Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma

Epithelial ovarian carcinoma is often diagnosed at an advanced stage of disease and is the leading cause of death from gynaecological neoplasia. The genetic changes that occur during the development of this carcinoma are poorly understood. It has been proposed that IGFIIR, TGF beta1 and TGF beta RII act as a functional unit in the TGF beta growth inhibitory pathway, and that somatic loss-of-function mutations in any one of these genes could lead to disruption of the pathway and subsequent loss of cell cycle control. We have examined these 3 genes in 25 epithelial ovarian carcinomas using single-stranded conformational polymorphism analysis and DNA sequence analysis. A total of 3 somatic missense mutations were found in the TGF beta RII gene, but none in IGFRII or TGF beta1. An association was found between TGF beta RII mutations and histology, with 2 out of 3 clear cell carcinomas having TGF beta RII mutations. This data supports other evidence from mutational analysis of the PTEN and beta -catenin genes that there are distinct developmental pathways responsible for the progression of different epithelial ovarian cancer histologic subtypes. (C) 2001 Cancer Research Campaign.

Replacing prelinguistic behaviors with functional communication

This study evaluated the effectiveness of a teacher-implemented intervention package designed to replace prelinguistic behaviors with functional communication. Four young children with autism participated in a multiple-probe design across three communicative functions. Initially, three existing communication functions were selected for each child. Next, the existing prelinguistic behaviors that the children used to achieve these functions were identified. Replacement forms that were considered more recognizable and symbolic were defined to achieve these same functions. After a baseline phase, teachers received inservice training, consultation, and feedback on how to encourage, acknowledge, and respond to the replacement forms. During intervention, the replacement forms increased and prelinguistic behaviors decreased in most cases. The results suggested that the teacher-implemented intervention was effective in replacing prelinguistic behaviors with alternative forms of functional communication.

Power of the joint segregation analysis method for testing mixed major-gene and polygene inheritance models of quantitative traits

Understanding the genetic architecture of quantitative traits can greatly assist the design of strategies for their manipulation in plant-breeding programs. For a number of traits, genetic variation can be the result of segregation of a few major genes and many polygenes (minor genes). The joint segregation analysis (JSA) is a maximum-likelihood approach for fitting segregation models through the simultaneous use of phenotypic information from multiple generations. Our objective in this paper was to use computer simulation to quantify the power of the JSA method for testing the mixed-inheritance model for quantitative traits when it was applied to the six basic generations: both parents (P-1 and P-2), F-1, F-2, and both backcross generations (B-1 and B-2) derived from crossing the F-1 to each parent. A total of 1968 genetic model-experiment scenarios were considered in the simulation study to quantify the power of the method. Factors that interacted to influence the power of the JSA method to correctly detect genetic models were: (1) whether there were one or two major genes in combination with polygenes, (2) the heritability of the major genes and polygenes, (3) the level of dispersion of the major genes and polygenes between the two parents, and (4) the number of individuals examined in each generation (population size). The greatest levels of power were observed for the genetic models defined with simple inheritance; e.g., the power was greater than 90% for the one major gene model, regardless of the population size and major-gene heritability. Lower levels of power were observed for the genetic models with complex inheritance (major genes and polygenes), low heritability, small population sizes and a large dispersion of favourable genes among the two parents; e.g., the power was less than 5% for the two major-gene model with a heritability value of 0.3 and population sizes of 100 individuals. The JSA methodology was then applied to a previously studied sorghum data-set to investigate the genetic control of the putative drought resistance-trait osmotic adjustment in three crosses. The previous study concluded that there were two major genes segregating for osmotic adjustment in the three crosses. Application of the JSA method resulted in a change in the proposed genetic model. The presence of the two major genes was confirmed with the addition of an unspecified number of polygenes.

Rehabilitation outcomes for brain injured patients in Australia: functional status, length of stay and discharge destination

This study describes the rehabilitation length of stay (LOS), discharge destination and discharge functional status of 149 patients admitted with traumatic brain injury (TBI) to an Australian hospital over a 5-year period. Hospital charts of patients admitted between 1993-1998 were reviewed. Average LOS over the 5-year time period was 61.8 days and only decreased nominally over this time. Longer LOS was predicted by lower admission motor FIM scores and presence of comorbidities. Mean admission and discharge motor FIM scores were 58 and 79, which represented a gain of 21 points. Higher discharge motor FIM scores were predicted by higher admission motor FIM scores and younger age. FIM gain was predicted by cognitive status and age. Most patients, 88%, were discharged back to the community, with 30% changing their living setting or situation. Changing living status was predicted by living alone and having poorer functional status on admission.

