The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

Modulation of dermal microvascular endithelial cell responses to growth factors and haemostatic factors in the presence of vitronectin

In order to effect permanent closure in burns patients suffering from full thickness wounds, replacing their skin via split thickness autografting, is essential. Dermal substitutes in conjunction with widely meshed split thickness autografts (+/- cultured keratinocytes) reduce scarring at the donor and recipient sites of burns patients by reducing demand for autologous skin (both surface area and thickness), without compromising dermal delivery at the wound face. Tissue engineered products such as Integra consist of a dermal template which is rapidly remodelled to form a neodermis, at which time the temporary silicone outer layer is removed and replaced with autologous split thickness skin. Whilst provision of a thick tissue engineered dermis at full thickness burn sites reduces scarring, it is hampered by delays in vascularisation which results in clinical failure. The ultimate success of any skin graft product is dependent upon a number of basic factors including adherence, haemostasis and in the case of viable tissue grafts, success is ultimately dependent upon restoration of a normal blood supply, and hence this study. Ultimately, the goal of this research is to improve the therapeutic properties of tissue replacements, through impregnation with growth factors aimed at stimulating migration and proliferation of microvascular endothelial cells into the donor tissue post grafting. For the purpose of my masters, the aim was to evaluate the responsiveness of a dermal microvascular endothelial cell line to growth factors and haemostatic factors, in the presence of the glycoprotein vitronectin. Vitronectin formed the backbone for my hypothesis and research due to its association with both epithelial and, more specifically, endothelial migration and proliferation. Early work using a platform technology referred to as VitroGro (Tissue Therapies Ltd), which is comprised of vitronectin bound BP5/IGF-1, aided keratinocyte proliferation. I hypothesised that this result would translate to another epithelium - endothelium. VitroGro had no effect on endothelial proliferation or migration. Vitronectin increases the presence of Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) receptors, enhancing cell responsiveness to their respective ligands. So, although Human Microvascular Endothelial Cell line 1 (HMEC-1) VEGF receptor expression is generally low, it was hypothesised that exposure to vitronectin would up-regulate this receptor. HMEC-1 migration, but not proliferation, was enhanced by vitronectin bound VEGF, as well as vitronectin bound Epidermal Growth Factor (EGF), both of which could be used to stimulate microvascular endothelial cell migration for the purpose of transplantation. In addition to vitronectin's synergy with various growth factors, it has also been shown to play a role in haemostasis. Vitronectin binds thrombin-antithrombin III (TAT) to form a trimeric complex that takes on many of the attributes of vitronectin, such as heparin affinity, which results in its adherence to endothelium via heparan sulfate proteoglycans (HSP), followed by unaltered transcytosis through the endothelium, and ultimately its removal from the circulation. This has been documented as a mechanism designed to remove thrombin from the circulation. Equally, it could be argued that it is a mechanism for delivering vitronectin to the matrix. My results show that matrix-bound vitronectin dramatically alters the effect that conformationally altered antithrombin three (cATIII) has on proliferation of microvascular endothelial cells. cATIII stimulates HMEC-1 proliferation in the presence of matrix-bound vitronectin, as opposed to inhibiting proliferation in its absence. Binding vitronectin to tissues and organs prior to transplant, in the presence of cATIII, will have a profound effect on microvascular infiltration of the graft, by preventing occlusion of existing vessels whilst stimulating migration and proliferation of endothelium within the tissue.

Stability analysis based on birfurcation theory of the DSTATCOM operating in current control mode

This paper presents the stability analysis for a distribution static compensator (DSTATCOM) that operates in current control mode based on bifurcation theory. Bifurcations delimit the operating zones of nonlinear circuits and, hence, the capability to compute these bifurcations is of important interest for practical design. A control design for the DSTATCOM is proposed. Along with this control, a suitable mathematical representation of the DSTATCOM is proposed to carry out the bifurcation analysis efficiently. The stability regions in the Thevenin equivalent plane are computed for different power factors at the point of common coupling. In addition, the stability regions in the control gain space, as well as the contour lines for different Floquet multipliers are computed. It is demonstrated through bifurcation analysis that the loss of stability in the DSTATCOM is due to the emergence of a Neimark bifurcation. The observations are verified through simulation studies.

