Is local adaptation in Mimulus guttatus caused by trade-offs at individual loci?

Local adaptation is considered to be the result of fitness trade-offs for particular phenotypes across different habitats. However, it is unclear whether such phenotypic trade-offs exist at the level of individual genetic loci. Local adaptation could arise from trade-offs of alternative alleles at individual loci or by complementary sets of loci with different fitness effects of alleles in one habitat but selective neutrality in the alternative habitat. To evaluate the genome-wide basis of local adaptation, we performed a field-based quantitative trait locus (QTL) mapping experiment on recombinant inbred lines (RILs) created from coastal perennial and inland annual races of the yellow monkeyflower (Mimulus guttatus) grown reciprocally in native parental habitats. Overall, we detected 19 QTLs affecting one or more of 16 traits measured in two environments, most of small effect. We identified 15 additional QTL effects at two previously identified candidate QTLs [DIVERGENCE (DIV)]. Significant QTL by environment interactions were detected at the DIV loci, which was largely attributable to genotypic differences at a single field site. We found no detectable evidence for trade-offs for any one component of fitness, although DIV2 showed a trade-off involving different fitness traits between sites, suggesting that local adaptation is largely controlled by non-overlapping loci. This is surprising for an outcrosser, implying that reduced gene flow prevents the evolution of individuals adapted to multiple environments. We also determined that native genotypes were not uniformly adaptive, possibly reflecting fixed mutational load in one of the populations.

Modern management of arrhythmias.

The specialist management of arrhythmias has changed significantly over the past decade. This article outlines current management strategies for atrial flutter and atrial fibrillation, with particular emphasis on curative strategies with catheter ablation and the recent data on rhythm compared with rate control strategies. The expanding role of catheter ablation in the treatment of a wide variety of supraventricular and ventricular arrhythmias is discussed. The current evidence for implantable cardioverter defibrillators in the prevention of sudden cardiac death is summarised. The article also highlights the increasing recognition of a number of inherited syndromes that predispose to sudden cardiac death (for example, Brugada and long QT syndromes).

Relative impact of radiographic osteoarthritis and pain on quadriceps strength, proprioception, static postural sway and lower limb function.

OBJECTIVE: To investigate the relative impact of radiographic osteoarthritis (ROA) and current knee pain on lower limb physical function, quadriceps strength, knee joint proprioception, and postural sway. METHODS: Using a 2x2 factorial design, 142 community derived subjects aged over 45 were divided into four subgroups based on the presence or absence of ROA (Kellgren & Lawrence>grade 2) and knee pain (as assessed by NHANES questions and a 100 mm visual analogue scale). Maximum isometric contraction of the quadriceps, knee joint proprioceptive acuity, static postural sway, and WOMAC index (both whole and function subscale) were assessed in all subjects. RESULTS: Compared with normal subjects, reported disability was greater for all other subgroups (p<0.01). Subjects with both ROA and knee pain reported the greatest disability, and those with knee pain only had greater disability than those with ROA only. Quadriceps weakness was observed in all groups compared with normal subjects (p<0.01), though they were no significant intergroup differences. Subjects with knee pain had a greater sway area than those without (p<0.05) but the presence of ROA was not associated with increased postural sway. No differences in proprioceptive acuity were observed between groups. CONCLUSIONS: The presence of knee pain has a negative association with quadriceps strength, postural sway, and disability compared with ROA. However, the presence of pain-free ROA has a significant negative influence on relative quadriceps strength and reported disability.

Effect of media, temperature and culture conditions on the species population and antibiotic resistance of enterococci from broiler chickens.

AIMS: The effect of media type, incubation temperature and enrichment period on the species population and antibiotic susceptibility of enterococci from poultry carcass rinsates was determined. METHODS AND RESULTS: Aliquots of rinsates, incubated in BBL Enterococcosel broth at 37 degrees C, 42 degrees C, or 45 degrees C for 24 and 48 h, were inoculated onto BBL Enterococcosel and M-enterococcus agar. Presumptive positive colonies were identified to species and tested for antibiotic resistance. Significant differences (P < or = 0.05) were observed for media and temperature. More Enterococcus faecalis were isolated from M-enterococcus media and at 37 degrees C while more E. faecium were isolated from Enterococcosel agar and at 45 degrees C. The number of antibiotic-resistant E. faecalis and E. faecium were also affected by media and temperature. CONCLUSIONS: Culture conditions for enterococci affect the observed species and antibiotic resistance patterns and therefore should be carefully considered. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that media and temperature can influence the recovery and selection of enterococcal species and antibiotic susceptibility.

