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Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds β-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl β-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We present the first measurement of elliptic (v(2)) and triangular (v(3)) flow in high-multiplicity (3)He+Au collisions at √(s(NN))=200  GeV. Two-particle correlations, where the particles have a large separation in pseudorapidity, are compared in (3)He+Au and in p+p collisions and indicate that collective effects dominate the second and third Fourier components for the correlations observed in the (3)He+Au system. The collective behavior is quantified in terms of elliptic v(2) and triangular v(3) anisotropy coefficients measured with respect to their corresponding event planes. The v(2) values are comparable to those previously measured in d+Au collisions at the same nucleon-nucleon center-of-mass energy. Comparisons with various theoretical predictions are made, including to models where the hot spots created by the impact of the three (3)He nucleons on the Au nucleus expand hydrodynamically to generate the triangular flow. The agreement of these models with data may indicate the formation of low-viscosity quark-gluon plasma even in these small collision systems.

The CombiMatrix antibody microarray is a versatile, sensitive detection platform based on the generation and transduction of electrochemical signals following antigen binding to surface antibodies. The sensor chip described herein is comprised of microelectrodes coupled to an adjacent bio-friendly matrix coated with antibodies to the biological pathogens Yersinia pestis and Bacillus anthracis, and the bacterial toxin staphylococcal enterotoxin B (SEB). Using this system, we were able to detect SEB and inactivated Y. pestis individually as well as in two-plex assays at concentrations as low as 5 pg/mL and 10(6) CFU/mL, respectively. We also introduce super avidin-biotin system (SABS) as a viable and effective means to enhance assay signal responses and lower detection limits. Together these technologies represent substantial advances in point-of-care and point-of-use detection applications.

The CombiMatrix microarray with 12,544 electrodes supports in situ electrochemical synthesis of user-defined DNA probes. As an alternative, we immobilized commercially synthesized DNA probes on individual electrodes coated with electropolymerized polypyrrole (Ppy). Hybridization was measured using a biotinylated target oligonucleotide and either Cy5-streptavidin and fluorescence detection or horseradish peroxidase-streptavidin and enzyme-enhanced electrochemical detection. Detection efficiencies were optimized by varying the deposition of the Ppy, the terminal groups on the DNA probes, and other factors that impacted fluorescence quenching and electrical conductivity. Optimized results were compared against those obtained using a microarray with the same DNA sequences synthesized in situ. Immobilized probes produced higher fluorescence signals, possibly by providing a greater stand off between the Cy5 on the target oligonucleotide and the quenching effects of the Ppy and the platinum electrode.

DNA microarrays are an important tool with a variety of applications in gene expression studies, genotyping, pharmacogenomics, pathogen classification, drug discovery, sequencing and molecular diagnostics. They are having a strong impact in medical diagnostics for cancer, toxicology and infectious disease applications. A series of papers have been published describing DNA biochips as alternative to conventional microarray platforms to facilitate and ameliorate the signal readout. In this review, we will consider the different methods proposed for biochip construction, focusing on electrochemical detection of DNA. We also introduce a novel single-stranded DNA platform performing high-throughput SNP detection and gene expression profiling.

Objective: Our qualitative study explored prenatal care providers' methods for identifying and counseling pregnant women to reduce or stop smoking, alcohol use, illicit drug use, and the risk of domestic violence., Methods: We conducted six focus groups (five with OB/Gyn physicians, one with nurse practitioners and certified nurse midwives), total N=49, using open-ended questions. Investigators analyzed transcripts to identify and describe themes., Results: Three major themes emerged: (1) specific risk-prevention tactics or strategies exist that are useful during pregnancy; (2) some providers address patients' isolation or depression; and (3) providers can adopt a policy of "just chipping away" at risks. Specific tactics included normalizing risk prevention, using specific assessment techniques and counseling strategies, employing a patient-centered style of smoking reduction, and involving the family., Conclusions: Providers generally agreed that addressing behavioral risks in pregnant patients is challenging. Patient-centered techniques and awareness of patients' social contexts help patients disclose and discuss risks., Practice Implications: Brief but routine assessment and risk reduction messages require little time of the provider, but can make a big difference to the patient, who may make changes later.

Background: Dentists have a unique opportunity to address the problem of domestic violence (DV). The authors tested the effectiveness of a tutorial designed to educate dentists in identifying and responding to DV., Methods: The authors developed a brief interactive multimedia tutorial for dentists and recruited practicing dentists (N = 174) for a randomized, controlled trial. A 24-question instrument assessed participants' knowledge, attitudes and practice behaviors regarding DV at two time points. The control group took the tutorial before completing a posttest. The authors also administered a 20-question empathy scale., Results: The experimental group demonstrated significantly greater improvement in scores on most items, including knowledge, attitudes and behaviors, relative to control subjects (P < .01). Empathy scores did not show significant correlation with change scores on the DV assessment instrument., Conclusions: The tutorial is effective in helping dentists learn how to identify and help patients who are experiencing abuse., Clinical Implications: Broad dissemination of the tutorial about DV would introduce dentists to simple strategies for responding to patients who experience DV.

