Molecular mechanisms involved in the enhancement of mitochondrial malate dehydrogenase activity by calcitriol in chick intestine

Mitochondrial malate dehydrogenase (mMDH) from the intestine is the NAD-linked oxidoreductase of the tricarboxylic acid cycle with the highest activity and response to vitamin D treatment in vitamin D-deficient chicks (?D). The aim of this study was to elucidate potential molecular mechanisms by which cholecalciferol or calcitriol enhances the activity of this enzyme. One group of animals used was composed of ?D and ?D treated with cholecalciferol or with calcitriol. A second group consisted of ?D and ?D supplemented with high Ca²⁺ diet. A third group included chicks receiving either a normal or a low Ca²⁺ diet. In some experiments, animals were injected with cycloheximide. Data showed that either vitamin D (cholecalciferol or calcitriol) or a low Ca²⁺ diet increases mMDH activity. High Ca²⁺ diet did not modify the intestinal mMDH activity from ?D. The mMDH activity from ?D remained unaltered when duodenal cells were exposed to 10^?8 mol/L calcitriol for 15 min. The enhancement of mMDH activity by calcitriol was completely abolished by simultaneous cycloheximide injection to ?D. mMDH mRNA levels, detected by RT-PCR, indicate that calcitriol did not affect gene expression. In contrast, Western blots show that calcitriol enhanced the protein expression. In conclusion, calcitriol stimulates intestinal mMDH activity by increasing protein synthesis. No response of mMDH activity by rapid effects of calcitriol or activation through increment of serum Ca²⁺ was demonstrated. Consequently, ATP production would be increased, facilitating the Ca²⁺ exit from the enterocytes via the Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger, which participate in the intestinal Ca²⁺ absorption.     

Application of homonuclear 3D NMR experiments and 1D analogs to study the conformation of sialyl Lewisx bound to E-selectin

The conformation of the sialyl Lewis ^x tetrasaccharidebound to E-selectin was previously determined from transfer NOE (trNOE)experiments in conjunction with a distance-geometry analysis. However, theorientation of the tetrasaccharide ligand in the binding site of E-selectinis still unknown. It can be predicted that the accurate quantitativeanalysis of all trNOEs, including those originating from spin diffusion, isone key to analyze the orientation of sialyl Lewis^x in thebinding pocket of E-selectin. Therefore, we applied homonuclear 3D NMRexperiments and 1D analogs to obtain trNOEs that could not unambiguously beassigned from previous 2D trNOESY spectra, due to severe resonance-signaloverlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a1D trNOESY-TOCSY experiment of the sialyl Lewis^x/E-selectincomplex furnished new interglycosidic trNOEs and provided additionalinformation for the interpretation of trNOEs that have been describedbefore. A 2D trROESY spectrum of the sialyl Lewis^x/E-selectincomplex allowed one to identify the amount of spin-diffusion contributionsto trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysisof spin-diffusion pathways, was obtained, creating a basis for aquantitative analysis of trNOEs in the sialylLewis^x/E-selectin complex.     

Dopamine receptor subtype density as a function of age in Aplysia californica

The age-associated changes in dopamine subtype receptors were examined in Aplysia californica. The density of the subtype receptors D1, D2, D3 and D4 was examined in the ganglia from 4.5-, 6-, 8-, 9- and 12-month animals. Receptor analysis was performed by examining the binding of radiolabeled ligands to the individual subtypes. [³H]SCH23390 and [³H]Clozapine were used to analyze D1 and D4 specific binding. [³H]Quinpirole was used for determining D2 and D3 specific binding. Specific binding was found to be present for all four receptor subtypes. All receptor subtypes showed an increase in density from 4.5 to 6 months. From 6 to 8 months D2 and D3 decreased, while D1 and D4 increased. D4 showed the strongest increase. All four subtypes examined showed decreases from 8 to 12 months. ANOVA results indicated age was a significant factor in the subtype receptor density for all receptor types.     

