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Background: There are no high-quality data to guide long-term mycophenolate mofetil (MMF) dosing in kidney transplant recipients (KTRs) to balance the long-term risks of allograft rejection with that of infections and malignancy. At our center, KTRs are managed with either a "preemptive" dose reduction strategy, where the MMF dose is reduced after the first year before the development of adverse events, or with a "reactive" dosing strategy, where they are maintained on the same MMF dose and only reduced if they develop an adverse event. We hypothesized that a preemptive MMF dosing strategy after the first year of transplantation is associated with decreased infections without increasing alloimmune complications., Methods: We conducted a retrospective cohort study of all KTRs receiving MMF from January 1, 2015, to December 31, 2020. The primary outcome was the incidence of infections requiring hospitalization., Results: One hundred forty-two KTRs met the inclusion criteria, of whom 44 (31%) were in the preemptive group and 98 (69%) were in the reactive group. The median follow-up was 4 y (interquartile range, 3.8-4.0). Multivariable analysis showed that a preemptive MMF dose reduction strategy was associated with a lower risk of infections requiring hospitalization (adjusted hazard ratio = 0.39; 95% confidence interval, 0.16-0.92). There was no difference in graft loss, rejection, or estimated glomerular filtration rate slope., Conclusions: Preemptive MMF dose reduction in KTRs may be an effective strategy to prevent infections without increasing the risk of allograft rejection. Randomized clinical trials are needed to confirm these findings., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Objectives: There is considerable variation in ordering practices for the initial laboratory evaluation of monoclonal gammopathies (MGs) despite clear society guidelines to include serum free light chain (sFLC) testing. We assessed the ability of a clinical decision support (CDS) alert to improve guideline compliance and analyzed its clinical impact., Methods: We designed and deployed a targeted CDS alert to educate and prompt providers to order an sFLC assay when ordering serum protein electrophoresis (SPEP) testing., Results: The alert was highly effective at increasing the co-ordering of SPEP and sFLC testing. Preimplementation, 62.8% of all SPEP evaluations included sFLC testing, while nearly 90% of evaluations included an sFLC assay postimplementation. In patients with no prior sFLC testing, analysis of sFLC orders prompted by the alert led to the determination that 28.9% (800/2,769) of these patients had an abnormal κ/λ ratio. In 452 of these patients, the sFLC assay provided the only laboratory evidence of a monoclonal protein. Moreover, within this population, there were numerous instances of new diagnoses of multiple myeloma and other MGs., Conclusions: The CDS alert increased compliance with society guidelines and improved the diagnostic evaluation of patients with suspected MGs., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

High expression of LIN28B is associated with aggressive malignancy and poor survival. Here, probing MYCN-amplified neuroblastoma as a model system, we showed that LIN28B expression was associated with enhanced cell migration in vitro and invasive and metastatic behavior in murine xenografts. Sequence analysis of the polyribosome fraction of LIN28B-expressing neuroblastoma cells revealed let-7-independent enrichment of transcripts encoding components of the translational and ribosomal apparatus and depletion of transcripts of neuronal developmental programs. We further observed that LIN28B utilizes both its cold shock and zinc finger RNA binding domains to preferentially interact with MYCN-induced transcripts of the ribosomal complex, enhancing their translation. These data demonstrated that LIN28B couples the MYCN-driven transcriptional program to enhanced ribosomal translation, thereby implicating LIN28B as a posttranscriptional driver of the metastatic phenotype.

