A numerical model for the prediction of radon flux from uranium mill tailings at Jaduguda, India.

Solid process fine waste or tailings of a uranium mill is a potential source of release of radiologically significant gaseous radon (222Rn). A number of variables such as radium (226Ra) content, porosity, moisture content, and tailings density can affect the extent of emanation from the tailings. Further, if a cover material is used for remediation purposes, additional challenges due to changes in the matrix characteristics in predicting the radon flux can be anticipated. The uranium mill tailings impoundment systems at Jaduguda have been in use for the long-term storage of fine process waste (tailings). A pilot-scale remediation exercise of one of the tailings ponds has been undertaken with 30 cm soil as a cover material. For the prediction of the radon flux, a numerical model has been developed to account for the radon exhalation process at the remediated site. The model can effectively be used to accommodate both the continuous and discrete variable inputs. Depth profiling and physicochemical characterization for the remediated site have been done for the required input variables of the proposed numerical model. The predicted flux worked out is well below the reference level of 0.74 Bq m-2 s-1 IAEA (2004).

Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in four malaria endemic states of India.

BACKGROUND: Malaria is a major public health problem in India and accounts for about 88% of malaria burden in South-East Asia. India alone accounted for 2% of total malaria cases globally. Anti-malarial drug resistance is one of the major problems for malaria control and elimination programme. Artemether-lumefantrine (AL) is the first-line treatment of uncomplicated Plasmodium falciparum in north eastern states of India since 2013 after confirming the resistance against sulfadoxine-pyrimethamine. In the present study, therapeutic efficacy of artemether-lumefantrine and k13 polymorphism was assessed in uncomplicated P. falciparum malaria. METHODS: This study was conducted at four community health centres located in Koraput district of Odisha, Bastar district of Chhattisgarh, Balaghat district of Madhya Pradesh and Gondia district of Maharashtra state. Patients with uncomplicated P. falciparum malaria were administered with fixed dose combination (6 doses) of artemether-lumefantrine for 3 days and clinical and parasitological response was recorded up to 28 days as per World Health Organization protocol. Nucleotide sequencing of msp1 and msp2 gene was performed to differentiate between recrudescence and reinfection. Amplification and sequencing of k13 propeller gene region covering codon 450-680 was also carried out to identify the polymorphism. RESULTS: A total 376 malaria patients who fulfilled the enrolment criteria as well as consented for the study were enrolled. Total 356 patients were followed up successfully up to 28 days. Overall, the adequate clinical and parasitological response was 98.9% and 99.4% with and without PCR correction respectively. No case of early treatment failure was observed. However, four cases (1.1%) of late parasitological failure were found from the Bastar district of Chhattisgarh. Genotyping of msp1 and msp2 confirmed 2 cases each of recrudescence and reinfection, respectively. Mutation analysis of k13 propeller gene showed one non-synonymous mutation Q613H in one isolate from Bastar. CONCLUSIONS: The study results showed that artemether-lumefantrine is highly effective in the treatment of uncomplicated P. falciparum malaria among all age groups. No functional mutation in k13 was found in the study area. The data from this study will be helpful in implementation of artemether-lumefantrine in case of treatment failure by artesunate plus sulfadoxine-pyrimethamine.

Periodic trends and complexation chemistry of tetravalent actinide ions with a potential actinide decorporation agent 5-LIO(Me-3,2-HOPO): A relativistic density functional theory exploration.

A relativistic density functional theory (DFT) study is reported which aims to understand the complexation chemistry of An4+ ions (An = Th, U, Np, and Pu) with a potential decorporation agent, 5-LIO(Me-3,2-HOPO). The calculations show that the periodic change of the metal binding free energy has an excellent correlation with the ionic radii and such change of ionic radii also leads to the structural modulation of actinide-ligand complexes. The calculated structural and binding parameters agree well with the available experimental data. Atomic charges derived from quantum theory of atoms in molecules (QTAIM) and natural bond order (NBO) analysis shows the major role of ligand-to-metal charge transfer in the stability of the complexes. Energy decomposition analysis, QTAIM, and electron localization function (ELF) predict that the actinide-ligand bond is dominantly ionic, but the contribution of orbital interaction is considerable and increases from Th4+ to Pu4+ . A decomposition of orbital contributions applying the extended transition state-natural orbital chemical valence method points out the significant π-donation from the oxygen donor centers to the electron-poor actinide ion. Molecular orbital analysis suggests an increasing trend of orbital mixing in the context of 5f orbital participation across the tetravalent An series (Th-Pu). However, the corresponding overlap integral is found to be smaller than in the case of 6d orbital participation. An analysis of the results from the aforementioned electronic structure methods indicates that such orbital participation possibly arises due to the energy matching of ligand and metal orbitals and carries the signature of near-degeneracy driven covalency.