Spliceosome Sm proteins D1, D3, and B/B' are asymmetrically dimethylated at arginine residues in the nucleus

We report a novel modification of spliceosome proteins Sm D1, Sm D3, and Sm B/B′. L292 mouse fibroblasts were labeled in vivo with [³H]methionine. Sm D1, Sm D3, and Sm B/B′ were purified from either nuclear extracts, cytosolic extracts or a cytosolic 6S complex by immunoprecipitation of the Sm protein-containing complexes and then separation by electrophoresis on a polyacrylamide gel containing urea. The isolated Sm D1, Sm D3 or Sm B/B′ proteins were hydrolyzed to amino acids and the products were analyzed by high-resolution cation exchange chromatography. Sm D1, Sm D3, and Sm B/B′ isolated from nuclear fractions were all found to contain ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine, modifications that have been previously described. In addition, Sm D1, Sm D3, and Sm B/B′ were also found to contain asymmetric ω-N^G,N^G-dimethylarginine in these nuclear fractions. Analysis of Sm B/B′ from cytosolic fractions and Sm B/B′ and Sm D1 from cytosolic 6S complexes showed only the presence of ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine. These results indicate that Sm D1, Sm D3, and Sm B/B′ are asymmetrically dimethylated and that these modified proteins are located in the nucleus. In reactions in which Sm D1 or Sm D3 was methylated in vitro with a hemagglutinin-tagged PRMT5 purified from HeLa cells, we detected both symmetric ω-N^G,N^G′-dimethylarginine and asymmetric ω-N^G,N^G-dimethylarginine when reactions were done in a Tris/HCl buffer, but only detected symmetric ω-N^G,N^G′-dimethylarginine when a sodium phosphate buffer was used. These results suggest that the activity responsible for the formation of asymmetric dimethylated arginine residues in Sm proteins is either PRMT5 or a protein associated with it in the immunoprecipitated complex.     

A model for the mechanism of chloride activation of oxygen evolution in photosystem II

A hypothesis is proposed to explain the function of Cl^- in activating the oxygenevolving complex (OEC) of photosystem II (PS II), based on the results of recent ³⁵Cl-NMR studies. The putative mechanism involves Cl^- binding to two types of sites. An intrinsic site is suggested to be composed of three histidyl residues (His 332 and His 337 from D1 and His 337 D2). It is proposed that Cl^- binding to this site accelerates the abstraction of H⁺ from water by raising the pK_a's of the histidine imidazole groups. Cl^- binding also stimulates the transfer of H⁺ from this intrinsic site to a set of extrinsic sites on the 33 kD extrinsic polypeptide. The extrinsic Cl^- binding sites are suggested to involve four protein domains that are linked together by salt-bridge contacts. Chloride and H⁺ donated from the intrinsic site attack these intramolecular salt-bridges in a defined sequence, thereby exposing previously inaccessible Cl^- and H⁺ binding sites and stimulating the oxidation of water. This hypothesis also proposes a possible structure for the Mn active site within the D1/D2 complex. Specific amino-acid residues that are likely to participate as Mn lignads are identified on the lumenal portions of the D1 and D2 proteins that are different from those in the L and M subunits of photosynthetic bacteria; the choice of these residues is based on the metal coordination chemistry of these residues, their location within the polypeptide chain, the regularity of their spacing, and their conservation through evolution. The catalytic Mn-binding residues are suggested to be D-61, E-65, E-92, E-98, D-103; D-308, E-329, E-342 and E-333 in D1, and H-62, E-70, H-88, E-97, D-101; E-313, D-334, E-338 and E-345 in D2. Finally, this hypothesis identifies sites on both D2 and the 33 kD extrinsic polypeptide that might be involved in high- and low-affinity Ca²⁺ binding.To whom correspondence should be addressed     

Influence of an Acrylic Polymer Blend on the Physical Stability of Film-Coated Theophylline Pellets

The purpose of this study was to investigate the physical stability of a coating system consisting of a blend of two sustained release acrylic polymers and its influence on the drug release rate of theophylline from coated pellets. The properties of both free films and theophylline pellets coated with the polymer blend were investigated, and the miscibility was determined via differential scanning calorimetry. Eudragit^® RS 30 D was plasticized by the addition of Eudragit^® NE 30 D, and the predicted glass transition temperature (T _g) of the blend was similar to the experimental values. Sprayed films composed of a blend of Eudragit^® NE 30 D/Eudragit^® RS 30 D (1:1) showed a water vapor permeability six times greater than films containing only Eudragit^® NE 30 D. The presence of quaternary ammonium functional groups from the RS 30 D polymer increased the swellability of the films. The films prepared from the blend exhibited stable permeability values when stored for 1 month at both 25°C and 40°C, while the films which were composed of only Eudragit^® NE 30 D showed a statistically significant decrease in this parameter when stored under the same conditions. Eudragit^® NE 30 D/Eudragit^® RS 30 D (1:1)-sprayed films decreased in elongation from 180% to 40% after storage at 40°C for 1 month, while those stored at 25°C showed no change in elongation. In coated pellets, the addition of Eudragit^® RS 30 D to the Eudragit^® NE 30 D increased the theophylline release rate, and the pellets were stable when stored at 25°C for a period of up to 3 months due to maintenance of the physico-mechanical properties of the film. Pellets stored at 40°C exhibited a decrease in drug release rate over time as a result of changes in film physico-mechanical properties which were attributed to further coalescence and densification of the polymer. When the storage temperature was above the T _g of the composite, instabilities in both drug release rate and physical properties were evident. Stabilization in drug release rate from coated pellets could be correlated with the physico-mechanical stability of the film formulation when stored at temperatures below the T _g of the polymer.     