Anomalies in species identification of enterococci from veterinary sources using a commercial biochemical identification system.

AIMS: A commercial biochemical panel ID kit was used to identify presumptive enterococci isolates of veterinary or agricultural origin obtained during different steps of culture. METHODS AND RESULTS: Fifty isolates identified as enterococci using a genus PCR assay were tested for genus and species identification using the BBL Crystal Identification Gram-Positive ID kit (Becton Dickinson, Sparks, MD, USA). Following sub-culture of the isolates three times, 59% agreement with the original panel ID was obtained. After four and six sub-cultures, percentage agreement increased to 61 and 64%, respectively. Nineteen of the 50 cultures were identified as both Enterococcus faecalis and E. faecium. CONCLUSIONS: As a result of the variability between speciation of isolates following re-culture, additional methods for speciation are warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the identification of the genus and species of non-human enterococcal isolates can vary greatly during successive passages when using this kit.

A prospective quality-of-life study in men with clinically localized prostate carcinoma treated with radical prostatectomy, external beam radiotherapy, or interstitial brachytherapy.

PURPOSE: To prospectively assess the health-related quality of life (HRQOL) and changes in HRQOL during the first year after 3 different treatments for clinically localized prostate cancer. METHODS AND MATERIALS: Ninety men with T1-T2 adenocarcinoma of the prostate were treated with curative intent between May 1998 and June 1999 and completed a quality-of-life Functional Assessment of Cancer Therapy-Prostate (FACT-P) questionnaire before treatment (T0) and 1 month (T1), 3 months (T3), and 12 months (T12) after treatment. Forty-four men were treated with permanent source interstitial brachytherapy (IB), 23 received external beam radiotherapy (EBRT), and 23 men were treated with radical prostatectomy (RP). The mean age of the entire study population was 65.9 years (median 67, range 42-79). The mean pretreatment prostate-specific antigen level of the entire study population was 6.81 ng/mL (median 6.25, range 1.33-19.6). The Gleason score was

High affinity cooperative DNA binding by the yeast Mlh1-Pms1 heterodimer.

We demonstrate here that the Saccharomyces cerevisiae Mlh1-Pms1 heterodimer required for DNA mismatch repair and other cellular processes is a DNA binding protein. Binding was evaluated using a variety of single and double-stranded DNA molecules. Mlh1-Pms1 bound short substrates with low affinity and showed a slight preference for single-stranded DNA. In contrast, Mlh1-Pms1 exhibited a much higher affinity for long DNA molecules, suggesting that binding is cooperative. High affinity binding required a duplex DNA length greater than 241 base-pairs. The rate of association with DNA was rapid and dissociation of protein-DNA complexes following extensive dilution was very slow. However, in competition experiments, we observed a rapid active transfer of Mlh1-Pms1 from labeled to unlabeled DNA. Binding was non-sequence specific and highly sensitive to salt type and concentration, suggesting that Mlh1-Pms1 primarily interacts with the DNA backbone via ionic contacts. Cooperative binding was observed visually by atomic force microscopy as long, continuous tracts of Mlh1-Pms1 protein bound to duplex DNA. These images also showed that Mlh1-Pms1 simultaneously interacts with two different regions of duplex DNA. Taken together, the atomic force microscope images and DNA binding assays provide strong evidence that Mlh1-Pms1 binds duplex DNA with positive cooperativity and that there is more than one DNA binding site on the heterodimer. These DNA binding properties of Mlh1-Pms1 may be relevant to its participation in DNA mismatch repair, recombination and cellular responses to DNA damage.

Transrectal ultrasound and biopsy in the early diagnosis of prostate cancer.

