Biochemical and Structural Characterization of XoxG and XoxJ and Their Roles in Lanthanide-Dependent Methanol Dehydrogenase Activity.

Lanthanide (Ln)-dependent methanol dehydrogenases (MDHs) have recently been shown to be widespread in methylotrophic bacteria. Along with the core MDH protein, XoxF, these systems contain two other proteins, XoxG (a c-type cytochrome) and XoxJ (a periplasmic binding protein of unknown function), about which little is known. In this work, we have biochemically and structurally characterized these proteins from the methyltroph Methylobacterium extorquens AM1. In contrast to results obtained in an artificial assay system, assays of XoxFs metallated with LaIII , CeIII , and NdIII using their physiological electron acceptor, XoxG, display Ln-independent activities, but the Km for XoxG markedly increases from La to Nd. This result suggests that XoxG's redox properties are tuned specifically for lighter Lns in XoxF, an interpretation supported by the unusually low reduction potential of XoxG (+172 mV). The X-ray crystal structure of XoxG provides a structural basis for this reduction potential and insight into the XoxG-XoxF interaction. Finally, the X-ray crystal structure of XoxJ reveals a large hydrophobic cleft and suggests a role in the activation of XoxF. These studies enrich our understanding of the underlying chemical principles that enable the activity of XoxF with multiple lanthanides in vitro and in vivo.

Longitudinal evaluation of a household energy package on blood pressure, central hemodynamics, and arterial stiffness in China.

BACKGROUND: Cardiovascular diseases are the leading contributors to disease burden in China and globally, and household air pollution exposure is associated with risk of cardiovascular disease. OBJECTIVES: We evaluated whether subclinical cardiovascular outcomes in adult Chinese women would improve after distribution of an energy package comprised of a semi-gasifier cookstove, water heater, chimney, and supply of processed biomass fuel. METHODS: We enrolled 204 households (n = 205 women) from 12 villages into a controlled before- and after-intervention study on cardiovascular health and air pollution in Sichuan Province. The intervention was distributed to 124 households during a government-sponsored rural energy demonstration program. The remaining 80 households received the package 18 months later at the end of the study, forming a comparison group. One woman from each household had their blood pressure (BP), central hemodynamics, and arterial stiffness measured along with exposures to air pollution and demographic and household characteristics, on up to five visits. We used a difference-in-differences mixed-effects regression approach with Bayesian inference to assess the impact of the energy package on sub-clinical cardiovascular outcomes. RESULTS: Women who did not receive the energy package had greater mean decreases in brachial systolic (-4.1 mmHg, 95% credible interval (95%CIe) -7.3, -0.9) and diastolic BP (-2.0 mmHg, 95%CIe -3.6, -0.5) compared with women who received the package (systolic: -2.7, 95%CIe -5.0, -0.4; diastolic: -0.3, 95%CIe -1.4, 0.8) resulting in slightly positive but not statistically significant difference-in-differences effect estimates of 1.3 mmHg (95%CIe -2.5, 5.2) and 1.7 mmHg (95%CIe -0.3, 3.6), respectively. Similar trends were found for central BP, central pulse pressure, and arterial stiffness. Air pollution exposures decreased on average for both treatment groups, with a greater range of reductions among women who did not receive the package (with package: -30% to -50%; without package: +2% to -69%), likely as a result of increased use of gas fuel and electric stoves among this group. Outdoor air quality changed very little over time. CONCLUSIONS: Gasifier stoves have been widely promoted as the next generation of 'clean-cooking' technologies, however their effectiveness in improving health in real-world settings should be carefully evaluated and communicated before scaling up their implementation.

Efficacy, Immunogenicity, and Safety of a 9-Valent Human Papillomavirus Vaccine: Subgroup Analysis of Participants From Asian Countries.

