NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.     

Isolation and characterization of biologically active murine interleukin-1 alpha derived from expression of a synthetic gene in Escherichia coli

A murine interleukin-1 alpha (mIL-1 alpha) gene coding for amino acids 115 to 270 of the precursor protein (Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Giri, J.G., Pan, Y.E., Collier, K., Semionow, R., Chua, A.O. and Mizel, S.B. (1984) Nature 312, 458-462) was chemically synthesized and expressed in Escherichia coli. mIL-1 alpha, in the form of insoluble inclusion bodies, accounted for approx. 30% of total cellular protein produced by the recombinant strain. A simple isolation protocol was developed in which inclusion body material was first solubilized in 3 M guanidine hydrochloride, and the mIL-1 alpha was then simultaneously purified and allowed to fold to its active conformation by dialysis against distilled water. This procedure yielded pure, biologically active mIL-1 alpha with 41% recovery of the mIL-1 alpha present in the guanidine hydrochloride extract. The purified preparation had the expected amino acid composition, a molar absorptivity of 28,200 M-1.cm-1 and a pI of 5.2. No methionyl-mIL-1 alpha was detected by N-terminal sequence analysis, and the endotoxin level was less than 10 pg per micrograms of mIL-1 alpha. The specific biological activity was 3.10(7) units/mg in a co-mitogenic thymocyte proliferation assay. In addition to full-length mIL-1 alpha, the preparation contained N-terminally truncated mIL-1 alpha species (mainly des-4 and des-6 amino acid forms). The truncated species were isolated and found to have the same biological activity as the complete polypeptide. Thus, the active fragment of mIL-1 alpha appears to consist of a proteinase-sensitive N-terminal region which is not essential for activity, and a proteinase-resistant core which harbors the essential determinants of its cytokine function.     

Modulation of human chondrocyte metabolism by recombinant human interferon gamma: in-vitro effects on basal and IL-1-stimulated proteinase production, cartilage degradation and DNA synthesis

Human articular chondrocytes in monolayer culture and fragments of human articular cartilage were treated with recombinant human interferon gamma (IFN-gamma) both alone and in combination with interleukin 1 (IL-1). IFN-gamma alone inhibits metalloproteinase production, as measured in the caseinase assay, and decreases glycosaminoglycan release from cartilage fragments in culture. The synthesis of DNA, as measured by [3H]thymidine incorporation, is stimulated by IFN-gamma. Similar effects are seen in the presence of IL-1. Thus, IFN-gamma opposes the stimulatory effect of IL-1 on caseinase production and decreases IL-1-stimulated cartilage degradation, as measured by glycosaminoglycan release. In contrast, IFN-gamma has no effect on IL-1-stimulated prostaglandin production, and acts synergistically with IL-1 to cause a large stimulation of DNA synthesis. These results show that IFN-gamma has a number of effects on articular chondrocytes in-vitro and suggest a possible role for IFN-gamma in limiting cartilage degradation in inflammatory joint conditions.     

Developmental regulation of the human zeta globin gene in transgenic mice.

We have characterized the expression of the human zeta (zeta) gene, which encodes an embryonic alpha-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site II (HSII) from the distal 5. region of the human beta globin gene cluster potentiates expression of the zeta globin gene. In the absence of the HSII fragment, no zeta expression is observed. Expression of the human zeta gene in mice parallels expression of a murine embryonic alpha-like globin gene (x). Thus, expression of the human zeta gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult alpha globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in alpha globin gene expression can be analyzed in detail.     

Trypanosoma cruzi invade a mammalian epithelial cell in a polarized manner

We have determined that parasite entry into host cells can be influenced by cell polarity using a DNA probe to quantitate the infection of cultured Madin-Darby canine kidney (MDCK) epithelial cells by Trypanosoma cruzi, the agent of Chagas' disease. Confluent MDCK cells are polarized, with their plasma membrane separated by tight junctions into two domains, apical and basolateral. We show that T. cruzi forms corresponding to the insect infective stages (metacyclics) and the vertebrate blood stages (trypomastigotes) enter confluent MDCK cells preferentially through their basolateral domains. Sparsely plated MDCK cells are less polarized and are better infected than confluent cells. Scanning electron microscopy showed that 92% +/- 4% of the parasites entered at the edges of cells.     

Fine structure,mechanism of heart function and haemodynamics in the prosobranch gastropod molluscLittorina littorea (L.)

Summary The heart, main blood vessels, and associated structures ofLittorina littorea were examined by scanning and transmission electron microscopy. The auricle is subdivided into two compartments, one receiving blood from the gill and opening to the nephridial gland vein, the other connecting with the latter anteriorly and the ventricle posteriorly.Video recordings were made of the beating heart in vivo and revealed that the auricle expelled blood not only to the ventricle, but also the nephridial gland vein at systole and provided further evidence of tidal flow of blood in the vein. There is clear indication that the constant volume mechanism of auricular re-filling is not strictly true inLittorina.Blood pressure in the heart and major vessels was measured using a servo-nulling micropressure system. The rate of formation of urine (derived by filtration of blood through the auricular wall) was measured using [⁵¹Cr] EDTA as a blood marker.Basal blood pressure was slightly above ambient (0.7 cm H₂O). Peak systolic pressure in the ventricle (3.8 cm H₂O) was synchronised with a subambient trough in pericardial pressure (–1.0 cm H₂O); these pressure pulses were out of phase with that of the auricle (2.3 cm H₂O) at systole. The observations are consistent in broad terms with a constant volume mechanism, but this does not take into account urine formation or filling of the nephridial gland vein.A filtration pressure of 1.5 cm H₂O has been demonstrated across the auricular wall throughout the cardiac cycle. Colloidal back pressure appears to be negligible. The mean rate of urine formation is 0.26 l g^–1 min^–1.     