A DNA microarray with 12,000 features was integrated with a microfluidic cartridge to automate the fluidic handling steps required to carry out a gene expression study of the human leukemia cell line (K562). The fully integrated microfluidic device consists of microfluidic pumps/mixers, fluid channels, reagent chambers, and a DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated into the cartridge to provide pumping of liquid solutions. The device was completely self-contained: no external pressure sources, fluid storage, mechanical pumps, mixers, or valves were necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Fluidic experiments were performed to study the on-chip washing efficiency and uniformity. A single-color transcriptional analysis of K562 cells with a series of calibration controls (spiked-in controls) to characterize this new platform with regard to sensitivity, specificity, and dynamic range was performed. The device detected sample RNAs with a concentration as low as 0.375 pM. Experiment also showed that the performance of the integrated microfluidic device is comparable with the conventional hybridization chambers with manual operations, indicating that the on-chip fluidic handling (washing and reaction) is highly efficient and can be automated with no loss of performance. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps in genomic analysis.

Background: The purpose of this study was to evaluate the impact of a brief, interactive multimedia tutorial designed to prepare dentists to recognize and respond to domestic violence., Methods: The authors randomly assigned dentists and dental students to one of three groups: a control group or one of two experimental groups in a modified Solomon four-group design., Results: One hundred sixty-one dental students and 13 dentists completed the multimedia tutorial. At the posttest, subjects in both experimental groups demonstrated significantly better scores than did subjects in the control group on most items. The two experimental groups (pretest and posttest, posttest only) did not differ significantly from each other., Conclusion: An engaging, interactive tutorial presenting a simplified model for ways in which dental professionals can recognize and respond to domestic violence significantly improved dental students' knowledge of, and attitudes toward, the topic., Clinical Implications: Clinicians may improve the care they provide to patients by accessing this brief tutorial and following the lessons contained in it.

Objectives: To determine whether a diverse group of people would predominantly choose a white male physician regardless of group member's sex and ethnicity when given a choice among 6 actor-portrayed video doctors (males and females of Latino, European, and African descent) and whether further exposure would alter initial selections., Study Design: Participants selected a video doctor after viewing a brief introduction and again after viewing the delivery of a prevention message., Population: Three hundred ninety-five participants recruited at a shopping mall in the San Francisco Bay Area (61% female, 39% male; 30% Asian American, 29% European American, 26% Latino, 8% African American, and 7% other)., Outcomes Measured: Initial and final video doctor selections; ratings of video doctors on interpersonal qualities., Results: Most participants (85% of females and 63% of males) initially chose a female video doctor (P<.001) and even more did so at final selection. Approximately half initially chose a same-race video doctor (66% of European Americans, 51% of Latinos, and 50% of African Americans), but fewer did so at final selection (56% of European Americans, 44% of Latinos, and 52% of African Americans). In addition, at final selection 57% of Asian Americans and other-ethnicity participants chose a non-European American video doctor., Conclusions: Many healthcare consumers will accept physicians of both sexes and of different races. After observing the video doctors demonstrate a professional and warm affect, participants became even more receptive to choosing a video doctor of a different race. Video doctor technology holds promise for increasing our understanding of patients' preferences.

Background: Physicians routinely confront patient risk behaviors once considered private, including tobacco use, alcohol abuse, and HIV/STD-risk behavior. We compared physicians' behaviors and beliefs on screening and intervention for domestic violence with each other risk., Methods: Survey of nationwide, random sample of 610 primary care physicians from the American Medical Association Physician Masterfile., Results: Fewer primary care physicians screened for domestic violence than for other risks (p <0.001); once domestic violence was identified, however, physicians intervened with equal or greater frequency than for other risks. Fewer believed that they knew how to screen or intervene for domestic violence compared with other risks, and significantly fewer believed that domestic violence interventions were successful compared with interventions for tobacco and HIV/STD risks (Bonferroni adjusted p<0.001)., Conclusions: Lower domestic violence screening rates may reflect physicians' beliefs that they do not know how to screen or intervene, and that interventions are less successful for domestic violence than for other risks. We may improve screening rates by educating physicians that a simplified role, as for other risks, can be effective for domestic violence.

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

Squalene synthase catalyzes the biosynthesis of squalene, a key cholesterol precursor, through a reductive dimerization of two farnesyl diphosphate (FPP) molecules. The reaction is unique when compared with those of other FPP-utilizing enzymes and proceeds in two distinct steps, both of which involve the formation of carbocationic reaction intermediates. Because FPP is located at the final branch point in the isoprenoid biosynthesis pathway, its conversion to squalene through the action of squalene synthase represents the first committed step in the formation of cholesterol, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structures of recombinant human squalene synthase complexed with several different inhibitors. The structure shows that SQS is folded as a single domain, with a large channel in the middle of one face. The active sites of the two half-reactions catalyzed by the enzyme are located in the central channel, which is lined on both sides by conserved aspartate and arginine residues, which are known from mutagenesis experiments to be involved in FPP binding. One end of this channel is exposed to solvent, whereas the other end leads to a completely enclosed pocket surrounded by conserved hydrophobic residues. These observations, along with mutagenesis data identifying residues that affect substrate binding and activity, suggest that two molecules of FPP bind at one end of the channel, where the active center of the first half-reaction is located, and then the stable reaction intermediate moves into the deep pocket, where it is sequestered from solvent and the second half-reaction occurs. Five alpha helices surrounding the active center are structurally homologous to the active core in the three other isoprenoid biosynthetic enzymes whose crystal structures are known, even though there is no detectable sequence homology.