Background: A 9-valent human papillomavirus-6/11/16/18/31/33/45/52/58 (9vHPV) vaccine extends coverage to 5 next most common oncogenic types (31/33/45/52/58) in cervical cancer versus quadrivalent HPV (qHPV) vaccine. We describe efficacy, immunogenicity, and safety in Asian participants (India, Hong Kong, South Korea, Japan, Taiwan, and Thailand) from 2 international studies: a randomized, double-blinded, qHPV vaccine-controlled efficacy study (young women aged 16-26 years; NCT00543543; Study 001); and an immunogenicity study (girls and boys aged 9-15 years; NCT00943722; Study 002). Methods: Participants (N = 2519) were vaccinated at day 1 and months 2 and 6. Gynecological samples (Study 001 only) and serum were collected for HPV DNA and antibody assessments, respectively. Injection-site and systemic adverse events (AEs) were monitored. Data were analyzed by country and vaccination group. Results: 9vHPV vaccine prevented HPV-31/33/45/52/58-related persistent infection with 90.4%-100% efficacy across included countries. At month 7, ≥97.9% of participants seroconverted for each HPV type. Injection-site AEs occurred in 77.7%-83.1% and 81.9%-87.5% of qHPV and 9vHPV vaccine recipients in Study 001, respectively, and 62.4%-85.7% of girls/boys in Study 002; most were mild to moderate. Conclusions: The 9vHPV vaccine is efficacious, immunogenic, and well tolerated in Asian participants. Data support 9vHPV vaccination programs in Asia. Clinical Trials Registration: NCT00543543; NCT00943722.

Cavernous sinus syndrome in a Holstein bull.

A 13-month-old Holstein bull was presented for right-sided exophthalmos. Ophthalmologic examination noted that the animal was visual in both eyes, but that the right pupil was persistently dilated and very sluggish to constrict when stimulated with a bright light and that normal ocular motility was absent. Fundic examination of the right eye was normal as was a complete ophthalmologic examination of the left eye. Radiographs at presentation did not reveal the presence of sinusitis or other skull abnormalities. Initial treatment comprised intravenous antibiotics and anti-inflammatories for orbital inflammation over a 14-day period. There was no perceptible change in the appearance or neuro-ophthalmologic examination of the right eye during hospitalization. The animal was discharged to the owner's care, but 3 weeks later was found recumbent with unilateral strabismus of the left eye and a fixed right pupil. Due to the inability to rise and rapid deterioration, humane euthanasia was performed, and a full postmortem examination, preceded by a MRI, was performed that identified abscesses extending bilaterally through the round foramina obliterating the cavernous sinus region, as well as abscessation of the right mandible, right trigeminal neuritis, right-sided sinusitis, and right-sided otitis media. Cavernous sinus syndrome should be considered in cattle with a combination of exophthalmos and neuro-ophthalmologic abnormalities involving cranial nerves III, IV, V, and VI, whose branches are located within the cavernous sinus.

Pollutant emissions and energy efficiency of Chinese gasifier cooking stoves and implications for future intervention studies.

Household air pollution from solid fuel combustion is the leading environmental health risk factor globally. In China, almost half of all homes use solid fuel to meet their household energy demands. Gasifier cookstoves offer a potentially affordable, efficient, and low-polluting alternative to current solid fuel combustion technology, but pollutant emissions and energy efficiency performance of this class of stoves are poorly characterized. In this study, four Chinese gasifier cookstoves were evaluated for their pollutant emissions and efficiency using the internationally recognized water boiling test (WBT), version 4.1.2. WBT performance indicators included PM2.5, CO, and CO2 emissions and overall thermal efficiency. Laboratory investigation also included evaluation of pollutant emissions (PM2.5 and CO) under stove operating conditions designed to simulate common Chinese cooking practices. High power average overall thermal efficiencies ranged from 22 to 33%. High power average PM2.5 emissions ranged from 120 to 430 mg/MJ of useful energy, and CO emissions ranged from 1 to 30 g/MJ of useful energy. Compared with several widely disseminated "improved" cookstoves selected from the literature, on average, the four Chinese gasifier cookstoves had lower PM2.5 emissions and higher CO emissions. The recent International Organization for Standardization (ISO) International Workshop Agreement on tiered cookstove ranking was developed to help classify stove performance and identify the best-performing stoves. The results from this study highlight potential ways to further improve this approach. Medium power stove operation emitted nearly twice as much PM2.5 as was emitted during high power stove operation, and the lighting phase of a cooking event contributed 45% and 34% of total PM2.5 emissions (combined lighting and cooking). Future approaches to laboratory-based testing of advanced cookstoves could improve to include greater differentiation between different modes of stove operation, beyond those evaluated with the WBT.