Isolation and Characterization of Acidithiobacillus ferrooxidans Strain D3-2 Active in Copper Bioleaching from a Copper Mine in Chile

Acidithiobacillus ferrooxidans strain D3-2, which has a high copper bioleaching activity, was isolated from a low-grade sulfide ore dump in Chile. The amounts of Cu²⁺ solubilized from 1% chalcopyrite (CuFeS₂) concentrate medium (pH 2.5) by A. ferrooxidans strains D3-2, D3-6, and ATCC 23270 and 33020 were 1360, 1080, 650, and 600 mg·l ^?1·30 d^?1. The iron oxidase activities of D3-2, D3-6, and ATCC 23270 were 11.7, 13.2, and 27.9 μl O₂ uptake·mg protein^?1·min^?1. In contrast, the sulfite oxidase activities of strains D3-2, D3-6, and ATCC 23270 were 5.8, 2.9, and 1.0 μl O₂ uptake·mg protein^?1·min^?1. Both of cell growth and Cu-bioleaching activity of strains D3-6 and ATCC 23270, but not, of D3-2, in the chalcopyrite concentrate medium were completely inhibited in the presence of 5 mM sodium bisulfite. The sulfite oxidase of strain D3-2 was much more resistant to sulfite ion than that of strain ATCC 23270. Since sulfite ion is a highly toxic intermediate produced during sulfur oxidation that strongly inhibits iron oxidase activity, these results confirm that strain D3-2, with a unique sulfite resistant-sulfite oxidase, was able to solubilize more copper from chalcopyrite than strain ATCC 23270, with a sulfite-sensitive sulfite oxidase.     

Chronic ethanol alters the receptor binding characteristics of the delta opioid receptor ligand,DAla2DLeu5 enkephalin in mouse brain

The binding characteristics of the delta opioid receptor ligand, ³HDAla²DLeu⁵ enkephalin, were markedly altered in brains obtained from mice fed an ethanol-containing diet for five days. Control mice exhibited both a high and low affinity site for ³HDAla²DLeu⁵ enkephalin, whereas those consuming the ethanol diet were found to possess only one binding site. This singular site has an intermediate K_D value with an increase in receptor number when compared to the high and low affinity sites observed in control mice. The $\text{in}$$\text{vitro}$ addition of ethanol to a brain membrane preparation obtained from untreated mice, at a concentration equivalent to that found in the blood of the ethanol-treated mice, did not markedly affect DAla²DLeu⁵ enkephalin binding characteristics. No alteration in the binding characteristics of ³H-naloxone, a mu receptor ligand, was noted following five days of ethanol consumption. Mice maintained on the ethanol-containing diet were tolerant to the activity-stimulating effects of acute ethanol administration. These results suggest that mice consuming an ethanol diet in sufficient quantities to render them tolerant exhibit a specific loss of a ³HDAla²DLeu⁵ enkephalin binding site, while the binding of ³H-naloxone was unchanged.     