Background: Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate for glycolysis. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target., Results: The binding site in human liver glycogen phosphorylase (HLGP) for a class of promising antidiabetic agents was identified crystallographically. The site is novel and functions allosterically by stabilizing the inactive conformation of HLGP. The initial view of the complex revealed key structural information and inspired the design of a new class of inhibitors which bind with nanomolar affinity and whose crystal structure is also described., Conclusions: We have identified the binding site of a new class of allosteric HLGP inhibitors. The crystal structure revealed the details of inhibitor binding, led to the design of a new class of compounds, and should accelerate efforts to develop therapeutically relevant molecules for the treatment of diabetes.

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-A resolution and has a characteristic GST fold (Protein Data Bank entry code ). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.

The type RIIbeta regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain. Knockout mice suggest a role for RIIbeta in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity. A truncated version of the RIalpha subunit has been used in a crystallographic study and was used here to design an analogous RIIbeta construct. Despite substantial screening, conditions were not found for the crystallization of the truncated RIIbeta subunit. However, limited proteolysis of the full-length RIIbeta subunit identified boundaries of the 'hinge' region and a fragment containing the two cAMP-binding domains which did crystallize. A recombinant version of the fragment was expressed and crystallized for X-ray diffraction studies. The crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 91.6, b = 105.9, c = 85.8 A, and diffracted to at least 2.3 A.

Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we determined the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a. During activation, forty residues of the catalytic site undergo order/disorder transitions, changes in secondary structure, or packing to reorganize the catalytic site for substrate binding and catalysis. Knowing the inactive and active conformations of the liver enzyme and how each differs from its counterpart in muscle phosphorylase provides the basis for designing inhibitors that bind preferentially to the inactive conformation of the liver isozyme.

8 h) and high steady-state levels of protein accumulation, while the H2 intrabodies had a half-life of 2 h and less protein at steady state. These results suggest that the choice of sFv as an intrabody depends critically on the intracellular sFv protein having an extended half-life and elevated steady-state level. Thus, extended half-life must be considered together with sFv antibody specificity and affinity when choosing an optimal sFv intrabody for functional studies of cellular proteins.

Through the use of empirical and computational methods, phosphinate-based inhibitors of MMP-1 and MMP-13 that bind into the S2 pocket of these enzymes were designed. The synthesis and testing of 2 suggested that binding was occurring as hypothesized. Structure determination of a co-crystal of 2 bound to the catalytic domain of MMP-1 confirmed the binding mode. Substituents binding into S2, S1', S2' and S3', were optimized yielding compounds with low double-digit nM IC50's against these enzymes.

An inhibitor of human liver glycogen phosphorylase a (HLGPa) has been identified and characterized in vitro and in vivo. This substance, [R-(R*, S*)]-5-chloro-N-[3-(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)pr opyl]-1H-indole-2-carboxamide (CP-91149), inhibited HLGPa with an IC50 of 0.13 microM in the presence of 7.5 mM glucose. CP-91149 resembles caffeine, a known allosteric phosphorylase inhibitor, in that it is 5- to 10-fold less potent in the absence of glucose. Further analysis, however, suggests that CP-91149 and caffeine are kinetically distinct. Functionally, CP-91149 inhibited glucagon-stimulated glycogenolysis in isolated rat hepatocytes (P < 0.05 at 10-100 microM) and in primary human hepatocytes (2.1 microM IC50). In vivo, oral administration of CP-91149 to diabetic ob/ob mice at 25-50 mg/kg resulted in rapid (3 h) glucose lowering by 100-120 mg/dl (P < 0.001) without producing hypoglycemia. Further, CP-91149 treatment did not lower glucose levels in normoglycemic, nondiabetic mice. In ob/ob mice pretreated with 14C-glucose to label liver glycogen, CP-91149 administration reduced 14C-glycogen breakdown, confirming that glucose lowering resulted from inhibition of glycogenolysis in vivo. These findings support the use of CP-91149 in investigating glycogenolytic versus gluconeogenic flux in hepatic glucose production, and they demonstrate that glycogenolysis inhibitors may be useful in the treatment of type 2 diabetes.