Head-torso coordination in police officers wearing loaded tactical vests during running.

BACKGROUND: The influence of load carriage in operational police officers is not well understood despite a relatively high injury rate. Assessing load related changes in head and torso coordination may provide valuable insight into plausible injury mechanisms. RESEARCH QUESTION: Do typical police tactical vest loads alter head and torso coordination during running? METHODS: Thirty-eight UK police officers ran at a self-selected pace (>2 ms-1) on a non-motorised treadmill in four vest load conditions (unloaded, and low, high and evenly distributed loads). Peak head and torso tilt, and peak vest displacement were compared between all four conditions. Timings between vest and torso change of direction were compared between the three loaded conditions. The coupling angle between the head and torso calculated using modified vector coding were compared between unloaded and each loaded conditions using Statistical Parametric Mapping. RESULTS: No significant differences were found between conditions for peak head or torso tilt alone (p > 0.05). Loading equipment low on the vest led to significantly greater mediolateral vest displacements (38 mm) away from the torso than a high (34 mm) or evenly distributed (30 mm) conditions. The vest was found to change direction vertically before the torso in the anterior-posterior direction, and then influence torso motion. The loaded conditions changed the head-torso coupling from in-phase (with head-dominancy) to anti-phase (with torso dominancy) between 55% and 77% stance. Anti-phase with a relatively stationary head and the torso rotating forward likely places a greater concentric demand on the posterior neck muscles relative to unloaded running. SIGNIFICANCE: Current tactical vest designs allow significant extra displacement of load away from the body during running, altering coordination at the head and torso.

Indoor air pollution in developing countries: research and implementation needs for improvements in global public health.

Exposure to indoor air pollution (IAP) from the burning of solid fuels for cooking, heating, and lighting accounts for a significant portion of the global burden of death and disease, and disproportionately affects women and children in developing regions. Clean cookstove campaigns recently received more attention and investment, but their successes might hinge on greater integration of the public health community with a variety of other disciplines. To help guide public health research in alleviating this important global environmental health burden, we synthesized previous research on IAP in developing countries, summarized successes and challenges of previous cookstove implementation programs, and provided key research and implementation needs from structured discussions at a recent symposium.

Proteomic analysis of the supernatant of red blood cell units: the effects of storage and leucoreduction.

BACKGROUND: Red blood cell (RBC) transfusion is a life-saving intervention for critically ill patients; however, it has been linked to increased morbidity and mortality. We hypothesize that a number of important proteins accumulate during routine storage of RBCs, which may explain some of the adverse effects seen in transfused patients. STUDY DESIGN: Five RBC units were drawn and divided (half prestorage leucoreduced (LR-RBC) and half left as an unmodified control (RBC). The supernatant was separated on days 1 and 42 of storage and proteomic analyses completed with in-gel tryptic digestion and nano-liquid chromatography tandem mass spectrometry. RESULTS: In RBC supernatants, 401 proteins were identified: 203 increased with storage, 114 decreased, and 84 were unchanged. In LR-RBC supernatant, 231 proteins were identified: 84 increased with storage, 30 decreased, and 117 were unchanged. Prestorage leucoreduction removed many platelet- and leucocyte-derived structural proteins; however, a number of intracellular proteins accumulated including peroxiredoxins (Prdx) 6 and latexin. The increases were confirmed by immunoblotting, including the T-phosphorylation of Prdx-6, indicating that it may be functioning as an active phospholipase. Active matrix metalloproteinase-9 also increased with a coinciding decrease in the metalloproteinase inhibitor 1 and cystatin C. CONCLUSION: We conclude that a number of proteins increase with RBC storage, which is partially ameliorated with leucoreduction, and transfusion of stored RBCs may introduce mediators that result in adverse events in the transfused host.

