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How do intricate multi-residue features such as protein-protein interfaces evolve? To address this question, we evolved a new colicin-immunity binding interaction. We started with Im9, which inhibits its cognate DNase ColE9 at 10(-14) M affinity, and evolved it toward ColE7, which it inhibits promiscuously (Kd > 10(-8) M). Iterative rounds of random mutagenesis and selection toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to an approximately 10(5)-fold increase in affinity and a 10(8)-fold increase in selectivity. Analysis of intermediates along the evolved variants revealed that changes in the binding configuration of the Im protein uncovered a latent set of interactions, thus providing the key to the rapid divergence of new Im7 variants. Overall, protein-protein interfaces seem to share the evolvability features of enzymes, that is, the exploitation of promiscuous interactions and alternative binding configurations via 'generalist' intermediates, and the key role of compensatory stabilizing mutations in facilitating the divergence of new functions. Show less

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 A resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs. Show less

Spermatogenesis is the process that involves the division and differentiation of spermatogonial stem cells into spermatozoa. However, the autocrine molecules and signaling pathways controlling their fate remain unknown. This study was designed to identify novel growth factors and signaling pathways that regulate proliferation, differentiation, and survival of spermatogonial stem/progenitor cells. To this end, we have for the first time explored the expression, function, and signaling pathway of Nodal, a member of the transforming growth factor-beta superfamily, in mouse spermatogonial stem/progenitor cells. We demonstrate that both Nodal and its receptors are present in these cells and in a spermatogonial stem/progenitor cell line (C18-4 cells), whereas Nodal is undetected in Sertoli cells or differentiated germ cells, as assayed by reverse transcription-polymerase chain reaction, Western blots, and immunocytochemistry. Nodal promotes proliferation of spermatogonial stem/progenitor cells and C18-4 cells, whereas Nodal receptor inhibitor SB431542 blocks their propagation as shown by proliferation and bromodeoxyuridine incorporation assays. Nodal knockdown by RNA interference results in a marked increase of cell apoptosis and a reduction of cell division as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling and proliferation assays. Conversely, overexpression of Nodal leads to an increase of cell proliferation. Nodal activates Smad2/3 phosphorylation, Oct-4 transcription, cyclin D1, and cyclin E expression, whereas SB431542 completely abolishes their increase. Together, Nodal was identified as the first autocrine signaling molecule that promotes proliferation of mouse spermatogonial stem/progenitor cells via Smad2/3 and Oct-4 activation. This study thus provides novel and important insights into molecular mechanisms regulating proliferation and survival of spermatogonial stem/progenitor cells. Show less

In Poland, prenatal exposure to tobacco smoke is observed in about 30 percent of children and postnatal in over 50 percent of children. This exposure has serious health consequences, including the negative effect on child neurodevelopment. The aim of the study was to assess the effect of environmental tobacco smoke (ETS) exposure on child psychomotor development. The study population consisted of 63 children with well assessed prenatal ETS exposure (a threefold analysis of cotinine level in saliva of pregnant women). To assess ETS exposure in infants within one year after birth a questionnaire-based interview was conducted with mothers. The Bayley Scale for Infant and Toddler Development (BESID-III) was used for the evaluation of child neurodevelopment. Multivariate analysis (including gender, birth order of the child and parental educational status) indicated the statistically significant association between prenatal exposure to ETS and cognitive child development (b = -4.0; p = 0.04). ETS exposure has also a negative impact on motor (b = -2.7; p = 0.2) and language (b = -3.4; p = 0.08) abilities of the child although the results were not statistically significant. Maternal smoking was found to be related to a decrease in child neurodevelopment, however, it was impossible to separate the prenatal from postnatal exposure. A lot of effort should be made to eliminate ETS exposure of children. Show less