Loss of cytosolic Mg2+ binding sites in the Thermotoga maritima CorA Mg2+ channel is not sufficient for channel opening

The CorA Mg²⁺ channel is a homopentamer with five-fold symmetry. Each monomer consists of a large cytoplasmic domain and two transmembrane helices connected via a short periplasmic loop. In the Thermotoga maritima CorA crystal structure, a Mg²⁺ is bound between D89 of one monomer and D253 of the adjacent monomer (M1 binding site). Release of Mg²⁺ from these sites has been hypothesized to cause opening of the channel. We generated mutants to disrupt Mg²⁺ interaction with the M1 site. Crystal structures of the D89K/D253K and D89R/D253R mutants, determined to 3.05 and 3.3?Å, respectively, showed no significant structural differences with the wild type structure despite absence of Mg²⁺ at the M1 sites. Both mutants still appear to be in the closed state. All three mutant CorA proteins exhibited transport of ⁶³Ni²⁺, indicating functionality. Thus, absence of Mg²⁺ from the M1 sites neither causes channel opening nor prevents function. We also provide evidence that the T. maritima CorA is a Mg²⁺ channel and not a Co²⁺ channel.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates II. Folate Metabolism and Polyglutamate Cleavage Activity of Uninfected and Infected Escherichia coli Cells and Bacteriophage Particles

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28^ts or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28^ts production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28^ts was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29⁻, 26⁻, 27⁻, 51⁻, and 10⁻, but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28^ts. Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28^ts activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.     

Purification and Some Properties of a Novel Ethanol Dehydrogenase with High Activity against Dulcitol 1-Phosphate from Salmonella Typhimurium IFO 12529

A novel ethanol dehydrogenase with high activity against dulcitol 1-phosphate (D1P-EDH) was purified from Salmonella typhimurium IFO 12529 grown in a medium containing dulcitol as a carbon source. D1P-EDH was purified from a crude extract of S. typhimurium cells by (NH₄)₂SO₄ precipitation and column chromatographies on Blue-Cellulofine, Sephacryl S-300, and Zorbax GF-250. D1P-EDH was purified 277-fold with an activity yield of 21.3%. The purified preparation gave a single band on an electrophoregram. The activity staining of the electrophoregram of the (NH₄)₂SO₄ precipitate indicated that there was no isozyme of D1P-EDH in the extract. The molecular weight of D1P-EDH was estimated to be 158,000 by gel filtration and 40,000 by SDS-polyacrylamide gel electrophoresis. D1P-EDH showed its maximal activity in a pH range from 9.0 to 9.5. D1P-EDH was stable in a pH range from 6.0 to 10.0 and was also stable at 30°C for 120 min. The purified preparation oxidized fructose 6-phosphate and galactose 6-phosphate to the same extent as D1P and oxidized much more ethanol than D1P. D1P-EDH activity was strongly inhibited by p-chloromercuribenzoic acid and NaN₃ though it was activated by Al^{3 +} , Ba^{2 +} , Ca^{2 +}, and Fe^{2 +}.     

Mechanisms for Two-Step Proton Transfer Reactions in the Outward-Facing Form of MATE Transporter

Bacterial pathogens or cancer cells can acquire multidrug resistance, which causes serious clinical problems. In cells with multidrug resistance, various drugs or antibiotics are extruded across the cell membrane by multidrug transporters. The multidrug and toxic compound extrusion (MATE) transporter is one of the five families of multidrug transporters. MATE from Pyrococcus furiosus uses H⁺ to transport a substrate from the cytoplasm to the outside of a cell. Crystal structures of MATE from P. furiosus provide essential information on the relevant H⁺-binding sites (D41 and D184). Hybrid quantum mechanical/molecular mechanical simulations and continuum electrostatic calculations on the crystal structures predict that D41 is protonated in one structure (Straight) and, both D41 and D184 protonated in another (Bent). All-atom molecular dynamics simulations suggest a dynamic equilibrium between the protonation states of the two aspartic acids and that the protonation state affects hydration in the substrate binding cavity and lipid intrusion in the cleft between the N- and C-lobes. This hypothesis is examined in more detail by quantum mechanical/molecular mechanical calculations on snapshots taken from the molecular dynamics trajectories. We find the possibility of two proton transfer (PT) reactions in Straight: the 1st PT takes place between side-chains D41 and D184 through a transient formation of low-barrier hydrogen bonds and the 2nd through another H⁺ from the headgroup of a lipid that intrudes into the cleft resulting in a doubly protonated (both D41 and D184) state. The 1st PT affects the local hydrogen bond network and hydration in the N-lobe cavity, which would impinge on the substrate-binding affinity. The 2nd PT would drive the conformational change from Straight to Bent. This model may be applicable to several prokaryotic H⁺-coupled MATE multidrug transporters with the relevant aspartic acids.     

