Impact of symptom burden and health-related quality of life (HRQOL) on esophageal motor diagnoses.

BACKGROUND: High-resolution manometry (HRM) categorizes esophageal motor processes into specific Chicago Classification (CC) diagnoses, but the clinical impact of these motor diagnoses on symptom burden remain unclear. METHODS: Two hundred and eleven subjects (56.8±1.0 years, 66.8% F) completed symptom questionnaires (GERDQ, Mayo dysphagia questionnaire [MDQ], visceral sensitivity index, short-form 36, dominant symptom index, and global symptom severity [GSS] on a 100-mm visual analog scale) prior to HRM. Subjects were stratified according to CC v3.0 and by dominant presenting symptom; contraction wave abnormalities (CWA) were evaluated within "normal" CC. Symptom burden, impact of diagnoses, and HRQOL were compared within and between cohorts. KEY RESULTS: Major motor disorders had highest global symptom burden (P=.02), "normal" had lowest (P<.01). Dysphagia (MDQ) was highest with esophageal outflow obstruction (P=.02), but reflux symptoms (GERDQ) were similar in CC cohorts (P=ns). Absent contractility aligned best with minor motor disorders. Consequently, pathophysiologic categorization into outflow obstruction, hypermotility, and hypomotility resulted in a gradient of decreasing dysphagia and increasing reflux burden (P<.05 across groups); GSS (P=.05) was highest with hypomotility and lowest with "normal" (P=.002). Within the "normal" cohort, 33.3% had CWA; this subgroup had symptom burden similar to hypermotility. Upon stratification by symptoms, symptom burden (GSS, MDQ, HRQOL) was most profound with dysphagia. CONCLUSIONS AND INFERENCES: Chicago Classification v3.0 diagnoses identify subjects with highest symptom burden, but pathophysiologic categorization may allow better stratification by symptom type and burden. Contraction wave abnormalities are clinically relevant and different from true normal motor function. Transit symptoms have highest yield for a motor diagnosis.

The learning curve for interpretation of oesophageal high-resolution manometry: a prospective interventional cohort study.

BACKGROUND: High-resolution manometry has become the preferred choice of oesophagologists for oesophageal motor assessment, but the learning curve among trainees remains unclear. AIM: To determine the learning curve of high-resolution manometry interpretation. METHODS: A prospective interventional cohort study was performed on 18 gastroenterology trainees, naïve to high-resolution manometry (median age 32 ± 4.0 years, 44.4% female). An intake questionnaire and a 1-h standardised didactic session were performed at baseline. Multiple 1-h interpretation sessions were then conducted periodically over 15 months where 10 studies were discussed; 5 additional test studies were provided for interpretation, and results were compared to gold standard interpretation by the senior author. Hypothetical management decisions based on trainee interpretation were separately queried. Accuracy was compared across test interpretations and sessions to determine the learning curve, with a goal of 90% accuracy. RESULTS: Baseline accuracy was low for abnormal body motor patterns (53.3%), but higher for achalasia/outflow obstruction (65.9%). Recognition of achalasia reached 90% accuracy after six sessions (P = 0.01), while overall accurate management decisions reached this threshold by the 4th session (P < 0.001). Based on our data, the threshold of 90% accuracy for recognition of any abnormal from normal pattern was reached after 30 studies (3rd session) but fluctuated. Diagnosis of oesophageal body motor patterns remained suboptimal; accuracy of advisability of fundoplication improved, but did not reach 90%. CONCLUSIONS: High-resolution manometry has a steep learning curve among trainees. Achalasia recognition is achieved early, but diagnosis of other abnormal motor patterns and management decisions require further supervised training.

Distribution and function of polycystin-2 in mouse retinal ganglion cells.