Spermatogonial stem cells (SSCs) self-renew throughout life to produce progenitor cells that are able to differentiate into spermatozoa. However, the mechanisms underlying the cell fate determination between self-renewal and differentiation have not yet been delineated. Culture conditions and growth factors essential for self-renewal and proliferation of mouse SSCs have been investigated, but no information is available related to growth factors that affect fate determination of human spermatogonia. Wnts form a large family of secreted glycoproteins, the members of which are involved in cell proliferation, differentiation, organogenesis, and cell migration. Here, we show that Wnts and their receptors Fzs are expressed in mouse spermatogonia and in the C18-4 SSC line. We demonstrate that WNT3A induces cell proliferation, morphological changes, and cell migration in C18-4 cells. Furthermore, we show that beta-catenin is activated during testis development in 21-day-old mice. In addition, our study demonstrates that WNT3A sustained adult human embryonic stem (ES)-like cells derived from human germ cells in an undifferentiated stage, expressing essential human ES cell transcription factors. These results demonstrate for the first time that Wnt/beta-catenin pathways, especially WNT3A, may play an important role in the regulation of mouse and human spermatogonia. Show less

Small RNA molecules (small RNAs), including small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs), have recently emerged as important regulators of gene expression at the post-transcriptional or translation level. Significant progress has recently been made utilizing small RNAs in elucidating the molecular mechanisms regulating spermatogenesis. Spermatogenesis is a complex process that involves the division and eventual differentiation of spermatogonial stem cells into mature spermatozoa. The process of spermatogenesis is composed of several phases: mitotic proliferation of spermatogonia to produce spermatocytes; two meiotic divisions of spermatocytes to generate haploid round spermatids; and spermiogenesis, the final phase that involves the maturation of early-round spermatids into elongated mature spermatids. A number of miRNAs are expressed abundantly in male germ cells throughout spermatogenesis, while piRNAs are only present in pachytene spermatocytes and round spermatids. In this review, we first address the synthesis, mechanisms of action, and functions of siRNA, miRNA, and piRNA, and then we focus on the recent advancements in defining the small RNAs in the regulation of spermatogenesis. Concerns pertaining to the use of siRNAs in exploring spermatogenesis mechanisms and open questions in miRNAs and piRNAs in this field are highlighted. The potential applications of small RNAs to male contraception and treatment for male infertility and testicular cancer are also discussed. Show less

Recent reports have demonstrated that adult tissue cells can be induced to pluripotency, the iPS cells, mostly with the addition of genes delivered using viruses. Also, several publications both in mouse and in human have demonstrated that spermatogonial stem cells (SSCs) from testes can convert back to embryonic stem (ES)-like cells without the addition of genes. Furthermore, these pluripotent ES-like cells can differentiate into all three germ layers and organ lineages. Thus, SSCs have great potential for cell-based, autologous organ regeneration therapy for various diseases. We obtained testes from organ donors and using 1 g pieces of tissue (biopsy size) we demonstrate that testis germ cells (putative SSCs and/or their progenitors) reprogram to pluripotency when removed from their stem cell niche and when appropriate growth factors and reagents in embryonic stem cell medium are added. In addition, our method of obtaining pluripotent ES-like cells from germ cells is simpler than the described methods and may be more suitable if this procedure is developed for the clinic to obtain pluripotent cells to cure disease. Show less

Spermatogonial stem cells (SSCs) have unique characteristics in that they produce sperm that transmit genetic information from generation to generation and they can be reprogrammed spontaneously to form embryonic stem (ES)-like cells to acquire pluripotency. In rodents, it is generally believed that the A-single (A(s)) is the stem cell population, whereas the A-paired (A(pr)) and A-aligned (A(al)) represent the progenitor spermatogonial population. The A(1) to A(4) cells, intermediate, and type B spermatogonia are considered differentiated spermatogonia. In human, very little information is available about SSCs, except for the earlier work of Clermont and colleagues who demonstrated that there are two different types of A spermatogonia, the A(dark) and A(pale) spermatogonia. The A(dark) spermatogonia were referred to as the reserve stem cells, whereas the A(pale) were considered the renewing stem cells. In this review, we outline several spermatogonial renewal schemes for both rodents and primates, including man. We also compare phenotypic markers for spermatogonia/spermatogonial stem cells in rodents and humans and address SSC potential and therapeutic application. Show less