Squalene synthase catalyzes the first committed step in cholesterol biosynthesis and thus is important as a potential target for therapeutic intervention. In order to determine the important functional domains of the protein, the amino and carboxyl terminal regions thought to be involved in membrane association of the enzyme were removed genetically. The 30 N-terminal amino acids were deleted with no apparent effect on activity. Additional deletion of 81 or 97 amino acids from the C-terminus completely ablated activity. However, a protein with a C-terminal deletion of 47 amino acids retained full activity. The latter enzyme was readily overexpressed in Escherichia coli and purified to homogeneity. The pure, doubly truncated enzyme exhibited a specific activity similar to that reported for the protease-solubilized rat liver enzyme, had a KM for farnesyl diphosphate similar to that observed for native enzyme, and was inhibited by anionic compounds to the same degree as native enzyme. Using the vapor diffusion method, the protein was crystallized as an enzyme-inhibitor complex, yielding orthorhombic crystals which diffracted to 2.2 A., (Copyright 1998 Academic Press.)

Apoptosis plays an important role in regulating development and homeostasis of the immune system, yet the elements of the signaling pathways that control cell death have not been well defined. When expressed in Jurkat T cells, an activated form of the small GTPase Cdc42 induces cell death exhibiting the characteristics of apoptosis. The death response induced by Cdc42 is mediated by activation of a protein kinase cascade leading to stimulation of c-Jun amino terminal kinase (JNK). Apoptosis initiated by Cdc42 is inhibited by dominant negative components of the JNK cascade and by reagents that block activity of the ICE protease (caspase) family, suggesting that stimulation of the JNK kinase cascade can lead to caspase activation. The sequence of morphological events observed typically in apoptotic cells is modified in the presence of activated Cdc42, suggesting that this GTPase may account for some aspects of cytoskeletal regulation during the apoptotic program. These data suggest a means through which the biochemical and morphological events occurring during apoptosis may be coordinately regulated.

N,N-dimethylglycylamido (DMG) derivatives of 6-demethyl-6-deoxytetracycline and doxycycline bind 5-fold more effectively than tetracycline to the tetracycline high-affinity binding site on the Escherichia coli 70S ribosome, which correlates with a 10-fold increase in potency for inhibition of E. coli cell-free translation. The potencies of DMG-doxycycline and DMG-6-demethyl-6-deoxytetracycline were unaffected by the ribosomal tetracycline resistance factors Tet(M) and Tet(O) in cell-free translation assays and whole-cell bioassays with a conditional Tet(M)-producing E. coli strain.

GTPases of the Rho family regulate many aspects of inflammatory cell activity, including motility, formation of toxic oxygen metabolites, and generation of proinflammatory cytokines. Defective regulation of such signaling pathways leads to a variety of acute and chronic inflammatory disorders, although the mechanisms by which this occurs have not been well defined. We describe in this work specific proteolytic cleavage of D4 GDI, a critical regulator of Rho GTPase activity in inflammatory leukocytes, by IL-1 beta-converting enzyme (ICE). Cleavage of D4 GDI by ICE occurs at Asp55, leading to the formation of the truncated D4 that is unable to effectively bind and regulate GTPases of the Rho family. Our data suggest that activation of ICE protease(s) at inflammatory sites leads to defective Rho GTPase regulation. Release of these critical regulatory proteins may contribute substantially to the inflammatory response at these sites, exacerbating and perpetuating the resulting tissue damage.

To study the role interleukin (IL)-5 may play in altering airway function in asthma, we have produced recombinant protein for exogenous administration to guinea pigs. The guinea pig IL-5 (gpIL-5) cDNA was cloned by polymerase chain reaction (PCR) amplification of guinea pig spleen RNA and expressed as a secretion product from recombinant baculovirus-infected Sf9 insect cell cultures. The protein was purified to homogeneity by a four-step procedure that included immunoaffinity chromatography using polyclonal antipeptide antibodies against a region of the mature secreted cytokine. The cytokine was properly processed after the signal sequence by the Sf9 cells, was glycosylated with terminal mannose-containing oligosaccharide, and had proper disulfide-linked dimer structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation was active in vitro and in vivo as determined by its ability to prime human basophils to release leukotriene C4 in the presence of C5a and to induce airway eosinophilia in naive guinea pigs.

Apoptosis (programmed cell death) is a fundamental process for normal development of multicellular organisms, and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis, ICE/CED-3 family cysteine proteases have been implicated directly in apoptosis, but relatively few of the substrates through which their action is mediated have been identified. Here we report that D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, is a substrate of the apoptosis protease CPP32/Yama/Apopain. D4-GDI was rapidly truncated to a 23-kDa fragment in Jurkat cells with kinetics that parallel the onset of apoptosis following Fas cross-linking with agonistic antibody or treatment with staurosporine. Fas- and staurosporine-induced apoptosis as well as cleavage of D4-GDI were inhibited by the ICE inhibitor, YVAD-cmk. D4-GDI was cleaved in vitro by recombinant CPP32 expressed in Escherichia coli to form a 23-kDa fragment. The CPP32-mediated cleavage of D4-GDI was completely inhibited by 1 microM DEVD-CHO, a reported selective inhibitor of CPP32. In contrast, the ICE-selective inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit CPP32-mediated D4-GDI cleavage at concentrations up to 50 microM. N-terminal sequencing of the 23-kDa D4-GDI fragment demonstrated that D4-GDI was cleaved between Asp19 and Ser20 of the poly(ADP-ribose) polymerase-like cleavage sequence DELD19S. These data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by CPP32-mediated cleavage of the GDI protein.