Immunologic risk stratification of pediatric heart transplant patients by combining HLA-EMMA and PIRCHE-II.

Human leukocyte antigen (HLA) molecular mismatch is a powerful biomarker of rejection. Few studies have explored its use in assessing rejection risk in heart transplant recipients. We tested the hypothesis that a combination of HLA Epitope Mismatch Algorithm (HLA-EMMA) and Predicted Indirectly Recognizable HLA Epitopes (PIRCHE-II) algorithms can improve risk stratification of pediatric heart transplant recipients. Class I and II HLA genotyping were performed by next-generation sequencing on 274 recipient/donor pairs enrolled in the Clinical Trials in Organ Transplantation in Children (CTOTC). Using high-resolution genotypes, we performed HLA molecular mismatch analysis with HLA-EMMA and PIRCHE-II, and correlated these findings with clinical outcomes. Patients without pre-formed donor specific antibody (DSA) (n=100) were used for correlations with post-transplant DSA and antibody mediated rejection (ABMR). Risk cut-offs were determined for DSA and ABMR using both algorithms. HLA-EMMA cut-offs alone predict the risk of DSA and ABMR; however, if used in combination with PIRCHE-II, the population could be further stratified into low-, intermediate-, and high-risk groups. The combination of HLA-EMMA and PIRCHE-II enables more granular immunological risk stratification. Intermediate-risk cases, like low-risk cases, are at a lower risk of DSA and ABMR. This new way of risk evaluation may facilitate individualized immunosuppression and surveillance.

Gas-phase formaldehyde adsorption isotherm studies on activated carbon: correlations of adsorption capacity to surface functional group density.

Formaldehyde (HCHO) adsorption isotherms were developed for the first time on three activated carbons representing one activated carbon fiber (ACF) cloth, one all-purpose granular activated carbon (GAC), and one GAC commercially promoted for gas-phase HCHO removal. The three activated carbons were evaluated for HCHO removal in the low-ppm(v) range and for water vapor adsorption from relative pressures of 0.1-0.9 at 26 °C where, according to the IUPAC isotherm classification system, the adsorption isotherms observed exhibited Type V behavior. A Type V adsorption isotherm model recently proposed by Qi and LeVan (Q-L) was selected to model the observed adsorption behavior because it reduces to a finite, nonzero limit at low partial pressures and it describes the entire range of adsorption considered in this study. The Q-L model was applied to a polar organic adsorbate to fit HCHO adsorption isotherms for the three activated carbons. The physical and chemical characteristics of the activated carbon surfaces were characterized using nitrogen adsorption isotherms, X-ray photoelectron spectroscopy (XPS), and Boehm titrations. At low concentrations, HCHO adsorption capacity was most strongly related to the density of basic surface functional groups (SFGs), while water vapor adsorption was most strongly influenced by the density of acidic SFGs.

Do non-rearfoot runners experience greater second metatarsal stresses than rearfoot runners?

Stress fracture of the second metatarsal is a common and problematic injury for runners. The choice of foot strike pattern is known to affect external kinetics and kinematics but its effect on internal loading of the metatarsals is not well understood. Models of various complexities can be used to investigate the effects of running characteristics on metatarsal stresses. This study aimed to compare second metatarsal stress between habitual rearfoot and non-rearfoot strikers during barefoot running, using a novel participant-specific finite element model, including accurate metatarsal and soft tissue geometry. Synchronised force and kinematic data were collected during barefoot overground running from 20 participants (12 rearfoot strikers). Stresses were calculated using a previously evaluated and published 3D finite element model. Non-rearfoot strikers demonstrated greater external loading and joint contact forces than rearfoot runners, but there were no differences in stresses between groups. Additionally, the study allowed for a qualitative assessment of bone geometries and stresses. No correlation was found between bone volume and stresses, however, there was found to be a large variation in metatarsal shapes, possibly accounting for the lack of difference in stresses. This emphasises the importance of bone geometry when estimating bone stress and supports the suggestion that external forces should not be assumed to be representative of internal loading.

