Amino acid sequences of stomach and nonstomach lysozymes of ruminants

Summary Complete amino acid sequences are presented for lysozymesc from camel and goat stomachs and compared to sequences of other lysozymesc. Tree analysis suggests that the rate of amino acid replacement went up as soon as lysozyme was recruited for the stomach function in early ruminants. The two lysozymes from goat stomach are the products of a gene duplication that probably took place before the divergence of cow, goat, and deer about 25 million years ago. Partial sequences of three lysozymes from goat tears indicated that (a) the goat tear family of lysozymes may have diverged from the stomach lysozyme family by an ancient duplication and (b) later duplications are probably responsible for the multiple forms of tear and milk lysozymes in ruminants.     

Relationship of indole-3-butyric acid and adventitious rooting in M.26 apple (Malus Pumila mill.) shoots cultured in vitro

The role of indole-3-butyric acid (IBA) in adventitious root formation was studied by analyzing the uptake and subsequent metabolism of IBA in shoots of M.26 apple (Malus pumila Mill.) rootstock grown in vitro. Roots were induced by exposing shoots to 4 M IBA and [³H]IBA for 5 days in the dark and then transferring them to plant growth regulator (PGR)-free medium in the light until roots formed. Approximately 50% of the total radioactivity applied was taken up from the agar medium by the shoots during the 5-day incubation period in IBA. Indole-3-butyric acid metabolism was studied by extraction and high-performance liquid chromatographic (HPLC) separation of [³H]IBA and metabolites from the basal sections of treated shoots. The major [³H]IBA metabolite co-eluted with authentic [¹⁴C]indole-3-acetic acid (IAA) suggesting that IBA was converted to IAA in the shoots. The proportion of newly synthesized IAA present as conjugates was higher at the end of the 5-day IBA treatment period than after 13 days in PGR-free medium. There appeared to be no conjugation of IBA at any time.     

Progesterone interacts with P-glycoprotein in multidrug-resistant cells and in the endometrium of gravid uterus

P-Glycoprotein (P-GP) plays a pivotal role in maintaining the multidrug-resistant (MDR) phenotype. This membrane glycoprotein is overproduced in MDR cells and the endometrium of the mouse gravid uterus (Arceci, R.J., Croop, J.M., Horwitz, S.B., and Housman, D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4350-4354). This latter observation and an interest in endogenous substrates for P-GP led to a study of the interaction of steroids with P-GP found in the endometrium of the mouse gravid uterus and in MDR cells derived from the murine macrophage-like cell J774.2. [3H]Azidopine labeling of P-GP from these two sources was inhibited by various steroids, particularly progesterone. Progesterone also markedly inhibited [3H]vinblastine binding to membrane vesicles prepared from MDR cells, enhanced vinblastine accumulation in MDR cells, and increased the sensitivity of MDR cells to vinblastine. In addition, we have demonstrated that the hydrophobicity of a steroid is important in determining its effect on inhibition of drug binding to P-GP. It is concluded that progesterone, a relatively nontoxic endogenous steroid, interacts with P-GP and is capable of reversing drug resistance in MDR cells.     

Effect of high light on the efficiency of photochemical energy conversion in a variety of lichen species with green and blue-green phycobionts

Exposure to high light induced a quantitatively similar decrease in the rate of photosynthesis at limiting photon flux density (PFD) and of photosystem II (PSII) photochemical efficiency, F_V/F_M, in both green and blue-green algal lichens which were fully hydrated. Such depressions in the efficiency of photochemical energy conversion were generally reversible in green algal lichens but rather sustained in blue-green algal lichens. This greater susceptibility of blue-green algal lichens to sustained photoinhibition was not related to differences in the capacity to utilize light in photosynthesis, since the light-and CO₂-saturated rates of photosynthetic O₂ evolution were similar in the two groups. These reductions of PSII photochemical efficiency were, however, largely prevented in lichen thalli which were fully desiccated prior to exposure to high PFD. Thalli of green algal lichens which were allowed to desiccate during the exposure to high light exhibited similar recovery kinetics to those which were kept fully hydrated, whereas bluegreen algal lichens which became desiccated during a similar exposure exhibited greatly accelerated recovery compared to those which were kept fully hydrated. Thus, green algal lichens were able to recover from exposure to excessive PFDs when thalli were in either the hydrated or desiccated state during such an exposure, whereas in blue-green algal lichens the decrease in photochemical efficiency was reversible in thalli illuminated in the desiccated state but rather sustained subsequent to illumination of thalli in the hydrated state.Abbreviations and Symbols F_o yield of instantaneous fluorescence - F_M maximum yield of fluorescence induced by pulses of saturating light - F_V variable yield of fluorescence - PFD photon flux density (400–700 nm) - PSII photosystem II This work was supported by the Deutsche Forschungsgeneinschaft (Forscherguppe Ökophysiologic and Sonderforschungsbereich 251 of the University of Würzburg) and the Fonds der Chemischen Industrie. W.W.A. gratefully acknowledges the support of a fellowship from the Alexander von Humboldt Foundation. We thank Professor T.G.A. Green for identifying and supplying all of the New Zealand lichen material and Professor F.-C. Czygan for advice concerning the chlorophyll analyses which were performed by Johanna Leisner.     