The molecular mechanisms that govern the choreographed timing of organ development remain poorly understood. Our investigation of the role of the Lin28a and Lin28b paralogs during the developmental process of branching morphogenesis establishes that dysregulation of Lin28a/b leads to abnormal branching morphogenesis in the lung and other tissues. Additionally, we find that the Lin28 paralogs, which regulate post-transcriptional processing of both mRNAs and microRNAs (miRNAs), predominantly control mRNAs during the initial phases of lung organogenesis. Target mRNAs include Sox2, Sox9, and Etv5, which coordinate lung development and differentiation. Moreover, we find that functional interactions between Lin28a and Sox9 are capable of bypassing branching defects in Lin28a/b mutant lungs. Here, we identify Lin28a and Lin28b as regulators of early embryonic lung development, highlighting the importance of the timing of post-transcriptional regulation of both miRNAs and mRNAs at distinct stages of organogenesis., Competing Interests: Declaration of interests G.Q.D. holds equity in and receives consulting fees for service on the scientific advisory board of 28/7 Therapeutics, Inc. He holds a patent on technology related to LIN28, and J.K.O., A.V.Y., and G.Q.D. have filed a provisional patent related in part to the current work., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mechanobiology is focused on how the physical forces and mechanical properties of proteins, cells, and tissues contribute to physiology and disease. Although the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single-molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. We demonstrate the broad applicability and versatility through in vitro and in cellulo approaches. Overall, our methodology allows, for the first time (to our knowledge), ensemble biochemical and cell-based assays to be performed under force in high-throughput format. This approach substantially increases the availability of mechanobiology measurements., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The LIN28:pre-let-7:TUTase ternary complex regulates pluripotency and oncogenesis by controlling processing of the let-7 family of microRNAs. The complex oligouridylates the 3' ends of pre-let-7 molecules, leading to their degradation via the DIS3L2 exonuclease. Previous studies suggest that components of this complex are potential therapeutic targets in malignancies that aberrantly express LIN28. In this study we developed a functional epitope selection approach to identify nanobody inhibitors of the LIN28:pre-let-7:TUT4 complex. We demonstrate that one of the identified nanobodies, Nb-S2A4, targets the 106-residue LIN28:let-7 interaction (LLI) fragment of TUT4. Nb-S2A4 can effectively inhibit oligouridylation and monouridylation of pre-let-7g in vitro. Expressing Nb-S2A4 allows maturation of the let-7 species in cells expressing LIN28, highlighting the therapeutic potential of targeting the LLI fragment., Competing Interests: Competing interest statement: G.Q.D. declares that he is a cofounder, consultant, and member of the Scientific Advisory Board of Twentyeightseven Therapeutics, a company that designs therapeutics to proteins that modulate RNA. He also holds a patent related to targeting LIN28 to prevent and treat cancer. P.S. declares that he is a cofounder, consultant, and member of the Scientific Advisory Board of Twentyeightseven Therapeutics. He also holds a patent related to LIN28/let-7 structure, purification, and use in screening for therapeutics. A.C.K., H.W., P.S., and T.U.S. were coauthors on a Nature Communications article in 2016 (PMID:26947396)., (Copyright © 2020 the Author(s). Published by PNAS.)

In humans and in mice the formation of nephrons during embryonic development reaches completion near the end of gestation, after which no new nephrons are formed. The final nephron complement can vary 10-fold, with reduced nephron number predisposing individuals to hypertension, renal, and cardiovascular diseases in later life. While the heterochronic genes lin28 and let-7 are well-established regulators of developmental timing in invertebrates, their role in mammalian organogenesis is not fully understood. Here we report that the Lin28b/let-7 axis controls the duration of kidney development in mice. Suppression of let-7 miRNAs, directly or via the transient overexpression of LIN28B, can prolong nephrogenesis and enhance kidney function potentially via upregulation of the Igf2/H19 locus. In contrast, kidney-specific loss of Lin28b impairs renal development. Our study reveals mechanisms regulating persistence of nephrogenic mesenchyme and provides a rationale for therapies aimed at increasing nephron mass.

