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PubMed2001

Protein and lysine content, grain yield, and other technological traits in durum wheat under Mediterranean conditions.

A major problem for durum wheat production in the Mediterranean region is yield fluctuation. This fluctuation is a result of year-to-year variation in precipitation and heat stress during grain growth, which is typical of the Mediterranean climate. Both yield stability and good quality are needed in adapted durum wheat ideotypes. Ten durum wheat cultivars differing in drought resistance were grown during 1998, under both rainfed and irrigated conditions, at three sites in southern Spain. The main traits studied were protein and lysine content, grain yield, test weight, SDS sedimentation, semolina color, and grain vitreousness. Results show a high influence of site on all traits. Only test weight (TW), SDS sedimentation, grain vitreousness, and protein per kernel appeared to be determined also by cultivar effect. Vitreousness was positively correlated with TW (r = 0.48**) and semolina color (r = 0.46**). An inverse relationship was found between grain yield and protein content. Regression of cultivar mean values of protein content and grain yield showed a negative correlation (r = -0.72***), probably due to dilution of protein by non-nitrogen compounds and reduced starch accumulation in the grain under drought conditions. Lysine content was negatively associated with protein content (r = -0.86***), indicating the difficulty of a simultaneous breeding for both characteristics.

Climate↗

PubMed2000

The Bbeta-sheet in the PAI-1 molecule plays an important role for its stability.

We have investigated the B beta-sheet in PAI-1 regarding its role for the stability of the molecule. The residues from His(219) to Tyr(241) (except for Gly(230) and Pro(240)), covering the s2B and s3B strands, and in addition His(185) and His(190)) were substituted by amino acids with opposite properties. The 23 generated single-site changed mutants and also wild type PAI-1 (wtPAI-1) were expressed in E. coli. Subsequently they were purified by heparin-Sepharose and anhydrotrypsin agarose affinity chromatographies. The stability of the purified PAI-1 variants was analyzed at 37 degrees C and at different pHs (5.5, 6.5 or 7.5). At pH 7.5 and 37 degrees C, single substitutions of the residues in the central portions of both strands 2 and 3 in the B beta-sheet (Ile(223) to Leu(226) on s2B and Met(235) to Ile(237) on s3B), caused a significant decrease in stability, yielding half-lives of about 10-25% as compared to wtPAI-1. On the other hand, mutations at both sides of the central portion of the B beta-sheet (Tyr(221), Asp(222), Tyr(228) and Thr(232)) frequently resulted in an increased PAI-1 stability (up to 7-fold). While wtPAI-1 exhibited prolonged half-lives at pH 6.5 and 5.5, the PAI-1 variant Y228S was more stable at neutral pH (half-life of 9.6 h at pH 7.5) as compared to its half-life at pH 5.5 (1.1 h). One of the 4 modified histidine residues (His(229)) resulted in a variant with a clearly affected stability as a function of pH, suggesting that it may, at least in part, be of importance for the pH dependence of the PAI-1 stability. Thus, our data demonstrate that the B beta-sheet is of great importance for the stability of the molecule. Modifications in this part causes decreased or increased stability in a certain pattern, suggesting effects on the insertion rate of the reactive center loop into the A beta-sheet of the molecule.

Drug Stability↗

PubMed2001

Increased risk for falling associated with obesity: mathematical modeling of postural control.

Recent epidemiological studies report that obesity is positively related to fracture incidence. In the present experiment, a model of postural control was used to examine the impact of an abnormal distribution of body fat in the abdominal area upon postural stability. Obese and lightweight humanoids were destabilized by imposing a small initial angular speed from a neutral standing position. To avoid a loss of stability yielding a stepping reaction or a fall, an ankle torque is necessary to counteract the perturbation. Three torque parameters--ankle torque onset, time to peak torque, and muscular ankle torque--were entered in a program to simulate the intrinsic variability of the human postural control system. A loss of stability was detected when the center of pressure exceeded stability margins. The most striking observation is the nonlinear increase of torque needed to stabilize the humanoid when the motor response was characterized by delayed temporal parameters. The effect was more pronounced when an anterior position of the center of mass was included in the simulations. This suggests that, when submitted to daily postural stresses and perturbations, obese persons (particularly those with an abnormal distribution of body fat in the abdominal area) may be at higher risk of falling than lightweight individuals.

