GFP-tagged CFTR transgene is functional in the G551D cystic fibrosis mouse colon

Trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to its function, with the most common mutation, DeltaF508, resulting in abnormal processing and trafficking. Therefore, there is a significant need to develop tools, which enable the trafficking of CFTR to be studied in vitro and in vivo. In previous studies it has been demonstrated that fusion of the green fluorescent protein (GFP) to the N-terminus of CFTR does lead to functional expression of CFTR chloride channels in epithelial cell lines. The aim of the present study was to examine whether it is possible to express GFP-tagged CFTR as a transgene in colonic and airway epithelial cells of cystic fibrosis (CF) mice and to correct the CF defect. Using the epithelial-specific human cytokeratin promoter K18, we generated bitransgenic mice cftr(G551D/G551D) K18-GFP-CFTR+/-, designated GFP mice. Transcripts for GFP-CFTR could be detected in bitransgenic mice by use of RT-PCR techniques. Expression of GFP-CFTR protein was detected specifically in the colonic epithelium by both direct GFP fluorescence and the use of an anti-GFP antibody. Ussing chamber studies showed that the ion transport defect in colon and airways observed in cftr(G551D/G551D) mice was partially corrected in the bitransgenic animals. Thus, K18-GFP-CFTR is functionally expressed in transgenic mice, which will be a valuable tool in studies on CFTR synthesis, processing and ion transport in native epithelial tissues.

Prolotherapy injections, saline injections, and exercises for chronic low-back pain: A randomized trial

Objectives. To assess the efficacy of a prolotherapy injection and exercise protocol in the treatment of chronic nonspecific low back pain. Design. Randomized controlled trial with two- by- two factorial design, triple- blinded for injection status, and single- blinded for exercise status. Setting. General practice. Participants. One hundred ten participants with nonspecific low- back pain of average 14 years duration were randomized to have repeated prolotherapy ( 20% glucose/ 0.2% lignocaine) or normal saline injections into tender lumbo- pelvic ligaments and randomized to perform either flexion/ extension exercises or normal activity over 6 months. Main outcome measures: Pain intensity ( VAS) and disability scores ( Roland- Morris) at 2.5, 4, 6, 12, and 24 months. Results. Follow- up was achieved in 96% at 12 months and 80% at 2 years. Ligament injections, with exercises and with normal activity, resulted in significant and sustained reductions in pain and disability throughout the trial, but no attributable effect was found for prolotherapy injections over saline injections or for exercises over normal activity. At 12 months, the proportions achieving more than 50% reduction in pain from baseline by injection group were glucose- lignocaine: 0.46 versus saline: 0.36. By activity group these proportions were exercise: 0.41 versus normal activity: 0.39. Corresponding proportions for > 50% reduction in disability were glucose- lignocaine: 0.42 versus saline 0.36 and exercise: 0.36 versus normal activity: 0.38. There were no between group differences in any of the above measures. Conclusions. In chronic nonspecific low- back pain, significant and sustained reductions in pain and disability occur with ligament injections, irrespective of the solution injected or the concurrent use of exercises.

The nature of anterior knee pain following injection of hypertonic saline into the infrapatellar fat pad

The infrapatellar fat pad has been implicated as a possible source of anterior knee pain. This study examined the nature, distribution and time-course of experimentally induced pain in the infrapatellar fat pad. Hypertonic saline (5%) was injected into the medial fat pad of 11 healthy individuals with no history of knee pain. Severity of pain was assessed at rest and during activity using an 11 point numerical rating scale (NRS) at regular intervals over 15-30 min following injection. Participants described the size of the pain region from a series of different sized circles while the area and type of pain was established from a body chart and the McGill pain questionnaire. The effect of pain on temperature-pain threshold and sensory thresholds of the anterior knee was assessed. Participants generally reported a deep aching pain that peaked in severity around 3 min and gradually declined over 15 min. Pain levels were not altered by clinical manoeuvres designed to impinge the fat pad. The size of the pain region was related to pain intensity. Pain was most commonly felt in the region of the fat pad medial to the patella, although some individuals reported proximal referred pain as far as the groin region. Thermal and sensory thresholds were not altered at a region close to the injection site during the experimental pain. These results suggest that nociceptive stimulation of the infrapatellar fat pad may cause anterior knee pain that is not necessarily confined locally particularly if pain is severe. This has implications for the investigation of pathological structures in patients presenting clinically with anterior knee pain and provides an experimental model of anterior knee pain. (C) 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Levels of physical activity for colon cancer prevention compared with generic public health recommendations: Population prevalence and sociodemographic correlates