Marker concentration patterns of labelled leaf and stem particles in the rumen of cattle grazing bermuda grass (Cynodon dactylon) analysed by reference to a raft model

Large (>1600 mum), ingestively masticated particles of bermuda grass (Cynodon dactylon L. Pers.) leaf and stem labelled with Yb-169 and Ce-144 respectively were inserted into the rumen digesta raft of heifers grazing bermuda grass. The concentration of markers in digesta sampled from the raft and ventral rumen were monitored at regular intervals over approximately 144 h. The data from the two sampling sites were simultaneously fitted to two pool (raft and ventral rumen-reticulum) models with either reversible or sequential flow between the two pools. The sequential flow model fitted the data equally as well as the reversible flow model but the reversible flow model was used because of its greater application. The reversible flow model, hereafter called the raft model, had the following features: a relatively slow age-dependent transfer rate from the raft (means for a gamma 2 distributed rate parameter for leaf 0.0740 v. stem 0.0478 h(-1)), a very slow first order reversible flow from the ventral rumen to the raft (mean for leaf and stem 0.010 h(-1)) and a very rapid first order exit from the ventral rumen (mean of leaf and stem 0.44 h(-1)). The raft was calculated to occupy approximately 0.82 total rumen DM of the raft and ventral rumen pools. Fitting a sequential two pool model or a single exponential model individually to values from each of the two sampling sites yielded similar parameter values for both sites and faster rate parameters for leaf as compared with stem, in agreement with the raft model. These results were interpreted as indicating that the raft forms a large relatively inert pool within the rumen. Particles generated within the raft have difficulty escaping but once into the ventral rumen pool they escape quickly with a low probability of return to the raft. It was concluded that the raft model gave a good interpretation of the data and emphasized escape from and movement within the raft as important components of the residence time of leaf and stem particles within the rumen digesta of cattle.

Functional roles of abdominal and back muscles during isometric axial rotation of the trunk

Electromyographic (EMG) studies have shown that a large number of trunk muscles are recruited during axial rotation. The functional roles of these trunk muscles in axial rotation are multiple and have not been well investigated. In addition, there is no information on the coupling torque at different exertion levels during axial rotation. The aim of the study was to investigate the functional roles of rectus abdominis. external oblique. internal oblique, latissimus dorsi, iliocostalis lumborum and multifidus during isometric right and left axial rotation at 100%, 70%, 50% and 30% maximum voluntary contractions (MVC) in a standing position. The coupling torques in sagittal and coronal planes were measured during axial rotation to examine the coupling nature of torque at different levels of exertions. Results showed that the coupled sagittal torque switches from nil to flexion at maximum exertion of axial rotation. Generally, higher EMG activities were shown at higher exertion levels for all the trunk muscles. Significant differences in activity between the right and left axial rotation exertions were demonstrated in external oblique, internal oblique, latissimus dorsi and iliocostalis lumborum while no difference was shown in rectus abdominis and multifidus. These results demonstrated the different functional roles of trunk muscles during axial rotation. This is important considering that the abdominal and back muscles not only produce torque but also maintain the spinal posture and stability during axial rotation exertions. The changing coupling torque direction in the sagittal plane when submaximal to maximal exertions were compared may indicate the complex nature of the kinetic coupling of trunk muscles. (C) 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

An easy and reliable method for establishment and maintenance of leaf and root cell cultures of Arabidopsis thaliana

Cell suspension cultures are useful for a wide range of biochemical and physiological studies, yet their production can be technically demanding and often unreliable. Here we describe a protocol for producing Arabidopsis cell suspension cultures that is reliable and easy to use.

Protein targeting to the plasma membrane of adult skeletal muscle fiber: An organized mosaic of functional domains

The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta -dystroglycan, dystrophin, and caveolin-3, These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta -dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta -dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expressed domain-specific markers, We found that each marker protein was targeted to distinct microdomains, The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division. (C) 2001 Academic Press.

