyrin. The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron. Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria. Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.     

Regulation of Harvest-induced Senescence in Broccoli (Brassica oleracea var. italica) by Cytokinin, Ethylene, and Sucrose

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. The two plant hormones ethylene and cytokinin are known to act antagonistically on harvest-induced senescence in broccoli: ethylene by accelerating the process, and cytokinin by delaying it. To determine the level at which these hormones influenced senescence, we isolated and monitored the expression of genes normally associated with senescence in broccoli florets treated with exogenous 6-benzyl aminopurine (6-BAP), 1-aminocyclopropane-1-carboxylic acid (ACC), a combination of 6-BAP and ACC, and sucrose, in the five days following harvest. Exogenous 6-BAP caused both a reduction (BoACO) and an increase (BoACS) in ethylene biosynthetic gene expression. The expression of genes used as senescence markers, BoCP5 and BoMT1, was reduced, whereas BoCAB1 levels were maintained after harvest in response to exogenous 6-BAP. In addition, the expression of genes encoding sucrose transporters (BoSUC1 and BoSUC2) and carbohydrate metabolizing enzymes (BoINV1 and BoHK1) was also reduced upon 6-BAP feeding. Interestingly, the addition of ACC prevented the 6-BAP-induced increase in expression of BoACS, but 6-BAP negated the ACC-induced increase in expression of BoACO. The culmination of these results indicates a significant role for cytokinin in the delay of senescence. The implication that cytokinin regulates postharvest senescence in broccoli by inhibiting ethylene perception and/or biosynthesis, thus regulating carbohydrate transport and metabolism, as well as senescence-associated gene expression, is discussed and a model presented.     

Plant regeneration in <Emphasis Type="Italic">Arachis stenosperma</Emphasis> Krapov. and W. C. Gregory from roots and calluses derived from leaflets of <Emphasis Type="Italic">in vitro</Emphasis> plants

Seed explants of A. stenosperma were cultured on MS medium supplemented with 6-benzylaminopurine with the aim of rescuing nonviable accessions stored in seed bank conditions. The regeneration potential of leaf explants from in vitro plants derived from embryonic axes was studied by using whole leaflets and leaflet segments. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzylaminopurine and naphthalene acetic acid. Indirect organogenesis was observed in response to 6-benzylaminopurine, either alone or in association with naphthalene acetic acid, in both explant types. Media supplemented with naphthalene acetic acid as the sole growth regulator induced rhizogenesis in whole leaflets and leaflet segments, with subsequent shoot production directly from the roots.