Abdomen↗

PubMedDate not supplied

Preparation, 99mTc-labeling, and in vitro characterization of HYNIC and N3S modified RC-160 and [Tyr3]octreotide.

The synthesis of conjugates of two somatostatin analogues, RC-160 and [Tyr3]octreotide with different bifunctional chelators for labeling with Tc-99m, is described. Conjugates with hydrazinonicotinamide (HYNIC) and two N3S compounds (benzoyl MAG3 and a N3S adipate derivative) were prepared on a small scale with high purity allowing evaluation of different bifunctional chelators on the same peptide without extensive peptide synthesis. High in vitro stability and retained binding affinity was found for all conjugates except for the N3S adipate. Peptide conjugates could be labeled at high specific activities (>1 Ci/micromol) with 99mTc, and different coligands were explored for the HYNIC conjugates. The resulting radiolabeled complexes were highly stable and showed binding affinity to somatostatin receptors in the nanomolar range. Varying labeling yield, stability, lipophilicity, and isomerism were found for different coligands used for labeling HYNIC conjugates, with lower lipophilicity, higher stability, and fewer coordination isomers for EDDA and tricine/nicotinic acid as ternary coligand compared to tricine. In particular, HYNIC complexes showed promising results for further in vivo evaluation.

Animals↗

PubMed1980

Stoichiometric model of alpha-cyclodextrin complex formation.

The solubility, spectral, and kinetic methods were used to study complexing between alpha-cyclodextrin (ligand, L) and 3,5-dimethoxycinnamic acid, benzalacetone, and methyl cinnamate (substrates, S). In aqueous solution at 25 degrees and with an ionic strength of 0.01 M, the following stability constants were found (K11 for SL and K12 for SL2): 3,5-dimethoxycinnamic acid, K11=1965 M-1 and K12=0; benzalacetone, K11=105 M-1 and K12=15 M-1; and methyl cinnamate, K11=1200 M-1 and K12=50 M-1. A model of complex formation is proposed that can account for the observed stoichiometry and that yields stability estimates for two isomeric 1:1 complexes in the systems in which a 1:2 complex forms. For cinnamic acid, benzalacetone, and methyl cinnamate, stability constants are inversely correlated with the substrate dipole moment.

Butanones↗

PubMed2001

Challenges for meeting the global food and nutrient needs in the new millennium.

Major advances have occurred in food production during the last 30 years as a result of the adoption of green revolution technology. The price of rice and wheat is 40 % lower than it was in the 1950s. This lower price has helped the poorer sections of society, who spend 50-60% of their income on food. The proportion of the population in the developing world that is malnourished fell from 46.5% in the early 1960s to 31% in 1995. However, there are still 1.3 billion of the population who go to bed hungry every day. Deficiencies of micronutrients such as Fe, Zn and vitamin A affect millions of the population in the developing world. The world population is increasing at the rate of 1.4%, or an increase of eighty million per year. It is estimated that we will have to produce 50% more food grains by 2025. Various strategies for meeting this challenge exist, including the development of cereal varieties with a higher yield potential and yield stability, and farmer-friendly public policies. In order to tackle hidden hunger, efforts are underway to develop crop varieties with higher concentrations of Fe and Zn. Recently, a breakthrough has occurred in the introduction of the genes for the pathway leading to the biosynthesis of beta-carotene, a precursor of vitamin A, in rice. Various conventional approaches and modem tools of biotechnology are being employed in the development of crop varieties with higher yields and higher levels of micronutrients.

Biotechnology↗

PubMed1982

Reliability and validity of clinical assessments of malocclusion.

The purpose of this research was to determine the reliability and validity of selected clinical judgments of malocclusion, including general evaluations of occlusal status and more specific aspects of dentofacial malrelations. Study casts of twenty-one adolescents planning orthodontic treatment and twenty-nine not planning treatment were examined and rated. The examiners were five dentists in an orthodontic specialty-training program. They completed ratings on six dimensions: (1) need for treatment, (2) degree of malocclusion, (3) potential for tissue loss, (4) negative effect on occlusal stability, (5) negative effect on dental-facial attractiveness, and (6) negative effect on masticatory function. Six weeks later the same five rates scored the fifty casts, using the standardized Treatment Priority Index (TPI). Three weeks later, or 9 weeks after the initial ratings, the casts were again rated on the two general dimensions: need for treatment and degree of malocclusion. Correlations among all the measures were examined. Inter-rater reliability was highest for the ratings of impact on dental-facial attractiveness (r = 0.88). The two general assessments also yielded relatively high rater reliabilities, and the second rating yielded stability coefficients of 0.84 for both of these ratings. Correlations with total TPI scores were 0.70 for the dental-facial attractiveness measure and 0.65 and 0.64, respectively, for assessments of need for treatment and degree of malocclusion. The data indicate that clinical evaluations of the severity of malocclusions are comparable to objective measures in terms of inter-rater reliability. Clinical evaluations are also relatively stable over time. Correlations with the TPI scores also provide evidence of the concurrent validity of clinical judgments.