Functional heterogeneity within rhodamine123(lo) Hoechst33342(lo/sp) primitive hemopoietic stem cells revealed by pyronin Y

Objective. The aim of this study was to determine the function of primitive hematopoietic stem cells (PHSC) at phases G(0) and G(1) of the cell cycle. Materials and Methods. A combination of supravital dyes rhodamine123 (Rh), Hoechst33342 (Ho), and pyronin (PY) was used to isolate the G(0) and G(1) subsets of PHSC. A competitive repopulation assay was used to evaluate their in vivo function. Results. We confirmed that the Rh(lo)Lin(-)Kit(+)Sca-1(+) PHSC were relatively quiescent when compared with the more mature Rh(hi)Lin(-)Kit(+)Sca-1 HSC and Rh(hi)Lin(-)Kit(+)Sca-1(-) progenitors. In addition, cells with Rh(lo)Lin(-)Kit(+)Sca-1(+), Rh(lo)Ho(lo)Lin(-)Sca-1(+), or Rh(lo)Ho(sp)Lin(-)Sca-1(+) phenotypes identified the same cell population. We further subfractionated the Rh(lo)Ho(lo/sp)Lin(-)Sca-1(+) PHSC using PY into PYlo and PYhi subsets. Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the (PYRhHolo/sp)-Rh-lo-Ho-lo PHSC was 1 in 10 cells, whereas there was at least a three-fold lower frequency in those isolated at the G(1) phase (PYhi) We found a dose-dependent PY-mediated cytotoxicity that at moderate concentration affected most of the murine hematopoietic compartment but spared the early HSC compartment. Conclusion. Our data confirm that the HSC compartment is hierarchically ordered on the basis of quiescence and further extend this concept to PY-mediated cytotoxicity. PY supravital dye can be used to reveal functional heterogeneity within the (RhHolosp)-Ho-lo PHSC population but is of limited use in dissecting the relatively more mature hematopoietic stem/progenitor cell population. (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Functional annotation of a full-length mouse cDNA collection

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Cloning and functional analysis of the Sry-related HMG box gene, Sox18

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii);ln intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor-2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue. (C) 2001 Elsevier Science B.V. All rights reserved.

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.

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.

The number of long-lasting functional memory CD8(+) T cells generated depends on the nature of the initial nonspecific stimulation

The mechanism of generation of memory cytotoxic T cells (CTL) following immunization remains controversial. Using tumor protection and IFN-gamma ELISPOT assays in mice to detect functional CTL, we show that the initial effector CTL burst size after immunization is not directly related to the amount of functional memory CTL formed, suggesting that memory CTL are unlikely to arise stochastically from effector CTL. Induction of MHC class II-restricted T helper cells at the time of immunization by inclusion of a T helper peptide or protein in the immunogen, is necessary to generate memory CTL, although no T helper cell induction is required to generate effector CTL to a strong MHC class I-binding peptide. Host protective T cell memory correlates with the number of CTL epitope responsive IFN-gamma-secreting memory T cells as measured in an ELISPOT assay at the time of tumor challenge. We conclude that a different antigen presenting environment is required to induce long-lasting functional memory CTL, and non-cognate stimulation of the immune system is essential to allow generation of a long-lasting host protective memory CTL response.

The life-history basis of latitudinal diversity gradients: how do species traits vary from the poles to the equator.

1. Latitudinal variation among species in life-history traits is often suggested to contribute to high tropical species richness. However, traditional methods of analysing such variation rarely control for phylogeny and latitudinal range overlap between species, potentially giving misleading results. 2. Using a method of pairwise independent contrasts which overcomes these problems, I tested for latitudinal variation among bird species in a number of traits which have been linked, theoretically or empirically, with both latitude and species richness. 3. This method indicates strong support for Rapoport's Rule and decreasing clutch size towards the equator in both hemispheres, but only partial support for decreasing body size and ecological generalism towards the equator. 4. Indirect measures of sexual selection (sexual dichromatism and size dimorphism) show no variation with latitude; an apparent increase in dichromatism towards the equator is shown to be an artefact of phylogeny. 5. Many of the associations between life history and latitude were not detected by traditional cross-species analyses, highlighting the importance of incorporating phylogeny and overlap in studies of geographical life-history variation. Establishing associations between life-history traits and latitude does not prove, but is a necessary prerequisite for., a link between these traits and latitudinal diversity gradients.