Two sides of the same coin? Transient hybridization in refugia and rapid postglacial ecological divergence ensures the evolutionary persistence of sister Nothofagus.

Glacial periods have been considered inhospitable environments that consisted of treeless vegetation at higher latitudes. The fossil record suggests many species survived the Last Glacial Maximum (LGM) within refugia, usually at lower latitudes. However, phylogeographic studies have given support to the existence of previously unknown high-latitude refugia not detected in the fossil record. Here we test the hypothesis that cold-tolerant trees of Patagonia survived cold periods in microclimatically favourable locales where hybridisation occurred between sister taxa. To study local presence through glacial periods in multiple refugia we used pollen records and genetic information (isozymes, microsatellites, and combined nuclear and chloroplast DNA sequences) of population pairs of Nothofagus antarctica and N. pumilio, that belong to the ancient subgenus Nothofagus which can potentially hybridize in nature, along their entire latitudinal range in Patagonia. Studied species share the N. dombeyi type pollen which was abundant >20% at the northern-most latitudinal bands (35-43°S), even during the LGM. Mid- and southern latitudinal records (44-55°S) yielded lower abundances of ~10% that increased after c. 15.0 cal. ka BP. Therefore, fossil pollen evidence suggests a long-lasting local presence of Nothofagus throughout glacial-interglacial cycles but mostly as small populations between 44-51°S. We found species-specific and shared genetic variants, the latter of which attained relatively high frequencies thus providing evidence of ancestral polymorphisms. Populations of each species were similarly diverse suggesting survival throughout the latitudinal range. Estimates of coalescent divergence times were broadly synchronous across latitudes suggesting that regional climates similarly affected populations and species that hybridized through climate cycles fostering local persistence.

Comparing the responses of grain-fed feedlot cattle under moderate heat load and during subsequent recovery with those of feed-restricted thermoneutral counterparts: blood cells and inflammatory markers.

Given the climate projections for livestock rearing regions globally, understanding the inflammatory status of livestock under various heat loads will be informative to animal welfare and management. A survey of plasma inflammatory markers was conducted, and blood leucocyte counts followed to investigate the capacity of the ~ 500 kg grain fed Black Angus steer to respond to and recover from a moderate heat load challenge. Two sequential cohorts of 12 steers were housed in climate-controlled rooms (CCR) for 18 days. A thermally challenged (TC) group (n = 2 × 6) experienced five consecutive periods: PreChallenge, Challenge, and Recovery within the CCR, and 40 days in outdoor pens (PENS and Late PENS). PreChallenge (5 days) and Recovery (7 days) delivered thermoneutral conditions, whereas in Challenge the TC steers experienced a diurnal temperature range of 28-35 °C. A feed-restricted thermoneutral (FRTN) treatment (n = 2 × 6) was run concurrently to differentiate between responses to reduced feed intake alone and moderate heat stress. Blood neutrophil counts were particularly sensitive to moderate heat load with higher numbers during Challlenge and in PENs. The plasma concentrations of TNFα and IL-1ß were depressed in the TC group compared to the FRTN counterparts and remained so for 40 days after Challenge. Linear relationships of the concentrations of IL-1ß, IL-10, and haptoglobin with rumen temperature or dry matter intake detected in the FRTN group were altered or absent in the TC group. The findings suggest significant impacts of moderate heat load on the inflammatory status of feedlot cattle.

Comparing the responses of grain fed feedlot cattle under moderate heat load and during subsequent recovery with those of feed restricted thermoneutral counterparts: metabolic hormones.