The polycystin family of transient receptor potential (TRP) channels form Ca(2+) regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, polycystins control intracellular Ca(2+) signals and more generally the translation of extracellular signals and stimuli to intracellular responses. Polycystin-2 channels have been cloned from retina, but their distribution and function in retinal ganglion cells (RGCs) have not yet been established. In the present study, we determined cellular and subcellular localization as well as functional properties of polycystin-2 channels in RGCs. Polycystin-2 expression and distribution in RGCs was assessed by immunohistochemistry on vertical cryostat section of mouse retina as well as primary cultured mouse RGCs, using fluorescence microscopy. Biophysical and pharmacological properties of polycystin-2 channels isolated from primary cultured RGCs were determined using planar lipid bilayer electrophysiology. We detected polycystin-2 immunoreactivity both in the ganglion cell layer as well as in primary cultured RGCs. Subcellular analysis revealed strong cytosolic localization pattern of polycystin-2. Polycystin-2 channel current was Ca(2+) activated, had a maximum slope conductance of 114 pS, and could be blocked in a dose-dependent manner by increasing concentrations of Mg(2+). The cytosolic localization of polycystin-2 in RGCs is in accordance with its function as intracellular Ca(2+) release channel. We conclude that polycystin-2 forms functional channels in RGCs, of which biophysical and pharmacological properties are similar to polycystin-2 channels reported for other tissues and organisms. Our data suggest a potential role for polycystin-2 in RGC Ca(2+) signaling.

Production of wood decay enzymes, loss of mass, and lignin solubilization in wood by diverse tropical freshwater fungi.

In vitro production of cellulase and xylanase was common among diverse freshwater ascomycetes and their hyphomycetous anamorphs. Production of enzymes involved in lignin degradation was rare. Most isolates were capable of causing mass loss in angiosperm wood, although values were low, at approximately 10% during a 24-week period. A few isolates caused higher mass loss of up to 26.5%, and five of these were shown to solubilize significant amounts of lignin. This is the first report of lignin solubilization by freshwater fungi. Torula herbarum (hyphomycete) and Ophioceras dolichostomum (ascomycete) produced indices of lignin solubilization equivalent to those of terrestrial white-rot basidiomycetes. In all cases wood decay was 2.2- to 3-fold higher in exposed rather than submerged conditions.

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis.

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.

Short-course intensity-modulated radiotherapy (70 GY at 2.5 GY per fraction) for localized prostate cancer: preliminary results on late toxicity and quality of life.

PURPOSE: To present our preliminary observations on the late toxicity and quality of life (QOL) of patients treated with short-course intensity-modulated radiotherapy (SCIM-RT). METHODS AND MATERIALS: Fifty-one patients were treated with SCIM-RT at the Cleveland Clinic Foundation between October 1998 and May 1999. The technique consisted of intensity-modulated radiotherapy using 5 static fields (anterior, 2 laterals, and 2 anterior obliques). Inverse plans were generated by the Corvus treatment-planning system. The treatment delivery was performed with a dynamic multileaf collimator. A total of 70.0 Gy was prescribed in all cases at 2.5 Gy per fraction to be delivered in 28 fractions over 5 and a half weeks. The location of the prostate gland was verified and adjusted daily with the BAT transabdominal ultrasound system. The median follow-up was 18 months (range: 11 to 26 months). The Radiation Therapy Oncology Group (RTOG) scales were used to evaluate late toxicity. The Expanded Prostate Cancer Index Composite (EPIC) was used to evaluate QOL. A total of 24 patients completed the EPIC questionnaire at approximately 2 years after therapy (median time from treatment to questionnaire administration: 24 months; range: 21 to 26 months). The results from the EPIC questionnaires were compared to scores from 46 patients treated during the same time period with conformal radiotherapy (CRT) to 78 Gy at 2 Gy per fraction. RESULTS: The dose was prescribed to an isodose line ranging from 82.0% to 90.0% (mean: 87.2%). The range of the individual prostate mean doses was 73.5 to 78.5 Gy (average: 75.3 Gy). To date, only 1 patient had Grade 1 late urinary toxicity. To date, only 4 patients had Grade 1 late rectal toxicity. No Grade 2 or 3 late urinary or rectal complications have occurred. The actuarial rectal bleeding rate observed at 18 months was 7%. There were no differences in scores from the urinary, bowel, hormonal, and overall QOL domains between SCIM-RT patients and patients treated with CRT. The overall physical and mental QOL scores were also nearly identical to scores reported for the general U.S. population. CONCLUSION: Preliminary late toxicity results up to 2 years after SCIM-RT are encouraging, with a median follow-up of 18 months (range 11 to 26 months). Late toxicity assessed by the physicians using RTOG late toxicity scores has been excellent. QOL reported by the patients using the EPIC questionnaire reveals no difference between patients treated with high-dose CRT at standard fractionation and patients treated with SCIM-RT. SCIM-RT is an alternative method of dose escalation in the treatment of localized prostate cancer. The proposed schedule significantly increases convenience to patients due to the decrease in overall treatment time.

