Identification of mitochondrial proteins on two-dimensional electrophoresis gels of extracts of adult Drosophila melanogaster

Several hundred proteins have been resolved on two-dimensional gels of extracts of [³⁵S]methionine-labeled adult Drosophila melanogaster. 27 of these polypeptides disappear from the gel pattern after feeding the K⁺ ionophore nonactin. These proteins have been identified as mitochondrial, since the two-dimensional gel pattern of extracts of isolated mitochondria correlates well with the pattern of the proteins missing from that of nonactin-treated flies. Nine new proteins also appear on the two-dimensional gels of the extracts from the nonactin-treated flies. Apparently, these nine proteins are precursors of the mature mitochondrial forms. These particular data support the concept that processing of many of the cytoplasmically synthesized mitochondrial proteins requires a specific membrane potential, and that some of these proteins are modified intramitochondrially. However, using [³⁵S]methionine incorporation techniques, not all labeled polypeptides disappear from mitochondria during such treatment. Feeding similarly radiolabeled flies with chloramphenicol, an inhibitor of mitochondrial protein synthesis, results in the disappearance of only one protein from the gel pattern with the concurrent appearance of a ‘new’ high-molecular-weight polypeptide. Collectively, these data show that a specific group of [³⁵S]methionine-labeled mitochondrial proteins can be identified by selective inhibition of mitochondrial function in whole cell protein maps of adult D. melanogaster.     

Reproductive strategies of Atlantic salmon: ecology and evolution

Atlantic salmon (Salmo solar, Salmonidae) show a diversity of life history, behavioural and morphological adaptations for reproduction which have evolved as an outcome of competition to maximize reproductive success. Reproductive traits of females have been shaped principally by natural selection for offspring production and survival, those of males by sexual selection for access to matings. Female Atlantic salmon invest approximately six times more energy in offspring production (i.e. gonads) than males and face an important trade-off between number and size of eggs to produce that will maximize the number of surviving offspring. Timing of breeding and the construction of nests appear adapted to increase offspring survival. The most important determinant of female breeding success is body size because it affords high fecundity, access to breeding territories and decreased probability of nest destruction. Asynchronous female spawning and the male ability to spawn rapidly and repeatedly results in male-biased operational sex ratios that generate intense male competition for mates. This has likely been responsible for the evolution of elaborate male secondary sexual characters associated with fighting and status signalling. Furthermore, it has given rise, through frequency-dependent selection, to two alternative male breeding phenotypes: (1) large, anadromous males; and (2) small, mature male parr. Anadromous males invest heavily in behavioural activity on the spawning grounds, searching and fighting for mates and courting them, with body size being an important determinant of their breeding success. This behavioural activity carries a heavy cost, as anadromous males have significantly reduced survival relative to females. In contrast, mature male parr invest proportionally more in testes for sperm competition and attempt to sneak access to matings. While this behaviour also carries costs in terms of subsequent growth and survival, male parr are more likely to breed again, either prior to or following a migration to sea, than anadromous males. While knowledge about the breeding of Atlantic salmon is detailed, we are only beginning to understand the ultimate causes and/or functional significances of their reproductive strategies. Predictive models of the life history variation are developing, focusing on the need for empirical study and testing of life history and reproductive patterns.     

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm.Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

Development of alfalfa strains with differential tolerance to aluminum toxicity

Summary Aluminum toxicity limits root growth in acid subsoils that are difficult to lime. An alternative to subsoil liming is the development of plants having greater tolerance to Al. Alfalfa (Medicago sativa L.) is considered an Al-susceptible species. Preliminary studies indicated that alfalfa cultivars differ in Al tolerance, but the extreme plant-to-plant variation within cultivars prevented the establishment of clearcut cultivar differences.Tolerant and susceptible plants were selected from each of six cultivars (DuPuits, Atlantic, Team, Buffalo, Grimm, and Sirsa 9) grown on an Al-toxic Bladen soil at pH 4.1 to 4.3. The tolerant selections were repotted and interpollinated to form one population of polycross seed. Susceptible selections were treated similarly to form a second population. These two populations, tolerant and susceptible, were subjected to an additional cycle of recurrent phenotypic selection for tolerance and susceptibility, respectively, to Al-toxic Bladen soil at pH 4.6.Plants from the population selected for tolerance to the acid Bladen soil were significantly higher in both root and top vigor on Al-toxic Tatum soil than plants from the population selected for susceptibility. The results indicated that Al tolerance is a heritable trait in these alfalfa populations and that recurrent selection can be used effectively to develop strains having differential tolerance to Al-toxic soils. The observation that only 2% of the plants from the tolerant population were in the most tolerant class suggests a good opportunity for more progress in selecting toward Al tolerance.     