Adolescent↗

PubMed2026

Clinical evidence on non-viral CAR-T cell therapies for solid tumors: a scoping review.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy in solid tumors is hindered by the immunosuppressive tumor microenvironment and by toxicities associated with viral-vector manufacturing. Non-viral gene delivery platforms have emerged as a potential alternative, though clinical evidence remains fragmented. METHODS: Following an a priori protocol registered on the Open Science Framework (OSF; https://doi.org/10.17605/OSF.IO/2TPQS) and adhering to JBI/PRISMA-ScR guidelines, a systematic search was conducted across four databases from inception through May 15, 2026. Patient-level data were extracted to describe cellular persistence and clinical outcomes across strictly non-viral delivery platforms. RESULTS: Four early-phase studies met the inclusion criteria, encompassing 28 heavily pretreated patients with metastatic solid tumors. Two non-viral platforms were identified: mRNA electroporation (n=19; intravenous in 13, intratumoral in 6) and the piggyBac transposon system (n=9). Across both mRNA routes, transient CAR-T persistence (<7 days) was observed, with no objective responses (ORR 0%), though disease stabilization yielded a disease control rate (DCR) of 53%; cross-route comparison is limited by differing distribution profiles. The piggyBac system showed longer persistence (~28 days) and a DCR of 78%, including the only documented objective response (ORR 11%). No Grade ≥3 cytokine release syndrome or neurotoxicity was reported in any of the 28 patients, and no tocilizumab or systemic corticosteroids were required. CONCLUSIONS: Within this limited early-phase evidence base, no severe toxicities attributable to non-viral platforms were reported, and the evidence identifies knowledge gaps warranting prospective investigation. mRNA platforms showed transient persistence and disease stabilization in 53% of patients. One partial response was documented with the piggyBac platform in a single patient; however, this outcome cannot be attributed to the delivery platform given simultaneous differences in target antigen, tumor histology, route of administration, and geographic setting. No firm conclusions regarding comparative platform performance can be drawn from this evidence base. SYSTEMATIC REVIEW REGISTRATION: https://doi.org/10.17605/OSF.IO/2TPQS, identifier OSF.IO/2TPQS.

Humans↗

PubMed2026

Beyond the salt barrier: CRISPR-mediated DNA reprogramming to uncouple yield from tolerance in Rice: A review.

Rice (Oryza sativa L.) feeds half of humanity, yet its cultivation is increasingly threatened by soil salinization, which now affects 1.4 billion hectares globally. Decades of breeding and engineering have focused on Na+ exclusion, principally through the Saltol QTL and the xylem-unloading transporter OsHKT1;5, yet this strategy has reached a physiological ceiling. Excluder genotypes survive salinity but fail to fill grain, because the ATP-intensive cost of continuous ion extrusion starves reproductive sinks, while ABA-mediated stomatal closure imposes chronic carbon limitation. The resulting "survival-yield gap" exposes a fundamental flaw in single-trait approaches to a polygenic stress. In this review, we argue that durable, yield-stable salt tolerance requires a coordinated systems-level intervention spanning five mechanistic tiers: (i) CRISPR/Cas9-mediated removal of negative regulatory brakes (OsRR22, RST1, PC1) that suppress plant's latent stress-adaptive capacity; (ii) reinforcement of actin-myosin cytoskeletal transport to sustain SOS1, NHX1, and HKT1;5 delivery under ionic stress; (iii) importation of halophyte design principles from Oryza coarctata, including salt gland architecture and superior Na+ compartmentalization; (iv) recalibration of the ROS-photosynthesis axis via the DHHC09-STRK1-CatC molecular switch and stomatal density engineering; and (v) pyramiding these modules into a "Salt-Shield Rice" genotype through multiplex editing, marker-assisted introgression, speed breeding, and genomic selection. We propose a phased ten-year roadmap that integrates synthetic biology circuit design with conventional breeding to deliver field-ready, multi-module varieties with greater than 70% yield stability at 8-10 dS m-1. This remains an aspirational design target rather than a demonstrated outcome, as three of the five tiers-halophyte-derived structural traits, cytoskeletal reinforcement, and full multi-module pyramiding-remain unvalidated in rice.