BACKGROUND: Historically, the prostate was evaluated for cancer by simple digital rectal examination, and biopsy to obtain a tissue diagnosis of cancer was performed blindly. The advent of ultrasound technology offered a new way to evaluate the prostate, and biopsy techniques were soon developed to incorporate ultrasound guidance. METHODS: The authors review the role of transrectal ultrasound (TRUS) of the prostate and ultrasound-guided biopsy of the prostate in the diagnosis of prostate cancer. These techniques are traced from their origins to the current standards of care, with attention paid to developments and controversies in recent literature. RESULTS: Early experience with TRUS led to the description of "classic" sonographic findings of prostate cancer. To obtain a tissue diagnosis of cancer, these regions were initially targeted in ultrasound-guided biopsies. Concomitant with the development of TRUS, though, was the development of the prostate-specific antigen (PSA) assay. Over the past decade, there has been a profound stage migration due to earlier detection of prostate cancer. Most patients now diagnosed with prostate cancer have no palpable abnormality or specific sonographic findings. In response, ultrasound-guided biopsies have become more systematic, rather than lesion-specific, in nature. CONCLUSIONS: TRUS continues to play an important role in the evaluation of the prostate when malignancy is suspected. Although the optimal method of prostate biopsy is controversial, ultrasound is critical in ensuring accurate sampling of the gland.

Purification of eukaryotic MutL homologs from Saccharomyces cerevisiae using self-affinity technology.

Self-cleaving affinity technology is an effective tool for rapid purification of native sequence recombinant proteins overproduced in Escherichia coli. In this report, we describe the adaptation of this technology to purify DNA mismatch repair proteins overproduced in the eukaryote Saccharomyces cerevisiae. Mlh1 and Pms1 are homologs of the E. coli MutL protein that participate in a variety of DNA transactions in cells, including correction of DNA replication errors, recombination, excision repair, and checkpoint control. Difficulties in preparing substantial quantities of highly purified MutL homologs have impeded descriptions of their biophysical and biochemical properties and mechanisms of action. To overcome this limitation, here we use self-cleaving affinity technology to purify to apparent homogeneity the yeast Mlh1--Pms1 heterodimer and the individual yeast and human Mlh1 subunit. The availability of these proteins should accelerate an understanding of their multiple functions in mismatch repair and other DNA transactions. The general approach is a valid alternative for simple, rapid purification of recombinant proteins in yeast when expression in bacteria is unsuitable.

Inactivation of DNA mismatch repair by increased expression of yeast MLH1.

Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mol. Cell. Biol. 19:3177-3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1. Here we investigate the mechanism of this mutator effect. Hybridization of poly(A)(+) mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1 overexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1 overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3'-->5' exonuclease activity of replicative DNA polymerases delta and epsilon but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, or PMS1. This suggests that overexpression of MLH1 inactivates mismatch repair of replication errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K(d) of 3.14 microM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressing MLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.

Ureteroscopic management of patients with upper tract transitional cell carcinoma.

Endoscopic therapy for the management of upper urinary tract TCC is mainly indicated for patients with an anatomically or functionally solitary kidney, renal insufficiency, bilateral tumors, or severe medical comorbidity. It may be a reasonable alternative to distal ureterectomy with bladder-cuff resection in individuals with low-grade superficial distal ureteral tumors. Although use of this approach has been suggested for treating standard patients with low-grade, low-stage collecting system tumors, this recommendation should not be embraced until more supporting evidence is generated. The efficacy of adjuvant therapy for the prevention of recurrent or progressive disease needs to be defined. If current adjuvant strategies prove ineffective, alternative ones will need to be developed. It is anticipated that advancements in endoscopic technology will facilitate the performance of this type of surgery in the future.

Thoracoabdominal radical nephrectomy: is a postoperative thoracostomy tube necessary?