Radiation dose response in patients with favorable localized prostate cancer (Stage T1-T2, biopsy Gleason < or = 6, and pretreatment prostate-specific antigen < or = 10).

PURPOSE: To study the radiation dose response as determined by biochemical relapse-free survival in patients with favorable localized prostate cancers, i.e., Stage T1-T2, biopsy Gleason score (bGS) < or = 6, and pretreatment prostate-specific antigen (iPSA) < or = 10 ng/mL. METHODS AND MATERIALS: A total of 292 patients with favorable localized prostate cancer were treated with radiotherapy alone between 1986 and 1999. The median age was 69 years. Sixteen percent of cases (n = 46) were African-American. The distribution by clinical T stage was as follows: T1/T2A, 243 (83%); and T2B/T2C, 49 (17%). The distribution by iPSA was as follows: < or = 4 ng/mL, 49 (17%); and > 4 ng/mL, 243 (83%). The mean iPSA level was 6.2 (median, 6.4). The distribution by bGS was as follows: or = 5 in 89 cases (30%) and 6 in 203 cases (70%). The median radiation dose was 70.0 Gy (range, 63.0-78.0 Gy). Doses of < or = 70.0 Gy were delivered in 175 cases, 70.2-72.0 Gy in 24 cases, 74 Gy in 30 cases, and 78 Gy in 63 cases. For patients receiving < 72 Gy, the median dose was 68 Gy, vs. 78 Gy for patients receiving > or = 72 Gy. A conformal technique was used in 129 (44%) of cases. The median follow-up was 43 months (range, 3-153). RESULTS: For the entire cohort, the projected 5- and 8-year biochemical relapse-free survival (bRFS) rates were both 81%. For patients receiving > or = 72 Gy, the 5- and 8-year bRFS rates were both 95% vs. only 77% for patients receiving < 72 Gy, p = 0.010. For patients receiving 74 Gy, the 4-year bRFS rate was 94% vs. 96% for patients receiving 78 Gy, p = 0.90. A multivariate analysis for factors affecting bRFS rates using Cox proportional hazards was performed for all cases using the following variables: age (continuous variable), race (black vs. white), iPSA (continuous variable), bGS (< or = 5 vs. 6), Stage (T1-2A vs. T2B-C), radiation dose (continuous variable), and radiation technique (conformal vs. standard). From the multivariate analysis, only iPSA (p = 0.017, chi(2) = 5.7), and radiation dose (p = 0.021, chi(2) = 5.3) were independent predictors of outcome. Age (p = 0.94), race (p = 0.89), stage (p = 0.45), biopsy GS (p = 0.40), and radiation technique (p = 0.45) were not. CONCLUSION: There is a clear radiation dose response in patients with favorable localized prostate cancers (i.e., Stage T1-T2, biopsy Gleason score < or = 6, and iPSA < or = 10 ng/mL). At least 74 Gy should be delivered to the prostate and periprostatic tissues. With our cohort of patients, longer follow-up will be needed to assess the importance of doses exceeding 74 Gy.

Biotransformation of linear alkylbenzene sulfonate (LAS) by Phanerochaete chrysosporium: oxidation of alkyl side-chain.

