Restoration of normal stamen development and pollen formation by fusion of different cytoplasmic male-sterile cultivars of Nicotiana tabacum

Summary Fusion of two cytoplasmic male-sterile cultivars of Nicotiana tabacum, one with N. bigelovii cytoplasm and one with N. undulata cytoplasm, resulted in the restoration of male fertility in cybrid plants. All male-fertile cybrids exhibited fused corollas, which is characteristic for the cultivar with N. undulata cytoplasm, while their stamen structures varied from cybrid to cybrid, some producing stamens with anthers fused to petal-like appendages and one producing stamens of a normal appearance for N. tabacum. Restriction enzyme digestion and agarose gel electrophoresis of mitochondrial DNA showed that mitochondrial DNA of the fertile cybrids was more similar to the male-sterile cultivar with the cytoplasm of N. undulata than to the cultivar with N. bigelovii cytoplasm. Some restriction fragments were unique to the male-fertile cybrids. Comparisons between stamen structure and mitochondrial DNA for eight fertile progeny from one cybrid plant led to the identification of several restriction fragments that appeared at enhanced levels in connection with normal stamen development.     

Effect of cyclophosphamide on the candidacidal activity of rabbit peritoneal macrophages

Single and repeated intravenous administration of cyclophosphamide significantly decreased the candidacidal activity of rabbit peritoneal macrophages. Using higher doses of the drug, a more pronounced decrease, persisting up to 10 d, was observed. The phagocytic index has not changed significantly 10 d after cyclophosphamide injection as compared with controls. No changes in the phagocytic activity were recorded. The decreased candidacidal activity may be one of the causes of serious microbial infections in cyclophosphamide-treated patients.     

The interaction of ammonia with the photosynthetic oxygen-evolving system

The reaction of ammonia with the oxygen-evolving system was investigated using EPR. Two sites with distinct binding properties were found. One site, previously known to be responsible for the modification by ammonia of the multiline EPR signal from the S₂ state and believed to be accessible in this state only, was found to bind ammonia also in the S₁ state although weaker. The second binding site, identified by the effect of bound ammonia on the shape and position of the g = 4.1 EPR signal, was also found to be accessible in both the S₁ and S₂ states. The apparent dissociation constants for ammonia at the two sites in the S₁ and S₂ states were determined. In neither state did the binding the ammonia account for the observed inhibition of oxygen evolution, suggesting that binding to other S states plays an important role in the inhibition. Chloride, which is known to interfere with ammonia-induced inhibition of oxygen evolution, was found to compete with ammonia at the site associated with the modification of the g = 4.1 EPR signal. The broadening of the hyperfine lines of the multiline EPR signal, seen in the presence of ¹⁷O-labeled water, was still observed after the modification of the signal by ammonia. This indicates that ammonia has not completely displaced water bound to the catalytic site in the S₂ state. The results of the binding studies are interpreted in terms of a two state — two site model, where the two states are identified by their EPR signals, the multiline and the g = 4.1 signal, respectively, and the two sites identified by the effects of ammonia on these signals and where the equilibrium between the two states is regulated by the binding of ligands to the sites.     

Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation

Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity "specific" for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg- p -nitroanilide, Suc-Ala-Pro-Phe- p -nitro-anilide and Bz-Phe-Val-Arg- p -nitroanilide) was absent, whereas the embryos were able to hydrolyze them.     

High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae

Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea ) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).     

New pUC-derived expression vectors for rapid construction of cDNA libraries

We have constructed a new series of the pUC-derived plasmids with an extended polylinker obtained from M13tg131 [Kieny et al., Gene 26 (1983) 91-99]. These vectors allowed us to design a simplified version of the method of Okayama and Berg [Mol. Cell. Biol. 2 (1982) 161-170] for establishing complete cDNA libraries. Improvements included easy recovery of the inserted cDNA due to the extended polylinkers; use of these vectors for gene expression in Escherichia coli, and amenability to supercoil sequencing with the universal primers of the M13 system [Chen and Seeburg, DNA 4 (1985) 165-170], which speeds up the identification of positive clones. Moreover, there is no need for an additional linker fragment, as was required by the Okayama and Berg [Mol. Cell. Biol. 2 (1982) 161-170] method. We have successfully used this system to obtain cDNA clones coding for the different chains of the large basement membrane proteins type IV collagen and laminin.     

Bemerkungen zur Taxonomie vonVicia sepium L.

In der ArtVicia sepium L. kann man in Mitteleuropa und in südlichen Teilen von Nordeuropa drei Varietäten unterscheiden: 1. var.sepium mit breiten Blättchen und einem kahlen bis angedrückt behaarten Kelch, 2. var.montana Koch mit schmalen Blättchen und einem kahlen oder zerstreut angedrückt behaarten Kelch, und 3. var.eriocalyx ?elak. mit breiten bis mittelbreiten Blättchen und einem abstehend langbehaarten Kelch, noch mit einer niedrigen und einer hohen Wuchsform. Bei allen Proben wurde die Chromosomezahl 2n=14 festgestellt.     

Bemerkungen zur Taxonomie und Nomenklatur vonLotus krylovii Schischk. etSerg. undL. corniculatus L. subsp.frondosus Freyn

Lotus krylovii Schischk. etSerg. undL. corniculatus L. subsp.frondosus Freyn sind zwei unterschiedliche Taxa, gekennzeichnet durch einige morphologische Merkmale.L. krylovii ist am nächsten mitL. tenuis Waldst. etKit. verwandt;L. corniculatus subsp.frondosus gehört in den Umkreis der Unterart vonL. corniculatus L., die im östlichen Teil des Areals der Art verbreitet ist. Nach ihrer Haupt-Verbreitung gehören die beiden Taxa zu den westasiatischen Arten.     

Some chromosome numbers of the CzechoslovakLeguminosae

Chromosome numbers of the Czechoslovak species of the genusLotus, from various localities have been determined. The paper includes the speciesLotus uliginosus Schkuhr,L.tenuis Waldst. etKit. andL. borbásii Ujhelyi.     

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.