Optimal rates of 2,4‐dichlophenoxyacetic acid foliar application for control of common scab in potato

Applications of the synthetic auxin 2,4‐dichlorophenoxyacetic acid (2,4‐D) to the foliage of potato plants can reduce common scab, a tuber disease. However, in prior research effective applications at 200 mg L^?1 2,4‐D resulted in phytotoxic side effects with reduced tuber yield and quality. This study showed that minimal significant threshold rates from 8.3 to 23.6 mg L^?1 2,4‐D reduced disease incidence in pot trials, and from 10.8 to 41.0 mg L^?1 minimised disease severity in both pot and field trials. In only one pot trial, significant phytotoxicity was found with rates of 100 mg L^?1 or greater, reducing mean total tuber mass per plot and 38 mg L^?1 or greater, reducing mean mass per tuber. Notably, within the field trial, a more reliable plant growth system for estimation of yield, no significant impacts were observed. Disease control was associated with decreased sensitivity of tubers to thaxtomin A, the phytotoxin produced by the common scab pathogen essential for disease induction. The amount of residual 2,4‐D in tubers at harvest varied with cultivar, Russet Burbank accumulating more 2,4‐D than Desiree. Application rates less than 100 mg L^?1 resulted in levels of 2,4‐D below the Australian standard maximum residue limit. These data suggest that applications of 2,4‐D at low rates could provide a commercially suitable control strategy for common scab.     

Comprehensive determination of <Superscript>3</Superscript>J<Subscript>HNHα</Subscript> for unfolded proteins using <Superscript>13</Superscript>C′-resolved spin-echo difference spectroscopy

An experiment is presented to determine ³J_HNHα coupling constants, with significant advantages for applications to unfolded proteins. The determination of coupling constants for the peptide chain using 1D ¹H, or 2D and 3D ¹H-¹⁵N correlation spectroscopy is often hampered by extensive resonance overlap when dealing with flexible, disordered proteins. In the experiment detailed here, the overlap problem is largely circumvented by recording ¹H-¹³C′ correlation spectra, which demonstrate superior resolution for unfolded proteins. J-coupling constants are extracted from the peak intensities in a pair of 2D spin-echo difference experiments, affording rapid acquisition of the coupling data. In an application to the cytoplasmic domain of human neuroligin-3 (hNlg3cyt) data were obtained for 78 residues, compared to 54 coupling constants obtained from a 3D HNHA experiment. The coupling constants suggest that hNlg3cyt is intrinsically disordered, with little propensity for structure.     

Growth pattern and NGF-dependent survival of dorsal root ganglia neurons of distinct glyco-phenotype

Cell-surface glyco-phenotypes of dorsal root ganglia (DRG) neurons were specified with monoclonal antibodies (mABs) D1 and E1. D1 demarcated sensory afferents in skin but not muscle target. More than 90% of the drg neurons supported by nerve growth factor (NGF) in vitro were D1 positive (D1⁺). A fraction of these D1⁺ neurons, those of small to intermediate soma size, coexpressed a PNGase-sensitive glycoepitope E1, defined by mAB E1. In situ and in vitro, E1⁺/D1⁺ and E1⁻/D1⁺ neurons and nerve fibers were affiliated. After separation of the two glyco-phenotypes, NGF-dependent survival of E1⁻/D1⁺ neurons was no longer observed. Two interrelated concepts emerge from these findings: (a) NGFs survival functions for cutaneous sensory neurons are in part indirect and appear to be based on interneuronal cooperation for survival; and (b) interneuronal survival dependencies are likely to be a decisive factor governing nerve fiber assemblages. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 193–207, 1998     

Molecular Cloning,Stable Expression and Desensitization of the Human Dopamine D1B / D5 Receptor

Abstract

The sub-family of dopamine D1-like receptors is now known to be comprised of at least two members: the originally cloned D1 receptor (herein referred to as the D1a receptor) and a related receptor referred to as the D1b, D1β or D5 dopamine receptor (herein referred to as the D1b/D5 receptor). Here, we characterize the D1b/D5 receptor expressed transiently in COS-7 cells and permanently in Ltk^? cells.