Objective: The objective of this study was to develop an evidence-based method with a set of metrics that could be used to assess an information search tool., Methods: This pilot study analyzed a two-group convenience sample of graduate nursing students and resident physicians. The intervention group received ten minutes of instruction on a familiar search tool (eSearcher). Each group was provided one prompt to search for clinical guidelines on a given topic within their scope of practice and asked to find the best result using only eSearcher (intervention group) or specifically excluding eSearcher (comparison group). Three measurements of search results were employed: time elapsed to complete the search, an accuracy score, and a participant-reported score of confidence in the result., Results: Forty-two students participated in this study (23 graduate nursing students and 19 resident physicians). The intervention group consisted of 22 participants (12 graduate nursing students and 10 resident physicians), and the comparison group consisted of 20 participants (11 graduate nursing students and 9 resident physicians). The intervention group had lower mean ranks in both accuracy and confidence compared to the comparison (not eSearcher) group, although these differences were not significant. However, the intervention (eSearcher) group had significantly longer search times compared to the comparison (not eSearcher) group., Discussion: These findings provided new insights into the performance of the search tool and how users felt about their search experience. The quantitative evidence gained from this study led directly to an informed decision to explore other options for search tools. The evidence-based methods and process developed in this pilot study will enable similar studies to test other student groups and other search tools, leading to better informed purchasing and instructional decisions., Competing Interests: DISCLOSURES None of the authors has any conflict of interest to disclose. This study was approved as exempt by the Mount Carmel Health System Office of Research Affairs on April 9, 2015, IRB protocol #150305-1.

LIN28 is an RNA-binding protein that regulates the maturation of the let-7 family of microRNAs by bipartite interactions with let-7 precursors through its two distinct cold shock and zinc-knuckle domains. Through inhibition of let-7 biogenesis, LIN28 functions as a pluripotency factor, as well as a driver of tumorigenesis. Here, we report a fluorescence polarization assay to identify small-molecule inhibitors for both domains of LIN28 involved in let-7 interactions. Of 101,017 compounds screened, six inhibit LIN28:let-7 binding and impair LIN28-mediated let-7 oligouridylation. Upon further characterization, we demonstrate that the LIN28 inhibitor TPEN destabilizes the zinc-knuckle domain of LIN28, while LI71 binds the cold shock domain to suppress LIN28's activity against let-7 in leukemia cells and embryonic stem cells. Our results demonstrate selective pharmacologic inhibition of individual domains of LIN28 and provide a foundation for therapeutic inhibition of the let-7 biogenesis pathway in LIN28-driven diseases., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Signalling and post-transcriptional gene control are both critical for the regulation of pluripotency, yet how they are integrated to influence cell identity remains poorly understood. LIN28 (also known as LIN28A), a highly conserved RNA-binding protein, has emerged as a central post-transcriptional regulator of cell fate through blockade of let-7 microRNA biogenesis and direct modulation of mRNA translation. Here we show that LIN28 is phosphorylated by MAPK/ERK in pluripotent stem cells, which increases its levels via post-translational stabilization. LIN28 phosphorylation had little impact on let-7 but enhanced the effect of LIN28 on its direct mRNA targets, revealing a mechanism that uncouples LIN28's let-7-dependent and -independent activities. We have linked this mechanism to the induction of pluripotency by somatic cell reprogramming and the transition from naive to primed pluripotency. Collectively, our findings indicate that MAPK/ERK directly impacts LIN28, defining an axis that connects signalling, post-transcriptional gene control, and cell fate regulation.

For appropriate development, tissue and organ system morphogenesis and maturation must occur in synchrony with the overall developmental requirements of the host. Mistiming of such developmental events often results in disease. The hematopoietic system matures from the fetal state, characterized by robust erythrocytic output that supports prenatal growth in the hypoxic intrauterine environment, to the postnatal state wherein granulocytes predominate to provide innate immunity. Regulation of the developmental timing of these myeloerythroid states is not well understood. In this study, we find that expression of the heterochronic factor Lin28b decreases in common myeloid progenitors during hematopoietic maturation to adulthood in mice. This decrease in Lin28b coincides with accumulation of mature let-7 microRNAs, whose biogenesis is regulated by Lin28 proteins. We find that inhibition of let-7 in the adult hematopoietic system recapitulates fetal erythroid-dominant hematopoiesis. Conversely, deletion of Lin28b or ectopic activation of let-7 microRNAs in the fetal state induces a shift toward adult-like myeloid-dominant output. Furthermore, we identify Hmga2 as an effector of this genetic switch. These studies provide the first detailed analysis of the roles of endogenous Lin28b and let-7 in the timing of hematopoietic states during development., (© 2016 Rowe et al.)