OBJECTIVES: To report our results of patients undergoing thoracoabdominal radical nephrectomy without intraoperative placement of a thoracostomy tube. It has been routine in our hospital to not place a thoracostomy tube in patients undergoing thoracoabdominal radical nephrectomy since 1988. METHODS: We conducted a retrospective review of 47 thoracoabdominal radical nephrectomies performed from January 1988 through November 1998 at our institution. Of the 47 patients, 39 did not have a thoracostomy tube placed intraoperatively; the other 8 patients did. The development of all postoperative complications, length of hospital stay, and hospital charges were noted. RESULTS: No postoperative mortality was noted in our study. Of the 47 patients in the study, 20 patients had a total of 29 complications. The overall number of complications was not increased in the group without a thoracostomy tube compared with the group with a thoracostomy tube (P = 0.104). No patient treated without a thoracostomy tube required subsequent placement of a tube for persistent pneumothorax. The mean length of hospital stay in patients with a thoracostomy tube after radical nephrectomy was 9.14 +/- 2.65 days; in patients without a thoracostomy tube, the mean length of stay was 7.07 +/- 3.97 days (P = 0.071). CONCLUSIONS: In patients without parietal pleural injury, thoracoabdominal radical nephrectomy without the placement of a thoracostomy tube can be performed safely and effectively, with a low risk of postoperative complications and a decrease in the overall hospital stay and hospital charges.

Bladder cancer.

There is a need for the development of reliable tumor markers in bladder cancer. A number of studies this past year focused on the evaluation of urinary markers that hold promise as noninvasive adjuncts to traditional diagnostic or surveillance techniques, principally urinary cytology and cystoscopy. Tests for bladder tumor antigen, NMP22, and fibrin degradation products, as well as the Immunocyt test, are commercially available. Other urinary marker tests discussed in this review include telomerase, cytokeratins, and vascular endothelial growth factor. Although these tests in many instances have improved sensitivity in detecting bladder cancer compared with urinary cytology, none have become widely accepted in routine clinical practice. Nonetheless, with further refinement and prospective validation in multicenter trials, markers such as these may provide information that would permit tailoring on an individual basis the type of as well as interval of surveillance examinations. Furthermore, they may also provide information allowing the appropriate selection of therapy based on predicted response. In addition to urinary markers, intense research efforts have also focused on developing clinically useful molecular prognostic markers. A number of cell-cycle regulatory proteins, including p53 and p21, have received much attention in this regard. Emerging data suggests that it may soon be possible to determine the molecular phenotype of both superficial and invasive bladder cancers, thereby providing information regarding tumor behavior on an individual basis. As with urinary markers, however, no molecular markers have been incorporated as yet into day-to-day patient care. Assurances of reproducibility, standardization, and prospective validation studies are urgently needed. It is only through this type of rigorous evaluation that the level of confidence sufficient to base treatment decisions on marker status will be attained.

Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer.

The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction. It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags). Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity. Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search. This uncertainty, however, is overcome by MS/MS analysis of the peptides. Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein. This procedure is thus, highly sensitive, accurate, and rapid. In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling. Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1.

The growth inhibitory effect of p21 adenovirus on human bladder cancer cells.

PURPOSE: To evaluate whether p21 (WAF-1/CIP1) should be considered a potential candidate for human bladder cancer gene therapy, we determined: (1) the basal level of p21 expression in bladder cancer cell lines, (2) the response of bladder cancer cells to increased p21 expression following p21 adenovirus infection, and (3) the mechanism of growth inhibition produced by p21 overexpression. MATERIALS AND METHODS: Five established human bladder cancer cell lines and one primary culture derived from an invasive transitional cell carcinoma were used in this study. To examine the effect of p21 protein on the growth of human bladder cancer cells, a recombinant adenovirus vector system containing p21 cDNA, under the control of cytomegalovirus promoter, was constructed. A control virus containing p21 in an antisense orientation was used to eliminate potential artifacts caused by viral toxicity. RESULTS: Human bladder cancer cell lines exhibit variable endogenous p21 levels which correlate with the in vitro growth status. Significant, but highly variable increases in the steady-state level of p21 were detected in p21 adenovirus infected cells. Human bladder cancer cell lines responded heterogeneously to p21 adenovirus infection. Growth of the WH cell line was substantially inhibited in a dose and time-course dependent fashion. The mechanism of p21 growth inhibition was found to be due to G0/G1 arrest and not the induction of apoptosis. In contrast, p21 adenovirus failed to inhibit the growth of T24 bladder cancer cells because T24 cells were resistant to viral infection. The 253J bladder cancer cells exhibited marked sensitivity to adenovirus; substantial growth inhibition was seen with both sense and antisense p21 very early in the time course of infection. CONCLUSIONS: We found significant variation in the basal level of p21 protein expression in several human bladder cancer cell lines. Increased p21 expression as a result of adenoviral infection may be a potent growth suppressor in some human bladder cancer because it elicits cell cycle arrest in G0/G1 stage, but not the induction of apoptosis. Bladder cancer cells exhibit a wide spectrum of sensitivity to adenoviral infection that may be caused by the presence of viral receptor heterogeneity. This wide spectrum of sensitivity has significant basic scientific and clinical implications and warrants further study.