Development of accident modification factors on intersection crash maneuvers

In order to estimate the safety impact of roadway interventions engineers need to collect, analyze, and interpret the results of carefully implemented data collection efforts. The intent of these studies is to develop Accident Modification Factors (AMF's), which are used to predict the safety impact of various road safety features at other locations or in upon future enhancements. Models are typically estimated to estimate AMF's for total crashes, but can and should be estimated for crash outcomes as well. This paper first describes data collected with the intent estimate AMF's for rural intersections in the state of Georgia within the United Sates. Modeling results of crash prediction models for the crash outcomes: angle, head-on, rear-end, sideswipe (same direction and opposite direction) and pedestrian-involved crashes are then presented and discussed. The analysis reveals that factors such as the Annual Average Daily Traffic (AADT), the presence of turning lanes, and the number of driveways have a positive association with each type of crash, while the median width and the presence of lighting are negatively associated with crashes. The model covariates are related to crash outcome in different ways, suggesting that crash outcomes are associated with different pre-crash conditions.

Safety-restraint use rate as function of law enforcement and other factors : preliminary analysis

Persistent use of safety restraints prevents deaths and reduces the severity and number of injuries resulting from motor vehicle crashes. However, safety-restraint use rates in the United States have been below those of other nations with safety-restraint enforcement laws. With a better understanding of the relationship between safety-restraint law enforcement and safety-restraint use, programs can be implemented to decrease the number of deaths and injuries resulting from motor vehicle crashes. Does safety-restraint use increase as enforcement increases? Do motorists increase their safety-restraint use in response to the general presence of law enforcement or to targeted law enforcement efforts? Does a relationship between enforcement and restraint use exist at the countywide level? A logistic regression model was estimated by using county-level safety-restraint use data and traffic citation statistics collected in 13 counties within the state of Florida in 1997. The model results suggest that safety-restraint use is positively correlated with enforcement intensity, is negatively correlated with safety-restraint enforcement coverage (in lanemiles of enforcement coverage), and is greater in urban than rural areas. The quantification of these relationships may assist Florida and other law enforcement agencies in raising safety-restraint use rates by allocating limited funds more efficiently either by allocating additional time for enforcement activities of the existing force or by increasing enforcement staff. In addition, the research supports a commonsense notion that enforcement activities do result in behavioral response.

Conducting statistical tests of hypotheses : five common misconceptions found in transportation research

Most statistical methods use hypothesis testing. Analysis of variance, regression, discrete choice models, contingency tables, and other analysis methods commonly used in transportation research share hypothesis testing as the means of making inferences about the population of interest. Despite the fact that hypothesis testing has been a cornerstone of empirical research for many years, various aspects of hypothesis tests commonly are incorrectly applied, misinterpreted, and ignored—by novices and expert researchers alike. On initial glance, hypothesis testing appears straightforward: develop the null and alternative hypotheses, compute the test statistic to compare to a standard distribution, estimate the probability of rejecting the null hypothesis, and then make claims about the importance of the finding. This is an oversimplification of the process of hypothesis testing. Hypothesis testing as applied in empirical research is examined here. The reader is assumed to have a basic knowledge of the role of hypothesis testing in various statistical methods. Through the use of an example, the mechanics of hypothesis testing is first reviewed. Then, five precautions surrounding the use and interpretation of hypothesis tests are developed; examples of each are provided to demonstrate how errors are made, and solutions are identified so similar errors can be avoided. Remedies are provided for common errors, and conclusions are drawn on how to use the results of this paper to improve the conduct of empirical research in transportation.