The white rot fungus Phanerochaete chrysosporium, which generally mineralizes substituted aromatics to CO2, transformed linear alkylbenzene sulfonate (LAS) surfactants mainly at their alkyl side chain. Degradation of LAS was evidenced by a zone of clearing on LAS-containing agar plates and colorimetric analysis of liquid cultures. Disappearance of LAS was virtually complete within 10 days in low nitrogen (2.4 mM N), high nitrogen (24 mM N) and malt extract (ME) liquid media. After 5 days of incubation in ME medium, transformation of LAS was complete at concentrations < or = 4 mg l(-1), but decreased at higher concentrations. The LAS degradation was not dependent on lignin peroxidases (LiPs) and manganese-dependent peroxidases (MnPs). Mineralization of 14C-ring-LAS to 14CO2 by P. chrysosporium was < 1% regardless of the culture conditions used. Thin layer chromatography and mass spectral analyses indicated that P. chrysosporium transformed LAS to sulfophenyl carboxylates (SPCs) through oxidative shortening of the alkyl side-chains. While LAS disappearance in the cultures was not dependent on LiPs and MnPs, transformation of the parent LAS moieties to SPCs was more extensive in low N medium that favors expression of these enzymes. The SPCs produced in LN cultures were shorter in chain- length than those produced in ME cultures. Also there was a notable shift in the relative abundance of odd and even chain length metabolites compared to the starting LAS particularly in the low N cultures suggesting the possible involvement of processes other than or in addition to beta-oxidation in the chain-shortening process.

Application of recursive partitioning analysis and evaluation of the use of whole brain radiation among patients treated with stereotactic radiosurgery for newly diagnosed brain metastases.

PURPOSE: To evaluate the usefulness of whole brain radiotherapy (WBRT) and of the Radiation Therapy Oncology Group recursive partitioning analysis (RPA) for brain metastases among patients receiving stereotactic radiosurgery (SRS). METHODS AND MATERIALS: A retrospective analysis was performed on 135 patients who underwent linear accelerator (Linac) (n = 73) or Gamma Knife (n = 62) SRS for newly diagnosed brain metastases at the Cleveland Clinic Foundation between 8/89 and 12/98. Univariate and multivariate analyses were performed to evaluate the effects of age, primary site, control of the primary, interval to development of brain metastases (disease-free interval [DFI]), number of brain metastases, presence of extracranial metastases, Karnofsky performance status (KPS), treatment of brain metastases, and RPA class on overall survival. RESULTS: Application of the RPA classification revealed 29 patients fit the criteria for class I, 96 for class II, and 10 for class III. All of the patients underwent SRS. Fifty-seven patients also received WBRT at the time of initial presentation (SRS and immediate WBRT), and 78 patients received WBRT only if CNS relapse occurred (SRS alone). The median survival for all patients was 7.9 months (range: 1.1-90.1), and was 11.2 months for RPA class I compared to 6. 9 months for RPA classes II-III (p = 0.016). Median survival was 10. 5 months following SRS alone compared to 6.4 months following SRS and WBRT (p = 0.07). On univariate analysis, KPS >/= 80% (p = 0.002) and absence of systemic disease (p = 0.013) were also associated with longer survival, whereas control of the primary, DFI, and number of brain metastases did not have an impact. Multivariate analysis revealed only RPA class (p = 0.023) to be an independent predictor for overall survival, whereas treatment group (p = 0.079) was only marginally significant. At 2 years, immediate WBRT improved control at the original site of metastases (80% vs. 52%, p = 0.03) and prevention of new metastatic sites within the brain, 74% vs. 48% (p = 0.06). The 2-year intracranial disease-free survival was 60% following SRS and WBRT compared to only 34% following SRS alone (p = 0.03). CONCLUSIONS: Despite the inherent biases to select more favorable patients for SRS, the RPA class retains its prognostic value. Omission of WBRT from the initial management was not detrimental in terms of overall survival; however, progressive disease occurred in over 50% of patients treated in this manner. Further studies are required to determine which, if any, patients should be considered for SRS with WBRT held in reserve.

The role of whole brain radiotherapy and stereotactic radiosurgery on brain metastases from renal cell carcinoma.

