Cryopreservation of non-encapsulated embryogenic tissue of sweet potato (Ipomoea batatas)

Embryogenic tissue of the sweet potato (Ipomoea batatas (L) LAM) genotype TIB 10 was established from in vitro axillary shoot tips on Murashige and Skoog (1962) medium supplemented with 5 M 2,4-dichlorophenoxyacetic acid. Embryogenic aggregates of fresh mass 9.0–12 mg were subjected to a rapid freezing protocol in liquid nitrogen following sucrose preculture and varying degrees of dehydration. Up to 50% of embryogenic explants survived rapid freezing after preculture on 0.4 or 0.7M sucrose only. Dehydration with silica gel to moisture contents in the range 18–41% improved the survival after cryopreservation of embryogenic tissue. Tissue dehydrated for intermediate periods exhibited poor survival. Following freezing, embryogenic tissue appeared to develop normally, retaining its competence to produce mature embryos and plantlets.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Germ-Line Mutations in the von Hippel–Lindau Tumor-Suppressor Gene Are Similar to Somatic von Hippel–Lindau Aberrations in Sporadic Renal Cell Carcinoma

von Hippel–Lindau (VHL) disease is a hereditary tumor syndrome predisposing to multifocal bilateral renal cell carcinomas (RCCs), pheochromocytomas, and pancreatic tumors, as well as angiomas and hemangioblastomas of the CNS. A candidate gene for VHL was recently identified, which led to the isolation of a partial cDNA clone with extended open reading frame, without significant homology to known genes or obvious functional motifs, except for an acidic pentamer repeat domain. To further characterize the functional domains of the VHL gene and assess its involvement in hereditary and nonhereditary tumors, we performed mutation analyses and studied its expression in normal and tumor tissue. We identified germ-line mutations in 39% of VHL disease families. Moreover, 33% of sporadic RCCs and all (6/6) sporadic RCC cell lines analyzed showed mutations within the VHL gene. Both germ-line and somatic mutations included deletions, insertions, splice-site mutations, and missense and nonsense mutations, all of which clustered at the 3' end of the corresponding partial VHL cDNA open reading frame, including an alternatively spliced exon 123 nt in length, suggesting functionally important domains encoded by the VHL gene in this region. Over 180 sporadic tumors of other types have shown no detectable base changes within the presumed coding sequence of the VHL gene to date. We conclude that the gene causing VHL has an important and specific role in the etiology of sporadic RCCs, acts as a recessive tumor-suppressor gene, and appears to encode important functional domains within the 3' end of the known open reading frame.     

β-Amylase fromBacillus megaterium

Bacillus megaterium strain B₆ producing extracellular β-amylase was isolated and grown in a medium supplemented with waste potato starch. It showed highest enzyme synthesis in the early stationary phase. The partially purified β-amylase had a temperature optimum at 60°C and a pH optimum at 6.9 and was not affected by Schardinger dextrins. These properties would allow its application in sugar industry.     

Microbial production and purification of arachidonic acid

Summary Mortierella alpina S-17 cultivated in shaker flasks in the semisynthetic liquid medium with 3% glucose at 28 °C for 14 days accumulated 2.1 g/l of intracellular lipid rich in arachidonic acid (ARA). The content of ARA was 50 % of the total fatty acids. The simple method for high pure methyl arachidonate preparation was developed.     

The effect of propargylic substitution on the activation and aromatization of enediynes

The thiol activation and aromatization of bicyclo[7.1.0]enediynes was found to be dependent of the nature of the propargyl substituent. These effects are correlated to antitumor activity.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Conjugates of adenosine mimetics and arginine-rich peptides serve as inhibitors and fluorescent probes but not as long-lifetime photoluminescent probes for protein arginine methyltransferases

The conjugates of an adenosine mimetic and oligo-l -arginine or oligo-d -arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l -methionine (SAM) and S-adenosyl-l -homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.     

Successful synthesis of a glial-specific blood–brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin

Successful manual synthesis of the TD2.2 peptide acting as a blood–brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.     

Thermal biology and locomotor performance of the Andean lizard Liolaemus fitzgeraldi (Liolaemidae) in Argentina

Ectotherms thermoregulate to maintain their body temperature within the optimal range needed for performing vital functions. The effect of climate change on lizards has been studied as regards the sensitivity of locomotor performance to environmental temperatures. We studied thermoregulatory efficiency and locomotor performance for Liolaemus fitzgeraldi in the Central Andes of Argentina. We determined body temperature, micro-environmental temperatures and operative temperatures in the field. In the laboratory, we measured preferred temperatures and calculated the index of thermoregulatory efficiency. We estimated the thermal sensitivity of locomotion by measuring sprint speed (initial velocity and long sprint) and endurance at five different body temperatures. Body temperature was not associated with either micro-environmental temperature, nor did it show differences with preferred temperatures. Thermoregulatory efficiency was moderate (0.61). Initial velocity and long sprint trials showed differences at different temperatures; however, endurance did not. Moreover, the optimal temperatures for the performance trials showed no significant differences among themselves. We conclude that Liolaemus fitzgeraldi has thermal sensitivity in locomotor performance with respect to body temperature and that it is an eurythermic lizard that experiences a large variation in body temperature and that has thermal flexibility in the cold.     

Patterns of Integration and Expression of Retroviral, Non-Replicative Vectors in Avian Embryos: Embryo Developmental Stage and Virus Subgroup Envelope Modulate Tissue-Tropism

We previously demonstrated that Avian Leukemia Viruses (ALV) carrying the v-myc gene specifically induce two types of tumors, cardiomyocytic tumors when the virus is injected before embryonic day 3 (E3), skin tumors when the virus is injected at E3 or E5.

Aiming to elucidate the mechanisms which determine this time-dependent change in target, we infected chick and quail embryos at E3 and E5 with replication-deficient, lacZ gene-carrying, ALV-based viruses produced by a packaging cell line. Three constructs driven by 3 different Long Terminal Repeats (LTRs) were tested and yielded similar results. When the constructs were inoculated at E3 and the lacZ gene product revealed 5 days later, around 70% of the embryos carried lacZ+ clones in the heart, around 50% had positive clones in the skin anywhere on the body, while a few embryos displayed clones in internal organs (liver, stomach, lungs). Immunocytological identification of the heart cell type(s) expressing the virus revealed that the only cells infected were cardiomyocytes. When the constructs were inoculated at E5, no lacZ+ clones appeared in the heart but all were located in the cephalic skin. In order to examine the relationship between viral integration and expression, DNA of different organs or tissues from lacZ stained embryos was analyzed by PCR. A tight correlation between integration and expression in the heart and in the skin was revealed in most cases. In contrast, a significant PCR signal was often detected in the liver or the stomach despite weak or absent expression as revealed by lacZ+ clones.

We then investigated the influence of envelope glycoprotein subgroups on the tropism of these constructs. The lacZ vector driven by RAV-2 LTRs was packaged as subgroups A, B or E viral particles. The A subgroup, used in the part of the study described above, infects both chick and quail while the B and E subgroups are specific for chick or quail respectively. These B and E subgroups induced lacZ+ clones in the heart (after E3 injection) while no clones or only a few were detected in the skin either after E3 or E5 injection. The following conclusions can be drawn: 1) cardiomyocytes are at E3 the major target for integration and expression of ALV-derived viruses in vivo; 2) targets change rapidly with embryonic age; and 3) tissue-specific infections depend on the envelope subgroup, thus presumably on the presence of the cognate receptor. This study clearly indicates that E3 inoculation of ALV-based retroviral vectors is a simple and powerful method to transfer gene sequences into cardiomyocytes and epidermal cells.