Postresection CA19-9 and margin status as predictors of recurrence after adjuvant treatment for pancreatic carcinoma: Analysis of NRG oncology RTOG trial 9704.

PURPOSE: NRG Oncology RTOG 9704 was the first adjuvant trial to validate the prognostic value of postresection CA19-9 levels for survival in patients with pancreatic carcinoma. The data resulting from this study also provide information about predictors of recurrence that may be used to tailor individualized management in this disease setting. This secondary analysis assessed the prognostic value of postresection CA19-9 and surgical margin status (SMS) in predicting patterns of disease recurrence. METHODS AND MATERIALS: This multicenter cooperative trial included participants who were enrolled as patients at oncology treatment sites in the United States and Canada. The study included 451 patients analyzable for SMS, of whom 385 were eligible for postresection CA19-9 analysis. Postresection CA19-9 was analyzed at cut points of 90, 180, and continuously. Patterns of disease recurrence included local/regional recurrence (LRR) and distant failure (DF). Multivariable analyses included treatment, tumor size, and nodal status. To adjust for multiple comparisons, a P value of ≤ .01 was considered statistically significant and > .01 to ≤ .05 to be a trend. RESULTS: For CA19-9, 132 (34%) patients were Lewis antigen-negative (no CA19-9 expression), 200 (52%) had levels <90, and 220 (57%) had levels <180. A total of 188 patients (42%) had negative margins, 152 (34%) positive, and 111 (25%) unknown. On univariate analysis, CA19-9 cut at 90 was associated with increases in LRR (trend) and DF. Results were similar at the 180 cut point. SMS was not associated with an increase in LRR on univariate or multivariate analyses. On multivariable analysis, CA19-9 ≥ 90 was associated with increased LRR and DF. Results were similar at the 180 cut point. CONCLUSIONS: In this prospective evaluation, postresection CA19-9 was a significant predictor of both LRR and DF, whereas SMS was not. These findings support consideration of adjuvant radiation therapy dose intensification in patients with elevated postresection CA19-9.

Wartime toxin exposure: recognising the silent killer.

Wartime toxin exposures have been implicated in the genesis of malignancy in war veterans. Agent Orange, one toxin among many, has been linked to malignancy and the subcomponent phenoxyacetic acid has been associated with soft tissue sarcomas (STSs). This case demonstrates the association between a wartime toxin exposure (Agent Orange) and subsequent cancer development. Ultimately, we aim to highlight the importance of simple, specific questions in the patient history to account for previous wartime toxin exposures.

Acute disseminated encephalomyelitis: Extremely rare presentation of pediatric human immunodeficiency virus infection.

Acquired human immunodeficiency virus (HIV) infection in a 10-year-old child, presenting with monoparesis, progressing to triplegia over 4 weeks is an extremely rare feature. The child had left upper motor neurone facial palsy with left hemiplegia, paralyzed right lower limb, grade zero power, exaggerated deep tendon reflexes and bilateral extensor plantars. Child tested positive for HIV by ELISA. CD3(+) absolute count was 431. CD3(+) CD4 count was 28, and CD45 absolute count was 478. Magnetic resonance imaging of brain and spine showed multiple ill-defined foci of hyperintensity in white matter suggestive of ADEM. Acute demyelinating encephalomyelitis (ADEM) is an extremely rare presenting feature of perinatally acquired HIV infection in paediatrics. Clinically child remained same even with methylprednisolone, intravenous immunoglobulin, antituberculosis therapy, trimethoprim-sulfamethoxazole prophylaxis and supportive therapy. Child had sudden clinical deterioration and death before antiretroviral therapy could be initiated. This case emphasizes that pediatricians and neurophysicians should suspect HIV as an etiology of ADEM in cases with atypical clinical presentation and social risk factors, in spite of its very rare occurrence.

Role of ultrasound in evaluation of peripheral nerves.

Ultrasonography (USG) is an excellent cost-effective modality in imaging of peripheral nerves. With the newer high-frequency probes with different footprints which allow high-resolution imaging at relatively superficial location, USG can detect and evaluate traumatic, inflammatory, infective, neoplastic, and compressive pathologies of the peripheral nerves. This article describes the technique for evaluation of nerves by USG as well as the USG appearances of normal and diseased peripheral nerves.