Determination of parameters for enzyme therapy using L-asparaginase entrapped in canine erythrocytes

The antitumor agent L-asparaginase was entrapped in canine erythrocytes by a single dialysis encapsulation (efficiency mean = 30%). Concentration of asparaginase in carrier cells was about 240 IU/ml, with an average of 62% cell recovery. Use of a double dialysis procedure increased the L-asparaginase concentration within carrier cells to 530 IU/ml, with an overall cell recovery of 53.9%. In vitro efflux experiments showed L-asparaginase-loaded canine carriers were stable at both 4 and 37 degrees C for an 18-h period. In vivo cell survival studies showed that carrier cells did circulate and that L-asparaginase had a half-life of 6.5 days. No evidence suggesting that the enzyme left the cell was found. Carrier cells prepared with [3H]inulin and [14C]sucrose were stored at 4 degrees C for 2 weeks and began to show signs of deterioration after 2 days.     

Encapsulation of interleukin-2 in murine erythrocytes and subsequent deposition in mice receiving a subcutaneous injection

Radiolabeled recombinant human interleukin-2 (IL-2) was successfully encapsulated in both mouse and sheep erythrocytes. Of the added IL-2, 70% was recovered bound to or encapsulated within the carrier cells. Erythrocytes containing IL-2 were stable in vitro and most of the IL-2 remained associated with the cells following a 16-h incubation at 37 degrees C. When carrier erythrocytes containing IL-2 were injected subcutaneously into mice, intact [35S]IL-2 was detectable in a number of tissues 3 days after injection.     

Biologic markers in ethylene oxide-exposed workers and controls

Ethylene oxide (EtO) is an alkylating agent and a model direct-acting mutagen and carcinogen. This study has evaluated a panel of biologic markers including EtO-hemoglobin adducts (EtO-Hb), sister-chromatid exchanges (SCEs), micronuclei, chromosomal aberrations (CAs), DNA single-strand breaks (SSB) and an index of DNA repair (ratio of UDS to NA-AAF-DNA binding) in the peripheral blood cells of 34 workers at a sterilization unit of a large university hospital and 23 controls working in the university library. Comprehensive environmental histories were obtained on each subject including detailed occupational and smoking histories. Industrial hygiene data obtained prior to the study and personal monitoring during the 8 years preceding the study showed that workers were subject to low-level exposure near or below the current Occupational Safety and Health Administration (OSHA) standard of 1 ppm (TWA). Personal monitoring data obtained during 2 weeks prior to blood sampling were uniformly less than 0.3 ppm (TWA). After adjusting for smoking, EtO workplace exposure was significantly (p less than 0.001) associated with EtO-Hb (a carcinogen-protein adduct) and 2 measures of SCEs [the average number of SCEs/cell (SCE50) and the number of high frequency cells (SCEHFC)]. There was an apparent suppression of DNA repair capacity in EtO-exposed individuals as measured by the DNA repair index; i.e., the ratio of unscheduled DNA synthesis (UDS) and NA-AAF-DNA binding (p less than 0.01). No association of DNA repair index with smoking was found. Another important finding of this study is the highly significant correlation between EtO-Hb adduct levels and SCEHFC (p less than 0.01) and SCEs (p less than 0.02) which provides evidence of a direct link between a marker of biologically effective dose and markers of genotoxic response. In contrast, micronuclei, CAs and SSBs were not significantly elevated in the workers. The activity of the u-isoenzyme of glutathione-S-transferase (GT) was measured as a possible genetic marker of susceptibility and a modulator of biomarker formation. However, possibly because of confounding by age, no significant relationships were found between GT and any of the exposure-related markers by ANOVA or among other independent variables by regression. This study demonstrates significant effects of low-level EtO exposure, independent of smoking history, near or below 1 ppm on multiple biomarkers and suggests that the current OSHA standard may not be adequately protective. Previously described effects of smoking on EtO-Hb adducts, SCEs and SCEHFC were also seen in this study.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of protein release from yeast using enzymatic lysis for selective product recovery

The kinetics of release of four intracellular enzymes from different yeast cell locations using the Differential Product Release (DPR) method has been investigated. The method uses a combination of physical, chemical and biological agents such as lytic enzymes, an osmotic support and a spheroplast stabilizer. Using the DPR technique a wall enzyme, invertase, was released with a very high specific activity in the first step from a breadmaking strain ofS. cerevisiae. Maximum release could be obtained in this step when the incubation time was extended from 60 min to 100 min. Two cytosol enzymes, α-D-glucosidase and alcohol dehydrogenase were released in the second step. Fumarase was released in the third step almost instantaneously after disruption of the mitochondria which reduces considerably, by ca. 1 hour, the total incubation time of DPR. This paper investigates the kinetics of enzyme release during the 3 steps of DPR.