Single nucleotide polymorphism (SNP) - genotyping of Community Acquired Methicillin-Resistant Staphylococcus aureus, including the subtyping of PVL toxin producers using Real-Time PCR

Staphylococcus aureus is a common pathogen that causes a variety of infections including soft tissue infections, impetigo, septicemia toxic shock and scalded skin syndrome. Traditionally, Methicillin-Resistant Staphylococcus aureus (MRSA) was considered a Hospital-Acquired (HA) infection. It is now recognised that the frequency of infections with MRSA is increasing in the community, and that these infections are not originating from hospital environments. A 2007 report by the Centers for Disease Control and Prevention (CDC) stated that Staphylococcus aureus is the most important cause of serious and fatal infections in the USA. Community-Acquired MRSA (CA-MRSA) are genetically diverse and distinct, meaning they are able to be identified and tracked by way of genotyping. Genotyping of MRSA using Single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring MRSA, specifically ST93 (Queensland Clone) dissemination in the community. It has been shown that a large proportion of CA-MRSA infections in Queensland and New South Wales are caused by ST93. The rationale for this project was that SNP analysis of MLST genes is a rapid and cost-effective method for genotyping and monitoring MRSA dissemination in the community. In this study, 16 different sequence types (ST) were identified with 41% of isolates identified as ST93 making it the predominate clone. Males and Females were infected equally with an average patient age of 45yrs. Phenotypically, all of the ST93 had an identical antimicrobial resistance pattern. They were resistant to the β-lactams – Penicillin, Flu(di)cloxacillin and Cephalothin but sensitive to all other antibiotics tested. Virulence factors play an important role in allowing S. aureus to cause disease by way of colonising, replication and damage to the host. One virulence factor of particular interest is the toxin Panton-Valentine leukocidin (PVL), which is composed of two separate proteins encoded by two adjacent genes. PVL positive CA-MRSA are shown to cause recurrent, chronic or severe skin and soft tissue infections. As a result, it is important that PVL positive CA-MRSA is genotyped and tracked. Especially now that CA-MRSA infections are more prevalent than HA-MRSA infections and are now deemed endemic in Australia. 98% of all isolates in this study tested positive for the PVL toxin gene. This study showed that PVL is present in many different community based ST, not just ST93, which were all PVL positive. With this toxin becoming entrenched in CA-MRSA, genotyping would provide more accurate data and a way of tracking the dissemination. PVL gene can be sub-typed using an allele-specific Real-Time PCR (RT-PCR) followed by High resolution meltanalysis. This allows the identification of PVL subtypes within the CA-MRSA population and allow the tracking of these clones in the community.

Work-related driving safety in light vehicle fleets : a review of past research and the development of an intervention framework

This paper presents a critical review of past research in the work-related driving field in light vehicle fleets (e.g., vehicles < 4.5 tonnes) and an intervention framework that provides future direction for practitioners and researchers. Although work-related driving crashes have become the most common cause of death, injury, and absence from work in Australia and overseas, very limited research has progressed in establishing effective strategies to improve safety outcomes. In particular, the majority of past research has been data-driven, and therefore, limited attention has been given to theoretical development in establishing the behavioural mechanism underlying driving behaviour. As such, this paper argues that to move forward in the field of work-related driving safety, practitioners and researchers need to gain a better understanding of the individual and organisational factors influencing safety through adopting relevant theoretical frameworks, which in turn will inform the development of specifically targeted theory-driven interventions. This paper presents an intervention framework that is based on relevant theoretical frameworks and sound methodological design, incorporating interventions that can be directed at the appropriate level, individual and driving target group.

What factors are associated with cyclists getting injured? Correlates of cyclist injuries in Queensland

Pedal cyclists are over-represented in traffic crash injuries in Australia. This study examined correlates of cycling injuries in a sample of Queensland cyclists. Members of Bicycle Queensland (n=1976) were asked about cycling injuries as part of an online survey. They also reported demographic characteristics, reasons for cycling, years of cycling as an adult, and cycling frequency. Multivariate logistic regression modelling was used to examine the association between these variables and experiencing cycling injuries last year (yes/no). Thirty-one percent of respondents (n=617) reported at least one cycling injury. Respondents had greater likelihood of injury if they cycled more frequently, had cycled <5 years, or cycled for recreation or competition. These findings suggest that injuries are mostly likely to occur among less experienced cyclists, those cycling the most, and those cycling for sport and recreation. Injury prevention interventions should include cycle skills training along with fostering safer cycling environments.