CRISPR/Cas9↗

PubMed2006

[The synthesis, characterization and in situ intestinal absorption of different molecular weight scutellarin-PEG conjugates].

In this report, highly water soluble esters of scutellarin with different molecular weight polyethylene glycol (PEG) were synthesized in order to improve the bioavailability of scutellarin. The physicochemical properties, the stabilities under different conditions and the in situ intestinal absorption in rats of the conjugates were investigated. By PEG modification, the greatly increased water solubility and desirable partition coefficient of scutellarin were obtained. These compounds function as prodrugs i. e. breakdown occurred in a predictable fashion: the t1/2 of them in PBS buffer at pH7.4 was above 12 h (37 degrees C) in vitro, while in plasma a rapid breakdown was observed, with a t1/2 of about 1. 5-3 h. The stabilities of the prodrugs were improved according to the increase of the molecular weight of PEG, thus, PEGylated prodrugs with desirable rates of hydrolysis could be obtained by the use of variable molecular weight PEGs. The PEGylation could enhance the absorption of scutellarin in rat intestine, and the absorption of scutellarin and its PEG conjugates by intestine was mainly via passive transport, for when the concentration was raised, the uptake did not appear saturable, and the permeability coefficient kept at an equilibrium level. When the molecular weight of PEG increased from 200 to 1000 Da, the absorption of the conjugates decreased. In conclusion, by overall consideration of the yield, stability and absorption, the present authors estimate that the PEG molecular weight used for the PEGylation of scutellarin should be within the range of 400-1 000 Da.

Animals↗

PubMed2025

The Clinician-reported Genetic Testing Utility InDEx for Neonatal Intensive Care (C-GUIDE NICU): Quantifying genome-wide sequencing utility in the NICU.

PURPOSE: Use of genomic sequencing (GS) in neonatal intensive care units (NICUs) has increased with improved diagnostic yield. However, uncertainty persists regarding when and for whom GS is most useful. Because a standardized approach to assessing utility is lacking, we developed a novel version of the Clinician-reported Genetic testing Utility InDEx (C-GUIDE) to quantify the utility of GS in NICUs. METHODS: Informed by a scoping review, we developed a draft C-GUIDE NICU tool to quantify utility, which underwent iterative revisions through feedback from clinician interviews and questionnaires on item relevance, comprehensibility, and comprehensiveness. We finalized the expert-informed C-GUIDE NICU using an international Delphi consensus process. RESULTS: Scoping review (n = 25 articles) and interviews (n = 21) revealed key themes of utility. Guided by qualitative feedback and item scoring, C-GUIDE was iteratively reduced to include 21, 18, and 14 items. The Delphi consensus process with 22 experts achieved item consensus and stability, yielding a final 10-item tool. CONCLUSION: Using a rigorous process, we developed a consensus-based standardized method for capturing the clinical utility of GS in NICUs. C-GUIDE NICU can be used by clinicians, researchers, and payers to assess GS value to patient care and will be available for licensed use following reliability and validity testing.

Humans↗

PubMed2026

Whole genome sequence data on Ethiopian key sorghum landraces and founder lines.

Sorghum (Sorghum bicolor (L.) Moench) is the fifth most important cereal globally. Its genetic diversity is key to improving yield stability, stress tolerance, and adaptation to different environments. Ethiopia is one of the centers for the crop's origin, diversity, and use in both human food and livestock feed. However, genomic data on Ethiopian sorghum remain limited, especially for landraces preferred by local farmers. This dataset consists of whole-genome sequencing data for 188 Ethiopian sorghum accessions, including founder lines and important landraces from major agroecological zones. Sequencing was performed using the Complete Genomics DNBSEQ-T7 platform, generating high-coverage whole-genome data (20 × coverage). On average, each accession produced 64.7 million reads. Reads were aligned to the Sorghum bicolor NCBIv3 reference genome and variants called using GATK HaplotypeCaller with joint genotyping (GATK v4.6.1.0). Hard-filtering followed GATK best-practice thresholds (QD <2.0, FS> 60.0, MQ <40.0, MQRankSum <-12.5, ReadPosRankSum <-8.0), retaining biallelic SNPs with mean depth 10-50×, missingness ≤20%, and MAF ≥0.05, yielding 6095,752 high-confidence SNPs across 185 accessions. Both the raw FASTQ files and processed VCF files are publicly available to support studies of sorghum genetic diversity, population structure, selection, and the genetic basis of important traits.

