Plant growth analysis: an evaluation of experimental design and computational methods

Journal of Experimental Botany, Vol. 47, No. 302, pp. 1343–1351,September 1996 On page 1345, equation (2) should have read:      

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

EPISTASIS AS A SOURCE OF INCREASED ADDITIVE GENETIC VARIANCE AT POPULATION BOTTLENECKS

The role of epistasis in evolution and speciation has remained controversial. We use a new parameterization of physiological epistasis to examine the effects of epistasis on levels of additive genetic variance during a population bottleneck. We found that all forms of epistasis increase average additive genetic variance in finite populations derived from initial populations with intermediate allele frequencies. Average additive variance continues to increase over many generations, especially at larger population sizes (N = 32 to 64). Additive-by-additive epistasis is the most potent source of additive genetic variance in this situation, whereas dominance-by-dominance epistasis contributes smaller amounts of additive genetic variance. With additive-by-dominance epistasis, additive genetic variance decreases at a relatively high rate immediately after a population bottleneck, rebounding to higher levels after several generations. Empirical examples of epistasis for murine adult body weight based on measured genotypes are provided illustrating the varying effects of epistasis on additive genetic variance during population bottlenecks.     

Perinatal Steroid Treatments Alter Alloparental and Affiliative Behavior in Prairie Voles

This experiment was designed to examine the hypothesis that perinatal manipulation of gonadal or adrenal steroids can alter the subsequent expression of juvenile parental (alloparenting) and affiliative behavior in prairie voles (Microtus ochrogaster). Corticosterone (PRECORT), testosterone (PRE-TP), or oil injections (PRESES) were given on Prenatal Days 12–20 or on Postnatal Days 1–6 (CORT6, TP6, or SES6, respectively). Alloparenting was reduced significantly in females in the CORT6 group and in males in the TP6 group. Sibling affiliative preferences were increased significantly in PRE-TP females and stranger preferences were increased in TP6 and CORT6 females. The results suggest timing is a critical factor determining whether hormones have a facilitative or inhibitory effect on alloparental and affiliative behavior in prairie voles. In this species, corticosterone and testosterone have similar organizational effects on affiliative behavior in females. Alloparental behavior is inhibited by postnatal corticosterone administration in females and by postnatal testosterone administration in males, whereas prenatal steroid administration had no significant effect on alloparenting in either gender.     

Life-history variations of the Lesser Sheathbill Chionis minor in contrasting habitats

From 1987 until 1995, life-history traits of the Lesser Sheathbill Chionis minor in the Kerguelen Archipelago were compared with those at other localities, where this species breeds in seabird colonies. At Kerguelen Island only, some pairs breed on shores free of penguin and cormorant colonies. Moreover, pairs, including nonbreeders, and solitary individuals maintain territories all the year round. Site and mate changes were not linked, and all divorces were permanent. Fidelity rates were similar in all localities and so were the annual survival rates of adults. The survival rate of immature individuals was highest at Kerguelen Island, where sheathbills laid smaller clutches, produced fewer fledglings and had lower breeding success than in the other islands. At Kerguelen Island, pairs breeding on shores had similar clutch sizes but fledged fewer offspring than those breeding in seabird colonies. Further, the proportion of vacant territories and nonbreeders was higher on shores. Kerguelen sheathbills devoted less time to food acquisition than those on Marion Island, being exceptionally kleptoparasitic, and spent a smaller part of their foraging time exploiting seabird colonies. Their diet was mainly algae. The differences on Kerguelen Island as compared with other localities were caused by the presence of an extensive intertidal zone on the former, which reduced competition, making many more sites suitable and the environment more predictable. These life-history and behavioural traits are discussed in relation to life-history theory. As differences were also found, although less extensive, within the Kerguelen Archipelago, we suggest that some traits represent an adaptive response to external constraints and that the life-history strategy of the Lesser Sheathbill is particularly opportunistic.     

Effect of riparian land use on contributions of terrestrial invertebrates to streams

Since terrestrial invertebrates are often consumed by stream fishes, land-use practices that influence the input of terrestrial invertebrates to streams are predicted to have consequences for fish production. We studied the effect of riparian land-use regime on terrestrial invertebrate inputs by estimating the biomass, abundance and taxonomic richness of terrestrial invertebrate drift from 15 streams draining catchments with three different riparian land-use regimes and vegetation types: intensive grazing — exotic pasture grasses (4 streams), extensive grazing — native tussock grasses (6 streams), reserve — native forest (5 streams). Terrestrial invertebrate drift was sampled from replicated stream reaches enclosed by two 1 mm mesh drift nets that spanned the entire channel. The mean biomass of terrestrial invertebrates that entered tussock grassland (12 mg ash-free dry mass m^–2 d^–1) and forest streams (6 mg AFDM m^–2 d^–1) was not significantly different (p > 0.05). However, biomass estimated for tussock grassland and forest streams was significantly higher than biomass that entered pasture streams (1 mg AFDM m^–2 d^–1). Mean abundance and richness of drifting terrestrial invertebrates was not significantly different among land-use types. Winged insects contributed more biomass than wingless invertebrates to both pasture and tussock grassland streams. Winged and wingless invertebrates contributed equally to biomass entering forest streams. Land use was a useful variable explaining landscape-level patterns of terrestrial invertebrate input for New Zealand streams. Evidence from this study suggests that riparian land-use regime will have important influences on the availability of terrestrial invertebrates to stream fishes.     

Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep

Abstract We have previously identified a Brucella melitensis 28 kDa cytosoluble protein (CP28) which was highly immunogenic in infected sheep and which in addition made possible the serological differentiation between infected and B. melitensis Rev.l vaccinated sheep. Monoclonal antibodies against CP28 were used to screen a B. melitensis 16M genomic library and to clone the corresponding gene. DNA sequencing of the gene encoding CP28 of B. melitensis 16M revealed that it was nearly identical to that of the recently published bp26 gene of Brucella abortus vaccine strain S19 coding for a periplasmic protein. The differences between the B. melitensis 16M gene and that of B. abortus S19 consisted of single nucleotide substitutions, one or two codon deletions, one codon addition, and most importantly a 21-bp deletion. The corresponding region of B. abortus S19 contains two 10-bp direct repeats which could have been involved in the genesis of the deletion. Expression of the B. melitensis 16M bp26 gene in Escherichia coli studied by the use of the monoclonal antibodies showed the same characteristics as reported for the B. abortus S19 bp26 gene, i.e. the presence of a higher molecular mass preprotein and a lower molecular mass band which probably corresponds to the mature protein exported to the periplasm. Immunoblotting performed with sera from either naturally infected or B. melitensis H38 experimentally infected sheep confirmed the importance of the B. melitensis CP28/BP26 protein as diagnostic antigen.     

Molecular analysis of tlrD, an MLS resistance determinant from the tylosin producer, Streptomyces fradiae

The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonym ermSF, and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant. However, the T1rA and T1rD proteins differ in their action; the former dimethylates, and the latter monomethylates, the target nucleotide. Here, 2.2 kb of DNA from the tylLM region of the tylosin biosynthetic gene cluster of S. fradiae has been sequenced and shown to encompass tlrD. Comparison of the sequences of tlrA and tlrD (and of their deduced products) with those of related (‘erm-type’) genes from other actinomycetes suggests that the combined presence of tlrA and tlrD in S. fradiae is not the result of recent gene duplication.     

Improvements in the transformation of Arabidopsis thaliana C24 leaf-discs by Agrobacterium tumefaciens

Summary We report here an efficient Arabidopsis leafdisc transformation protocol yielding an average transformation frequency of 1.6 transgenic shoots per leaf explant 4 weeks after the bacterial infection period. Subsequent cultivation in vitro is such that a high percentage (85–90%) of the primary transformants produces seeds with an average seed yield of 100–300 seeds per plant. This improved transformation protocol yields mainly (70%) transformants segregating for a single T-DNA locus of which 68% actually contain one T-DNA insert. The objective is to generate a pool of independent transformants harboring an activator T-DNA construct in a gene tagging approach to isolate genes involved in morphogenesis and auxin signal transduction.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AGM Arabidopsis growth medium - BAP benzylaminopurine - CaMV Cauliflower Mosaic Virus - CTAB Hexadecyltrimethylammoniumbromide - DIG digoxigenin - FeNaEDTA Iron-sodium-ethylenedinitrilo tetraacetic acid complex - GUS ß-Glucuronidase - IBA indole-3-butyric acid - LB left T-DNA border - MES 2-(N-morpholino) ethane sulfonic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - RB right T-DNA border     

Constitutively hyposialylated human T-lymphocyte clones in the Tn-syndrome: binding characteristics of plant and animal lectins

Previously, 1,3-galactosyltransferase-deficient (Tn+) and normal (TF+) T-lymphocyte clones have been established from a patient suffering from Tn-syndrome [Thurnheret al. (1992)Eur J Immunol 22: 1835–42], Tn+ T lymphocytes express only Tn antigen (GalNAc1-O-R) while other O-glycan structures such as sialosyl-Tn (Neu5Ac2,6GalNAc1-O-R) or TF (Gal1-3GalNAc1-O-R) antigens are absent from these cells as shown by flow cytometry using specific mABs for TF and sialosyl-Tn antigen, respectively. Normal T lymphocytes express the TF antigen and derivatives thereof. The surface glycans of Tn+ and TF+ cells were then analysed by flow cytometry using the following sialic acid-binding lectins:Amaranthus caudatus (ACA),Maackia amurensis (MAA),Limax flavus (LFA),Sambucus nigra (SNA) andTriticum vulgare (WGA). Equal and weak binding of MAA and SNA to both TF+ and Tn+ cells was found. WGA, LFA and ACA bound more strongly to TF+ cells than to Tn+ cells. Binding of ACA to TF+ cells was enhanced after sialidase treatment. To investigate the possible biological consequences of hyposialylation, binding of three sialic acid-dependent adhesion molecules to Tn+ and TF+ cells was estimated using radiolabelled Fc-chimeras of sialoadhesin (Sn), myelin-associated glycoprotein (MAG) and CD22. Equal and strong binding of human CD22 to both TF+ and Tn+ cells was found. Whereas binding of Sn and MAG to TF+ cells was strong (100%), binding to Tn+ cells amounted only to 33% (Sn) and 19% (MAG). These results indicate that thein vivo interactions of T lymphocytes in the Tn syndrome with CD22 are not likely to be affected, whereas adhesion mediated by Sn or MAG could be strongly reduced.