Background: The proportion of Australian adults achieving physical activity levels believed to be sufficient for colon cancer prevention was estimated, and sociodemographic correlates (age, gender, educational attainment, occupation, marital status, and children in household) of meeting these levels of activity were analyzed. Methods: Data from the 2000 National Physical Activity Survey were used to estimate the prevalence of participation in physical activity in relation to three criteria: generic public health recommendations, weekly amount of at least moderate-intensity physical activity currently believed to reduce risk of colon cancer, and weekly amount of vigorous-intensity physical activity believed to reduce risk of colon cancer. Results: Overall, 46% of adults met the generic public health criterion, 26% met the colon cancer criterion based on participation in at least moderate-intensity physical activity, and 10% met the colon cancer criterion based on vigorous-intensity physical activity. Women were less likely than men to meet the colon cancer criteria. Younger and more educated persons were more likely to meet all three criteria. The most pronounced differences between gender, age, and educational attainment groups were found for meeting the amount of vigorous-intensity physical activity believed to reduce risk of colon cancer. Conclusions: The population prevalence for meeting proposed physical activity criteria for colon cancer prevention is low and much lower than that related to the more generic public health recommendations. If further epidemiologic studies confirm that high volumes and intensities of activity are required, the public health challenges for colon cancer will be significant.

Isoform specific changes in PPAR alpha and beta in colon and breast cancer with differentiation

To investigate the role of peroxisome proliferator-activated receptors (PPARs) chi and beta in the differentiation of colon cancer cells, we differentiated HT-29 cells using sodium butyrate (NaB) and culturing post-confluence and assessed differentiation using the marker intestinal alkaline phosphatase. While PPAR chi levels only changed with culturing post confluence, PPAR beta levels increased independent of the method of differentiation. To explore further the differences induced by NaB. we assessed changes in both PPAR isoforms in MCF-7 breast cancer cells cultured in the presence of NaB over 48 h. Again a very different expression pattern was observed with PPAR-1 increasing after 4 h and remaining elevated, while PPAR beta increased transiently. Our studies suggest that the expression of PPARs is dependent upon both the method of differentiation and on time. Moreover, these studies show that changes in levels are not required for the differentiation of colon cancer cell lines, whereas changes in PPAR beta are more closely associated with differentiation. (c) 2005 Elsevier Inc. All rights reserved.

Identification of three gene candidates for multicellular resistance in colon carcinoma

Solid tumours display elevated resistance to chemo- and radiotherapies compared to individual tumour derived cells. This so-called multicellular resistance (MCR) phenomenon can only be partly explained by reduced diffusion and altered cell cycle status; even fast growing cells on the surface of solid tumours display MCR. Multicellular spheroids (MCS) recapture this phenomenon ex vivo and here we compare gene expression in exponentially growing MCS with gene expression in monolayer culture. Using an 18,664 gene microarray, we identified 42 differentially expressed genes and three of these genes can be linked to potential mechanisms of MCR. A group of interferon response genes were also up-regulated in MCS, as were a number of genes that that are indicative of greater differentiation in three-dimensional cultures.

Mutations in the MYB intron I regulatory sequence increase transcription in colon cancers

Although MYB overexpression in colorectal cancer (CRC) is known to be a prognostic indicator for poor survival, the basis for this overexpression is unclear. Among multiple levels of MYB regulation, the most dynamic is the control of transcriptional elongation by sequences within intron I. The authors have proposed that this regulatory sequence is transcribed into an RNA stem-loop and 19-residue polyuridine tract, and is subject to mutation in CRC. When this region was examined in colorectal and breast carcinoma cell lines and tissues, the authors found frequent mutations only in CRC. It was determined that these mutations allowed increased transcription compared with the wild type sequence. These data suggest that this MYB regulatory region within intron I is subject to mutations in CRC but not breast cancer, perhaps consistent with the mutagenic insult that occurs within the colon and not mammary tissue. In CRC, these mutations may contribute to MYB overexpression, highlighting the importance of noncoding sequences in the regulation of key cancer genes. (c) 2006 Wiley-Liss, Inc.