Adaptation↗

PubMed2001

Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation.

Methods for measuring rates of DNA synthesis, and thus cell proliferation, in humans had not been available until recently. We (D. C. Macallan, C. A. Fullerton, R. A. Neese, K. Haddock, S. S. Park, and M. K. Hellerstein, 1998, Proc. Natl. Acad. Sci. USA 95, 708-713) recently developed a stable isotope-mass spectrometric technique for measuring DNA synthesis by labeling the deoxyribose (dR) moiety of purine deoxyribonucleotides through the de novo nucleotide synthesis pathway. The original analytic approach had limitations, however. Here, we describe technical improvements that increase yield, stability, sensitivity, and reproducibility of the method. The purine deoxyribonucleoside, deoxyadenosine (dA), is directly isolated from hydrolysates of DNA by using an LC18 SPE column. Two derivatives were developed for analyzing the dR moiety of dA alone (without the base), an aldonitrile-triacetate derivative, and a reduced pentose-tetraacetate (PTA) derivative. The PTA derivative in particular exhibited greater stability (no degradation after several weeks), greater GC/MS signal, and much less abundance sensitivity of isotope ratios (i.e., less dependence of mass isotopomer abundances on the amount of material injected into the mass spectrometer source), compared to previous derivatives of dA. The need for complex, multidimensional abundance corrected standard curves was thereby avoided. Using the PTA derivative, dR enrichments from DNA of fully turned over cells of rodents with 2H2O enrichments in body water of 2.2-2.8% were 9.0-9.5%, and less than 1.0 microg DNA (ca. 2 x 10(5) cells) was required for reproducible analyses. In summary, these methodologic advances allow measurement of stable isotope incorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and experimental animals, with fewer cells, greater reproducibility, and less labor. Many applications of this approach can be envisioned.

Animals↗

PubMed2002

QTLs for grain carbon isotope discrimination in field-grown barley.

In several crops including cereals, carbon isotope discrimination (Delta) has been associated with drought tolerance in terms of water-use efficiency and yield stability in drought-prone environments. By using a complete genetic map generated from 167 recombinant inbred lines from a cross between Tadmor and Er/Apm, QTLs associated with grain Delta have been detected in barley grown in three Mediterranean field environments, two differing only in water availability. Ten QTLs were identified: one was specific to one environment, two presented interaction with the environment, six presented main effects across three or two environments and one presented both effects. Heading date did not contribute to the environment (E) and G x E effects acting on Delta. Seasonal rainfall and the ratio of rainfall to evapo-transpiration made large contributions to the environmental effect, but their influence on G x E was weaker. Eight QTLs for Delta co-located with QTLs for physiological traits related to plant water status and/or osmotic adjustment, and/or for agronomic traits previously measured on the same population. Some perspectives in terms of characterising drought tolerance are evoked.

Biomarkers↗

PubMed2005

A high-density genetic map of hexaploid wheat (Triticum aestivum L.) from the cross Chinese Spring x SQ1 and its use to compare QTLs for grain yield across a range of environments.