PURPOSE: We reviewed our experience with patients who have undergone stereotactic radiosurgery (SRS) for brain metastases secondary to renal cell carcinoma (RCC). Analysis was performed to determine the survival, local control, distant brain failure (DBF), and then to define which tumors may not require upfront whole-brain radiotherapy (WBRT). METHODS AND MATERIALS: Twenty-nine patients with 66 tumors underwent SRS from 1991 to 1998. Median follow-up from time of brain metastases diagnoses relative to each tumor was 12.5 months and 6.8 months from the time of SRS. Median SRS dose was 1,800 cGy to the 60% isodose line. Three patients had undergone SRS for previously treated tumors. RESULTS: Median survival time from diagnosis was 10.0 months. Overall survival was not affected by age, addition of WBRT, number of lesions, tumor volume, or the presence of systemic disease. Of the 23 patients with follow-up neuroimaging, 4 of 47 (9%) tumors recurred. The addition of WBRT did not improve local control. Of the 13 patients who presented with a single lesion, 3 went on to develop DBF (23%), while 6 of the 10 patients who presented with multiple metastases developed DBF (60%). CONCLUSION: Patients with brain metastases secondary to RCC treated by SRS alone have excellent local control. The decision of whether or not to add WBRT to SRS should depend on whether the patient has a high likelihood of developing DBF. Our study suggests that patients who present with multiple brain lesions may be more likely to benefit from the addition of WBRT because they appear to be more than twice as likely to develop DBF as compared to patients with a single lesion.

Higher than standard radiation doses (> or =72 Gy) with or without androgen deprivation in the treatment of localized prostate cancer.

PURPOSE: To study the effect on biochemical relapse-free survival (bRFS) and clinical disease-free survival of radiation doses delivered to the prostate and periprostatic tissues for localized prostate cancer. METHODS AND MATERIALS: A total of 1041 consecutive localized prostate cancer cases treated with external beam radiotherapy (RT) at our institution between 7/86 and 2/99 were reviewed. All cases had available pretreatment parameters including pretreatment prostate-specific antigen (iPSA), biopsy Gleason score (bGS), and clinical T stage. The median age was 69 years. Twenty-three percent of cases (n = 238) were African-American. The distribution by clinical T stage was as follows: T1 in 365 cases (35%), T2 in 562 cases (54%), and T3 in 114 cases (11%). The median iPSA level was 10.1 ng/ml (range: 0.4-692.9). The distribution by biopsy Gleason score (bGS) was as follows: < or =6 in 580 cases (56%) and > or =7 in 461 cases (44%). Androgen deprivation (AD) in the adjuvant or neoadjuvant setting was given in 303 cases (29%). The mean RT dose was 71.9 Gy (range: 57.6-78.0 Gy). The median RT dose was 70.2 Gy, with 458 cases (44%) receiving at least 72.0 Gy. The average dose in patients receiving <72 Gy was 68.3 Gy (median 68.4) versus 76.5 Gy (median 78.0) for patients receiving > or =72 Gy. The mean follow-up was 38 months (median 33 months). The number of follow-up prostate-specific antigen (PSA) levels available was 5998. RESULTS: The 5- and 8-year bRFS rates were 61% (95% CI 55-65%) and 58% (95% CI 51-65%), respectively. The 5-year bRFS rates for patients receiving radiation doses > or =72 Gy versus <72 Gy were 87% (95% CI 82-92%) and 55% (95% CI 49-60%), respectively. The 8-year bRFS rates for patients receiving radiation doses > or =72 Gy versus <72 Gy were 87% (95% CI 82-92%) and 51% (95% CI 44-58%), respectively (p < 0.001). A multivariate analysis of factors affecting bRFS was performed using the following parameters: age (continuous variable), race, T-stage (T1-T2 vs. T3), iPSA (continuous variable), bGS (< or =6 vs. > or =7), use of AD (yes vs. no), radiation technique (conformal versus standard), and radiation dose (continuous variable). T-stage (p < 0.001), iPSA (p < 0.001), bGS (p < 0.001), and RT dose (p < 0.001) were independent predictors of outcome. Age (p = 0.74), race (p = 0.96), radiation technique (p = 0.15), and use of AD (p = 0.31) were not. We observed 11% clinical failures (local, distant, or both) at 5 years and 15% at 8 years for the entire cohort. There was a statistically significant improvement with higher radiation doses (p = 0.032). The 5-year clinical relapse rates for patients receiving > or =72 Gy versus <72 Gy were 5% and 12%, respectively. The 8-year clinical relapse rates for patients receiving radiation doses > or =72 Gy versus <72 Gy were 5% and 17%, respectively (p = 0.026). CONCLUSION: Patients receiving radiation doses exceeding 72 Gy had significantly better bRFS and clinical disease-free survival rates. Although results need to be confirmed with longer follow-up, these preliminary results are extremely encouraging. If these results are confirmed by other institutions and by longer follow-up, RT doses exceeding 72 Gy should be considered as standard of care.