We set out to determine the impact of moderate heat load on the plasma concentrations of a suite of hormones involved in regulating energy metabolism and feed intake. The responses of the thermally challenged (TC) feedlot steers were compared to those of feed restricted thermoneutral (FRTN) steers. Two sequential cohorts of twelve 518 ± 23 kg Black Angus steers on finisher grain ration were housed in climate-controlled rooms (CCR) for 18 days and returned to outdoor pens for 40 days. The TC group was subjected to a diurnal range of 28-35 °C for 7 days (Challenge) but held in thermoneutral conditions beforehand (PreChallenge), and in Recovery (after Challenge). The FRTN group was held in thermoneutral conditions and feed restricted throughout. Blood was collected over the three periods in CCR and two periods in outdoor pens for 40 days (PENS and Late PENS). Plasma concentrations of prolactin, thyroid stimulating hormone, insulin, leptin, adiponectin and thyroxine (T4) were determined during the five periods. Whilst the pituitary hormones were relatively stable, there were differences in plasma leptin, adiponectin and T4 between the two groups during Challenge and Recovery, and occasionally in PENS. The interaction of the plasma hormone concentrations and rumen temperature and DMI were also investigated. Whilst the positive relationship between DMI and leptin was confirmed, we found a strong negative relationship between adiponectin and rumen temperature, and a strong positive relationship between adiponectin and dry matter intake (DMI) in the TC steers only.

Comparing the responses of grain fed feedlot cattle under moderate heat load and during subsequent recovery with those of feed restricted thermoneutral counterparts: plasma biochemistry.

Responses to heat stress in ruminants reflect the integration of local climatic conditions, environment/production system and the animal's homeostatic and homeorhetic capacities. Thus, the goal of ameliorating heat stress requires experimental settings that, within limits, closely resemble the target production system and cohort. We investigated the blood biochemical changes of two sequential cohorts of twelve 518 ± 23 kg grain fed Black Angus steers. Each cohort consisted of two treatments of 6 head/group: a thermally challenged (TC) treatment and a feed restricted thermoneutral (FRTN) treatment. Both groups were housed in climate controlled rooms for 19 days, with the TC group experiencing three distinct periods: PreChallenge, Challenge and Recovery. PreChallenge and Recovery delivered thermoneutral conditions, while Challenge consisted of 7 days of moderate diurnal heat load. The FRTN group was maintained in thermoneutral conditions at all times. Both groups were then relocated to outdoor pens for a further 40 days to detect any enduring change to metabolism as a consequence of the treatments. We compared blood biochemical responses of the treatments and inferred likely metabolic changes. Relative to the FRTN group, the TC animals experienced limited supply of triglycerides, cholesterol and glutamine during moderate heat load, suggesting constraints to energy metabolism. Lower blood urea during Recovery and in outdoor pens implied a requirement to capture N rather than allow its excretion. Altered liver enzyme profiles indicated a higher level of hepatic stress in the TC group. By the completion of feedlot finishing, the groups were not separable on most measures.

Articular cartilage gene expression patterns in the tissue surrounding the impact site following applications of shear and axial loads.

BACKGROUND: Osteoarthritis is a degradative joint disease found in humans and commercial swine which can develop from a number of factors, including prior joint trauma. An impact injury model was developed to deliver in vitro loads to disease-free porcine patellae in a model of OA. METHODS: Axial impactions (2000 N normal) and shear impactions (500 N normal with induced shear forces) were delivered to 48 randomly assigned patellae. The patellae were then cultured for 0, 3, 7, or 14 days following the impact. Specimens in the tissue surrounding the loading site were harvested and expression of 18 OA related genes was studied via quantitative PCR. The selected genes were previously identified from published work and fell into four categories: cartilage matrix, degradative enzymes, inflammatory response, and apoptosis. RESULTS: Type II collagen (Col2a1) showed significantly lower expression in shear vs. axial adjacent tissue at day 0 and 7 (fold changes of 0.40 & 0.19, respectively). In addition, higher expression of degradative enzymes and Fas, an apoptosis gene, was observed in the shear specimens. CONCLUSIONS: The results suggest that a more physiologically valid shear load may induce more damage to surrounding articular cartilage than a normal load alone.

Brachygnathia, cardiomegaly and renal hypoplasia syndrome (BCRHS) in Merino sheep maps to a 1.1-megabase region on ovine chromosome OAR2.