The use of hypertonic saline for treating intracranial hypertension after traumatic brain injury

The past decade has witnessed a resurgence of interest in the use of hypertonic saline for low-volume resuscitation after trauma. Preliminary studies suggested that benefits are limited to a subgroup of trauma patients with brain injury, but a recent study of prehospital administration of hypertonic saline to patients with traumatic brain injury failed to confirm a benefit. Animal and human studies have demonstrated that hypertonic saline has clinically desirable physiological effects on cerebral blood flow, intracranial pressure, and inflammatory responses in models of neurotrauma. There are few clinical studies in traumatic brain injury with patient survival as an end point. In this review, we examined the experimental and clinical knowledge of hypertonic saline as an osmotherapeutic agent in neurotrauma.

The effect of organic mulch amendments on the physical and chemical properties and revegetation success of a saline-sodic minespoil from central Queensland, Australia

Saline-sodic clay minespoil materials excavated during open-cut coal mining in central Queensland, Australia, pose significant challenges for revegetation, particularly where suitable topsoil capping is not available. We examined the ability of sawdust or straw mulch amendments to ameliorate the adverse properties of these minespoils and improve the success of revegetation efforts. In laboratory studies, mulch application improved infiltration, increased soil moisture retention and reduced surface crust strength. In the field, mulches incorporated to a depth of 0.15 m at application rates of at least 20 t/ha straw or 80 t/ha sawdust were needed to mitigate against capillary rise of salts during drying cycles and support satisfactory vegetation cover. Further research is needed to determine whether improvements are maintained beyond the 4-year trial period reported here.

Adenocarcinoma of colon differentiating as dome epithelium of gut-associated lymphoid tissue

Aims: An early adenocarcinoma of the ascending colon was confined to a mass of gut-associated lymphoid tissue (GALT). The first description of an adenocarcinoma of colon differentiating as dome epithelium is presented. Methods and results: A plaque-like carcinoma was identified opposite the ileocaecal valve in an asymptomatic 56-year-old man with a family history of colorectal cancer. Malignant epithelium was confined to a mass of GALT filling but limited to the submucosa, Characterization of the neoplasm was undertaken by means of mucin histochemistry, immunohistochemistry, electron microscopy and assessment of DNA microsatellite instability status. The malignant epithelium comprised well differentiated columnar cells with a microvillous brush border and expressing MUC1, but no goblet cells or expression of MUC2. The demonstration of focal clusters of intraepithelial B-lymphocytes supported the presence of functioning M-cells within the malignant neoplasm. The cancer was DNA microsatellite stable despite the finding of tumour infiltrating lymphocytes. Conclusions: There is evidence for the origin of colorectal neoplasia from dome epithelium in both experimental models and microreconstruction studies of early adenomas in nonpolypotic human colorectal mucose, It is suggested that the lymphocyte-rich subset of colorectal cancer that expresses MUC1 but not MUC2 may be differentiating as dome epithelium of gut-associated lymphoid tissue.

Electrolyte transport in the mammalian colon: mechanisms and implications for disease Kunzelmann and Marcus Mall

Electrolyte Transport in the Mammalian Colon: Mechanisms and Implications for Disease. Physiol. Rev. 82: 245-289, 2002.The colonic epithelium has both absorptive and secretory functions. The transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium does secret mucus, bicarbonate, and KCl. Polarized distribution of transport proteins in both luminal and basolateral membranes enables efficient salt transport in both directions, probably even within an individual cell. Meanwhile, most of the participating transport proteins have been identified, and their function has been studied in detail. Absorption of NaCl is a rather steady process that is controlled by steroid hormones regulating the expression of epithelial Na+ channels (ENaC), the Na+-K+-ATPase, and additional modulating factors such as the serum- and glucocorticoid-regulated kinase SGK. Acute regulation of absorption may occur by a Na+ feedback mechanism and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl- secretion in the adult colon relies on luminal CFTR, which is a cAMP-regulated Cl- channel and a regulator of other transport proteins. As a consequence, mutations in CFTR result in both impaired Cl- secretion and enhanced Na+ absorption in the colon of cystic fibrosis (CF) patients. Ca2+- and cAMP-activated basolateral K+ channels support both secretion and absorption of electrolytes and work in concert with additional regulatory proteins, which determine their functional and pharmacological profile. Knowledge of the mechanisms of electrolyte transport in the colon enables the development of new strategies for the treatment of CF and secretory diarrhea. It will also lead to a better understanding of the pathophysiological events during inflammatory bowel disease and development of colonic carcinoma.

