Characterization of the growth and auxin physiology of roots of the tomato mutant,diageotropica

Roots of the tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([³H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [³H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.Abbreviations BCA bicinchoninic acid - IAA indole 3-acetic acid - dgt diageotropica - IC₅₀ concentration for 50% inhibition of growth - NPA N-1-naphthylphthalamic acid - SCB-1 semicarbazone 1 This research was supported by grants from Sandoz Agro, Inc. (G.K.M), the National Aeronautics and Space Administration (NASA) and the National Science Foundation (T.L.L), and NASA (D.L.R.).     

Body composition by air-displacement plethysmography by using predicted and measured thoracic gas volumes

The BOD POD, anew air-displacement plethysmograph for measuring human bodycomposition, utilizes the inverse relationship between pressure andvolume (Boyle's law) to measure body volume directly. The quantity ofair in the lungs during tidal breathing, the average thoracic gasvolume (Vtg), is also measured by the BOD POD by using a standardplethysmographic technique. Alternatively, the BOD POD provides the useof a predicted Vtg (Vtg_pred).The validity of using Vtg_pred inplace of measured Vtg (Vtg_meas)to determine the percentage of body fat (%BF) was evaluated in 50 subjects (36 women, 14 men; ages 18-56 yr). There wasno significant difference betweenVtg_meas andVtg_pred (mean difference ± SE, 53.5 ± 63.3 ml) nor in %BF by usingVtg_meas vs.Vtg_pred (0.2 ± 0.2 %BF). Onan individual basis, %BF measured by usingVtg_meas vs.Vtg_pred differed within ±2.0%BF for 82% of the subjects; maximum differences were 2.9 to+3.0% BF. For comparison, data from 24 subjects who had undergonehydrostatic weighing were evaluated for the validity of using predictedvs. measured residual lung volume(VR_pred vs.VR_meas,respectively). Differences between VR_meas andVR_pred andin %BF calculated by usingVR_meas vs.VR_pred weresignificant (187 ± 46 ml and 1.4 ± 0.3% BF, respectively; P < 0.001). On an individual basis,%BF determined by usingVR_meas vs.VR_preddiffered within ±2.0% BF for 46% of the subjects; maximum differences were 2.9 to +3.8% BF. With respect to %BF measured by air displacement, our findings support the use ofVtg_pred for group mean comparisonsand for purposes such as screening in young to middle-aged individuals.This contrasts with the use ofVR_pred inhydrostatic weighing, which leads to significant errors in theestimation of %BF. Furthermore, although the use ofVtg_pred has some application,determining Vtg_meas is relativelysimple in most cases. Therefore, we recommend that the use ofVtg_meas remain as standardexperimental and clinical practice.

     

Mesohabitat-specific macroinvertebrate assemblage responses to water quality variation in mid-continent (North America) great rivers

We compared the responsiveness of macroinvertebrate assemblages to variation in water quality (ions, nutrients, dissolved metals, and suspended sediment) in two mesohabitats within the main channel of three North American great rivers, the Upper Mississippi, Missouri, and Ohio. Based on about 400 paired samples, we examined the responsiveness of benthic assemblages sampled in the littoral zone and assemblages sampled from the surface of woody snags in the main channel. The assemblages in the two mesohabitats were different in all rivers. Taxa richness was much higher in the benthos than on snags. Macroinvertebrate assemblage response to water quality variation was weak on the Mississippi River, but the reasons for this are unknown. Based on analysis of the similarity between the composition of assemblages from groups of sites with high and low concentrations of water quality variables, benthic assemblages were only slightly more sensitive to water chemistry variation than were snag assemblages. Results of two-sample comparisons between groups of sites with high and low concentrations of water quality variables were consistent with rank correlations of assemblage metrics with water quality. In general, there was little difference between habitats in response to variation in water quality on any river. Our simple method of snag sampling in great rivers is usually much easier than littoral benthic sampling because it does not require wading. Snag sampling in large rivers has some limitations (e.g., natural snags are sometimes absent, samples are semi-quantitative), but lack of sensitivity to water quality gradients compared to the benthos is not among them.     

Reproductive rates of Russian wheat aphid (Hemiptera: Aphididae) biotypes 1 and 2 on a susceptible and a resistant wheat at three temperature regimes

The reproductive rates of Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), Biotype 1 (RWA 1) and Biotype 2 (RWA 2) were compared in the laboratory at three temperature regimes on a Russian wheat aphid resistant cultivar ('Prairie Red') and a susceptible cultivar ('TAM 107'). The objective of this study was to expose RWA 1 and RWA 2 to three temperature regimes and two levels of resistance to find whether there were reproductive differences that may occur within each biotype as well as differences in reproduction between biotypes. In addition, temperature effects of the Dn4 gene on biotype reproduction were noted. Differences in reproductive rates between the two biotypes seem to be driven by temperature. For both biotypes, longevity and reproductive rate parameters, except for intrinsic rate of increase, were lower at the 24-29 degree C temperature regime than the 13-18 degree C and 18-24 degree C temperature regimes. The intrinsic rate of increase was higher for both biotypes at the 18-24 degree C and 24-29 degree C temperature regimes than at the 13-18 degree C temperature regime. Reproductive rates between biotypes were similar at the two higher temperature regimes, but the fecundity for RWA 1 was less than RWA 2 at the 13-18 degree C temperature. The change in fecundity rates between RWA 1 and RWA 2 at lower temperatures could have ecological and geographical implications for RWA 2.     

Nanoscale Investigation of Pathogenic Microbial Adhesion to a Biomaterial

Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. In many cases, the device infections are caused by the adhesion of microbes to the implant, ensuing growth, pathogenesis, and dissemination. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an atomic force microscope (AFM) cantilever, and a biomaterial or biofilm substrate. The two model microbes used in this study, Candida parapsilosis (ATCC 90018) and Pseudomonas aeruginosa (ATCC 10145), were chosen for both their clinical relevance and their ease of acquisition and handling in the laboratory setting. Attractive interactions exist between C. parapsilosis and both unmodified silicone rubber and P. aeruginosa biofilms. Using C. parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive forces of 4.3 ± 0.25 nN in the approach portion of the force cycle. On P. aeruginosa biofilms, the magnitude of the attractive force decreases to 2.0 ± 0.40 nN and is preceded by a 2.0-nN repulsion at approximately 75 nm from the cell surface. These data suggest that C. parapsilosis may adhere to both silicone rubber and P. aeruginosa biofilms, possibly contributing to patient morbidity and mortality. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative link between the physicomechanical and physicochemical properties of implant materials and the nanoscale interactions leading to microbial colonization and infection.     

Comparative analysis of gonadal and adrenal activity in the black and white rhinoceros in North America by noninvasive endocrine monitoring

Patterns of fecal reproductive steroid metabolites and adrenal corticoids were characterized for 12‐ to 24‐month periods in black (n = 10 male, 16 female) and white (n = 6 male, 13 female) rhinoceroses at 14 institutions. All black rhinoceros females exhibited at least some ovarian cyclicity on the basis of fecal progestogen analysis (range, 2–12 cycles/yr). However, cycles often were erratic, with many being shorter (<20 days; 18% of cycles) or longer (>32 days; 21%) than the average of 26.8 ± 0.5 days (n = 104 cycles). Five females exhibited periods of acyclicity of 2–10‐month duration that were unrelated to season. One complete and seven partial pregnancies were evaluated in the black rhinoceros. Fecal progestogens increased over luteal phase concentrations after 3 months of gestation. Females resumed cyclicity within 3 months postpartum, before calves were weaned (n = 5). Approximately half of white rhinoceros females (6 of 13) showed no evidence of ovarian cyclicity. Of the cycles observed, 5 were “short” (32.8 ± 1.2 days) and 24 were “long” (70.1 ± 1.6 days). Only two females cycled continuously throughout the study. One had both long (n = 9) and short (n = 2) cycles, whereas the other exhibited long cycles only (n = 5). Fecal estrogen excretion was variable, and profiles were not useful for characterizing follicular activity or diagnosing pregnancy in either species. Males of both species showed no evidence of seasonality on the basis of fecal androgen profiles. Androgen metabolite concentrations were higher (P < 0.05) in the black (27.6 ± 6.9 ng/g) than in the white (16.8 ± 3.1 ng/g) rhinoceros. An adrenocorticotropin hormone challenge in four black rhinoceros males demonstrated that the clearance rate of corticoid metabolites into feces was ～24 hours. Fecal corticoid concentrations did not differ between males and females, but overall means were higher in the black (41.8 ± 3.1 ng/g) than in the white (31.2 ± 1.7 ng/g) rhinoceros. In summary, fecal steroid analysis identified a number of differences in hormonal secretory dynamics between the black and white rhinoceros that may be related to differences in reproductive rates in captivity. Most black rhinoceros females exhibited some cyclic ovarian activity. In contrast, few white rhinoceroses demonstrated evidence of regular estrous cyclicity, and those females that were active had comparatively long cycles. Results also suggest that fecal corticoid concentrations reflect adrenal activity and may be species specific. Continued studies are needed to determine whether fecal corticoid measurements will be useful for understanding the cause of inconsistent gonadal activity in these two species. Because all but three (15.8%) of the white rhinoceroses evaluated in this study were less than 20 years of age compared to 73.1% (19 of 26) of the black rhinoceroses, the impact of age on reproductive and adrenal activity also needs to be evaluated further. Zoo Biol 20:463–486, 2001. © 2002 Wiley‐Liss, Inc.     

Length of the linker and the interval between immunizations influences the efficacy of Vibrio cholerae O1, Ogawa hexasaccharide neoglycoconjugates

Ogawa hexasaccharide neoglycoconjugates induce protective antibodies in mice. Similar Ogawa conjugates but with a longer linker that connects the carrier to shorter saccharides are immunogenic, but generally ineffective at inducing vibriocidal or protective antibodies. The efficacy of Ogawa hexasaccharide neoglycoconjugates of different linker lengths were tested. The majority of mice given immunizations separated by a 14-day gap did not produce vibriocidal or protective antibodies. Mice immunized 28 days apart with immunogens containing the shortest or medium length linker, but not the longest, produced vibriocidal and protective antibodies. A nonprotective, priming dose of purified Ogawa LPS followed 5 days later with a booster of the Ogawa neoglycoconjugates (di-, tetra-, or hexasaccharide) resulted in vibriocidal antibodies at day 10.     

Pseudotyping Incompatibility between HIV-1 and Gibbon Ape Leukemia Virus Env Is Modulated by Vpu

The Env protein from gibbon ape leukemia virus (GaLV) has been shown to be incompatible with human immunodeficiency virus type 1 (HIV-1) in the production of infectious pseudotyped particles. This incompatibility has been mapped to the C-terminal cytoplasmic tail of GaLV Env. Surprisingly, we found that the HIV-1 accessory protein Vpu modulates this incompatibility. The infectivity of HIV-1 pseudotyped with murine leukemia virus (MLV) Env was not affected by Vpu. However, the infectivity of HIV-1 pseudotyped with an MLV Env with the cytoplasmic tail from GaLV Env (MLV/GaLV Env) was restricted 50- to 100-fold by Vpu. A Vpu mutant containing a scrambled membrane-spanning domain, Vpu_RD, was still able to restrict MLV/GaLV Env, but mutation of the serine residues at positions 52 and 56 completely alleviated the restriction. Loss of infectivity appeared to be caused by reduced MLV/GaLV Env incorporation into viral particles. The mechanism of this downmodulation appears to be distinct from Vpu-mediated CD4 downmodulation because Vpu-expressing cells that failed to produce infectious HIV-1 particles nonetheless continued to display robust surface MLV/GaLV Env expression. In addition, if MLV and HIV-1 were simultaneously introduced into the same cells, only the HIV-1 particle infectivity was restricted by Vpu. Collectively, these data suggest that Vpu modulates the cellular distribution of MLV/GaLV Env, preventing its recruitment to HIV-1 budding sites.The gammaretrovirus gibbon ape leukemia virus (GaLV) has been widely used for gene therapy because of its wide host cell tropism and nonpathogenicity (1, 6, 10, 12, 13, 20). The host cell receptor for GaLV Env has been cloned and identified as a sodium-dependent phosphate transporter protein (25, 26). Like other retroviruses, GaLV encodes a single transmembrane surface glycoprotein (GaLV Env), which is cleaved into surface (SU) and transmembrane (TM) subunits (Fig. ​(Fig.1).1). The TM domain of GaLV Env contains a short 30-amino-acid C-terminal cytoplasmic tail. Although GaLV Env functions well when coupled (pseudotyped) with murine leukemia virus (MLV)-based retroviral vectors, it has been shown to be completely incompatible with HIV-1 (4, 35). When GaLV Env is expressed with HIV-1, essentially no infectious HIV-1 particles are produced (4, 35). The mechanism for this infectivity downmodulation is unknown, but the component of GaLV Env responsible for the restriction has been mapped to the cytoplasmic tail. Replacing the cytoplasmic tail of GaLV Env with the equivalent sequence from MLV Env ameliorates the restriction. Likewise, replacing the cytoplasmic tail of MLV Env with that from GaLV Env confers the restriction (4).Open in a separate windowFIG. 1.Schematic of MLV Env protein. Sequences are the C-terminal cytoplasmic tails of MLV Env, GaLV Env, and human CD4. GaLV sequences in boldface are residues that have been shown to modulate the HIV-1 incompatibility (4). Underlined sequences in CD4 are amino acids required for Vpu-mediated downmodulation (2, 15). Arrows denote the location of MLV/GaLV tail substitution. SU, surface domain; TM, transmembrane domain.Vpu is an 81-amino-acid HIV-1 accessory protein produced from the same mRNA as the HIV-1 Env gene. The N terminus of Vpu contains a membrane-spanning domain, followed by a 50-amino-acid cytoplasmic domain. Vpu is unique to HIV-1 and a few closely related SIV strains. The best-characterized roles for Vpu in the HIV-1 life cycle are modulation of host proteins CD4 and tetherin (also known as BST-2, CD317, and HM1.24) (24, 38, 39). Vpu promotes the degradation of CD4 in the endoplasmic reticulum through a proteasome-dependent mechanism (29). The cytoplasmic tail of Vpu physically interacts with the cytoplasmic tail of CD4 and recruits the human β-transducing repeat-containing protein (β-TrCP) and E3 ubiquitin ligase components to polyubiquitinate and ultimately trigger the degradation of CD4 (18). Two serine residues at positions 52 and 56 of Vpu are phosphorylated by casein kinase-2 and are required for CD4 degradation (31, 32). The membrane-spanning domain of Vpu is not specifically required for CD4 degradation. A mutant protein containing a scrambled membrane-spanning sequence, Vpu_RD, is still able to trigger the degradation of CD4 (32). The region of CD4 that is targeted by Vpu is approximately 17 to 13 amino acids from the C terminus in the cytoplasmic tail (Fig. ​(Fig.1)1) (2, 15).In addition to degrading CD4, Vpu has also long been known to result in enhanced viral release (EVR) in certain cell lines (14, 36). Recently, the type I interferon-induced host protein tetherin was identified as being responsible for this Vpu-modulated restriction (24, 38). In the absence of Vpu, tetherin causes particles to remain tethered (hence the name) to the host cell postfission. Although Vpu counteracts the function of tetherin, the exact mechanism has not been fully elucidated. However, the mechanism for tetherin antagonism appears to be distinct from that for modulating CD4. Mutation of the serines 52 and 56 of Vpu abolish CD4 degradation, but only reduce EVR activity (5, 17, 21, 32). Some EVR activity remains even when much of the Vpu cytoplasmic tail is deleted (30). In addition, many mutations in the membrane-spanning domain, such as Vpu_RD, do not affect CD4 degradation and yet completely abolish EVR activity (27, 30, 37). The critical residues in tetherin for recognition by Vpu appear to be in the membrane-spanning domain and not the cytoplasmic tail (9, 19, 28). Although β-TrCP is required for complete EVR activity, there is no consensus whether the degradation of tetherin is proteasome or lysosome mediated (5, 7, 21) or whether degradation is required at all. In some cases there can be some EVR activity in the absence of tetherin degradation (17, 22).We demonstrate here that Vpu is responsible for the incompatibility between HIV-1 and GaLV Env. Glycoproteins containing the cytoplasmic tail from GaLV Env are prevented from being incorporated into HIV-1 particles by Vpu, effectively reducing infectious particle production by 50- to 100-fold. The serines at positions 52 and 56 are required for this restriction, but the membrane-spanning domain is not. Although the mechanism for this restriction appears similar to CD4 degradation, there are apparent differences. Vpu does not prevent surface expression, and it does not prevent its incorporation into MLV particles. Therefore, the mechanism of restriction appears to involve a system that does not rely directly on global protein degradation.