Effects of matriconditioning on onion seed germination, seedling emergence and associated physical and metabolic events

The effect of matriconditioning, the physiological presowing seed technique, using Micro-Cel E on Allium cepa L. cv. Czerniakowska seed quality was studied. Several ratios of seeds, carrier, water and time of priming were tested. The most effective treatment for improving onion seed germination at most tested temperatures was priming to a ratio of 2 g seed:1 g Micro-Cel:3 g water for 5 days in light at 15 °C. Matriconditioning greatly improved the germination and emergence percentage, seedling fresh and dry weight and reduced electrolyte leakage compared to that of untreated seeds; this beneficial effect was especially evident at suboptimal temperatures. Matriconditioning improved the germinability of aged seeds, the effect being more pronounced in the more aged seeds. No significant differences in ethylene production by primed and non-primed seeds were observed in the absence of its precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), but its presence during imbibition caused an increase in ethylene production; an enhanced activity of in vivo ACC oxidase in Allium cepa matriconditioned seeds in comparison to untreated seeds, indicates that the endogenous level of ACC is a limiting factor of ethylene production. Likewise, the activity of ACC oxidase isolated from matriconditioned seeds was higher than that from untreated seeds. Higher endo--mannanase and total dehydrogenase activities were observed in primed air-dried seeds in comparing to non-primed seeds.     

Cytotoxic activity of flavonoids and extracts from Retama sphaerocarpa Boissier

Seven flavonoids isolated from chloroform, ethyl acetate and butanol extracts, obtained from the aerial parts of Retama sphaerocarpa, have been assessed for cytotoxic activity against three human cancer cell lines: TK-10 (renal adenocarcinoma), MCF-7 (breast adenocarcinoma) and UACC-62 (melanoma), using the SRB assay. All of them, extracts and flavonoids, were actives in, at least, one of the three cell lines at the recommended National Cancer Institute doses. They produce a dose-dependent inhibition of cell growth at concentrations in the 10(-6)-10(-4) M and 25-250 microg/ml range for the flavonoids and extracts respectively, being the flavonol rhamnazin the most cytotoxic.     

Role of glycosphingolipid microdomains in CD4-dependent HIV-1 fusion

The fusion of HIV-1 with the plasma membrane of CD4+ cells is triggered by the interaction of HIV-1 surface envelope glycoprotein gp120 with the CD4 receptor, and requires coreceptors (CCR5 and CXCR4). Recent advances in the study of HIV-1 entry into CD4+ cells suggest that glycosphingolipids (GSL) may also participate in the fusion process. GSL are organized in functional microdomains which are associated with specific membrane proteins such as CD4. GSL-enriched microdomains were purified from human lymphocytes and reconstituted as a monomolecular film at the air-water interface of a Langmuir film balance. Surface pressure measurements allowed to characterize the sequential interaction of GSL with CD4 and with gp120. Using this approach, we identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main lymphocyte GSL recognized by gp120. In both cases, the interaction was saturable and dramatically increased by CD4. We propose that GSL microdomains behave as moving platforms allowing the recruitment of HIV-1 coreceptors after the initial interaction between the viral particle and CD4. According to this model, the GSL microdomain may: i) stabilize the attachment of the virus with the cell surface through multiple low affinity interactions between the V3 domain of gp120 and the carbohydrate moiety of GSL, and ii) convey the virus to an appropriate coreceptor by moving freely in the outer leaflet of the plasma membrane. This model can be extrapolated to all envelope viruses (e.g. influenza virus) that use cell surface GSL of the host cells as receptors or coreceptors.     

Doxorubicin treatment activates a Z-VAD-sensitive caspase, which causes deltapsim loss, caspase-9 activity, and apoptosis in Jurkat cells

Doxorubicin induces caspase-3 activation and apoptosis in Jurkat cells but inhibition of this enzyme did not prevent cell death, suggesting that another caspase(s) is critically implicated. Western blot analysis of cell extracts indicated that caspases 2, 3, 4, 6, 7, 8, 9, and 10 were activated by doxorubicin. Cotreatment of cells with the caspase inhibitors Ac-DEVD-CHO, Z-VDVAD-fmk, Z-IETD-fmk, and Z-LEHD-fmk alone or in combination, or overexpression of CrmA, prevented many morphological features of apoptosis but not loss of mitochondrial membrane potential (delta(psi)m), phospatidilserine exposure, and cell death. Western blot analysis of cells treated with doxorubicin in the presence of inhibitors allowed elucidation of the sequential order of caspase activation. Z-IETD-fmk or Z-LEHD-fmk, which inhibit caspase-9 activity, blocked the activation of all caspases studied, lamin B degradation, and the development of apoptotic morphology, but not cell death. All morphological and biochemical features of apoptosis, as well as cell death, were prevented by cotreatment of cells with the general caspase inhibitor Z-VAD-fmk or by overexpression of Bcl-2. Doxorubicin cytotoxicity was also blocked by the protein synthesis inhibitor cycloheximide. Delayed addition of Z-VAD-fmk after doxorubicin treatment, but prior to the appearance of cells displaying a low delta(psi)m, prevented cell death. These results, taken together, suggest that the key mediator of doxorubicin-induced apoptosis in Jurkat cells may be an inducible, Z-VAD-sensitive caspase (caspase-X), which would cause delta(psi)m loss, release of apoptogenic factors from mitochondria, and cell death.     