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis., Competing Interests: GQD holds options and intellectual property relating to 28/7 Therapeutics, a company seeking to develop inhibitors of the LIN28/let-7 pathway.

MicroRNAs (miRNAs) are important regulators and potential therapeutic targets of metabolic disease. In this study we show by in vivo administration of locked nucleic acid (LNA) inhibitors that suppression of endogenous miR-29 lowers plasma cholesterol levels by ~40%, commensurate with the effect of statins, and reduces fatty acid content in the liver by ~20%. Whole transcriptome sequencing of the liver reveals 883 genes dysregulated (612 down, 271 up) by inhibition of miR-29. The set of 612 down-regulated genes are most significantly over-represented in lipid synthesis pathways. Among the up-regulated genes are the anti-lipogenic deacetylase sirtuin 1 (Sirt1) and the anti-lipogenic transcription factor aryl hydrocarbon receptor (Ahr), the latter of which we demonstrate is a direct target of miR-29. In vitro radiolabeled acetate incorporation assays confirm that pharmacologic inhibition of miR-29 significantly reduces de novo cholesterol and fatty acid synthesis. Our findings indicate that miR-29 controls hepatic lipogenic programs, likely in part through regulation of Ahr and Sirt1, and therefore may represent a candidate therapeutic target for metabolic disorders such as dyslipidemia.

Mast cells (MCs) are critical components of the innate immune system and important for host defense, allergy, autoimmunity, tissue regeneration and tumor progression. Dysregulated MC development leads to systemic mastocytosis (SM), a clinically variable but often devastating family of hematologic disorders. Here we report that induced expression of Lin28, a heterochronic gene and pluripotency factor implicated in driving a fetal hematopoietic program, caused MC accumulation in adult mice in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in LIN28B-expressing hematopoietic progenitors, with increased levels of LIN28B in common myeloid and basophil-MC progenitors altering gene expression patterns to favor cell fate choices that enhanced MC specification. In addition, LIN28B-induced MCs appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of MC terminal differentiation in the context of LIN28B upregulation. Finally, interrogation of human MC leukemia samples revealed upregulation of LIN28B in abnormal MCs from patients with SM. This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in MC disease.

Unlabelled: Cellular and plasma lipid levels are tightly controlled by complex gene regulatory mechanisms. Elevated plasma lipid content, or hyperlipidemia, is a significant risk factor for cardiovascular morbidity and mortality. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and have emerged as important modulators of lipid homeostasis, but the extent of their role has not been systematically investigated. In this study we performed high-throughput small RNA sequencing and detected ≈ 150 miRNAs in mouse liver. We then employed an unbiased, in silico strategy to identify miRNA regulatory hubs in lipid metabolism, and miR-27b was identified as the strongest such hub in human and mouse liver. In addition, hepatic miR-27b levels were determined to be sensitive to plasma hyperlipidemia, as evidenced by its ≈ 3-fold up-regulation in the liver of mice on a high-fat diet (42% calories from fat). Further, we showed in a human hepatocyte cell line (Huh7) that miR-27b regulates the expression (messenger RNA [mRNA] and protein) of several key lipid-metabolism genes, including Angptl3 and Gpam. Finally, we demonstrated that hepatic miR-27b and its target genes are inversely altered in a mouse model of dyslipidemia and atherosclerosis., Conclusion: miR-27b is responsive to lipid levels and controls multiple genes critical to dyslipidemia., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Identifying cis-regulatory elements is important to understanding how human pancreatic islets modulate gene expression in physiologic or pathophysiologic (e.g., diabetic) conditions. We conducted genome-wide analysis of DNase I hypersensitive sites, histone H3 lysine methylation modifications (K4me1, K4me3, K79me2), and CCCTC factor (CTCF) binding in human islets. This identified ∼18,000 putative promoters (several hundred unannotated and islet-active). Surprisingly, active promoter modifications were absent at genes encoding islet-specific hormones, suggesting a distinct regulatory mechanism. Of 34,039 distal (nonpromoter) regulatory elements, 47% are islet unique and 22% are CTCF bound. In the 18 type 2 diabetes (T2D)-associated loci, we identified 118 putative regulatory elements and confirmed enhancer activity for 12 of 33 tested. Among six regulatory elements harboring T2D-associated variants, two exhibit significant allele-specific differences in activity. These findings present a global snapshot of the human islet epigenome and should provide functional context for noncoding variants emerging from genetic studies of T2D and other islet disorders., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The conversion of the cellular form of the prion protein (PrP(C)) to an altered disease state, generally denoted as scrapie isoform (PrP(Sc)), appears to be a crucial molecular event in prion diseases. The details of this conformational transition are not fully understood, but it is perceived that they are associated with misfolding of PrP or its incapacity to maintain the native fold during its cell cycle. Here we present a tryptophan mutant of PrP (F198W), which has enhanced fluorescence sensitivity to unfolding/refolding transitions. Equilibrium folding was studied by circular dichroism and fluorescence. Pressure-jump experiments were successfully applied to reveal rapid submillisecond folding events of PrP at temperatures not accessed before.