A population of 96 doubled haploid lines (DHLs) was prepared from F1 plants of the hexaploid wheat cross Chinese Spring x SQ1 (a high abscisic acid-expressing breeding line) and was mapped with 567 RFLP, AFLP, SSR, morphological and biochemical markers covering all 21 chromosomes, with a total map length of 3,522 cM. Although the map lengths for each genome were very similar, the D genome had only half the markers of the other two genomes. The map was used to identify quantitative trait loci (QTLs) for yield and yield components from a combination of 24 site x treatment x year combinations, including nutrient stress, drought stress and salt stress treatments. Although yield QTLs were widely distributed around the genome, 17 clusters of yield QTLs from five or more trials were identified: two on group 1 chromosomes, one each on group 2 and group 3, five on group 4, four on group 5, one on group 6 and three on group 7. The strongest yield QTL effects were on chromosomes 7AL and 7BL, due mainly to variation in grain numbers per ear. Three of the yield QTL clusters were largely site-specific, while four clusters were largely associated with one or other of the stress treatments. Three of the yield QTL clusters were coincident with the dwarfing gene Rht-B1 on 4BS and with the vernalisation genes Vrn-A1 on 5AL and Vrn-D1 on 5DL. Yields of each DHL were calculated for trial mean yields of 6 g plant(-1) and 2 g plant(-1) (equivalent to about 8 t ha(-1) and 2.5 t ha(-1), respectively), representing optimum and moderately stressed conditions. Analyses of these yield estimates using interval mapping confirmed the group-7 effects on yield and, at 2 g plant(-1), identified two additional major yield QTLs on chromosomes 1D and 5A. Many of the yield QTL clusters corresponded with QTLs already reported in wheat and, on the basis of comparative genetics, also in rice. The implications of these results for improving wheat yield stability are discussed.

Biomass↗

PubMed2006

Performance of wheat crops with different chromosome ploidy: root-sourced signals, drought tolerance, and yield performance.

Pot-culture experiments were carried out to estimate the role of non-hydraulic root signals (nHRS) and the relation of these signals to drought tolerance and grain yield formation under drought stress in six wheat varieties. These were two modern hexaploid wheat (Triticum aestivum L., AABBDD) Plateau602 and Longchun8139-2, two diploid wheat (Triticum monococcum L., AB) MO1 and MO4, and two tetraploid wheat (Triticum dicoccum Schuebl L., AABB) DM22 and DM31. In the two diploid relatives, the nHRS was switched on and off at a soil water content (SWC) of approximately 53-45% field water capacity (FWC). In contrast, in the modern hexaploid varieties, Longchun8139-2 and Plateau602 the nHRS occurred between a SWC of about 71 and 35% FWC, a much wider soil moisture range. The two tetraploid relatives, DM22 and DM31, were generally intermediate. The nHRS threshold range in SWC also narrowed as all six varieties went through successive developmental stages from shooting to grain filling. The two hexaploid wheat varieties had the longest duration of survival after the water supply ceased, and the best yield stability under drought stress, similar to with tetraploid wheat varieties; the diploid wheat varieties were least robust. These two parameters were both significantly correlated with the nHRS soil moisture threshold range (r=0.9456** and 0.8608*, respectively). Based on these patterns, we propose a "triple Z" model to describe the features of non-hydraulic stomatal sensitivity versus soil drought in wheat growth.

Crops, Agricultural↗

PubMed1996

Bulk-freezing of Theileria parva stabilates for vaccine production.

The freezing and thawing characteristics of different volume samples of freezing medium and Theileria parva stabilates were studied in order to identify suitable conditions for freezing large volumes of stabilates for use in the preparation of vaccines. Conventionally, 1.8 ml aliquots are used but are cumbersome to handle in the field preparation of trivalent vaccines. In this study, 75 ml aliquots of the stabilates were found to exhibit freezing and thawing characteristics similar to those of the 1.8 ml aliquots. Bulk freezing of 2 stabilates yielded sporozoites which remained infective for cattle upon thawing, induced a range of clinical reactions in vaccinated cattle and stimulated the development of immune reactions.

Animals↗

PubMed1993

Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents.

A series of variants of kanamycin nucleotidyltransferase (KNTase), isolated previously on the basis of enhanced thermostability by cloning and selection for enzymatic activity in the thermophile Bacillus stearothermophilus, was used to systematically test the hypothesis that thermostable enzymes would also be more resistant to other forms of protein denaturation. The purified KNTases were treated with proteinase K or assayed at 37 degrees C in the presence of urea, N-lauroylsarcosine, Triton X-100, tetrahydrofuran, ethanol, or dimethylformamide. With all these agents, the KNTases displayed increasing resistance to denaturation in the order: wild type, mutant TK9 (with a Thr130-->Lys substitution), TK1 (Asp80-->Tyr), and TK101 (both substitutions). This is the same order in which their thermostability increases, indicating that the structural mechanism(s) whereby the mutations yield enhanced resistance to heat denaturation also yield stabilization towards chemical forms of enzymatic inactivation. These results suggest that selection in thermophiles is a useful method to obtain enzyme variants with increased overall stability, even at nonthermophilic temperatures.

Detergents↗