A feasibility study of metabolic phenotyping of dried blood spot specimens in rural Chinese women exposed to household air pollution.

BACKGROUND: Exposure-response studies and policy evaluations of household air pollution (HAP) are limited by current methods of exposure assessment which are expensive and burdensome to participants. METHODS: We collected 152 dried blood spot (DBS) specimens during the heating and non-heating seasons from 53 women who regularly used biomass-burning stoves for cooking and heating. Participants were enrolled in a longitudinal study in China. Untargeted metabolic phenotyping of DBS were generated using ultra-high performance liquid chromatography coupled with mass spectrometry to exemplify measurement precision and assessment for feasibility to detect exposure to HAP, evaluated by season (high pollution vs. low pollution) and measured personal exposure to fine particulate matter <2.5 µm diameters (PM2.5) and black carbon (BC) in the 48-h prior to collecting the DBS specimen. RESULTS: Metabolites e.g., amino acids, acyl-carnitines, lyso-phosphorylcholines, sphinganine, and choline were detected in the DBS specimens. Our approach is capable of detecting the differences in personal exposure to HAP whilst showing high analytical reproducibility, coefficient of variance (CV) <15%, meeting the U.S. Food and Drug Administration criteria. CONCLUSIONS: Our results provide a proof of principle that high-resolution metabolic phenotypic data can be generated using a simple DBS extraction method thus suitable for exposure studies in remote, low-resource settings where the collection of serum and plasma is logistically challenging or infeasible. The analytical run time (19 min/specimen) is similar to most global phenotyping methods and therefore suitable for large-scale application.

Burns in young children: a retrospective matched cohort study of health and developmental outcomes.

BACKGROUND: Unintentional injuries are a major cause of death and disability in childhood. Most burns are unintentional, the majority occurring in pre-school children. Little is known about the outcomes of young children following burns. The purpose of this study was to examine the presenting features of burned children and compare their health and developmental outcomes with controls. METHODS: Children under 3 years admitted to the Welsh Regional Burns Centre between September 1994 and August 1997 were studied up to their sixth birthday (final data collection 2003) to ascertain the nature, course and cause of their burn. One hundred and forty-five burned children were matched with 145 controls. Their physical, psychosocial and educational health status was compared. Retrospective data were gathered from hospital notes, social services, emergency department databases, child health surveillance records and schools. RESULTS: Burns peaked at age 13-18 months were typically sustained by scalding, drink spillage and contact with hot objects. They occurred most frequently at mealtimes and 89.7% were judged to be unintentional. There was a high rate of non-attendance for follow-up - 24%. The families of children admitted with burns were more likely to have moved home than those of controls (P = 0.001). By age 6 significantly more cases were admitted to hospital with an unrelated condition (P = 0.018). There were no differences between the cases and controls in immunization status, development, school attendance and educational progress up to the age of 6 years (P > 0.05). CONCLUSIONS: We found important findings in relation to unintentional injury prevention and also noted markers that may indicate inequalities in health service utilization between cases and controls. There were no major differences between developmental and educational outcomes in the two groups.

Three dimensional finite element modelling of metatarsal stresses during running.

Second metatarsal stress fractures are a problematic injury for runners and are formed when the rate of repair of bone is outpaced by the damage accumulated during loading. Measuring the peak stresses on the bone during running gives an indication of damage accumulation but direct measurement is invasive. Finite element modelling is a viable alternative method of accurately estimating bone stresses but tends to be too computationally expensive for use in applied research. This study presents a novel and simple finite element model which can estimate bone stresses on the second metatarsal during the stance phase of walking and running, accounting for joint reaction forces and soft tissue effects. The influence of the forces and kinematic inputs to the model and the presence of the soft tissues was quantified using a sensitivity analysis. The magnitudes of maximum stress from the model are similar to existing finite element models and bone staple strain gauge values collected during walking and running. The model was found to be most sensitive to the pitch angle of the metatarsal and the joint reaction forces and was less sensitive to the ground reaction forces under the metatarsal head, suggesting that direct measurement of external forces should not be assumed to represent internal stresses.