Helicase motifs: the engine that powers DNA unwinding.

Helicases play essential roles in nearly all DNA metabolic transactions and have been implicated in a variety of human genetic disorders. A hallmark of these enzymes is the existence of a set of highly conserved amino acid sequences termed the 'helicase motifs' that were hypothesized to be critical for helicase function. These motifs are shared by another group of enzymes involved in chromatin remodelling. Numerous structure-function studies, targeting highly conserved residues within the helicase motifs, have been instrumental in uncovering the functional significance of these regions. Recently, the results of these mutational studies were augmented by the solution of the three-dimensional crystal structure of three different helicases. The structural model for each helicase revealed that the conserved motifs are clustered together, forming a nucleotide-binding pocket and a portion of the nucleic acid binding site. This result is gratifying, as it is consistent with structure-function studies suggesting that all the conserved motifs are involved in the nucleotide hydrolysis reaction. Here, we review helicase structure-function studies in the light of the recent crystal structure reports. The current data support a model for helicase action in which the conserved motifs define an engine that powers the unwinding of duplex nucleic acids, using energy derived from nucleotide hydrolysis and conformational changes that allow the transduction of energy between the nucleotide and nucleic acid binding sites. In addition, this ATP-hydrolysing engine is apparently also associated with proteins involved in chromatin remodelling and provides the energy required to alter protein-DNA structure, rather than duplex DNA or RNA structure.

In vivo and in vitro expression of steroid-converting enzymes in human breast tumours: associations with interleukin-6.

Enzymes modulating local steroid availability play an important role in the progression of human breast cancer. These include isoforms of 17beta-hydroxysteroid dehydrogenase (17-HSD), aromatase and steroid sulphatase (STS). The aim of this study was to investigate the expression, by reverse transcription polymerase chain reaction, of 17-HSD types I-IV, aromatase and steroid STS in a series of 51 human breast tumour biopsies and 22 primary cultures of epithelial and stromal cells derived from these tumours, giving a profile of the steroid-regulating network for individual tumours. Correlations between enzyme expression profiles and expression of the interleukin (IL)-6 gene were also sought. All except one tumour expressed at least one isoform of 17-HSD, either alone or in combination with aromatase and STS. Expression of 17-HSD isoforms I-IV were observed in nine tumours. Of the 15 tumours which expressed three isoforms, a combination of 17-HSD II, III and IV was most common (6/15 samples). The majority of tumours (n = 17) expressed two isoforms of 17-HSD with combinations of 17-HSD II and IV predominant (7/17 samples). Eight tumours expressed a single isoform and of these, 17-HSD I was in the majority (5/8 samples). In primary epithelial cultures, enzyme expression was ranked: HSD I (86%) > STS (77%) > HSD II (59%) > HSD IV (50%) = aromatase (50%) > HSD III (32%). Incidence of enzyme expression was generally reduced in stromal cultures which were ranked: HSD I (68%) > STS (67%) > aromatase (48%) > HSD II (43%) > HSD IV (28%) > HSD III (19%). Expression of IL-6 was associated with tumours that expressed > or = 3 steroid-converting enzymes. These tumours were of higher grade and tended to come from patients with family history of breast cancer. In conclusion, we propose that these enzymes work in tandem with cytokines thereby providing sufficient quantities of bioactive oestrogen from less active precursors which stimulates tumour growth.