An infrared reflection-absorption spectroscopy study of the secondary structure in (KL4)4K,a therapeutic agent for respiratory distress syndrome,in aqueous monolayers with phospholipids

KLLLLKLLLLKLLLLKLLLLK (KL(4)) has been suggested to mimic some aspects of the pulmonary surfactant protein SP-B and has been tested clinically as a therapeutic agent for respiratory distress syndrome in premature infants [Cochrane, C. G., and Revak, S. D. (1991) Science 254, 566-568]. It is of obvious interest to understand the mechanism of KL(4) function as a guide for design of improved therapeutic agents. Attenuated total reflection (ATR) IR measurements have indicated that KL(4) is predominantly alpha-helical with a transmembrane orientation in lipid multilayers (1), a geometry quite different from the originally proposed peripheral membrane lipid interaction. However, the lipid multilayer model required for ATR may not be the best experimental paradigm to mimic the in vivo function of KL(4). In the current experiments, IR reflection-absorption spectroscopy (IRRAS) was used to evaluate peptide secondary structure in monolayers at the air/water interface, the physical state that best approximates the alveolar lining. In contrast to the ATR-IR results, KL(4) (2.5-5 mol %) films with either DPPC or DPPC/DPPG (7/3 mol ratio) adopted an antiparallel beta-sheet structure at all surface pressures studied > or =5 mN/m, including pressures physiologically relevant for lung function (40-72 mN/m). In contrast, in DPPG/KL(4) films, the dominant conformation was the alpha-helix over the entire pressure range, a possible consequence of enhanced electrostatic interactions. IRRAS has thus provided unique molecular structure information and insight into KL(4)/lipid interaction in a physiologically relevant state. A structural model is proposed for the response of the peptide to surface pressure changes.     

Genotype-by-environment interaction for salinity tolerance in the freshwater-invading copepod Eurytemora affinis

This study examined the extent of phenotypic plasticity for salinity tolerance and genetic variation in plasticity in the invasive copepod Eurytemora affinis. Euryemora affinis is a species complex inhabiting brackish to hypersaline environments but has invaded freshwater lakes and reservoirs within the past century. Reaction norm experiments were performed on a relatively euryhaline population collected from a brackish lake with fluctuating salinity. Life history traits (hatching rate, survival, and development time) were measured for 20 full-sib clutches that were split and reared at four salinities (fresh, 5, 10, and 27 practical salinity units [PSU]). On average, higher salinities (10 and 27 PSU) were more favorable for larval growth, yielding greater survival and faster development rate. Clutches differed significantly in their response to salinity, with a significant genotype-by-environment interaction for development time. In addition, genetic (clutch) effects were evident in response to low salinity, given that survival in fresh (lake) water was significantly positively correlated with survival at 5 PSU for individual clutches. Clutches raised in fresh water could not survive beyond metamorphosis, suggesting that acclimation to fresh water could not occur in a single generation. Results suggest the importance of natural selection during freshwater invasion events, given the inability of plasticity to generate a freshwater phenotype, and the presence of genetic variation for plasticity upon which natural selection could act.     

Strongyloidiasis and Infective Dermatitis Alter Human T Lymphotropic Virus-1 Clonality in vivo

Human T-lymphotropic Virus-1 (HTLV-1) is a retrovirus that persists lifelong by driving clonal proliferation of infected T-cells. HTLV-1 causes a neuroinflammatory disease and adult T-cell leukemia/lymphoma. Strongyloidiasis, a gastrointestinal infection by the helminth Strongyloides stercoralis, and Infective Dermatitis associated with HTLV-1 (IDH), appear to be risk factors for the development of HTLV-1 related diseases. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the HTLV-1-infected T-cell population (i.e. the number of distinct clones and abundance of each clone). A newly developed biodiversity estimator called “DivE” was used to estimate the total number of clones in the blood. We found that the major determinant of proviral load in all subjects without leukemia/lymphoma was the total number of HTLV-1-infected clones. Nevertheless, the significantly higher proviral load in patients with strongyloidiasis or IDH was due to an increase in the mean clone abundance, not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone. In patients co-infected with Strongyloides there was an increased degree of oligoclonal expansion and a higher rate of turnover (i.e. appearance and disappearance) of HTLV-1-infected clones. In Strongyloides co-infected patients and those with IDH, proliferation of the most abundant HTLV-1⁺ T-cell clones is independent of the genomic environment of the provirus, in sharp contrast to patients with HTLV-1 infection alone. This implies that new selection forces are driving oligoclonal proliferation in Strongyloides co-infection and IDH. We conclude that strongyloidiasis and IDH increase the risk of development of HTLV-1-associated diseases by increasing the rate of infection of new clones and the abundance of existing HTLV-1⁺ clones.     