Angiosarcoma of the thyroid: a case report with review of the literature.

Angiosarcoma is a rare and aggressive tumor of the thyroid gland, mainly seen in the Alpine regions. We present such a case with literature review. We present the case of a 60-year-old man with cough, dyspnea, and hemoptysis along with slow increase in the size of his long-standing goiter. Computed tomography of the neck showed a large thyroid mass and chest imaging revealed multiple pulmonary nodules. Fine needle aspiration cytology and tru-cut biopsy of the thyroid were notable for poorly differentiated malignant cells. Diagnosis of angiosarcoma of the thyroid was made after total thyroidectomy. Patient died of continued hemoptysis and respiratory failure 3 weeks after admission. We searched the literature for previous case reports using Pubmed and Ovid. Forty-seven reported cases were identified and our case was added to make a database of 48 cases. Demographic and tumor characteristics were analyzed. Angiosarcoma was found to be more common in females and at age of 60 or above. Results were consistent with previously reported series of 14 and 17 cases from Austria. This review provides information on various characteristics angiosarcoma of the thyroid which can be used as baseline data for future reference and research studies for this cancer.

Spectral and in vitro antimicrobial properties of 2-oxo-4-phenyl-6-styryl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid transition metal complexes.

2-oxo-4-phenyl-6-styryl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid (ADP) was complexed with acetates of Mn(II), Ni(II), Cu(II) and Zn(II). The structures of the ligand and its metal complexes were characterized by microanalysis, IR, NMR, UV-vis spectroscopy, magnetic susceptibility and TGA-DTA analyses. Octahedral and square planar geometries were suggested for the complexes in which the central metal ion coordinated with O donors of ligand and acetate ions. Each ligand binds the metal using carboxylate oxygens. The ligand and complexes were evaluated for their antimicrobial activities against different species of pathogenic bacteria and fungi. The present novel pyrimidine containing complexes could constitute a new group of antibacterial and antifungal agents.

Image-guided stereotactic radiosurgery for locally advanced pancreatic adenocarcinoma results of first 85 patients.

BACKGROUND: Locally advanced unresectable pancreatic adenocarcinoma is characterized by poor survival despite chemotherapy and conventional radiation therapy (RT). Recent advances in real-time image-guided stereotactic radiosurgery (SRS) have made it possible to treat these cancers in two to four fractions followed by systemic chemotherapy. AIMS: The aims of this study includes the following: (1) obtain local control of the disease; (2) improve the survival of these unresectable patients; (3) evaluate the toxicity of SRS; and (4) report results of the largest series from a single center. METHODS: Pancreatic SRS involves delivery of high doses of accurately targeted radiation given non-invasively in two to four fractions. We treated 85 consecutive patients with locally advanced and recurrent pancreatic adenocarcinoma from February 2004 to November 2009. Age range: 36-88 years, median 66 years; sex: 50 males, 35 females; race: 79 Caucasian, five African American, one Asian; histology: 80 adenocarcinoma, three islet cell, two other. Pre-SRS staging: T(3-4) 85; N(+) 16, N(x) 57, N(0) 12; M(0) 64, M(1) 21. All patients were unresectable at the time of SRS. Seventy-one had no prior surgical resection, and 14 had local recurrence after prior surgical resection. Twenty-nine patients had progression of disease after prior conventional RT. Location of the tumor: head, 57; body and tail, 28. Pre-SRS chemotherapy was given in 48 patients. All patients received gemcitabine-based chemotherapy regimen after SRS. Median tumor volume was 60 cm(3). PET/CT scans done in 55 patients were positive in 52 and negative in three patients. Average maximum standard uptake value was 6.9. Pain score on a scale of 1-10 was: 0-3 in 54, 4-7 in 18, and 8-10 in 13 patients. SRS doses ranged from 15 to 30 Gy with a mean dose of 25.5 Gy delivered in 3 days divided in equal fractions. Mean conformality index was 1.6, and mean isodose line was 80%. RESULTS: Tumor control: complete, partial, and stable disease were observed in 78 patients for the duration of 3-36 months with median of 8 months. Pain relief was noted in majority of patients lasting for 18-24 weeks. Most of the patients died of distant disease progression while their primary tumor was controlled. Overall median survival from diagnosis was 18.6 months and from SRS it was 8.65 months. For the group of 35 patients with adenocarcinoma without prior surgical resection or RT and no distant metastases, the average and 1-year survival from diagnosis was 15 months and 50%, respectively, and from SRS it was 11.15 months and 30.5%, respectively. TOXICITY: A total of 19 (22.37%) patients developed grades III/IV GI toxicity including duodenitis, 12 (14.1%); gastritis, 11 (12.9%); diarrhea, three (3.5%); and renal failure was noted in one (1.2%). Three patient had both gastritis and duodenitis. Toxicity was significantly more prevalent in the first 40 patients compared with the last 45 patients (32.5 vs 13.9%). CONCLUSIONS: SRS for unresectable pancreatic carcinoma can be delivered in three fractions with minimal morbidity and a local tumor control rate of 91.7%. The survival is comparable or better than the reported results for advanced pancreatic cancer, specifically for the group of previously untreated patients with unresectable tumors. Development of distant metastases remains a significant factor.