A genome scan was conducted to map the autosomal recessive lethal disorder brachygnathia, cardiomegaly and renal hypoplasia syndrome (BCRHS) in Poll Merino sheep. The scan involved 10 affected and 27 unaffected animals from a single Poll Merino/Merino sheep flock, which were genotyped with the Illumina Ovine SNP50 BeadChip. Association and homozygosity mapping analyses located the disorder in a region comprising 20 consecutive SNPs spanning 1.1 Mb towards the distal end of chromosome OAR2. All affected animals and none of the unaffected animals were homozygous for the associated haplotype in this region. These results provide the basis for identifying the causative mutation(s) and should enable the development of a DNA test to identify carriers in the Poll Merino sheep population. Understanding the molecular control of BCRHS may provide insight into the fundamental genetic control and regulation of the affected organ systems.

Evidence for pleiotropism and recent selection in the PLAG1 region in Australian Beef cattle.

A putative functional mutation (rs109231213) near PLAG1 (BTA14) associated with stature was studied in beef cattle. Data from 8199 Bos taurus, Bos indicus and Tropical Composite cattle were used to test the associations between rs109231213 and various phenotypes. Further, 23 496 SNPs located on BTA14 were tested for association with these phenotypes, both independently and fitted together with rs109231213. The C allele of rs109231213 significantly increased hip height, weight, net food intake, age at puberty in males and females and decreased IGF-I concentration in blood and fat depth. When rs109231213 was fitted as a fixed effect in the model, there was an overall reduction in associations between other SNPs and these traits but some SNPs remained associated (P < 10(-4) ). Frequency of the mutant C allele of rs109231213 differed among B. indicus (0.52), B. taurus (0.96) and Tropical Composite (0.68). Most chromosomes carrying the C allele had the same surrounding 10 SNP haplotype, probably because the C allele was introgressed into Brahman from B. taurus cattle. A region of reduced heterozygosity surrounds the C allele; this is small in B. taurus but 20 Mb long in Brahmans, indicating recent and strong selection for the mutant allele. Thus, the C allele appears to mark a mutation that has been selected almost to fixation in the B. taurus breeds studied here and introduced into Brahman cattle during grading up and selected to a frequency of 0.52 despite its negative effects on fertility.

Satisfaction with life among veterans with spinal cord injuries completing multidisciplinary rehabilitation.

STUDY DESIGN: This is a single-group, retrospective study. OBJECTIVES: The objective of this study was to understand the factors contributing to satisfaction with life (SWL) among veterans with a spinal cord injury (SCI) completing rehabilitation. SETTING: This study was conducted at Veterans Administration Medical Center, San Diego.MethodsBetween 1998 and 2010, N=118 Veterans participated in a Commission on Accreditation of Rehabilitation Facilities (CARF)-accredited rehabilitation program after a new SCI. Pre-rehabilitation measures of impairment at the organ/body level, activity limitation at the person level and participation restriction at the societal level were used to predict Satisfaction with Life Scale (SWLS) scores upon discharge. RESULTS: Although overall mean SWLS admission and discharge scores were not significantly different (P>0.10), individual change in SWLS scores during rehabilitation was notable, ranging from a 17-point improvement to a 22-point decline across veterans (mean Δ=+1.18, s.d.=6.04). Veterans who exhibited less activity limitation (higher cognitive functioning, r=0.31, P<0.01) and less participation restriction (greater social integration, r=0.21, P<0.05; a trend toward greater economic sufficiency, r=0.16, P<0.10) at baseline had higher SWLS scores after rehabilitation. When these factors were entered together into a single regression model, only cognitive functioning remained statistically significant (P<0.05). CONCLUSION: Findings highlight potential targets for interventions, aiming to improve SWL post SCI among US veterans. In addition to directly targeting SWL with psychosocial interventions, results suggest that rehabilitation settings should continue and/or expand upon programs targeting cognitive functioning (activity limitation) and social integration (participation restriction). Nevertheless, additional research is warranted to identify the biopsychosocial factors most reliably associated with SWL and/or other aspects of quality of life.

Genomewide association for a dominant pigmentation gene in sheep.