Evaluation of risk factors leading to cost overrun in delivery of highway construction projects

Accurate owner budget estimates are critical to the initial decision-to-build process for highway construction projects. However, transportation projects have historically experienced significant construction cost overruns from the time the decision to build has been taken by the owner. This paper addresses the problem of why highway projects overrun their predicted costs. It identifies the owner risk variables that contribute to significant cost overrun and then uses factor analysis, expert elicitation, and the nominal group technique to establish groups of importance ranked owner risks. Stepwise multivariate regression analysis is also used to investigate any correlation of the percentage of cost overrun with risks, together with attributes such as highway project type, indexed cost, geographics location, and project delivery method. The research results indicate a correlation between the reciprocal of project budgets size and percentage cost overrun. This can be useful for owners in determining more realistic decision-to-build highway budget estimates by taking into account the economies of scale associated with larger projects.

Deriving grassland management factors for a carbon accounting method developed by the intergovernmental panel on climate change

Grassland management affects soil organic carbon (SOC) storage and can be used to mitigate greenhouse gas emissions. However, for a country to assess emission reductions due to grassland management, there must be an inventory method for estimating the change in SOC storage. The Intergovernmental Panel on Climate Change (IPCC) has developed a simple carbon accounting approach for this purpose, and here we derive new grassland management factors that represent the effect of changing management on carbon storage for this method. Our literature search identified 49 studies dealing with effects of management practices that either degraded or improved conditions relative to nominally managed grasslands. On average, degradation reduced SOC storage to 95% +/- 0.06 and 97% +/- 0.05 of carbon stored under nominal conditions in temperate and tropical regions, respectively. In contrast, improving grasslands with a single management activity enhanced SOC storage by 14% 0.06 and 17% +/- 0.05 in temperate and tropical regions, respectively, and with an additional improvement(s), storage increased by another 11% +/- 0.04. We applied the newly derived factor coefficients to analyze C sequestration potential for managed grasslands in the U.S., and found that over a 20-year period changing management could sequester from 5 to 142 Tg C yr(-1) or 0.1 to 0.9 Mg C ha(-1) yr(-1), depending on the level of change. This analysis provides revised factor coefficients for the IPCC method that can be used to estimate impacts of management; it also provides a methodological framework for countries to derive factor coefficients specific to conditions in their region.

Environmental factors controlling soil respiration in three semiarid ecosystems

Previous research suggests that soil organic C pools may be a feature of semiarid regions that are particularly sensitive to climatic changes. We instituted an 18-mo experiment along an elevation gradient in northern Arizona to evaluate the influence of temperature, moisture, and soil C pool size on soil respiration. Soils, from underneath different free canopy types and interspaces of three semiarid ecosystems, were moved upslope and/or downslope to modify soil climate. Soils moved downslope experienced increased temperature and decreased precipitation, resulting in decreased soil moisture and soil respiration las much as 23 acid 20%, respectively). Soils moved upslope to more mesic, cooler sites had greater soil water content and increased rates of soil respiration las much as 40%), despite decreased temperature. Soil respiration rates normalized for total C were not significantly different within any of the three incubation sites, indicating that under identical climatic conditions, soil respiration is directly related to soil C pool size for the incubated soils. Normalized soil respiration rates between sites differed significantly for all soil types and were always greater for soils incubated under more mesic, but cooler, conditions. Total soil C did not change significantly during the experiment, but estimates suggest that significant portions of the rapidly cycling C pool were lost. While long-term decreases in aboveground and belowground detrital inputs may ultimately be greater than decreased soil respiration, the initial response to increased temperature and decreased precipitation in these systems is a decrease in annual soil C efflux.