Acutely sprained ankles represent a frequent and common injury among active duty troops in training, and are a significant source of morbidity with respect to days lost to training. Swelling in the form of periarticular edema limits motion, causes pain, prevents wearing of normal foot gear, and slows the healing process. Reduction of edema was attempted in acutely sprained ankles by the use of pulsed electromagnetic energy (Diapulse). In a randomized, prospective, double blind study of 50 grade I and II (no gross instability) sprained ankles, a statistically significant (p < 0.01) decrease in edema was noted following one treatment with Diapulse. The application of this modality in acutely sprained ankles could result in significant decreases in time lost to military training.

Human plasminogen activator inhibitor-1 (PAI-1) is the fast-acting inhibitor of tissue plasminogen activator and urokinase and is a member of the serpin family of protease inhibitors. Serpins normally form complexes with their target proteases that dissociate very slowly as cleaved species and then fold into a highly stable inactive state in which the residues that flank the scissile bond (P1 and P1';) are separated by about 70 A. PAI-1 also spontaneously folds into a stable inactive state without cleavage; this state is termed 'latent' because inhibitory activity can be restored through denaturation and renaturation. Here we report the structure of intact latent PAI-1 determined by single-crystal X-ray diffraction to 2.6 A resolution. The three-dimensional structure reveals that residues on the N-terminal side of the primary recognition site are inserted as a central strand of the largest beta sheet, in positions similar to the corresponding residues in the cleaved form of the serpin alpha 1-proteinase inhibitor (alpha 1-PI). Residues C-terminal to the recognition site occupy positions on the surface of the molecule distinct from those of the corresponding residues in cleaved serpins or in the intact inactive serpin homologue, ovalbumin, and its cleavage product, plakalbumin. The structure of latent PAI-1 is similar to one formed after cleavage in other serpins, and the stability of both latent PAI-1 and cleaved serpins may be derived from the same structural features.

Interleukin-6 (IL-6) is a multi-functional cytokine produced and secreted by several different cell types, including those of the immune system. A cDNA coding for the mature murine IL-6 (mIL-6), which extends from amino acid (aa) 25 through 211, was cloned into a prokaryotic vector and then expressed in Escherichia coli. The recombinant mIL-6 (remIL-6) was isolated from bacterial inclusion bodies by solubilization in 4 M guanidine hydrochloride followed by gel-filtration chromatography. The protein was refolded to an active conformation by dialysis against 25 mM Na. acetate pH 5.5. A final step of purification and concentration on a cation exchange resin yielded pure and biologically active remIL-6. The purified preparation had the expected aa composition, as confirmed by aa analysis and pI of 7.0-7.1. The biological activity of the recombinant protein was measured in two systems; a proliferation assay employing 7TD1 cells, and a fibrinogen biosynthesis assay employing primary rat hepatocytes. Both assay systems demonstrated that the remIL-6 was active in the range of 10(8) units/mg, which is similar to that estimated for native cytokine. Antibodies raised in rabbits against remIL-6 neutralized the biological activity of both recombinant and native IL-6.

Murine interleukin-6 (mIL-6) was expressed in Escherichia coli in the insoluble fraction of cell lysates. Approximately equal amounts of two polypeptide species, reactive with anti-IL-6 antibodies, were produced. The two forms of mIL-6 were isolated and found to have identical N-terminal sequences initiated by Met-Phe-Pro-Thr-Ser-Gln-. Peptide mapping after endoproteinase glu-C digestion led to isolation and characterization of the C-terminal peptides from each of the two forms and allowed the source of the heterogeneity to be identified as a C-terminal addition of three amino acids, Gln-Lys-Leu, to authentic mIL-6. Inspection of the nucleotide sequence of the plasmid containing the mIL-6 gene and expression of the plasmid in other strains suggested that the addition of three amino acids was caused by a readthrough of the termination codon arising from an unexpected suppressor mutation in the original host strain. Although the C-terminus of IL-6 is critical for the activity of this cytokine, the IL-6 variant with extended C-terminus was fully active in two separate bioassays. This suggests that the additional amino acids do not disrupt the structure or function of this important region of the molecule.

Crystals of bacterially expressed plasminogen activator inhibitor (PAI-1) suitable for X-ray diffraction analysis have been obtained from 8% (w/v) PEG 1500, pH 8.25. The space group is P1, and the lattice constants are a = 82.17 A, b = 47.82 A, c = 62.89 A, alpha = 90.00 degrees, beta = 106.90 degrees, gamma = 106.84 degrees. The diffraction limit is 2.3 A, and the unit cell contains two molecules of PAI-1. The crystals contain latent PAI-1 which can be partly reactivated by exposure to denaturants.