Expression and function of KCNH2 (HERG) in the human jejunum

Previous studies suggest that ether-a-go-go related gene (ERG) KCNH2 potassium channels contribute to the control of motility patterns in the gastrointestinal tract of animal models. The present study examines whether these results can be translated into a role in human gastrointestinal muscles. Messages for two different variants of the KCNH2 gene were detected: KCNH2 V1 human ERG (HERG) (28) and KCNH2 V2 (HERG(USO)) (13). The amount of V2 message was greater than V1 in both human jejunum and brain. The base-pair sequence that gives rise to domains S3-S5 of the channel was identical to that previously published for human KCNH2 V1 and V2. KCNH2 protein was detected immunohistochemically in circular and longitudinal smooth muscle and enteric neurons but not in interstitial cells of Cajal. In the presence of TTX (10(-6) M), atropine (10(-6) M). and l-nitroarginine (10(-4) M) human jejunal circular muscle strips contracted phasically (9 cycles/min) and generated slow waves with superimposed spikes. Low concentrations of the KCNH2 blockers E-4031 (10(-8) M) and MK-499 (3 x 10(-8) M) increased phasic contractile amplitude and the number of spikes per slow wave. The highest concentration of E-4031 (10(-6) M) produced a 10-20 mV depolarization, eliminated slow waves, and replaced phasic contractions with a small tonic contracture. E-4031 (10(-6) M) did not affect [(14)C]ACh release from enteric neurons. We conclude that KCNH2 channels play a fundamental role in the control of motility patterns in human jejunum through their ability to modulate the electrical behavior of smooth muscle cells.     

Tight junction biogenesis during early development

The tight junction (TJ) is an essential component of the differentiated epithelial cell required for polarised transport and intercellular integrity and signalling. Whilst much can be learnt about how the TJ is constructed and maintained and how it functions using a wide range of cellular systems, the mechanisms of TJ biogenesis within developmental models must be studied to gain insight into this process as an integral part of epithelial differentiation. Here, we review TJ biogenesis in the early mammalian embryo, mainly considering the mouse but also including the human and other species, and, briefly, within the amphibian embryo. We relate TJ biogenesis to inherent mechanisms of cell differentiation and biosynthesis occurring during cleavage of the egg and the formation of the first epithelium. We also evaluate a wide range of exogenous cues, including cell-cell interactions, protein kinase C signalling, gap junctional communication, Na⁺/K⁺-ATPase and cellular energy status, that may contribute to TJ biogenesis in the embryo and how these may shape the pattern of early morphogenesis.     

Fluorescence studies of rat cellular retinol binding protein II produced in Escherichia coli: an analysis of four tryptophan substitution mutants.

Rat intestinal cellular retinol binding protein II (CRBP II) is an abundant 134-residue protein that binds all-trans-retinol which contains 4 tryptophans in positions 9, 89, 107, and 110. Our ability to express CRBP II in Escherichia coli and to construct individual tryptophan substitution mutants by site-directed mutagenesis has provided a useful model system for studying the fluorescence of a multi-tryptophan protein. Each of the four mutant proteins binds all-trans-retinol with high affinity, although their affinities are less than that of the wild-type protein. Steady-state and time-resolved fluorescence analyses of these proteins indicate that W107 is at the hydrophobic binding site, W110 is in a polar environment, and the remaining two tryptophans are in a hydrophobic environment. Time-resolved fluorescence study indicates that excited-state energy transfer occurs from the hydrophobic tryptophans to W110. The Stern-Volmer analysis with acrylamide of these proteins reveals that static quenching occurs in the W9F mutant protein while others do not. The fluorescence of rat intestinal fatty acid binding protein (I-FABP), a related protein of known X-ray structure, was also studied for comparison. The results of these findings, coupled with those derived from NMR studies and molecular graphics, suggest that CRBP II undergoes minor structural changes in all of the mutant proteins. Since these effects may be cumulative on the protein structure and function, any conclusions derived from higher mutants in this family of proteins must be treated with caution.