Rats learn to prefer flavors paired with the post-oral effects of glucose. The present study examined how rapidly they acquire this preference. In Experiment 1, food-restricted rats were given repeated three-session training/testing cycles: one 30-min session with a CS+ flavor paired with intragastric (IG) infusion of 16% glucose, another session with a CS- flavor paired with IG water, and a third session with a choice between the flavors with their infusates. The rats preferred the CS+ (69%) in the first choice session, and preference increased across the six cycles to 86%. These data demonstrate that the post-oral reinforcing action of glucose is potent enough to support one-trial learning. In Experiment 2, two groups of rats were trained in the same way, with the CS+ flavor paired with IG infusion of 16% glucose or 7.1% corn oil emulsion, but tests were conducted under extinction conditions, with both CS+ and CS- flavors paired with IG water. Significant preference for the CS+ was acquired more rapidly with glucose (71% CS+ in test 1) than with oil (69% CS+ in test 4). Consistent with previous work, the post-oral stimulation by glucose was more potent than that of isocaloric oil emulsion in conditioning preferences. The last experiment examined the acquisition rate for a flavor-taste conditioned preference. Rats were trained with a CS+ flavor mixed into an 8% fructose + 0.2% saccharin solution and a CS- flavor in 0.2% saccharin. The same three-session training/testing cycles were used, and in the tests the flavors were presented in saccharin. A significant 74% preference for the CS+ flavor was apparent by the second test. Together these studies show that the acquisition of flavor preferences, whether based on flavor-taste or flavor-nutrient associations, can be quite rapid. Show less

The aim of the study was to evaluate the nurses' exposure to active and passive smoking. The study population consisted of 299 nurses. Among the study population detailed questionnaire was conducted incusing sociodemographic characteristic, smoking profile and environmental tobacco smoke exposure. About 18% of nurses indicated current tobacco smoking and 25% smoking in the past. Only 13% of the study population declared complete ban of tobacco smoking in their homes and 20% indicated exposure to tobacco smoke in the workplaces. The women were most frequently exposed to inhaling tobacco smoke in bars and pubs (97%), restaurants (65%) and in private cars (64%). Show less

In the title compound, {[Ag(2)Pr(C(6)H(4)NO(2))(4)(H(2)O)(4)]ClO(4)·H(2)O}(n), the Pr(III) atom, lying on a twofold rotation axis, has a distorted square-anti-prismatic coordination geometry, defined by four O atoms from four nicotinate (nic) ligands and four water mol-ecules. The Ag(I) atom is coordinated in an almost linear fashion by two pyridyl N atoms from two nicotinate ligands. The linear coordination is augmented by weak inter-actions with three O atoms from one perchlorate anion, one uncoordinated water mol-ecule and one carboxyl-ate group. Two Pr atoms link two {Ag(nic)(2)}(+) units into a ring, which is further extended into an infinite zigzag chain by sharing the Pr atoms. These chains are further connected into a three-dimensional network via weak Ag⋯O inter-actions, O-H⋯O hydrogen bonds, Ag⋯Ag inter-actions [3.357 (2) Å] and π-π inter-actions between the pyridyl rings [centroid-centroid distance = 3.685 (4) Å]. Show less

Chronic peritoneal dialysis (PD) is one of the major therapies for uremic patients. However, the peritoneal mesothelial cells (PMCs) are subject to the injury by bioincompatible dialysates. The aim of this study is to investigate the protective roles and mechanisms of heat shock response in PMCs. Primary cultured human PMCs (HPMCs) were subjected to commercial peritoneal dialysates. The cell viability was assayed by MTT test and Annexin V assay. The expression of HSPs was detected by Western blots analysis. Intracellular hydrogen peroxide and superoxide anion were detected using H(2)DCFDA and dHE probe, respectively, with flow cytometry. The mitochondrial membrane potential (DeltaPsim) of HPMCs was evaluated using JC1 probe with flow-cytometry. Exposure of HPMCs to 1.5%, 2.5%, and 4.25% dextrose, and 7.5% icodextrin dialysates, respectively, for 60 min resulted in significantly accumulation of intracellular reactive oxygen species (ROS), DeltaPsim loss, and cell death in HPMCs. Amino acid dialysates exhibited no significant cytotoxicity. Adjusting the acidity in 1.5% dextrose and icodextrin dialysate significantly attenuated the dialysate-induced ROS generation and cell death in HPMCs. Heat pretreatment (41 degrees C, 30 minutes), which induced HSP 27 and 72 syntheses, significantly attenuated the dialysate-induced intracellular ROS accumulation, Dym loss, and cell death in HPMCs. In conclusion, the acidic bioincompatible dialysates induce oxidative stress, DeltaPsim loss, and subsequent cell death in HPMCs. Amino acid dialysates is more biocompatible than glucose and icodextrin dialysates to HPMCs. Heat shock response protects HPMCs from the bioincompatible dialysates-induced cellular damage. Show less