Most published genomewide association studies (GWAS) in sheep have investigated recessively inherited monogenic traits. The objective here was to assess the feasibility of performing GWAS for a dominant trait for which the genetic basis was already known. A total of 42 Manchega and Rasa Aragonesa sheep that segregate solid black or white coat pigmentation were genotyped using the SNP50 BeadChip. Previous analysis in Manchegas demonstrated a complete association between the pigmentation trait and alleles of the MC1R gene, setting an a priori expectation for GWAS. Multiple methods were used to identify and quantify the strength of population substructure between black and white animals, before allelic association testing was performed for 49,034 SNPs. Following correction for substructure, GWAS identified the most strongly associated SNP (s26449) was also the closest to the MC1R gene. The finding was strongly supported by the permutation tree-based random forest (RF) analysis. Importantly, GWAS identified unlinked SNP with only slightly lower p-values than for s26449. Random forest analysis indicated these were false positives, suggesting interpretation based on both approaches was beneficial. The results indicate that a combined analytical approach can be successful in studies where a modest number of animals are available and substantial population stratification exists.

Assessing the performance and learning curve of orthopaedic surgical trainees in primary total hip arthroplasty.

INTRODUCTION: The aims of this study were threefold: first, to review the surgical performance of trainees in our departments by reviewing postoperative radiographs and operative times; second, to investigate the effect of supervision and assistant grade on postoperative radiographs and operative times; and third, to monitor trainees over a 6-month period looking for changes in postoperative radiograph appearances and operative times to assess whether these parameters reflect a trainee's learning curve. METHODS: A retrospective evaluation of a continuous series of primary hip arthroplasty procedures performed by 12 trainee orthopaedic surgeons (StR) during their arthroplasty rotation. In total, 348 primary total hip replacement (pTHR) operations were performed by StRs. Operative time, acetabular cup inclination, radiological leg length discrepancy (rLLD), femoral stem alignment (FSA) and the Barrack score for cementation were evaluated. The mean number of pTHRs performed per 6-month placement was 29 (range 15-51). Operative times were available for 292 cases and all postoperative imaging was evaluated. RESULTS: The mean operative time for StRs as first-surgeon was 84.3 minutes (range 42-174 minutes). Significant differences in operative times were observed between individual StRs. As a cohort, the operative times were not affected by the level of supervision but were significantly slower when StRs were assisted by other StRs. Significant differences in rLLD, FSA and Barrack score for cementation were observed across the cohort of StRs, although this did not change at a group or individual level between the first and second halves of the 6-month placement. CONCLUSIONS: Used in isolation, postoperative radiographs and operative time are not an effective measure of the learning curve in primary hip arthroplasty, however, they may be a useful adjunct in assessing the performance of orthopaedic trainees when learning primary hip arthroplasty.

Characterization of gene expression in naturally occurring feline degenerative joint disease-associated pain.

Degenerative joint disease (DJD) associated-pain is a clinically relevant and common condition affecting domesticated cats and other species including humans. Identification of the neurobiological signature of pain is well developed in rodent pain models, however such information is lacking from animals or humans with naturally occurring painful conditions. In this study, identification of housekeeping genes (HKG) for neuronal tissue and expression levels of genes considered associated with chronic pain in rodent models were explored in cats with naturally occurring osteoarthritic pain. Fourteen adult cats were evaluated - seven without clinical signs of osteoarthritic pain, and seven with hind limb radiographic DJD and pain. Expression of an investigator-selected set of pain signaling genes (including ASIC3, ATF3, COX2, CX3CL1, NAV1.7, NAV1.8, NAV1.9, NGF, NK1R, TNFα, TRKA) in lumbar spinal cord dorsal horn and lumbar dorsal root ganglia tissues from clinically healthy cats and cats with DJD were studied using quantitative RT-PCR (qPCR). HKG identified as the most stable across all tissue samples were many of the ribosomal protein genes, such as RPL30 and RPS19. qPCR results showed ATF3 and CX3CL1 up-regulated in DJD-affected dorsal root ganglia compared to clinically healthy controls. In spinal cord, CX3CL1 was up-regulated and NGF was down-regulated when DJD-affected samples were compared to healthy samples. Further work is needed to understand the neurobiology of pain in naturally occurring disease and what rodent models are predictive of these changes in more heterogeneous populations such as domestic cats.

The DNA damage response is developmentally regulated in the African trypanosome.