Importance of high radiation doses (72 Gy or greater) in the treatment of stage T1-T3 adenocarcinoma of the prostate.

OBJECTIVES: To analyze the effect of total radiation dose on the outcome of patients treated with external beam radiotherapy for early-stage prostate cancer. METHODS: The records of a total of 738 patients with localized prostate cancer treated with external beam radiotherapy (RT) and no androgen deprivation at our institution between July 1986 and February 1999 were reviewed. Two risk groups were defined: favorable (Stage T1-T2, pretreatment prostate-specific antigen [PSA] level 10.0 ng/mL or less, and biopsy Gleason score 6 or less) and unfavorable (Stage T3 lesion or pretreatment PSA level greater than 10.0 ng/mL or biopsy Gleason score 7 or greater). The median RT dose was 70.0 Gy (range 57.6 to 78.0), with 192 patients (26%) receiving at least 72.0 Gy. The mean follow-up was 45 months. RESULTS: The 5-year biochemical relapse-free survival (bRFS) rate was 58%. The 5-year bRFS rate for patients who received radiation doses of 72 Gy or greater versus less than 72 Gy was 85% and 54%, respectively (P <0.001). On multivariate analysis of factors affecting bRFS rates, the number of follow-up PSA levels (P <0.001), tumor stage (P <0.001), pretreatment PSA (P <0.001), biopsy Gleason score (P <0.00 1), and RT dose (P = 0.001) were the only independent predictors of outcome. For favorable tumors, the 5-year bRFS rate for patients who received radiation doses of 72 Gy or greater versus less than 72 Gy was 98% and 81 %, respectively (P = 0.023). For unfavorable tumors, the 5-year bRFS rate for patients who received radiation doses of 72 Gy or greater versus less than 72 Gy was 75% and 41 %, respectively (P = 0.001). CONCLUSIONS: Patients receiving radiation doses of 72 Gy or higher had a significantly better outcome. The improvement was seen in all subgroups of patients. If these results are confirmed, radiation doses exceeding 72 Gy should be considered the standard of care. Inc.

Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum.

Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (M(r) of 40,000 and 66, 000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity ( approximately 100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.

Identification of bacterial isolates obtained from intestinal contents associated with 12,000-year-old mastodon remains.

Mastodon (Mammut americanum) remains unearthed during excavation of ancient sediments usually consist only of skeletal material, due to postmortem decomposition of soft tissues by microorganisms. Two recent excavations of skeletal remains in anoxic sediments in Ohio and Michigan, however, have uncovered organic masses which appear to be remnants of the small and large intestines, respectively. Macrobotanical examinations of the composition of these masses revealed assemblages of plant material radiocarbon dated to approximately 11,500 years before the present and thought to be incompletely digested food remains from this extinct mammal. We attempted to cultivate and identify bacteria from the intestinal contents, bone-associated sediments, and sediments not in proximity to the remains using a variety of general and selective media. In all, 295 isolates were cultivated, and 38 individual taxa were identified by fatty acid-methyl ester (FAME) profiles and biochemical characteristics (API-20E). The taxonomic positions of selected enteric and obligately anaerobic bacteria were confirmed by 16S ribosomal DNA (rDNA) sequencing. Results indicate that the intestinal and bone-associated samples contained the greatest diversity of bacterial taxa and that members of the family Enterobacteriaceae represented 41% of all isolates and were predominant in the intestinal masses and sediments in proximity to the skeleton but were uncommon in the background sediments. Enterobacter cloacae was the most commonly identified isolate, and partial rDNA sequencing revealed that Rahnella aquatilis was the correct identity of strains suggested by FAME profiles to be Yersinia enterocolitica. No Bacteroides spp. or expected intestinal anaerobes were recovered. The only obligate anaerobes recovered were clostridia, and these were not recovered from the small intestinal masses. Microbiological evidence from this study supports other, macrobotanical data indicating the intestinal origin of these masses. Whether these organisms are direct descendants of the original intestinal microbiota, however, cannot be established.