Anti-hepatitic C virus antibodies hidden in circulating antibody/antigen aggregates in HCV-RNA positive patients

The aim of this study was to determine whether antibodies to HCV can be hidden in immunocomplex aggregates in anti-hepatitis C virus (HCV) negative, HCV-RNA positive patients and whether their presence could be related to HCV viral load or HCV genotype. Sera (23 in toto) from patients with elevated alanine aminotransferase (ALT) levels and negative for anti-HCV but positive for HCV-RNA and the immunocomplex aggregates (precipitate with PEG 6000 and glycine 1 M) were studied. The sera were treated using a rapid, simple new ELISA which disrupted the immunocomplex aggregates. Sera from ten patients were tested anti-HCV positive after immunocomplex disruption. No correlation with age, sex, ALT level, viral load or HCV genotype was observed. In some patients anti-HCV antibodies were hidden in circulating antibody/antigen complexes which could be dissociated with a simple, inexpensive and rapid protocol; therefore it can provide a valuable addition to the diagnosis of HCV infection and it may prevent some cases of post-transfusion hepatitis.     

Extensor digitorum brevis free flap: anatomic study and further clinical applications

The extensor digitorum brevis muscle flap is reliable, safe, and can be used either as a pedicle or as a free flap with minimal donor site morbidity. To increase the existing knowledge of this flap and to establish further anatomic basis for the design and elevation of the extensor digitorum brevis flap, 26 specimens from 13 fresh cadavers were dissected under 3.5x loupes. The lateral tarsal artery was found to be the main blood supply to the muscle. It has an average diameter of 1.83+/-0.35 mm and a length of 1.89+/-0.69 cm. The dorsalis pedis artery has, at the level of the lateral tarsal artery takeoff, a diameter of 3.25+/-0.62 mm. From this point to the origin of the deep plantar branch, the dorsalis pedis artery has minimal branching, and the surgeon has available an artery homogeneous in diameter that is 6.77+/-0.99 cm in length. Related neurovascular structures (anterior tibial artery and the venae comitantes, dorsalis pedis and first dorsal metatarsal artery, and deep peroneal nerve) were also studied. A safe and reliable harvesting technique and the "T interposed extensor digitorum brevis" technique for sparing the anterior tibial artery are presented, as are clinical case examples on the use of this flap as a flow-through, extensor digitorum brevis-vascularized nerve graft, a combined extensor digitorum brevis-deep peroneal nerve graft, and a bilobed extensor digitorum brevis-dorsalis pedis fasciosubcutaneous free flap.     

Small-scale isolation of high molecular weight DNA from Leishmania braziliensis

In this paper, we report a method for isolation of high molecular weight DNA from Leishmania promastigotes. This technique is especially indicated for small-scale purification of DNA suitable for the construction of highly representative genomic libraries. In our protocol, lysis buffer is compatible with RNase treatment, avoiding an additional precipitation step and consequent shearing of DNA. In order to prove the quality of the DNA isolated by this method, a Leishmania braziliensis genomic library was constructed, and an L. braziliensis KMP-11 gene was cloned after screening the library with a heterologous probe.     

GA4 Does Not Require Conversion into GA1 to Delay Senescence of Alstroemeria hybrida Leaves

The biological activity and metabolism of applied GA₁ and GA₄ were studied in leaves of alstroemeria (Alstroemeria hybrida). It appeared that GA₄ was 2 orders of magnitude more active in delaying leaf senescence than GA₁. GA₃-13-OMe, a GA analog that cannot be hydroxylated on the 13-C position, also retarded chlorophyll loss, although less efficiently. Tritiated and deuterated GA₁, GA₄, and GA₉ were applied to leaves, and their metabolites were analyzed. According to high performance liquid chromatography and gas chromatography-mass spectrometry analyses, GA₉ was converted into GA₄ and GA₃₄, and GA₄ was converted into GA₃₄ and more polar components. No evidence was found for the conversion of both GA₉ and GA₄ into GA₁, even at the relatively high concentrations that were taken up by the leaf. The results strongly suggest that GA₄ is recognized directly by a receptor involved in regulation of leaf senescence in alstroemeria. Received November 24, 1997; accepted February 17, 1998     

Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA

In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. The primary sequence of ms RNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25?000?×?g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA.