Inorganic nitrogen requirements during shoot organogenesis in tobacco leaf discs

The role of nitrate, ammonium, and culture medium pH on shoot organogenesis in Nicotiana tabacum zz100 leaf discs was examined. The nitrogen composition of a basal liquid shoot induction medium (SIM) containing 39.4 mM NO3- and 20.6 mM NH4+ was altered whilst maintaining the overall ionic balance with Na+ and Cl- ions. Omission of total nitrogen and nitrate, but not ammonium, from SIM prevented the initiation and formation of shoots. When nitrate was used as the sole source of nitrogen, a high frequency of explants initiated and produced leafy shoots. However, the numbers of shoots produced were significantly fewer than the control SIM. Buffering nitrate-only media with the organic acid 2[N-morpholinol]thanesulphonic acid (MES) could not compensate for the omission of ammonium. Ammonium used as the sole source of nitrogen appeared to have a negative effect on explant growth and morphogenesis, with a significant lowering of media pH. Buffering ammonium-only media with MES stabilized pH and allowed a low frequency of explants to initiate shoot meristems. However, no further differentiation into leafy shoots was observed. The amount of available nitrogen appears to be less important than the ratio between nitrate and ammonium. Shoot formation was achieved with a wide range of ratios, but media containing 40 mM nitrate and 20 mM ammonium (70:30) produced the greatest number of shoots per explant. Results from this study indicate a synergistic effect between ammonium and nitrate on shoot organogenesis independent of culture medium pH.

The pathogenesis of oral lichen planus

Both antigen-specific and non-specific mechanisms may be involved in the pathogenesis of oral lichen planus (OLP). Antigen-specific mechanisms in OLP include antigen presentation by basal keratinocytes and antigen-specific keratinocyte killing by CD8(+) cytotoxic T-cells. Non-specific mechanisms include mast cell degranulation and matrix metalloproteinase (MMP) activation in OLP lesions. These mechanisms may combine to cause T-cell accumulation in the superficial lamina propria, basement membrane disruption, intra-epithelial T-cell migration, and keratinocyte apoptosis in OLP. OLP chronicity may be due, in part, to deficient antigen-specific TGF-beta1-mediated immunosuppression. The normal oral mucosa may be an immune privileged site (similar to the eye, testis, and placenta), and breakdown of immune privilege could result in OLP and possibly other autoimmune oral mucosal diseases. Recent findings in mucocutaneous graft-versus-host disease, a clinical and histological correlate of lichen planus, suggest the involvement of TNF-alpha, CD40, Fas, MMPs, and mast cell degranulation in disease pathogenesis. Potential roles for oral Langerhans cells and the regional lymphatics in OLP lesion formation and chronicity are discussed. Carcinogenesis in OLP may be regulated by the integrated signal from various tumor inhibitors (TGF-beta1, TNF-alpha, IFN-gamma, IL-12) and promoters (MIF, MMP-9). We present our recent data implicating antigen-specific and non-specific mechanisms in the pathogenesis of OLP and propose a unifying hypothesis suggesting that both may be involved in lesion development. The initial event in OLP lesion formation and the factors that determine OLP susceptibility are unknown.

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

Low-density koala (Phascolarctos cinereus) populations in the mulgalands of south-west Queensland. I. Faecal pellet sampling protocol

Koala (Phascolarctos cinereus) populations in eastern Australia are threatened by land clearing for agricultural and urban development. At the same time, conservation efforts are hindered by a dearth of information about inland populations. Faecal deposits offer a source of information that is readily available and easily collected non-invasively. We detail a faecal pellet sampling protocol that was developed for use in a large rangeland biogeographic region. The method samples trees in belt transects, uses a thorough search at the tree base to quickly identify trees with koala pellets under them, then estimates the abundance of faecal pellets under those trees using 1-m(2) quadrats. There was a strong linear relationship between these estimates and a complete enumeration of pellet abundance under the same trees. We evaluated the accuracy of our method in detecting trees where pellets were present by means of a misclassification index that was weighed more heavily for missed trees that had high numbers of pellets under them. This showed acceptable accuracy in all landforms except riverine, where some trees with large numbers of pellets were missed. Here, accuracy in detecting pellet presence was improved by sampling with quadrats, rather than basal searches. Finally, we developed a method to reliably age pellets and demonstrate how this protocol could be used with the faecal-standing-crop method to derive a regional estimate of absolute koala abundance.

Habitat requirements for the conservation of arboreal marsupials in dry sclerophyll forests of Southeast Queensland, Australia

The habitat requirements of arboreal marsupials were investigated in the dry sclerophyll forests of southeast Queensland, Australia. Species richness and abundance of arboreal marsupials was correlated to the proportion of total stand basal area occupied by lemon-scented gum (Corymbia citriodora), the height of the tallest trees, and density of hollow-bearing trees. The first two factors suggested that the most productive forests were also the most suitable habitats for arboreal marsupials. Importantly, the number of hollow-bearing trees was a significant factor in determining species richness and abundance of arboreal marsupials in this study, with the maximum number of species reached at sites containing greater than or equal to4 hollow-bearing trees/ha, and maximum abundance occurring at sites with :6 hollow-bearingtrees/ha. The proportion of C. citriodora was significant for the presence of the common brushtail possum (Trichosurus vulpecula), greater glider (Petauroides volans), and the yellow-bellied glider (Petaurus australis), while understory Acacia sp. density was important for the presence of the sugar glider (Petaurus breviceps). The yellow-bellied glider was also affected by two other variables: the density of hollow-bearing trees >50 cm diameter at breast height (dbh), and the time since the last logging. Current Codes of Practice regulating the density of hollow-bearing trees and silvicultural practices in state-owned timber production forests appear to provide adequate protection for arboreal marsupials, but the recently introduced increase in timber extraction rates within state forests may be detrimental to the animals. Also, protective prescriptions do not apply to the privately owned and leasehold estates, which contain the majority of the dry sclerophyll forests in southeast Queensland.

The adult ventral nerve cord as a phylogenetic character in brachyceran Diptera

Insect ganglia are often composed of fused segmental units or neuromeres. We estimated the evolution of the ventral nerve cord (VNC) in higher Diptera by comparing the patterns of neuromere fusion among 33 families of the Brachycera. Variation within families is uncommon, and VNC architecture does not appear to be influenced by body shape. The outgroup pattern, seen in lower Diptera, is fusion of neuromeres belonging to thoracic segments 1 and 2 (T1 and T2), and fusion of neuromeres derived from T3 and abdominal segment 1 (A1). In the abdomen, neuromeres A7-10 are fused into the terminal abdominal ganglion (TAG). Increased neuromere fusion is a feature of the Brachycera. No brachyceran shows less fusion than the outgroups. We established six pattern elements; (1) fusion of T1 and T2, (2) fusion of T3 and A1, (3) fusion of the T1/T2 andT3/A1 ganglia, (4) increase in the number of neuromeres comprising the TAG, (5) anteriorward fusion of abdominal neuromeres, and (6) the complete fusion of thoracic and abdominal neuromeres into a synganglion. States 1 and 2 are present in the outgroup lower Diptera, and state 3 in the Xylophagomorpha, Stratiomyomorpha, Tabanomorpha and Cyclorrhapha. State 4 is a feature of all Eremoneura. State 5 is present in Cyclorrhapha only, and state 6, fusion into a synganglion, has evolved at least 4 times in the Eremoneura. Synapomorphies are provided for the Cyclorrhapha and Muscoidea, and a grouping of three basal brachyceran infraorders Xylophagomorpha, Stratiomyomorpha and Tabanomorpha. The patterns of fusion suggest that VNC architecture has evolved irreversibly, in accordance with Dollo's law.

Spermatozoal morphology of the freshwater anomuran Aegla longirostri Bond-Buckup & Buckup, 1994 (Crustacea : Decapoda : Aeglidae) from South America

Unique sperm morphology is described for Aegla longirostri Bond-Buckup & Buckup, 1994. a representative of the freshwater anomuran family Aeglidae from South America. Comparisons of the spermatozoal ultrastructure of this species with that described for other anomurans indicate that A. longirostri has a distinct suite of spermatozoal characters. Within the Anomura, the aeglids share more spermatozoal characters with the superfamily Lomoidea. represented by the monotypic Australian endemic genus, Lomis, than to any previously described representative from the Galatheoidea, Hippoidea. or Paguroidea. A more basal ancestry, with an independent evolutionary lineage. within the Anomura is Postulated for the Aeglidae. A Superficial resemblance of the spermatozoal ultrastructure of A. longirostri to that described for a palinurid lobster, Jasus, and a thalassinidean mud shrimp, Neaxius, is also noted.

The ultrastructure of Macropodinium moiri and revised diagnosis of the Macropodiniidae (Litostomatea : Trichostomatia)

The ultrastructural features of Macropodinium moiri were investigated. The somatic cortex is composed of two lateral non-ciliated zones covered with trapezoidal plates and separated by a trough-like dorsoventral groove (DVG) which divides the cell into left and right halves. The somatic kineties occupy the margins of the DVG and are composed of monokinetids whose infraciliature shows a typical litostome pattern. The pellicular plates are lamellate, and separated by V-shaped grooves which are lined by thick-walled vacuoles. The DVG cortex is composed of electron-opaque U-shaped ribs which alternate with electron-lucent saccular structures. The DVG surface is composed of small regular pellicular sacs built up to form the ridges of the dorsal DVG. The vestibulum forms a laterally compressed cone with left/right differentiation. The basal section of its non-ciliated right side is internally lined (outer to innermost) by longitudinal fibres, nematodesmata and transverse microtubular ribbons. The left side bears the vestibular kineties and in its basal section is lined (outer to innermost) by small nematodesmata and transverse tubules. Cytoplasmic organelles include endoplasmic reticulum, starch granules and a single contactile vacuole surrounded by patches of nephridioplasm. Hydrogenosomes are absent and coccoid Gram-positive bacteria lie under the ciliated portions of the cell. This set of characteristics differs significantly from those of the all other trichostomes; Macropodiniidae is therefore designated Trichostomatia incertae sedis. A revised familial diagnosis of the Macropodiniidae is proposed.

The ultrastructure of Amylovorax dehorityi comb. nov and erection of the Amylovoracidae fam. nov (Ciliophora : Trichostomatia)

The ultrastructural features of the holotrichous ciliates inhabiting macropodid maruspials were investigated to resolve their morphological similarity to other trichostome ciliates with observed differences in their small subunit rRNA gene sequences. The ultrastructure of Amylovorax dehorityi nov. comb. (formerly Dasytricha dehorityi) was determined by transmission electron microscopy. The somatic kineties are composed of monokinetids whose microtubules show a typical litostome pattern. The somatic cortex is composed of ridges which separate kinety rows, granular ectoplasm and a basal layer of hydrogenosomes lining the tela corticalis. The vestibulum is an invagination of the pellicle lined down one side with kineties (invaginated extensions of the somatic kineties); transverse tubules line the surface of the vestibulum and small nematodesmata surround it forming a cone-like network of struts. Cytoplasmic organelles include hydrogenosomes, irregularly shaped contractile vacuoles surrounded by a sparse spongioplasm, food vacuoles containing bacteria and large numbers of starch granules. This set of characteristics differs sufficiently from those of isotrichids and members of the genus Dasytricha to justify the erection of a new genus (Amylovorax) and a new family (Amylovoracidae). Dasytricha dehorityi, D. dogieli and D. mundayi are reassigned to the new genus Amylovorax and a new species A. quokka is erected. While the gross morphological similarities between Amylovorax and Dasytricha may be explained by convergent evolution, ultrastructural features indicate that these two genera have probably diverged independently from haptorian ancestors by successive reduction of the cortical and vestibular support structures.

Determinants of tissue Doppler measures of regional diastolic function during dobutamine stress echocardiography

Background Diastolic dysfunction induced by ischemia may alter transmitral blood flow, but this reflects global ventricular function, and pseudonormalization may occur with increased preload. Tissue Doppler may assess regional diastolic function and is relatively load-independent, but limited data exist regarding its application to stress testing. We sought to examine the stress response of regional diastolic parameters to dobutomine echocardiography (DbE). Methods Sixty-three patients underwent study with DbE: 20 with low probability of coronary artery disease (CAD) and 43 with CAD who underwent angiography. A standard DbE protocol was used, and segments were categorized as ischemic, scar, or normal. Color tissue Doppler was acquired at baseline and peak stress, and waveforms in the basal and mid segments were used to measure early filling (Em), late filling (Am), and E deceleration time. Significant CAD was defined by stenoses >50% vessel diameter. Results Diastolic parameters had limited feasibility because of merging of Em and Am waves at high heart rates and limited reproducibility. Nonetheless, compared with normal segments, segments subtended with significant stenoses showed a lower Em velocity at rest (6.2 +/- 2.6 cm/s vs 4.8 +/- 2.2 cm/s, P < .0001) and peak (7.5 +/- 4.2 cm/s vs 5.1 +/- 3.6 cm/s, P < .0001), Abnormal segments also showed a shorter E deceleration time (51 +/- 27 ms vs 41 +/- 27 ms, P = .0001) at base and peak. No changes were documented in Am. The same pattern was seen with segments identified as ischemic with wall motion score. However, in the absence of ischemia, segments of patients with left ventricular hypertrophy showed a lower Em velocity, with blunted Em responses to stress. Conclusion Regional diastolic function is sensitive to ischemia. However, a number of practical limitations limit the applicability of diastolic parameters for the quantification of stress echocardiography.

Development of a fully quantitative approach to the interpretation of stress echocardiography using radial and longitudinal myocardial velocities

Background: Tissue Doppler may be used to quantify regional left ventricular function but is limited by segmental variation of longitudinal velocity from base to apex and free to septal walls. We sought to overcome this by developing a composite of longitudinal and radial velocities. Methods and Results. We examined 82 unselected patients undergoing a standard dobutamine echocardiogram. Longitudinal velocity was obtained in the basal and mid segments of each wall using tissue Doppler in the apical views. Radial velocities were derived in the same segments using an automated border detection system and centerline method with regional chords grouped according to segment location and temporally averaged. In 25 patients at low probability of coronary disease, the pattern of regional variation in longitudinal velocity (higher in the septum) was the opposite of radial velocity (higher in the free wall) and the combination was homogenous. In 57 patients undergoing angiography, velocity in abnormal segments was less than normal segments using longitudinal (6.0 +/- 3.6 vs 9.0 +/- 2.2 cm/s, P = .01) and radial velocity (6.0 +/- 4.0 vs 8.0 +/- 3.9 cm/s, P = .02). However, the composite velocity permitted better separation of abnormal and normal segments (13.3 +/- 5.6 vs 17.5 +/- 4.2 cm/s, P = .001). There was no significant difference between the accuracy of this quantitative approach and expert visual wall motion analysis (81% vs 84%, P = .56). Conclusion: Regional variation of uni-dimensional myocardial velocities necessitates site-specific normal ranges, probably because of different fiber directions. Combined analysis of longitudinal and radial velocities allows the derivation of a composite velocity, which is homogenous in all segments and may allow better separation of normal and abnormal myocardium.

Influence of left ventricular size and hemodynamics on the systolic longitudinal myocardial Doppler velocity response to stress

Background Systolic myocardial Doppler velocity accurately identifies coronary artery disease. However, these velocities may be affected by age, hemodynamic responses to stress, and left ventricular cavity size. We sought to examine the influences of these variables on myocardial velocity during dobutamine stress in patients with normal wall motion. Methods One hundred seventy-nine consecutive patients with normal dobutamine echocardiograms were studied. Color myocardial tissue Doppler data were obtained at rest and peak stress, and peak systolic myocardial velocity (PSV) was measured in all basal and midventricular segments. Velocities at rest and peak stress were compared with left ventricular diastolic and systolic volumes, blood pressure, heart rate, and age by Pearson correlation and interdecile analysis by use of analysis of variance. Results The only clinical variable correlating with velocity was age; PSV showed only mild correlation with age at rest (r(2) = 0.01, P = .001) and peak stress (r(2) = 0.02, P = .001), but the normal peak velocity was significantly different between the extremes of age (<44 years and >74 years). There was very weak correlation of PSV with systolic and diastolic blood pressure (r(2) < 0.01), heart rate (r(2) < 0.01), systemic vascular resistance (r(2) = 0.08), and left ventricular volumes (r(2) < 0.01). Conclusions Peak systolic velocity during dobutamine stress is relatively independent of hemodynamic factors and left ventricular cavity size. The extremes of age may influence peak systolic Doppler velocities. These results suggest that peak systolic velocity may be a robust quantitative measure during dobutamine echocardiography across most patient subgroups.

Automated regional myocardial displacement for facilitating the interpretation of dobutamine echocardiography

Quantification of stress echocardiography may overcome the training requirements and subjective nature of visual wall motion score (WMS) assessment, but quantitative approaches may be difficult to apply and require significant time for image processing. The integral of long-axis myocardial velocity is displacement, which may be represented as a color map over the left ventricular myocardium. This study was designed to explore the feasibility and accuracy of measuring long-axis myocardial displacement, derived from tissue Doppler, for the detection of coronary artery disease (CAD) during dobutamine stress echocardiography (DBE). One hundred thirty patients underwent standard DBE, including 30 patients at low risk of CAD, 30 patients with normal coronary angiography (both groups studied to define normal ranges of displacement), and 70 patients who underwent coronary angiography in whom the accuracy of normal ranges was tested. Regional myocardial displacement was obtained by analysis of color tissue Doppler apical images acquired at peak stress. Displacement was compared with WMS, and with the presence of CAD by angiography. The analysis time was 3.2 +/- 1.5 minutes per patient. Segmental displacement was correlated with wall motion (normal 7.4 +/- 3.2 mm, ischemia 5.8 +/- 4.2 mm, viability 4.6 +/- 3.0 mm, scar 4.5 +/- 3.5 mm, p <0.001). Reversal of normal base-apex displacement was an insensitive (19%) but specific (90%) marker of CAD. The sum of displacements within each vascular territory had a sensitivity and specificity of 89% and 79%, respectively, for prediction of significant CAD, compared with 86% and 78%, respectively, for WMS (p = NS). The displacements in the basal segments had a sensitivity and specificity of 83% and 78%, respectively (p = NS). Regional myocardial displacement during DBE is feasible and offers a fast and accurate method for the diagnosis of CAD. (C),2002 by Excerpta Medica, Inc.

Molecular phylogenetics of Lentibulariaceae inferred from plastid rps16 Intron and trnL-F DNA sequences: implications for character evolution and biogeography

Phylogenetic relationships among 75 species of Lentibulariaceae, representing the three recognized genera, were assessed by cladistic analysis of DNA sequences from the plastid rps16 intron and the trnL-F region. Sequence data from the two loci were analyzed both separately and in combination. Consensus trees from all analyses are congruent, and parsimony jackknife results demonstrate strong support for relationships both between and within each of the three demonstrably monophyletic genera. The genus Pinguicula is sister to a Genlisea-Utricularia clade, the phylogenetic structure within this clade closely follows Taylor's recent sectional delimitations based on morphology. Three principal clades are shown within Utricularia, with the basal sections Polypoinpholyx and Pleiochasia together forming the sister lineage of the remaining Utricularia species. Of the fundamental morphological specializations, the stoloniferous growth form apparently arose independently within Genlisea and Utricularia three times, and within Utricularia itself, perhaps more than once. The epiphytic habit has evolved independently at least three times, in Pinguicula, in Utricularia section Phyllaria, and within the two sections Orchidioides and Iperua (in the latter as bromeliad tank-epiphytes). The suspended aquatic habit may have evolved independently within sections Utricularia and Vesiculina. Biogeographic optimization on the phylogeny demonstrates patterns commonly associated with the boreotropics hypothesis and limits the spatial origin of Lentibulariaceae to temperate Eurasia or tropical America.

An embryo-rescue protocol for Vigna interspecific hybrids

A successful embryo-rescue and culture protocol was developed for use with several indigenous Vigna species and mungbean cultivars grown in Australia. Germination of Vigna immature embryos and their subsequent development into plants was influenced by the time at which the embryos were isolated and by which medium additives were placed in the embryo-rescue medium. A medium containing MS basal nutrients with sucrose (88 mM), casein hydrolysate (500 mg L-1) and agar (8 g L-1) but devoid of plant-growth regulators was found to be the best for germination of immature embryos for all four Vigna species investigated. The protocol for successful germination of non-hybrid immature embryos was applied to the recovery of interspecific hybrids involving mungbean and five native Vigna species that had previously been found difficult to hybridise. Several putative hybrid plants were obtained including a confirmed interspecific cross between V. luteola (Jacq.) Benth and V. marina (Burm.) Merrill.