Genomes are affected by a wide range of damage, which has resulted in the evolution of a number of widely conserved DNA repair pathways. Most of these repair reactions have been described in the African trypanosome Trypanosoma brucei, which is a genetically tractable eukaryotic microbe and important human and animal parasite, but little work has considered how the DNA damage response operates throughout the T. brucei life cycle. Using quantitative PCR we have assessed damage induction and repair in both the nuclear and mitochondrial genomes of the parasite. We show differing kinetics of repair for three forms of DNA damage, and dramatic differences in repair between replicative life cycle forms found in the testse fly midgut and the mammal. We find that mammal-infective T. brucei cells repair oxidative and crosslink-induced DNA damage more efficiently than tsetse-infective cells and, moreover, very distinct patterns of induction and repair of DNA alkylating damage in the two life cycle forms. We also reveal robust repair of DNA lesions in the highly unusual T. brucei mitochondrial genome (the kinetoplast). By examining mutants we show that nuclear alkylation damage is repaired by the concerted action of two repair pathways, and that Rad51 acts in kinetoplast repair. Finally, we correlate repair with cell cycle arrest and cell growth, revealing that induced DNA damage has strikingly differing effects on the two life cycle stages, with distinct timing of alkylation-induced cell cycle arrest and higher levels of damage induced death in mammal-infective cells. Our data reveal that T. brucei regulates the DNA damage response during its life cycle, a capacity that may be shared by many microbial pathogens that exist in variant environments during growth and transmission.

Gene conversions mediating antigenic variation in Trypanosoma brucei can occur in variant surface glycoprotein expression sites lacking 70-base-pair repeat sequences.

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid immune system-mediated killing by their mammalian host. An important mechanism for switching the expressed VSG gene is the duplicative transposition of a silent VSG gene into one of the telomeric VSG expression sites of the trypanosome, resulting in the replacement of the previously expressed VSG gene. This process appears to be a gene conversion reaction, and it has been postulated that sequences within the expression site may act to initiate and direct the reaction. All bloodstream form expression sites contain huge arrays (many kilobase pairs) of 70-bp repeat sequences that act as the 5' boundary of gene conversion reactions involving most silent VSG genes. For this reason, the 70-bp repeats seemed a likely candidate to be involved in the initiation of switching. Here, we show that deletion of the 70-bp repeats from the active expression site does not affect duplicative transposition of VSG genes from silent expression sites. We conclude that the 70-bp repeats do not appear to function as indispensable initiation sites for duplicative transposition and are unlikely to be the recognition sequence for a sequence-specific enzyme which initiates recombination-based VSG switching.

Genomic analyses of tropical beef cattle fertility based on genotyping pools of Brahman cows with unknown pedigree.

We introduce an innovative approach to lowering the overall cost of obtaining genomic EBV (GEBV) and encourage their use in commercial extensive herds of Brahman beef cattle. In our approach, the DNA genotyping of cow herds from 2 independent properties was performed using a high-density bovine SNP chip on DNA from pooled blood samples, grouped according to the result of a pregnancy test following their first and second joining opportunities. For the DNA pooling strategy, 15 to 28 blood samples from the same phenotype and contemporary group were allocated to pools. Across the 2 properties, a total of 183 pools were created representing 4,164 cows. In addition, blood samples from 309 bulls from the same properties were also taken. After genotyping and quality control, 74,584 remaining SNP were used for analyses. Pools and individual DNA samples were related by means of a "hybrid" genomic relationship matrix. The pooled genotyping analysis of 2 large and independent commercial populations of tropical beef cattle was able to recover significant and plausible associations between SNP and pregnancy test outcome. We discuss 24 SNP with significant association ( < 1.0 × 10) and mapped within 40 kb of an annotated gene. We have established a method to estimate the GEBV in young herd bulls for a trait that is currently unable to be predicted at all. In summary, our novel approach allowed us to conduct genomic analyses of fertility in 2 large commercial Brahman herds managed under extensive pastoral conditions.

Enhancement of platelet 12-HETE production in the presence of polymorphonuclear leukocytes during calcium ionophore stimulation.