Mechanisms of Injury-Induced Calcium Entry into Peripheral Nerve Myelinated Axons: Role of Reverse Sodium-Calcium Exchange

Abstract: To investigate the route of axonal Ca²⁺ entry during anoxia, electron probe x-ray microanalysis was used to measure elemental composition of anoxic tibial nerve myelinated axons after in vitro experimental procedures that modify transaxolemmal Na⁺ and Ca²⁺ movements. Perfusion of nerve segments with zero-Na⁺/Li⁺-substituted medium and Na⁺ channel blockade by tetrodotoxin (1 µM) prevented anoxia-induced increases in Na and Ca concentrations of axoplasm and mitochondria. Incubation with a zero-Ca²⁺/EGTA perfusate impeded axonal and mitochondrial Ca accumulation during anoxia but did not affect characteristic Na and K responses. Inhibition of Na⁺-Ca²⁺ exchange with bepridil (50 µM) reduced significantly the Ca content of anoxic axons although mitochondrial Ca remained at anoxic levels. Nifedipine (10 µM), an L-type Ca²⁺ channel blocker, did not alter anoxia-induced changes in axonal Na, Ca, and K. Exposure of normoxic control nerves to tetrodotoxin, bepridil, or nifedipine did not affect axonal elemental composition, whereas both zero-Ca²⁺ and zero-Na⁺ solutions altered normal elemental content characteristically and significantly. The findings of this study suggest that during anoxia, Na⁺ enters axons via voltage-gated Na⁺ channels and that subsequent increases in axoplasmic Na⁺ are coupled functionally to extraaxonal Ca²⁺ import. Intracellular Na⁺-dependent, extraaxonal Ca²⁺ entry is consistent with reverse operation of the axolemmal Na⁺-Ca²⁺ exchanger, and we suggest that this mode of Ca²⁺ influx plays a general role in peripheral nerve axon injury.     

Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1A neuropathy

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescence in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype associated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplication, thus providing further support for the PMP22 gene dosage mechanism for CMT1A. Received: 3 May 1995 / Revised: 1 August 1995     

Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events.     

In vitro testing of the biological activity ofRubia cordifolia leaves on primateStrongyloides species

ChimpanzeesPan troglodytes in Kibale National Park, Uganda occasionally swallow entire leaves ofRubia cordifolia (Rubiaceae) without chewing them. The leaves are subsequently egested with minimal damage and no sign of any significant digestion. Similar behavior reported elsewhere has been proposed to have medicinal effects. Here we test the hypothesis that chemical components in swallowed leaves have negative effects on intestinal nematodes. We used anin vitro assay to detect the effects of methanol extracts ofR. cordifolia leaves on nematodes,Strongyloides spp., cultured from feces. Control extracts were distilled water, methanol solution, and methanol extracts of food items that were chewed, rather than swallowed, by chimpanzees. Effects of experimental or control solutions were assayed by nematode motility. There was no difference in nematode motility among control and experimental extracts. This study therefore did not support the hypothesis of pharmacological self-medication via leaf swallowing.     

Analysis of Fusarium causing dermal toxicosis in marram grass planters

In the European coastal dunes, marram grass (Ammophila arenaria) is planted in order to control sand erosion. In the years 1986 to 1991, workers on the Wadden islands in the Netherlands planting marram grass showed lesions of skin and mucous membranes, suggesting a toxic reaction. Fusarium culmorum dominated the mycoflora of those marram grass culms that were used for planting. This plant material had been cut and stored for more than one week in the open. The Fusarium toxin deoxynivalenol (DON) was detected in the suspect marram grass culms. Isolated F. culmorum strains were able to produce DON in vitro in liquid culture as well as in experimentally inoculated wheat heads. Pathogenicity tests, toxin test as well as RAPD analysis showed that the F. culmorum strains were not specialized for marram grass but may form part of the West-European F. culmorum population infecting cereals and grasses. Storage on old sand-dunes with plant debris may have led to the high occurrence of F. culmorum and contamination with DON. Marram grass culms should be obtained from young plantings on dunes on the seaward slopes and cut culms should not be stored.     

Isolation of the MurineNbr1Gene Adjacent to the MurineBrca1Gene

The humanNBR1cDNA has previously been identified using polyclonal sera to CA125, an ovarian tumor antigen used in monitoring ovarian cancer. The gene was mapped to theBRCA1region on chromosome 17q21 and subsequently found to lie in close proximity to the recently identifiedBRCA1gene. The NBR1 protein has a B-box motif but the function of the protein is as yet unknown. To investigate the function and importance of this gene, we have studied the conservation of this gene in other species and in particular in the mouse. We have isolated murineNbr1cDNA and genomic clones. Translation of the cDNA sequence indicates that the protein is highly conserved, being 89% similar and 84% identical to the human. Analysis of the murineNbr1genomic clones indicates that it maps less than 1 kb from theBrca1gene and that, unlike that in human, this region is not duplicated.