Incorporating subject-specific geometry to compare metatarsal stress during running with different foot strike patterns.

Stress fracture of the second metatarsal is a common and problematic injury for runners. The choice of foot strike pattern is known to affect external kinetics and kinematics but its effect on internal loading of the metatarsals is not well understood. Subject-specific models of the second metatarsal can be used to investigate internal loading in a non-invasive manner. This study aimed to compare second metatarsal stress between habitual rearfoot and non-rearfoot strikers during barefoot running, using a novel subject-specific mathematical model, including accurate metatarsal geometry. Synchronised force and kinematic data were collected during barefoot overground running from 20 participants (12 rearfoot strikers). Stresses were calculated at the plantar and dorsal periphery of the midshaft of the metatarsal using a subject-specific beam theory model. Non-rearfoot strikers demonstrated greater external loading, bending moments and compressive forces than rearfoot strikers, but there were no differences in peak stresses between groups. Statistical parametric analysis revealed that non-rearfoot strikers had greater second metatarsal stresses during early stance but that there was no difference in peak stresses. This emphasises the importance of bone geometry when estimating bone stress and supports the suggestion that external forces should not be assumed to be representative of internal loading.

Silicon optical fiber.

Described herein are initial experimental details and properties of a silicon core, silica glass-clad optical fiber fabricated using conventional optical fiber draw methods. Such semiconductor core fibers have potential to greatly influence the fields of nonlinear fiber optics, infrared and THz power delivery. More specifically, x-ray diffraction and Raman spectroscopy showed the core to be highly crystalline silicon. The measured propagation losses were 4.3 dB/m at 2.936 microm, which likely are caused by either microcracks in the core arising from the large thermal expansion mismatch with the cladding or to SiO(2) precipitates formed from oxygen dissolved in the silicon melt. Suggestions for enhancing the performance of these semiconductor core fibers are provided. Here we show that lengths of an optical fiber containing a highly crystalline semiconducting core can be produced using scalable fiber fabrication techniques.

Safety profile of the 9-valent human papillomavirus vaccine: assessment in prior quadrivalent HPV vaccine recipients and in men 16 to 26 years of age.

A 9-valent HPV (9vHPV) vaccine has been developed to protect against HPV type 6/11/16/18/31/33/45/52/58-related infection and disease. Previous safety analyses from 7 clinical trials conducted in 9vHPV vaccine recipients 9-26 years of age, including comparisons of 9vHPV and quadrivalent HPV (qHPV) vaccines in girls and women 16-26 years of age, showed that the 9vHPV vaccine was generally well tolerated. Additional safety analyses were conducted to include the results of new clinical studies. The safety profile of the 9vHPV vaccine in prior qHPV vaccine recipients (n = 3756 from 1 randomized controlled trial and 2 open-label extension studies) and young men (n = 248 9vHPV and n = 248 qHPV vaccine recipients from 1 randomized controlled trial) was evaluated. Vaccine was administered as a 3-dose regimen (at Day 1 and Months 2 and 6), and adverse events (AEs) were monitored. The most common AEs were injection-site events (91.1% and 79.0% in prior qHPV vaccine recipients and young men, respectively), the majority of which were mild. Discontinuations due to an AE were rare (0.2% and 0.0% among prior qHPV vaccine recipients and young men, respectively). In young men, the AE profile of the 9vHPV vaccine was generally similar to that of the qHPV vaccine. Overall, the 9vHPV vaccine was generally well tolerated in prior qHPV vaccine recipients and in young men, with an AE profile generally consistent with that previously reported with the broader clinical program.

Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain.

The covalent attachment of ubiquitin (Ub) to short-lived or damaged proteins is believed to be the signal that initiates their selective degradation. In several cases, it has been shown that the proteolytic signal takes the form of a multi-Ub chain in which successive Ub molecules are linked tandemly at lysine 48 (K-48). Here we show that Ub molecules can be linked together in vivo at two other lysine positions, lysine 29 (K-29) and lysine 63 (K-63). The formation of these alternative linkages is strongly dependent on the presence of the stress-related Ub conjugating enzymes UBC4 and UBC5. Furthermore, expression of Ub carrying a K-63 to arginine 63 substitution in a strain of Saccharomyces cerevisiae that is missing the poly-Ub gene, UBI4, fails to compensate for the stress defects associated with these cells. Taken together, these results suggest that the formation of multi-Ub chains involving K-63 linkages plays an important role in the yeast stress response. In broader terms, these results also suggest that Ub is a versatile signal in which different Ub chain configurations are used for different functions.

Identification of a positive regulator of the cell cycle ubiquitin-conjugating enzyme Cdc34 (Ubc3).

The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerevisiae plays an essential role in the progression of cells from the G1 to S phase of the cell division cycle. Using a high-copy suppression strategy, we have identified a yeast gene (UBS1) whose elevated expression suppresses the conditional cell cycle defects associated with cdc34 mutations. The UBS1 gene encodes a 32.2-kDa protein of previously unknown function and is identical in sequence to a genomic open reading frame on chromosome II (GenBank accession number Z36034). Several lines of evidence described here indicate that Ubs1 functions as a general positive regulator of Cdc34 activity. First, overexpression of UBS1 suppresses not only the cell proliferation and morphological defects associated with cdc34 mutants but also the inability of cdc34 mutant cells to degrade the general amino acid biosynthesis transcriptional regulator, Gcn4. Second, deletion of the UBS1 gene profoundly accentuates the cell cycle defect when placed in combination with a cdc34 temperature-sensitive allele. Finally, a comparison of the Ubs1 and Cdc34 polypeptide sequences reveals two noncontiguous regions of similarity, which, when projected onto the three-dimensional structure of a ubiquitin-conjugating enzyme, define a single region situated on its surface. While cdc34 mutations corresponding to substitutions outside this region are suppressed by UBS1 overexpression, Ubs1 fails to suppress amino acid substitutions made within this region. Taken together with other findings, the allele specificity exhibited by UBS1 expression suggests that Ubs1 regulates Cdc34 by interaction or modification.

Creation of a pluripotent ubiquitin-conjugating enzyme.

We describe the creation of a pluripotent ubiquitin-conjugating enzyme (E2) generated through a single amino acid substitution within the catalytic domain of RAD6 (UBC2). This RAD6 derivative carries out the stress-related function of UBC4 and the cell cycle function of CDC34 while maintaining its own DNA repair function. Furthermore, it carries out CDC34's function in the absence of the CDC34 carboxy-terminal extension. By using sequence and structural comparisons, the residues that define the unique functions of these three E2s were found on the E2 catalytic face partitioned to either side by a conserved divide. One of these patches corresponds to a binding site for both HECT and RING domain proteins, suggesting that a single substitution in the catalytic domain of RAD6 confers upon it the ability to interact with multiple ubiquitin protein ligases (E3s). Other amino acid substitutions made within the catalytic domain of RAD6 either caused loss of its DNA repair function or modified its ability to carry out multiple E2 functions. These observations suggest that while HECT and RING domain binding may generally be localized to a specific patch on the E2 surface, other regions of the functional E2 face also play a role in specificity. Finally, these data also indicate that RAD6 uses a different functional region than either UBC4 or CDC34, allowing it to acquire the functions of these E2s while maintaining its own. The pluripotent RAD6 derivative, coupled with sequence, structural, and phylogenetic data, suggests that E2s have diverged from a common multifunctional progenitor.