Preference and intake of sucrose varies across inbred and outbred strains of mice. Pharmacological analyses revealed that the greatest sensitivity to naltrexone-induced inhibition of sucrose (10%) intake was observed in C57BL10/J and C57BL/6J strains, whereas 129P3/J, SWR/J and SJL/J strains displayed far less sensitivity to naltrexone-induced inhibition of sucrose intake. Given that dopamine D1 (SCH23390) and D2 (raclopride) receptor antagonism potently reduce sucrose intake in outbred rat and mouse strains, the present study examined the possibility of genetic variance in the dose-dependent (50-1600 nmol/kg) and time-dependent (5-120 min) effects of these antagonists upon sucrose (10%) intake in the eight inbred (BALB/cJ, C3H/HeJ, C57BL/6J, C57BL/10J, DBA/2J, SJL/J, SWR/J and 129P3/J) and one outbred (CD-1) mouse strains previously tested with naltrexone. SCH23390 significantly reduced sucrose intake across all five doses in 129P3/J and SJL/J mice, across four doses in C57BL/6J and BALB/cJ mice, across three doses in DBA/2J, SWR/J, C3H/HeJ and C57BL/10J mice, but only at the two highest doses in CD-1 mice. SCH23390 was 2-3-fold more potent in inhibiting sucrose intake in 129P3/J and SJL/J mice relative to CD-1 mice. In contrast, only the highest equimolar 1600 nmol/kg dose of raclopride significantly reduced sucrose intake in the BALB/cJ, C3H/HeJ, C57BL/6J, C57BL/10J, DBA/2J, SJL/J and 129P3/J, but not the SWR/J and CD-1 strains. The present and previous data demonstrate specific and differential patterns of genetic variability in inhibition of sucrose intake by dopamine and opioid antagonists, suggesting that distinct neurochemical mechanisms control sucrose intake across different mouse strains. Show less

The aim of this work was the assessment of the clinical condition, birth weight, frequency of premature birth and incidence of intrauterine growth restriction (IUGR) of the newborns whose mothers were active and passive smokers. This was a prospective study conducted in a group of 147 newborns born during the years 2003-2004 in the Princess Anna Mazowiecka Hospital, Warsaw, and hospitalized in the Neonatal and Intensive Care Department of Warsaw Medical University. Based on a questionnaire identifying the exposure to tobacco smoke and cotinine concentration in the mother's urine, the newborns were assigned to three groups: the newborns whose mothers were active smokers, the newborns whose mothers were passive smokers and the newborns of non-smoking mothers. There were no statistically significant differences in the Apgar score assessment at the 1st and 5th minute between the three groups of the newborns. Acid-base balance parameters (pH, BE) were also similar. The birth weight of the newborns of mothers who were active smokers was 325g lower than the birth weight of the newborns of non-smoking mothers. This difference was statistically significant p = 0.033. Maternal smoking in pregnancy was associated with an increased risk of deficit in birth weight 2.6 (1.0-6.9, CI 95%). In the group of the newborns whose mothers were active smokers, the incidence of lower birth weight (< 2500g) was also statistically significantly higher p = 0.01. There were no statistically significant differences in the incidence of premature birth and intrauterine growth restriction (IUGR). Show less