A proteinase is essential for replication of HIV. Cloning and chemical synthesis have provided a sufficient supply of HIV-1 proteinase for the determination of its three-dimensional structure. Analogies between the structures of HIV-1 proteinase and the mammalian enzyme renin, which is involved in the control of blood pressure, have given important clues concerning the design of specific inhibitors that have antiviral activity.

5-Dimethylaminoaphthalene-1-sulfonyl-Ser-Gln-Asn-Tyr-Pro-Ile-Val-T rp (Dns-SQNYPIVW) is a fluorescent substrate for the aspartyl protease of human immunodeficiency virus-1. In intact substrate, fluorescence of Trp (lambda ex 290 nm, lambda em 360 nm) was 60% quenched by energy transfer to the dansyl group. Protease-catalyzed cleavage at the Tyr-Pro bond abolished the energy transfer, and the consequent increase in Trp fluorescence was used to follow the enzymatic reaction. At substrate concentrations less than 60 microM, initial reaction velocity increased as a linear function of substrate concentration, with kcat/KM = 9700 M-1 s-1. Limited solubility and internal fluorescence quenching precluded a determination of KM for Dns-SQNYPIVW, but this was clearly greater than 100 microM.

Expression of human recombinant plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli has led to crystallization of 'latent' PAI-1. Cleavage with restriction endonucleases of a cDNA clone encoding PAI-1 yielded an 1127 base pair fragment encoding residues 2-376 of the 379 amino acid serpin. Synthetic DNA linkers were ligated to the 5' and 3' ends of the subclone to add an initiation codon and restore the full coding sequence, and the resulting semisynthetic gene was incorporated into an expression plasmid, pPAIST-7, under the control of the E. coli trp promoter. Transformation of E. coli GE81 with pPAIST-7 led to expression of unglycosylated PAI-1. Lysates of expression cultures contained PAI-1 activity and PAI-1 protein with the predicted Mr. Unglycosylated PAI-1 from E. coli exhibited characteristic properties of authentic PAI-1: (1) it was recovered in both active and inactive (latent) forms; (2) its activity declined during incubation at 37 degrees C; (3) latent PAI-1 was activated by treatment with 4 M guanidine hydrochloride; (4) reactivated PAI-1 formed a detergent-stable complex with tissue plasminogen activator. Latent PAI-1 accounted for more than 85% of PAI-1 in cell lysates and was purified by ammonium sulfate fractionation, anion-exchange chromatography and hydrophobic interaction chromatography. The purified latent PAI-1 was crystallized.

Steroid 21-hydroxylase activity has been demonstrated, previously, in microsome-enriched fractions prepared from a number of human fetal tissues. The finding that this enzyme activity was present in thymus and spleen is suggestive of the possibility that deoxycorticosterone is important in regulation of immunological processes. In the present investigation, we characterized steroid 21-hydroxylase activity in microsome-enriched preparations of guinea pig spleen. The activity of the enzyme was linear with time for 40 min and with protein concentrations up to 4.8 mg X ml-1 incubation mixture. The apparent Km of the enzyme for progesterone was 0.405 microM. Thus, the potential exists for the biosynthesis of DOC from progesterone in the spleen of the guinea pig as well as in the spleen of the human fetus. Therefore, the guinea pig may be an appropriate animal model for the study of the regulation of steroid 21-hydroxylase activity in the spleen as well as a model for the study of the role of this enzyme in immunologic processes.

Murine polymorphonuclear leukocytes (PMN) are stimulated to produce hydrogen peroxide (H2O2) by some unopsonized fungi, including Coccidioides immitis spherules, Candida albicans blastospores, and Saccharomyces sp. blastospores (zymosan). In this communication we have examined the basis for this stimulation by studying the effects of mannan and its primary constituent, D-mannose, on PMN function in vitro, since this polysaccharide is a significant component in many fungal cell walls. Our results show that mannan stimulated H2O2 production and iodination by PMN and enhanced H2O2 production by cells stimulated with zymosan. Moreover, the amount of H2O2 released by PMN was directly related to the concentration of mannan, and mannan obtained commercially or prepared in our laboratory worked equally well. Mannose, on the other hand, inhibited H2O2 release from PMN stimulated with zymosan or mannan and reduced oxygen consumption, carbon dioxide production, superoxide release, and iodination by these cells as well. With respect to specificity of inhibition, 18 different monosaccharides were examined by using 2 different assays for H2O2 release; and only 2-deoxy-D-glucose, a potent glycolytic inhibitor, reduced H2O2 release from zymosan-stimulated PMN. Furthermore, H2O2 release from cells stimulated with phorbol myristate acetate or opsonized Sephadex was not inhibited by mannose whereas H2O2 release was reduced when PMN were stimulated with C. albicans blastospores or C. immitis spherules in the presence of this sugar. From these data we propose that initiation of PMN oxidative metabolic burst in response to some unopsonized fungi occurs through a mannose-specific mechanism.