This study investigates the effect of platelet/neutrophil interactions on eicosanoid production. Human platelets and polymorphonuclear leukocytes (PMNs) were stimulated alone and in combination, with calcium ionophore A23187 and the resulting eicosanoids 12S-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), 12S-heptadecatrienoic acid (HHT), 5S,12R-dihydroxy-(6Z,8E,10E,14Z)-eicosatetraenoi c acid (LTB4) and 5S-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) were measured by HPLC. The addition of PMNs to platelet suspensions caused a 104% increase in 12-HETE, a product of 12-lipoxygenase activity, but had only a modest effect on the cyclooxygenase product HHT (increase of 18%). By using PMNs labelled with [14C]arachidonic acid it was shown that the increases in these platelet eicosanoids could be accounted for by translocation of released arachidonic acid from PMNs to platelets and its subsequent metabolism. The observation that 12-lipoxygenase was about five times more efficient than cyclooxygenase at utilising exogenous arachidonic acid during the platelet/PMN interactions was confirmed in experiments in which platelets were stimulated with A23187 in the presence of [14C]arachidonic acid. Stimulations of platelets with thrombin in the presence of PMNs resulted in a decrease in 12-HETE and HHT levels of 40% and 26%, respectively. The presence of platelets caused a small increase in neutrophil LTB4 output but resulted in a decrease in 5-HETE production of 43% during stimulation with A23187. This study demonstrates complex biochemical interactions between platelets and PMNs during eicosanoid production and provides evidence of a mechanism to explain the large enhancement in 12-HETE production.

Differential effect of aspirin on platelet aggregation in IDDM.

Diabetes mellitus is associated with a high incidence of cardiovascular diseases not directly attributable to hyperlipidemia, smoking, or hypertension, but which in part may be explained by an enhanced tendency to thrombosis due to increased platelet activity. The aim of this study was to evaluate platelet function and compare the effectiveness of the antiplatelet drug aspirin on platelet aggregation in diabetic and nondiabetic subjects. Platelet aggregation and composition were examined in 20 male insulin-dependent diabetes mellitus (IDDM) patients and 20 nondiabetic control subjects matched for age and body mass index. All were normotensive with serum total cholesterol less than 6.5 mM. Although within the clinically acceptable normal range, blood pressure was significantly higher in diabetic patients (130/75 mmHg) than in control subjects (123/70 mmHg) (P less than 0.05). Serum thromboxane B2 and ex vivo aggregation of platelets in response to two doses of the agonists collagen and platelet-activating factor (PAF) were similar to nondiabetic subjects. However, after taking 100 mg/day aspirin for 5 days, platelet aggregation to collagen was reduced by 76% in control subjects compared to 56% in IDDM patients (P less than 0.001). Aspirin treatment also reduced the slope of the aggregation curve and increased the lag time (the period between the addition of collagen and the start of irreversible aggregation) significantly more in control than in diabetic platelets. This difference in platelet aggregation could not be attributed to differences in platelet serotonin or thromboxane A2 secretion, the latter being almost completely suppressed by aspirin in each group. Platelet aggregation to PAF was similar in both groups and was not affected by aspirin.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of biphasic and monophasic defibrillation waveforms in an isolated rabbit heart preparation.

STUDY OBJECTIVE: The aims were to develop a Langendorff rabbit heart model and to compare monophasic and biphasic defibrillation pulses. DESIGN: Hearts were perfused with a Krebs-Henseleit solution and two 1.4 cm2 Pt-Ir mesh patch electrodes were sutured onto the ventricles. A 5 ms monophasic or 10 ms biphasic pulse, with randomly selected voltages of 30, 50, 70, 90, 110, or 130 V, defibrillated the heart after 10 s of fibrillation. SUBJECTS: 11 adult male New Zealand white rabbits weighing 2.8(0.27) kg, were used for the studies. MEASUREMENTS AND MAIN RESULTS: A total of 72 fibrillation and defibrillation sequences were conducted in each preparation. The results were fitted to a sigmoidal dose-response curve by logistic regression analysis. Voltage and energy values from the fitted data at 50% and 80% success (V50, V80, E50, E80) indicated a significantly lower (p less than 0.05) defibrillation threshold voltage and energy for the biphasic waveform [V50 = 48 (SD19) V, V80 = 87(27) V, E50 = 0.15(0.12) J, E80 = 0.48(0.29) J] compared with the monophasic waveform [V50 = 79(20) V, V80 = 110(20) V, E50 = 0.27(0.12) J, E80 = 0.5(0.12) J]. There was no observed difference in defibrillation success rate between the first and second halves of any study. CONCLUSIONS: The Langendorff rabbit heart model is suitable for assessing electrical fibrillation and defibrillation mechanisms. Defibrillation can be achieved with a lower energy when using a biphasic rather than a monophasic pulse.