Practical syntheses of 4-fluoroprolines.

4-Fluoroprolines are among the most useful nonnatural amino acids in chemical biology. Here, practical routes are reported for the synthesis of the 2S,4R, 2S,4S, and 2R,4S diastereomers of 4-fluoroproline. Each route starts with (2S,4R)-4-hydroxyproline, which is a prevalent component of collagen and hence readily available, and uses a fluoride salt to install the fluoro group. Hence, the routes provide process-scale access to these useful nonnatural amino acids.

Mechanistic studies on Hsp90 inhibition by ansamycin derivatives.

Heat shock protein 90 (Hsp90) is a molecular chaperone that is required for the maturation and activation of a number of client proteins, many of which are involved in cancer development. The ansamycin family of natural products and their derivatives, such as geldanamycin (GA), are well-known inhibitors of the essential ATPase activity of Hsp90. Despite structural studies on the complexes of ansamycin derivatives with the ATPase domain of Hsp90, certain aspects of their inhibitory mechanism remain unresolved. For example, it is known that GA in solution exists in an extended conformation with a trans amide bond; however, it binds to Hsp90 in a significantly more compact conformation with a cis amide bond. GA and its derivatives have been shown to bind to Hsp90 with low micromolar affinity in vitro, in contrast to the low nanomolar anti-proliferative activity that these drugs exhibit in vivo. In addition, they show selectivity towards tumour cells. We have studied both the equilibrium binding, and the association and dissociation kinetics of GA derivative, 17-DMAG, and the fluorescently labelled analogue BDGA to both wild-type and mutant Hsp90. The mutants were made in order to test the hypothesis that conserved residues near the ATP-binding site may catalyse the trans-cis isomerisation of GA. Our results show that Hsp90 does not catalyse the trans-cis isomerisation of GA, and suggests that there is no isomerisation step before binding to Hsp90. Experiments with BDGA measured over a wide range of conditions, in the absence and in the presence of reducing agents, confirm recent studies that have suggested that the reduced dihydroquinone form of the drug binds to Hsp90 considerably more tightly than the non-reduced quinone species.

wCLUTO: a Web-enabled clustering toolkit.

As structural and functional genomics efforts provide the biological community with ever-broadening sets of interrelated data, the need to explore such complex information for subtle relationships expands. We present wCLUTO, a Web-enabled version of the stand-alone application CLUTO, designed to apply clustering methods to genomic information. Its first application is focused on the clustering transcriptome data from microarrays. Data can be uploaded by the user into the clustering tool, a choice of several clustering methods can be made and configured, and data are presented to the user in a variety of visual formats, including a three-dimensional "mountain" view of the clusters. Parameters can be explored to rapidly examine a variety of clustering results, and the resulting clusters can be downloaded either for manipulation by other programs or to be saved in a format for publication.

Synthesis of spirocycles via ring closing metathesis of heterocycles carrying gem-diallyl substituents obtained via ring opening of (halomethyl)cyclopropanes with allyltributyltin.

In the presence of allyl tri-n-butyltin--AIBN, cyclopropylmethyl bromides/xanthates undergo ring-opening reaction with concomitant formation of geminal diallyl derivatives in good yields. The ring closing metathesis reactions on geminal diallyl derivatives with Grubbs' catalyst provided spirocyclopentenyl products. Combination of these two methodologies has been applied to the synthesis of mono-, bis-cyclopentyl-carbohydrates as well as spirocyclopentylproline derivatives.

Evaluation of the immunocontraceptive potential of Escherichia coli-expressed recombinant dog ZP2 and ZP3 in a homologous animal model.