A 54-year-old man with plasma cell myeloma had sustained bleeding develop after prophylactic hip hemiarthroplasty. Routine coagulation studies revealed significant prolongation of the prothrombin time, activated partial thromboplastin time, and thrombin time. Further evaluation showed failure of the activated partial thromboplastin time to correct in a 1:1 mixture with pooled normal plasma, correction of the prolonged thrombin time by addition of protamine, and a normal reptilase time. A purified preparation of the immunoglobulin component of patient plasma produced the same pattern of coagulation abnormalities, suggesting the paraprotein possessed heparin-like anticoagulant activity. This appears to be a rare mechanism of bleeding diathesis in plasma cell myeloma.

Knowledge of the tertiary structure of the proteinase from human immunodeficiency virus HIV-1 is important to the design of inhibitors that might possess antiviral activity and thus be useful in the treatment of AIDS. The conserved Asp-Thr/Ser-Gly sequence in retroviral proteinases suggests that they exist as dimers similar to the ancestor proposed for the pepsins. Although this has been confirmed by X-ray analyses of Rous sarcoma virus and HIV-1 proteinases, these structures have overall folds that are similar to each other only where they are also similar to the pepsins. We now report a further X-ray analysis of a recombinant HIV-1 proteinase at 2.7 A resolution. The polypeptide chain adopts a fold in which the N- and C-terminal strands are organized together in a four-stranded beta-sheet. A helix precedes the single C-terminal strand, as in the Rous sarcoma virus proteinase and also in a synthetic HIV-1 proteinase, in which the cysteines have been replaced by alpha-aminobuytric acid. The structure reported here provides an explanation for the amino acid invariance amongst retroviral proteinases, but differs from that reported earlier in some residues that are candidates for substrate interactions at P3, and in the mode of intramolecular cleavage during processing of the polyprotein.

We have identified two types of mutants of Chinese hamster ovary cells in which the unique ADP-ribose attachment site in elongation factor 2 (EF-2) is altered, thereby rendering them resistant to diphtheria and Pseudomonas toxins (TOXR). The first is mutant in the gene for EF-2 and possesses a permanently altered, TOXR gene product. The second lacks a component of a posttranslational modification system that converts TOXR EF-2 to the toxin-sensitive (TOXS) state. We postulate that this modification system is involved in the conversion of a single histidine residue in EF-2 to the specific target of toxin-catalyzed ADP-ribosylation, the novel amino acid X. We have designated the second type MOD- mutants. The missing of nonfunctional component in the MOD- mutants can be restored by hybridizing them with either normal TOXS cells or with EF-2 structural gene mutants. The TOXR EF-2 from MOD- mutants is also converted to toxin sensitivity in vitro by incubation with extracts of TOXS or EF-2 gene mutant cells in the presence of an energy-generating system. Our results demonstrate that EF-2 can be synthesized and released from ribosomes in a toxin-resistant form and then converted to toxin sensitivity by posttranslational modification.

Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).

Chymosin C, an autolysis product of chymosin A, is not formed from chymosin B (Foltmann, B. (1966) C. R. Trav. Lab. Carlsberg 35, 143-231) even though chymosins A and B differ in only a single residue (residue 286 is Asp in chymosin A but is Gly in chymosin B). Autolysis of recombinant-derived chymosin A yielded chymosin C for structural analysis. N-terminal sequencing revealed two peptide chains in chymosin C, one of which begins with Gly43, the N terminus of chymosin A; the other begins with Asp289. C-terminal sequencing, peptide mapping, and amino acid analysis showed that chymosin A undergoes autolytic excision of Asp286--Glu287--Phe288 in yielding chymosin C. As predicted from the established disulfide pattern and verified by size-exclusion chromatography under denaturing conditions, no disulfide bond cross-links the two chymosin A fragments which comprise chymosin C. The N-terminal fragment (243 residues) is termed the C-protein, and the C-terminal fragment (77 residues) is termed the C-peptide. Studies with synthetic peptide analogues of relevant regions of the chymosin sequences suggested that Tyr285--Asp286 is the first chymosin A peptide bond hydrolyzed during chymosin C formation and that Tyr285--Gly286 in chymosin B is not cleaved. Cleavage of Phe288--Asp289, a bond which is present in both chymosins A and B, most likely occurs in the A form as a secondary event following nicking at Tyr285--Asp286. This explains why chymosin A gives rise to chymosin C, but chymosin B does not. Examination of the sequence of a prochymosin gene (Hidaka, M., Sasaki, K., Uozumi, T., and Beppu, T. (1986) Gene (Amst.) 43, 197-203) showed that the autolysis-sensitive region of the polypeptide is encoded by genomic DNA located at the end of one exon and at the beginning of the next. Thus, chymosin C illustrates the correlation of protease-sensitive regions of protein sequences with genomic splice junctions.

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.