Regulation of EGF-receptor expression by EGF and TGF alpha in epidermoid cancer cells is cell type-specific.

The epidermal growth factor receptor (EGF-R) and its major ligands EGF and transforming growth factor alpha (TGF alpha) play an important role in the development of multiple human tumors. However, little is known of the comparative effects of each ligand on the regulation of EGF-R expression. To investigate this issue we used two similar human epidermoid cancer cell lines that overexpress EGF-Rs (KB and A431). In KB cells, EGF and TGF alpha increased EGF-R mRNA and protein levels by 2-3 fold over 8 h, associated with a greater than 4-fold stabilization of EGF-R mRNA half-life. EGF and TGF alpha also increased transcription of EGF-R mRNA 2-3-fold in KB cells. In contrast, EGF and TGF alpha only minimally increased EGF-R mRNA and protein in A431 cells, without changing EGF-R mRNA half-life. Basal EGF-R mRNA half-life was 2 fold greater in A431 cells than in KB cells (6-7 h versus 2-3 h), whilst the half-life of a mutant 2.6 kb EGF-R mRNA present in A431 cells, which lacks the 3-untranslated region (3'-UTR), was 2 fold greater than the full-length EGF-R mRNA. RNA gel-shift studies demonstrated that KB and A431 cells contain cytoplasmic proteins that bind specifically to an AU-rich sequence from the 3'-UTR of EGF-R mRNA. Taken together, these results demonstrate that in KB cells EGF and TGF alpha upregulate EGF-R expression at both transcriptional and post-transcriptional levels. The identification of AU-rich EGF-R mRNA-specific RNA-binding proteins from epidermoid cancer cells that overexpress EGF-Rs suggests that regulated RNA-protein interactions involving this region may play a central role in modulating EGF-R mRNA stability.

Partial androgen insensitivity caused by an androgen receptor mutation at amino acid 907 (Gly-->Arg) that results in decreased ligand binding affinity and reduced androgen receptor messenger ribonucleic acid levels.

Androgen insensitivity is an X-linked disorder of sexual differentiation resulting from mutations in the androgen receptor (AR) gene. In this paper, we report the clinical phenotype and molecular analysis of two siblings with severe partial androgen insensitivity due to a novel mutation in the ligand-binding domain of the AR gene. Binding studies using cultured genital skin fibroblasts demonstrated reduced AR affinity and binding capacity. Nucleotide sequence analysis of the AR gene of both siblings revealed a point mutation causing a glycine to arginine amino acid substitution at position 907 within a conserved region of the ligand-binding domain. A silent guanine to adenine substitution was also identified in the protein-coding region of exon 1. Using an expression vector in which the identified mutation was recreated by site-directed mutagenesis, the mutant receptor was found to have a reduced binding affinity (Kd = 3.06 nmol/L) for mibolerone compared with that of normal AR (Kd = 1.71 nmol/L) when expressed in COS-7 cells. In cotransfection experiments using CV-1 cells and a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter system, the concentration of dihydrotestosterone required to induce half-maximal chloramphenicol acetyltransferase gene expression was 50-fold higher in cells transfected with the mutant AR complementary DNA than in cells transfected with normal AR complementary DNA. AR messenger ribonucleic acid levels in genital skin fibroblasts determined by both competitive PCR amplification and ribonuclease protection assay were decreased compared with normal values. Our studies demonstrate the importance of this region of the AR gene in normal AR function and AR gene expression.