Location of the major barriers to water and ion movement in young roots of Zea mays L.

The main barriers to the movement of water and ions in young roots of Zea mays were located by observing the effects of wounding various cell layers of the cortex on the roots' hydraulic conductivities and root pressures. These parameters were measured with a root pressure probe. Injury to the epidermis and cortex caused no significant change in hydraulic conductivity and either no change or a slight decline in root pressure. Injury to a small area of the endodermis did not change the hydraulic conductivity but caused an immediate and substantial drop in root pressure. When large areas of epidermis and cortex were removed (15–38% of total root mass), the endodermis was always injured and root pressure fell. The hydraulic conductance of the root increased but only by a factor of 1.2–2.7. The results indicate that the endodermis is the main barrier to the radial movement of ions but not water. The major barrier to water is the membranes and apoplast of all the living tissue. These conclusions were drawn from experiments in which hydrostatic-pressure differences were used to induce water flows across young maize roots which had an immature exodermis and an endodermis with Casparian bands but no suberin lamellae or secondary walls. The different reactions of water and ions to the endodermis can be explained by the huge difference in the permeability of membranes to these substances. A hydrophobic wall barrier such as the Casparian band should have little effect on the movement of water, which permeates membranes and, perhaps, also the Casparian bands easily. However, hydrophobic wall depositions largely prevent the movement of ions. Several hours after wounding the endodermis, root pressure recovered to some extent in most of the experiments, indicating that the wound in the endodermis had been partially healed.Abbreviations Lp_r hydraulic conductivity of root; T_1/2 = half-time of water exchange between root xylem and external medium This research was supported by a grant from EUROSILVA (project no. 39473C) to E.S., and by a Bilateral Exchange Grant jointly funded by the Deutsche Forschungsgemeinschaft and the Natural Sciences and Engineering Research Council of Canada to C.A.P. We thank Mr. Burkhard Stumpf for his excellent technicial assistance.     

Suberized bundle sheaths in grasses (Poaceae) of different photosynthetic types. II. Apoplastic permeability

Summary The relative hydraulic conductivities of major and minor longitudinal veins, and the apoplastic permeability of the bundle sheaths surrounding all longitudinal and transverse veins were investigated in representatives of the C₃, C₄/NAD-ME, C₄/NAD-ME/PCK intermediate, C₄/PCK and C₄/NADP-ME photosynthetic types. Using the Hagen-Poiseuille equation and measurements of tracheary element diameters, the number of elements in each vein type and the numbers of each vein type, we calculated that 87–99% of the water flow in a longitudinal direction would be expected to occur in the major veins. The permeability of the mestome sheaths and parenchymatous bundle sheaths surrounding the veins was tested using the negatively-charged, fluorescent dye, trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS). This dye proved nontoxic to plant tissue at a concentration of 0.5%, according to a deplasmolysis test with onion epidermal strips. The PTS concentration achieved in the tested grass leaves was about 0.035%, well below the toxic limit. When a solution of PTS was fed to the leaves by means of a basal cut, the dye moved into the veins of all orders. From there, it moved outward into the surrounding tissues, indicating that the sheaths surrounding the veins of all orders in all species tested were permeable. Therefore, contrary to previous predictions based on structural observations and some tracer studies, bundle sheaths with suberized cell walls do not function as endodermal layers.     

Fetal programming: testosterone exposure of the female sheep during midgestation disrupts the dynamics of its adult gonadotropin secretion during the periovulatory period

Prenatal exposure of the female sheep to excess testosterone (T) leads to hypergonadotropism, multifollicular ovaries, and progressive loss of reproductive cycles. We have determined that prenatal T treatment delays the latency of the estradiol (E2)-induced LH surge. To extend this finding into a natural physiological context, the present study was conducted to determine if the malprogrammed surge mechanism alters the reproductive cycle. Specifically, we wished to determine if prenatal T treatment 1) delays the onset of the preovulatory gonadotropin surge during the natural follicular phase rise in E2, 2) alters pulsatile LH secretion and the dynamics of the secondary FSH surge, and 3) compromises the ensuing luteal function. Females prenatally T-treated from Day 60 to Day 90 of gestation (147 days is term) and control females were studied when they were approximately 2.5 yr of age. Reproductive cycles of control and prenatally T-treated females were synchronized with PGF2alpha, and peripheral blood samples were collected every 2 h for 120 h to characterize cyclic changes in E2, LH, and FSH and then daily for 14 days to monitor changes in luteal progesterone. To assess LH pulse patterns, blood samples were also collected frequently (each 5 min for 6 h) during the follicular and luteal phases of the cycle. The results revealed that, in prenatally T-treated females, 1) the preovulatory increase in E2 was normal; 2) the latencies between the preovulatory increase in E2 and the peaks of the primary LH and FSH surges were longer, but the magnitudes similar; 3) follicular-phase LH pulse frequency was increased; 4) the interval between the primary and secondary FSH surges was reduced but there was a tendency for an increase in duration of the secondary FSH surge; but 5) luteal progesterone patterns were in general unaltered. Thus, exposure of the female to excess T before birth produces perturbances and maltiming in periovulatory gonadotropin secretory dynamics, but these do not produce apparent defects in cycle regularity or luteal function. To reveal the pathologies that lead to the eventual subfertility arising from excess T exposure during midgestation, studies at older ages must be conducted to assess if there is progressive disruption of neuroendocrine and ovarian function.     

A heat-shock promoter fusion to the Ac transposase gene drives inducible transposition of a Ds element during Arabidopsis embryo development

A heat-shock promoter fusion to the Ac transposase gene (hs::TPase) was constructed and introduced into Arabidopsis. In five transformants containing the fusion the abundance of transposase mRNA increased approximately 120-fold on exposure to high temperatures. Hybrid plants containing hs::TPase and a Ds element inserted in a streptomycin resistance gene (Ds::SPT) were made and these plants were self-fertilized either after heat shocking at different stages in development or without exposure to high temperature. The progeny of these plants were sown on streptomycin-containing medium and the frequency with which variegated or streptomycin-resistant (strep^R) seedlings occurred was used as an indication of the frequency of Ds excision. Very few of the progeny of plants not exposed to heat shock or of those heat shocked only during vegetative development were variegated or strep^R. However, plants that were heat shocked after the appearance of flower buds and during seed development produced high frequencies (approaching 100%) of variegated, but very few strep^R, progeny. Furthermore, when variegated seedlings were grown to maturity and self-fertilized without further exposure to heat shock then strep^R seedlings often occurred at high frequency among their progeny. Southern analysis indicated that the majority of these strep^R plants contained a transposed Ds at a new location. These data indicate that in response to heat shock Ds excision frequently occurs in embryonic cells which ultimately give rise to the gametes, as well as in cells of the developing cotyledons. The importance of an inducible transposon system for transposon tagging is discussed.     

Interleukin-8 haplotype structure from nucleotide sequence variation in commercial populations of U.S. beef cattle

The aim of the present study was twofold: first, to design a panel of 96 sires that reflects the breadth of genetic diversity in U.S. beef cattle, and second, to use this panel to discover nucleotide sequence diversity and haplotype structures of interleukin (IL)-8 in commercial populations. The latter is a requisite for epidemiological studies designed to test whether IL8 alleles are risk factors for acquiring or maintaining bacterial infections in production environments. IL-8 encodes a proinflammatory cytokine that plays a central role in cell-mediated immunity by attracting and activating neutrophils in the early stages of host defense against bacterial invasion. Seven single-nucleotide polymorphism (SNP) markers were identified by sequencing two IL8 DNA segments amplified from the panel of 17 popular cattle breeds (MARC beef cattle diversity panel, version 2.1). Assays for automated genotype scoring by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were developed to independently verify the seven SNP alleles in the 96 bulls and 313 cattle from the MARC reference population. Five haplotype structures, spanning the two IL8 DNA segments, were unambiguously defined for the set of seven IL8 SNPs. Based on the breadth of germplasm in bovine diversity panel, the five haplotype structures for IL8 are estimated to represent >98% of those present in these DNA segments in commercial populations of U.S. beef cattle. The frequencies of the five respective haplotypes in the eight Angus sires of the diversity panel (0.75, 0.25, 0.00, 0.00, 0.00) were similar to those scored in 150 purebred Angus cattle from six herds in four Midwestern states (0.82, 0.18, 0.01, 0.00, 0.00), suggesting that the diversity panel may also be useful for estimating allele frequencies in commercial populations. Received: 29 August 2000 / Accepted: 17 November 2000