In vitro degradation of natural insoluble lignin in aqueous media by the extracellular peroxidases of Phanerochaete chrysosporium.

The lignin peroxidases (LIP) and manganese peroxidases (MNP) of Phanerochaete chrysosporium catalyze a wide range of lignin depolymerization reactions with lignin models and synthetic lignins in solution. However, their ability to degrade insoluble natural lignin in aqueous media has not been demonstrated. Insoluble isolated poplar lignin similar to natural lignin was treated in vitro in aqueous media for 12 h with LIP, MNP, and both. Treatment with MNP alone slightly increased the solid mass and produced measurable amounts of lignin-derived 2,6-dimethoxyhydroquinone and 2-methoxyhydroquinone but did not appreciably decrease the total lignin content. Treatment with LIP alone did not decrease the mass but produced measurable amounts of lignin-derived p-hydroxybenzoic acid and slightly decreased the lignin content. Finally, treatment with LIP and MNP together decreased the solid mass by 11%, decreased the lignin content by 5%, and released low-concentration compounds with mass spectra containing the typical lignin-derived electron-impact fragments of mass 107, 137, 151, 167, and 181. These results suggest that MNP increases the effectiveness of LIP-mediated lignin degradation.

In vitro degradation of insoluble lignin in aqueous media by lignin peroxidase and manganese peroxidase.

The abilities of lignin peroxidase (LIP) and manganese peroxidase (MNP) from Phanerochaete chrysosporium to degrade an insoluble hardwood lignin in vitro in aqueous media were tested. Neither LIP nor MNP appreciably changed the mass or lignin content, although both produced small amounts of unique solubilized lignin fragments. Treatment with both LIP and MNP, however, decreased the mass by 11%, decreased the lignin content by 5.1% (4.2% as total weight), and solubilized unique lignin-derived molecules. These results suggest that LIP and MNP synergistically degrade high molecular weight insoluble lignin, but singly, neither enzyme is sufficient to effect lignin degradation.

Isolation of saprophytic basidiomycetes from soil.

A method with the combined advantages of soil particle washing, selective inhibitors, and an indicator substrate was developed to isolate saprophytic basidiomycetes from soil. Organic particles were washed from soil and plated on a medium containing lignin, guaiacol, and benomyl, which reduced mold growth and allowed detection of basidiomycetes producing laccase or peroxidase. The 64 soil samples yielded 67 basidiomycete isolates, representing 51 groups on the basis of morphology and physiology. This method should facilitate investigations into the biodiversity of soil basidiomycetes and yield organisms that are useful in bioremediation of soils contaminated with pesticides or other recalcitrant aromatic compounds.

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR.

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases.

Demonstration of Laccase in the White Rot Basidiomycete Phanerochaete chrysosporium BKM-F1767.

It has been widely reported that the white rot basidiomycete Phanerochaete chrysosporium, unlike most other white rot fungi, does not produce laccase, an enzyme implicated in lignin biodegradation. Our results showed that P. chrysosporium BKM-F1767 produces extracellular laccase in a defined culture medium containing cellulose (10 g/liter) and either 2.4 or 24 mM ammonium tartrate. Laccase activity was demonstrated in the concentrated extracellular culture fluids of this organism as determined by a laccase plate assay as well as a spectrophotometric assay with ABTS [2,2(prm1)-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] as the substrate. Laccase activity was observed even after addition of excess catalase to the extracellular culture fluid to destroy the endogenously produced hydrogen peroxide, indicating that the observed activity is not due to a peroxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by activity staining with ABTS revealed the presence of a laccase band with an estimated M(infr) of 46,500.