We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s. Between pCa 8-5.4 and at troponin C concentrations of 8-28 muM, the slow relaxation times were invariant, indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps, respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium-sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200-300 muM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo.

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release.

The conserved switch 1 loop of P-loop NTPases is implicated as a central element that transmits information between the nucleotide-binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G-proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods. We found that in the presence of MgADP, two states of switch 1 exist in dynamic equilibrium. Actin binding shifts the equilibrium towards one of the MgADP states, whereas ATP strongly favors the other. In the light of electron cryo-microscopic and X-ray crystallographic results, these findings lead to a specific structural model in which the equilibrium constant between the two states of switch 1 is coupled to the strength of the actin-myosin interaction. This has implications for the enzymatic mechanism of G-proteins and possibly P-loop NTPases in general.

Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C. neoformans is confronted with and responds to dramatic variations in CO2 concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO2) to infected hosts (5% CO2) is the induction of capsule biosynthesis. In cells, CO2 is hydrated to bicarbonate in a spontaneous reaction that is accelerated by carbonic anhydrases. Here we show that C. neoformans contains two beta-class carbonic anhydrases, Can1 and Can2. We further demonstrate that CAN2, but not CAN1, is abundantly expressed and essential for the growth of C. neoformans in its natural environment, where CO2 concentrations are limiting. Structural studies reveal that Can2 forms a homodimer in solution. Our data reveal Can2 to be the main carbonic anhydrase and suggest a physiological role for bicarbonate during C. neoformans growth. Bicarbonate directly activates the C. neoformans Cac1 adenylyl cyclase required for capsule synthesis. We show that this specific activation is optimal at physiological pH.

Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP(-) anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP(-*) anion. Prolonged continuous wave illumination at the peak of YFP(-) absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP(-) in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP(-) within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.

PlasmoDB (http://PlasmoDB.org) is the official database of the Plasmodium falciparum genome sequencing consortium. This resource incorporates the recently completed P. falciparum genome sequence and annotation, as well as draft sequence and annotation emerging from other Plasmodium sequencing projects. PlasmoDB currently houses information from five parasite species and provides tools for intra- and inter-species comparisons. Sequence information is integrated with other genomic-scale data emerging from the Plasmodium research community, including gene expression analysis from EST, SAGE and microarray projects and proteomics studies. The relational schema used to build PlasmoDB, GUS (Genomics Unified Schema) employs a highly structured format to accommodate the diverse data types generated by sequence and expression projects. A variety of tools allow researchers to formulate complex, biologically-based, queries of the database. A stand-alone version of the database is also available on CD-ROM (P. falciparum GenePlot), facilitating access to the data in situations where internet access is difficult (e.g. by malaria researchers working in the field). The goal of PlasmoDB is to facilitate utilization of the vast quantities of genomic-scale data produced by the global malaria research community. The software used to develop PlasmoDB has been used to create a second Apicomplexan parasite genome database, ToxoDB (http://ToxoDB.org).

Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50 microl) pressure-jump apparatus, powered by a piezoelectric actuator, based on the original design of Clegg and Maxfield [(1976) Rev. Sci. Instrum. 47, 1383-1393]. This equipment can apply pressure changes of +/-20 MPa (maximally) in time periods as short as 80 micros and follow the resulting change in fluorescence signals. The system is relatively simple to use with fast (approx. 1 min) exchange of samples. In the present study, we show that this system can perturb the binding of 2'(3')-O-(N-methylanthraniloyl)-ADP to myosin subfragment-1(S1) from skeletal and smooth muscles. The kinetic data are consistent with previous work, and in addition show that, although 2'(3')-O-(N-methylanthraniloyl)-ADP binds with a similar affinity to both proteins, the increase in molar volume for the skeletal-muscle S1 binding to ADP is half of that for the smooth-muscle protein. This high-volume change for smooth-muscle S1 may be related to the ability of ADP to induce a 23 degrees tilt in the tail of S1 bound to actin.

The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin II motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximately 1000 s(-1) at 20 degrees C). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF(4) state is largely closed. At 20 degrees C the open-closed equilibria for the AMP.PNP and ADP.BeF(x) complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step.

In this study we have evaluated stress-inducible hsp90 mRNA accumulation as a potential molecular biomarker in Xenopus laevis. In order to obtain a probe for Northern blot analysis we employed a PCR-based approach using degenerate primers for the amplification and cloning of an hsp90 gene sequence from Xenopus laevis. The deduced amino acid sequence is 102 amino acids in length and exhibited the highest degree of identity with zebrafish and human hsp90 beta genes. Furthermore, the putative intron and exon boundaries of this fragment are the same as hsp90 beta in chicken, mouse and human, indicating that the fragment represents a Xenopus hsp90 beta-like gene. Northern blot analyses revealed that this gene was constitutively expressed in cultured A6 cells. While heat shock and sodium arsenite exposure resulted in the increased accumulation of hsp90 mRNA in A6 cells, treatment with cadmium chloride and zinc chloride did not. Also, exposure of A6 cells to concurrent heat shock and sodium arsenite produced a mild synergistic response with respect to hsp90 mRNA levels in contrast to hsp70 mRNA levels which displayed a strong synergistic effect. Finally, hsp90 mRNA was detected constitutively throughout early embryogenesis but was heat-inducible only in late blastula and later stages of development. Given the normal abundance and limited stress-induced accumulation of hsp90 mRNA, it may not have a great deal of potential as a molecular biomarker compared to hsp70 and hsp30 mRNA. However, it may be useful in conjunction with other stress protein mRNAs to establish a set of biomarker profiles to characterize the cellular response to a stressful or toxic agent.

Hsp47 is a major stress-inducible protein that is localized to the endoplasmic reticulum of avian and mammalian cells and is thought to act as a molecular chaperone specific for the processing of procollagen. Although hsp47 is coordinately expressed together with several collagen types, and vertebrate embryos are known to express collagen genes in complex spatial and temporal patterns, limited information is available regarding the function or regulation of hsp47 during early embryonic development. We have initiated an examination of hsp47 in the zebrafish, Danio rerio, which offers a number of features that make it attractive as a model developmental system with which to examine the expression and function of hsp47. A polymerase chain reaction (PCR)-based cloning strategy was used to isolate a hsp47 cDNA from an embryonic zebrafish cDNA library. The deduced translation product of the cDNA is a 404-amino-acid polypeptide that is 72% identical to chicken, 64% identical to mouse and rat, and 69% identical to human hsp47. The protein contains a typical hydrophobic signal sequence, an RDEL endoplasmic reticulum retention signal, and a serine protease inhibitor signature sequence, all of which are characteristic of hsp47 in higher vertebrates. Thus, it is likely that hsp47 in zebrafish is also localized to the endoplasmic reticulum and may play a similar role to its counterpart in higher vertebrates. Northern blot analysis revealed that the hsp47 gene is expressed at relatively low levels in embryos during normal development but is strongly induced following exposure to heat shock at the gastrula, midsomitogenesis, 2-day, and 3-day larval stages. The level of induction was much higher than has previously been reported in chicken and mouse cells.