DAP5/p97 (death-associated protein 5) is a member of the eukaryotic translation initiation factor 4G family. It functions as a scaffold protein promoting cap-independent translation of proteins. During apoptosis, DAP5/p97 is cleaved by caspases at position 792, yielding an 86-kDa C-terminal truncated isoform (DAP5/p86) that promotes translation of several mRNAs mediated by an internal ribosome entry site. In this study, we report the crystal structure of the C-terminal region of DAP5/p97 extending between amino acids 730 and 897. This structure consists of four HEAT-Repeats and is homologous to the C-terminal domain of eIF4GI, eIF5, and eIF2Bepsilon. Unlike the other proteins, DAP5/p97 lacks electron density in the loop connecting alpha3 and alpha4, which harbors the caspase cleavage site. Moreover, we observe fewer interactions between these two helices. Thus, previous mapping of this site by mutation analysis is confirmed here by the resolved structure of the DAP5/p97 C-terminus. In addition, we identified the position of two conserved aromatic and acidic boxes in the structure of the DAP5/p97 C-terminus. The acidic residues in the two aromatic and acidic boxes form a continuous negatively charged patch, which is suggested to make specific interactions with other proteins such as eIF2beta. The caspase cleavage of DAP5/p97 removes the subdomain carrying acidic residues in the AA-box motif, which may result in exposure of a hydrophobic surface. These intriguing structural differences between the two DAP5 isoforms suggest that they have different interaction partners and, subsequently, different functions. Show less

Series of novel broad excitation band phosphors M2 MgSis O7 : Eu, Dy(M = Ca, Sr) were prepared by a high temperature solid-state reaction method. The crystal structure of compound was characterized. And the effects of part substitution of alkaline-earth on crystal structure, photoluminescence spectra and luminescence properties were also investigated. It is found that the excitation band of silicate luminescent materials extend to visible region and they exhibit yellow, green and blue long after-glow luminescence after excited by ultraviolet or visible light. Ca MgSi O7 : Eu, Dy luminescent materials can be excited effectively under the 450-480 nm range and exhibit a strong emission at 536 nm, nicely combining with blue light emitted by InGaN chips to produce white light. This promises the silicate luminescent materials a potential yellow phosphor for white LED. Show less

Malpractice litigation is part of everyday clinical practice and is an area of which all dentists need to be aware. With proper forethought and planning, this vexing issue can be controlled and made less anxiety producing. The astute clinician must be as diligent in risk-reduction management and strategies as he/she is in practicing excellent dentistry. This article discusses various preventive measures that can be used to help mitigate malpractice claims and preclude them from developing. Good patient communication, rapport, and excellent documentation are the keys to minimizing, and possibly eliminating future lawsuits. Show less

Modern dental offices must be equipped to initiate prompt emergency care should the sudden need arise. With the elderly population in dental practices increasing, these emergencies will undoubtedly occur. This article discusses the basic emergency equipment the average dental office should possess to allow for an adequate initial response. It also discusses the policies and personnel needed for dealing with emergencies. Among the basic emergency equipment, an office should have syringes, an Ambu bag, a portable oxygen system, a sphygmomanometer (child and adult sizes), and an EKG/defibrillator. Emergency drugs that should be stocked include aromatic ammonia, aspirin, and nitroglycerine. The dentist should also develop a protocol and policy for his/her staff to follow when a medical emergency arise. Show less

Under the Health Insurance Portability and Accountability Act (HIPAA) of 1996, all dental offices are required to formulate policies and procedures to ensure and secure patient privacy of health information. This article reviews the essential points of such a plan and makes recommendations for implementation. Show less

Toxic tobacco smoke exposure to human organism is strictly related to progress of atherosclerosis changes. One of the mechanisms of these effects is a change of blood lipoprotein fraction concentrations. The concentrations of the lipid profile parameters (TCL, HDL, LDL, TG) and the chosen biomarkers (urine cotinine and 1-hydroxypyrene and blood carboxyhaemoglobine) were determined. It was studied whether the procedure of the groups determination (nonsmokers, passive and active smokers) affect the calculated average values of lipid profile parameters. The role of the applied biomarkers for the detection of the effects related to the tobacco smoke exposure is also discussed. It is concluded that there is no difference among the lipid profiles of passive smokers and nonsmokers. Cigarette smoking increases and lowers the TG and HDL concentrations, respectively. Urine cotinine seems to be the best indicator of tobacco smoke exposure among three chosen biomarkers. Study subjects were 300 male and female volunteers. Show less

Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in regulating the proliferation of spermatogonial stem cells (SSC). The signaling pathways mediating the function of GDNF in SSC remain unclear. This study was designed to determine whether GDNF signals via the Ras/ERK1/2 pathway in the C18-4 cells, a mouse SSC line. The identity of this cell line was confirmed by the expression of various markers for germ cells, proliferating spermatogonia, and SSC, including GCNA1, Vasa, Dazl, PCNA, Oct-4, GFRalpha1, Ret, and Plzf. Western blot analysis revealed that GDNF activated Ret tyrosine phosphorylation. All 3 isoforms of Shc were phosphorylated upon GDNF stimulation, and GDNF induced the binding of the phosphorylated Ret to Shc and Grb2 as indicated by immunoprecipitation and Western blotting. The active Ras was induced by GDNF, which further activated ERK1/2 phosphorylation. GDNF stimulated the phosphorylation of CREB-1, ATF-1, and CREM-1, and c-fos transcription. Notably, the increase in ERK1/2 phosphorylation, c-fos transcription, bromodeoxyuridine incorporation, and metaphase counts induced by GDNF, was completely blocked by pretreatment with PD98059, a specific inhibitor for MEK1, the upstream regulator of ERK1/2. GDNF stimulation eventually upregulated cyclin A and CDK2 expression. Together, these data suggest that GDNF induces CREB/ATF-1 family member phosphorylation and c-fos transcription via the Ras/ERK1/2 pathway to promote the proliferation of SSC. Unveiling GDNF signaling cascades in SSC has important implications in providing attractive targets for male contraception as well as for the regulation of stem cell renewal vs. differentiation. Show less

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators. Show less

Analysis of the three-dimensional structures of two closely related thermophilic and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Entamoeba histolytica (EhADH1) and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro275) at the center of the dimerization interface might be crucial for maintaining the thermal stability of TbADH. To assess the contribution of Pro275 to the thermal stability of the ADHs, we applied site-directed mutagenesis to replace Asp275 of EhADH1 with Pro (D275P-EhADH1) and conversely Pro275 of TbADH with Asp (P275D-TbADH). The results indicate that replacing Asp275 with Pro significantly enhances the thermal stability of EhADH1 (DeltaT(1/2) Show less

Ankylosing spondylitis (AS) is a systemic inflammatory disorder with frequent spinal axis symptoms. In this paper, the authors explored the spinal manifestations of AS and its characteristic anatomical lesions, radiological findings, and complications. They also offer a comprehensive report of the medical and surgical treatments with a focus on deformity correction. Show less

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future. Show less

Antizyme inhibitor (AzI) regulates cellular polyamine homeostasis by binding to the polyamine-induced protein, Antizyme (Az), with greater affinity than ornithine decarboxylase (ODC). AzI is highly homologous to ODC but is not enzymatically active. In order to understand these specific characteristics of AzI and its differences from ODC, we determined the 3D structure of mouse AzI to 2.05 A resolution. Both AzI and ODC crystallize as a dimer. However, fewer interactions at the dimer interface, a smaller buried surface area, and lack of symmetry of the interactions between residues from the two monomers in the AzI structure suggest that this dimeric structure is nonphysiological. In addition, the absence of residues and interactions required for pyridoxal 5'-phosphate (PLP) binding suggests that AzI does not bind PLP. Biochemical studies confirmed the lack of PLP binding and revealed that AzI exists as a monomer in solution while ODC is dimeric. Our findings that AzI exists as a monomer and is unable to bind PLP provide two independent explanations for its lack of enzymatic activity and suggest the basis for its enhanced affinity toward Az. Show less

Historians have assumed that alchemy had a close association with mining, but exactly how and why miners were interested in alchemy remains unclear. This paper argues that alchemical theory began to be synthesised with classical and Christian theories of the earth in mining books after 1500, and served an important practical function. The theory of metals that mining officials addressed spoke of mineral vapours (Witterungen) that left visible markings on the earth's surface. The prospector searched for mineral ore in part by studying these indications. Mineral vapours also explained the functioning of the dowsing rod, which prospectors applied to the discovery of ore. Historians of early chemistry and mining have claimed that mining had a modernising influence by stripping alchemy of its theoretical component, but this paper shows something quite to the contrary: mining officials may have been sceptical of the possibility of artificial transmutation, but they were interested in a theory of the earth that could translate into prospecting knowledge. Show less