Cryopreservation of grapevine (Vitis spp.) shoot tips from growth chamber-sourced plants and histological observations

Many genebanks rely on cryopreservation as a method to preserve vulnerable field collections of vegetatively propagated crops. Effective cryopreservation procedures have been identified for Vitis; however, they usually use in vitro plantlets as the shoot tip source materials. It is costly to establish Vitis collections in vitro prior to cryopreservation. We sought to determine if growth chamber derived Vitis plants could serve as the source of shoot tips for cryopreservation. Nodal sections from growth chamber derived plants were surface-disinfected and placed in tissue culture on pre-treatment medium for 2 weeks. Uniform apical shoot tips (1 mm) were first obtained from the nodal sections and then precultured for 3 days on medium containing 0.3 M sucrose, salicylic acid, glutathione (reduced form), ascorbic acid and plant preservative mixture. Half-strength PVS2 was applied for 30 min at 22 °C, prior to full-strength PVS2 treatment at 0 °C. Cryopreserved shoot tips had the highest average regrowth of 50 and 55 % without and with cold-acclimation followed with a full-strength PVS2 exposure duration of 40 and 30 min at 0 °C, respectively. This cryopreservation protocol achieved high percentages of regrowth in V. vinifera 'Chardonnay' and 'Riesling' and V. hybrid 'Oppenheim'. Histological observations revealed that shoot tips from growth chamber plants had apical as well as multiple lateral meristems that survived LN immersion. The preservation of multiple meristems in each shoot tip may increase the capacity of shoot tip regeneration in cryopreserved Vitis that originates from ex vitro sources. The high percentage of regrowth after shoot tip cryopreservation using Vitis shoot tips derived from growth chamber source plants suggest that it may be possible to cryopreserve Vitis shoot tips without first introducing each accession into tissue culture

Evolutionary History of Tissue Kallikreins

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs

Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity

Mutations in SPINK5, encoding the serine protease inhibitor LEKTI, cause Netherton syndrome, a severe autosomal recessive genodermatosis. Spink5(-/-) mice faithfully replicate key features of Netherton syndrome, including altered desquamation, impaired keratinization, hair malformation and a skin barrier defect. LEKTI deficiency causes abnormal desmosome cleavage in the upper granular layer through degradation of desmoglein 1 due to stratum corneum tryptic enzyme and stratum corneum chymotryptic enzyme-like hyperactivity. This leads to defective stratum corneum adhesion and resultant loss of skin barrier function. Profilaggrin processing is increased and implicates LEKTI in the cornification process. This work identifies LEKTI as a key regulator of epidermal protease activity and degradation of desmoglein 1 as the primary pathogenic event in Netherton syndrome

LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum

Nature Publishing Group, Journal of Investigative Dermatology, 124, 2, 2005, 360-366 authorLympho-epithelial Kazal-type-related inhibitor (LEKTI) is a putative serine protease inhibitor encoded by serine protease inhibitor Kazal-type 5 (SPINK5). It is strongly expressed in differentiated keratinocytes in normal skin but expression is markedly reduced or absent in Netherton syndrome (NS), a severe ichthyosis caused by SPINK5 mutations. At present, however, both the precise intracellular localization and biological roles of LEKTI are not known. To understand the functional role of LEKTI, we examined the localization of LEKTI together with kallikrein (KLK)7 and KLK5, possible targets of LEKTI, in the human epidermis, by confocal laser scanning microscopy and immunoelectron microscopy. In normal skin, LEKTI, KLK7, and KLK5 were all found in the lamellar granule (LG) system, but were separately localized. LEKTI was expressed earlier than KLK7 and KLK5. In NS skin, LEKTI was absent and an abnormal split in the superficial stratum granulosum was seen in three of four cases. Collectively, these results suggest that in normal skin the LG system transports and secretes LEKTI earlier than KLK7 and KLK5 preventing premature loss of stratum corneum integrity/cohesion. Our data provide new insights into the biological functions of LG and the pathogenesis of NS