Dog zona pellucida glycoprotein 2 (dZP2), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned and expressed as a polyhistidine fusion protein in Escherichia coli to evaluate the immunocontraceptive efficacy of ZP glycoproteins. The recombinant dZP2 (rec-dZP2) revealed a 70 kDa band corresponding to the full length transcript, as well as several low molecular mass fragments in western blot analysis. In addition to rec-dZP2, E. coli expressed recombinant dog ZP glycoprotein 3 (rec-dZP3), which has also been evaluated for its efficacy to block fertility in a homologous system. Three groups of female dogs (n = 4 per group) were immunized with rec-dZP2 conjugated to diphtheria toxoid (rec-dZP2-DT), rec-dZP3 conjugated to DT (rec-dZP3-DT) and DT alone. Immunization of female dogs with rec-dZP2-DT and rec-dZP3-DT led to generation of antibodies against the respective ZP proteins as well as to DT. Subsequent to mating, the four female dogs immunized with rec-dZP2-DT all conceived, which is indicative of failure of the anti-rec-dZP2 antibodies to block fertility. In the group of dogs immunized with rec-dZP3-DT, three of four animals did not conceive when mated with males of proven fertility. The block in fertility was associated with anti-dZP3 antibody titres. Ovarian histopathology revealed that the block in fertility in the group immunized with rec-dZP3-DT is probably manifested by inhibition in the development of follicles and is due to atretic changes in the zona pellucida. These results, although preliminary, indicate that immunization with dZP3 may be a feasible proposition to control dog populations provided that adequate antibody titres are achieved.

Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis.

The electronic and redox properties of the iron-sulfur cluster and tungsten center in the as-isolated and sulfide-activated forms of formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (Em=-368mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the "low-potential" W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25-30% of the total W and undergoes redox cycling between W(IV)/ W(V) and W(V)/W(VI) states at physiologically relevant potentials (Em= -335 and -280 mV, respectively). At higher potentials a minor "mid-potential" W(V) species, g= 1.983, 1.956, 1.932, accounting for less than 5 % of the total W, appears with a midpoint potential of -34 mV and persists up to at least + 300 mV. At potentials above 0 mV, a major "high-potential" W(V) signal, g= 1.981, 1.956, 1.883, accounting for 30-40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g= 1.981, 1.950, 1.931; Em = -265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; Em = +65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR.

Purification and molecular characterization of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus: the third of a putative five-member tungstoenzyme family.

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100 degreesC by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS-) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (Km = 100 microM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5 semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.

Purification and Characterization of Two Functional Forms of Intracellular Protease PfpI from the Hyperthermophilic Archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100(deg)C by the fermentation of peptides and carbohydrates. From this organism, we have purified to homogeneity an intracellular protease, previously designated PfpI (P. furiosus protease I) (S. B. Halio, I. I. Blumentals, S. A. Short, B. M. Merrill, and R. M. Kelly, J. Bacteriol. 178:2605-2612, 1996). The protease contains a single subunit with a molecular mass of approximately 19 kDa and exists in at least two functional conformations, which were purified separately. The predominant form from the purification (designated PfpI-C1) is a hexamer with a molecular mass of 124 (plusmn) 6 kDa (by gel filtration) and comprises about 90% of the total activity. The minor form (designated PfpI-C2) is trimeric with a molecular mass of 59 (plusmn) 3 kDa. PfpI-C1 hydrolyzed both basic and hydrophobic residues in the P1 position, indicating trypsin- and chymotrypsin-like specificities, respectively. The temperature optimum for Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-MCA) hydrolysis was (symbl)85(deg)C both for purified PfpI-C1 and for proteolytic activity in P. furiosus cell extract. In contrast, the temperature optimum for PfpI prepared by incubating a cell extract of P. furiosus at 98(deg)C in 1% sodium dodecyl sulfate for 24 h at 95 to 100(deg)C (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1255-1262, 1990), designated PfpI-H, was (symbl)100(deg)C. Moreover, the half-life of activity of PfpI-C1 at 98(deg)C was less than 30 min, in contrast to a value of more than 33 h measured for PfpI-H. PfpI-C1 appears to be a predominant serine-type protease in cell extracts but is converted in vitro, probably in part by deamidation of Asn and Gln residues, to a more thermally stable form (PfpI-H) by prolonged heat treatment. The deamination hypothesis is supported by the differences in the measured pI values of PfpI-C1 (6.1) and PfpI-H (3.8). High levels of potassium phosphate (>0.5 mM) were found to extend the half-life of PfpI-C1 activity towards AAF-MCA by up to 2.5-fold at 90(deg)C, suggesting that compatible solutes play an important role in the in vivo function of this protease.