Spleen cells from mice immunized with a variety of antigens and incubated in vitro with killed spherules of Coccidioides immitis lyse six to eight times more autologous murine erythrocytes than normal spleen cells and spherules. Cellular and biochemical events in this phenomenon were investigated to ascertain its significance. Kinetic studies suggested that hemolysis results from the activation of some immune cells by spherules. The capacity of spherules to activate these cells is rather unusual because, of the inert particles tested, only zymosan A and crude chitin demonstrated comparable activity. Furthermore, although the hemolytic phenomenon occurred in serum-free medium, more lysis was effected by immune cells and opsonized spherules or zymosan A than by immune cells and untreated fungal particles. Sheep, chicken, and human erythrocytes were not lysed in the hemolytic phenomenon; however, hemoglobin in chicken and sheep erythrocytes was oxidized. Both the murine erythrocyte lysis and oxidation of ovine hemoglobin correlated with the reduction of Nitro Blue Tetrazolium by immune cells adherent to spherules, and both phenomena appeared to be mediated by H2O2 released into the medium by activated cells. Spleen cells reactive with spherules could not be depleted by treatment with iron carbonyl, antiimmunoglobulin plus complement, or anti-brain-associated theta plus complement, but they were partially or completely depleted after rosette formation with erythrocytes coated with antibody or murine complement. Using light and electron microscopy, we noted that immune spleens contained more neutrophils than normal spleens, that these neutrophils reduced Nitro Blue Tetrazolium after stimulation with phorbol myristate acetate, and that they were the most prevalent cell type adherent to spherules after incubation in vitro.

To study the interaction between Candida albicans blastoconidia and human phagocytes, we incubated peripheral leukocytes with fungi for 1 h at 37 degrees C and stained the cells with fluorescent vital stains ethidium bromide (EB) and fluorescein diacetate. Fungi that had been phagocytosed showed little staining; however, some leukocytes containing blastoconidia exhibited nuclear staining with EB, even though their cell membranes showed no signs of penetration by fungi. The number of EB-positive leukocytes was related to viability of the yeast cells and the temperature at which they were maintained before use. Because efforts to quantitate EB-positive leukocytes microscopically were frustrated by cell aggregation, we labeled the leukocytes with 51Cr and measured isotope release. We determined that leukocytes incubated with viable fungi released significantly more isotope than cells incubated alone or with killed blastoconidia. Furthermore, 51Cr release correlated directly with concentration of fungi in the assay, time of incubation, and temperature at which fungi were maintained before use. Using a number of isolates of C. albicans and several other species of Candida, we found that all exhibited cytotoxic activity against leukocytes, but the level of activity varied among organisms. Finally, we depleted or enriched peripheral leukocytes for specific cell populations and determined that only monocytes released more 51Cr after incubation with viable blastoconidia. Blastoconidia can lyse phagocytic cells through germination and penetration of cell membranes within 1 to 2 h, but the cytotoxic phenomenon we describe occurs within 15 to 30 min after yeast cells have been phagocytosed. Therefore, this capacity may represent a more immediate response by blastoconidia against phagocytosis and killing by monocytes.

Binding of cyclic adenosine 3' ,5'-monophosphate (cAMP) by the cAMP receptor protein in crude cell-free extracts of Escherichia coli was characterized. When cell were grown in glucose, binding was inhibited 50% relative to extracts from cells grown with succinate as carbon source . This inhibition could be relieved by dialysis.

Penicillin G acylase from Proteus rettgeri is an 80,000- to 90,000-dalton enzyme composed of two nonidentical subunits. Both subunits were required for enzymatic activity. The 65,000-dalton beta subunit contained a phenylmethylsulfonyl fluoride-sensitive residue required for enzymatic activity, and the 24,500-dalton alpha subunit contained the domain that imparts specificity for the penicillin side chain.

Proteus rettgeri and Escherichia coli W were shown to express structurally different penicillin G acylases. The enzymes had similar substrate specificity but differed in molecular weight, isoelectric point, and electrophoretic mobility in polyacrylamide gels and did not antigenically cross-react. When the organisms were subjected to environmental conditions which made expression of this enzyme essential for growth, spontaneous mutants were isolated that used different amides as the only source of nitrogen. These mutants acquired the ability to use amides for growth by deregulating the penicillin G acylase and by their evolution to novel substrate specificities. The enzymes expressed by mutants isolated from each genus appeared to have evolved in parallel since each acylase attained similar new substrate specificities when the organisms were subjected to identical selection pressure.

This investigation analyzed the effect of labor on the phagocytic activity of neutrophils in women with uncomplicated term pregnancies. Nineteen healthy women who were not pregnant and did not use oral contraceptives or glucocorticoids served as controls. Peripheral venous blood samples were collected from 15 study patients who were in the active phase of labor (5-10 cm of dilation). Neutrophil phagocytic function was evaluated with the radioiodine fixation test. Assays were conducted utilizing both pooled homologous serum and autologous serum. There was no statistically significant difference in the neutrophil phagocytic function of laboring patients and controls. In addition, there was no evidence that serum from pregnant women exerted a depressant effect on phagocytosis.