Measurement of exchangeable cations in saline soils

The measurement of exchangeable cations in saline soils is limited by the difficulty in accurately separating soluble cations from exchangeable cations. A method is examined for saline soils in which exchangeable cations are calculated as the total extractable cations minus the concentration of soil solution (soluble) cations. In addition, a further two standard methods were investigated, one which assumes the total soil extractable cations are exchangeable, the other utilises a pretreatment to remove soluble salts prior to measurement of the remaining (exchangeable) cations. After equilibration with a range of sodium adsorption ratio (SAR) solutions at various ionic strengths, the exchangeable cation concentrations of two soils (Dermosol and Vertosol) were determined by these methods and compared to known values. The assumption that exchangeable cations can be estimated as the total soil extractable cations, although valid at low ionic strength, resulted in an overestimation of exchangeable Na and Ca concentrations at higher ionic strengths due to the presence of soluble salts. Pretreatment with ethanol and glycerol was found to effectively remove soluble salts thus allowing the accurate measurement of the effective cation exchange capacity (ECEC), however, dilution associated with the pretreatment process decreased concentrations of exchangeable Ca while simultaneously increasing exchangeable Na. Using the proposed method, good correlations were found between known and measured concentrations of exchangeable Na (Dermosol: y=0.873x and Vertosol: y=0.960x) and Ca (Dermosol: y=0.906x, and Vertosol: y=1.05x). Therefore, for soils with an ionic strength of approximately 50 mM (ECse 4 dS m-1) or greater (in which exchangeable cation concentrations are overestimated by assuming the total soil cations are exchangeable), concentrations can be calculated as difference between total extractable cations and soluble cations.

Nitrogen balance for an agroforestry system irrigated with saline, high nitrogen effluent

Land disposal is commonly used for urban and industrial wastewater, largely due to the high costs involved in alternative treatments or disposal systems. However, the viability of such systems depends on many factors, including the composition of the effluent water, soil type, the plant species grown, growth rate, and planting density. The objective of this study is to establish whether land disposal of nitrogen (N) rich effluent using an agroforestry system is sustainable, and determine the effect of irrigation rate and tree planting density on the N cycle and subsequent N removal. We examined systems for the sustainable disposal of a high strength industrial effluent. The challenge was to leach the salt, by using a sufficiently high rate of irrigation, while simultaneously ensuring that N did not leach from the soil profile. We describe the N balance for two plant systems irrigated with effluent, one comprising Eucalyptus tereticornis and Eucalyptus moluccana and a Rhodes grass (Chloris gayana) pasture, and the other, Rhodes grass pasture alone. Nitrogen balance was assessed from N inputs in effluent and rainfall, accumulation of N in the plant biomass, changes in soil N storage, N loss in run-off water, denitrification and N loss to the groundwater by deep-drainage. Biomass production was estimated from allometric relationships derived from yearly destructive harvesting of selected trees. The N content of that biomass was then calculated from measured N content of the various plant parts, and their mass. Approximately 300 kg N/ha/yr was assimilated into tree biomass at a planting density of 2500 tree/ha of E. moluccana. In addition to tree assimilation, pasture growth between the tree rows, which was regularly harvested, contributed substantially to N uptake. If the trees were harvested after two years of growth and grass harvested regularly, biomass removal of N by the mixed system would be about 700 kg N/ha/yr. The results of this study show that the current system of effluent disposal is not sustainable as the nitrate leaching from the soil profile far exceeds standards set out by the ANZECC guidelines. Hence additional means of N removal will need to be implemented. Biological N removal is an area that warrants further studies as it is aimed at reducing N levels in the effluent before irrigation. This will complement the current agroforestry system.