Transiently expressed human D1b/D5 receptors bind the D1 specific ligand [¹²⁵I]SCH 23982 saturably and with high affinity (K_D = 500 _pM). Competition for [¹²⁵I]SCH 23982 binding to rat D1b/D5 and human D1a and D1b/D5 receptors supports the contention that the two D1b/D5 receptors are species homologues. Furthermore, in COS-7 cells, as previously observed, dopamine competes for the binding of [¹²⁵I]SCH 23982 to human D1b/D5 receptors with a higher affinity than that seen at the human D1a receptor. These results are similar to those seen in Ltk^? cells permanently transfected with the human D1b/D5 receptor. In these cells, dopamine competition for [¹²⁵I]SCH 23982 binding is complex, sensitive to guanine nucleotides and of a higher affinity than that observed for dopamine binding to the human D1a receptor expressed in these same cells. In both D1a and D1b/D5 expressing Ltk^? cells, dopamine stimulates adenylyl cyclase with an EC₅₀ of = 200 nM. Furthermore, preincubation of Ltk^? cells expressing the D1a and D1b/D5 receptors with dopamine results in desensitization of the response of adenylyl cyclase to subsequent agonist stimulation.     

Mutants partially defective in excision repair at five autosomal loci in Drosophila melanogaster

Primary cell cultures derived from mutants in seventeen different genes were analyzed for their ability to excise pyrimidine dimers from DNA. Five of these mutagen-sensitive mutants [mus(2)205^A1, mus(3)302^D1, mus(3) 304^D3, mus(3)306^D1, mus(3)308^D2] display a significantly reduced excision capacity relative to control cultures. In addition, two of the five [mus(3)306^D1, mus(3)308^D2] are defective in the accumulation of single-strand breaks normally seen after ultraviolet irradiation. This study, therefore, brings the total number of Drosophila mutants known to be defective in excision repair to seven. The results are discussed relative to other genetic and biochemical properties of these mutants. This work is dedicated to Professor W. Beermann whose own contributions were instrumental in focusing a modern analysis of the eukaryotic genome on the diptera. Those of us who benefitted so much from his personal guidance recognize that we did so as a result of some sacrifice on his part. One of Boyd's contemporaries in Tübingen once remarked: “It's terrifying to think what Professor Beermann could do if he were in the lab full time.”     

Comparison of the Cathepsin D from Mackerel (Scomber australasicus) and Milkfish (Chanos chanos) Muscle

Muscle proteases from mackerel and milkfish were purified to electrophoretical homogeneity by concanavalin A-Sepharose and Sephadex G-100 chromatographies. Both proteases appear to be an aspartic protease, cathepsin D (EC 3.4.23.5). The molecular weights of the purified cathepsin D’s from mackerel and milkfish were 51,000 and 54,000, estimated by Sephadex G-100, and 59,000 and 61,000 by SDS–PAGE, respectively. Both cathepsin D’s were completely inhibited by pepstatin, but not affected by leupeptin, N-ethylmaleimide, dithiothreitol, or glutathione. ß-Mercaptoethanol, iodoacetic acid, p-chloromercuri-benzoate, phenylmethylsulfonyl fluoride, and sodium dodecyl sulfate partially or completely inhibited both cathepsin D’s. Na⁺ and K⁺ partially activated the cathepsin D from milkfish. Both cathepsin D’s were inhibited by Mg²⁺, Sr²⁺, Fe²⁺, and H²⁺, but activated by Ca²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Cd²⁺. The pI and optimal temperature of the cathepsin D’s from mackerel and milkfish were 5.04 and 4.91, 45°, and 50°C, respectively. The temperatures for inactivating 50% activity of the cathepsin D’s from mackerel and milkfish during 20 min of incubation were 53° and 48°C, respectively. Both cathepsin D’s had similar optimal pHs near 3. The activity of that from milkfish markedly decreased when the pH was higher than 4, and was almost completely lost at pH above 6, while that from mackerel still had at least 40% activity at pH 6.     

Freezing Injury and Phospholipid Degradation in Vivo in Woody Plant Cells: III. Effects of Freezing on Activity of Membrane-bound Phospholipase D in Microsome-enriched Membranes

Freeze-thawing of microsome-enriched membranes from living bark tissues of black locust trees, especially those from less hardy tissues, caused a drastic increase in sensitivity to Ca²⁺ and a complete loss of the regulatory action of Mg²⁺ in membrane-bound phospholipase D activity with endogenous (membrane-bound) substrates. Also, the freeze-thaw cycle made phospholipase D in these membranes more resistant to digestion by proteases. Thus, the regulatory properties of the membrane-bound phospholipase D seem to be dependent on the nature of the membranes and on the interaction between the enzyme and membranes as well. The alteration of regulatory properties by freezing was protected by sucrose, at lower concentrations, and more effectively for membranes from hardy tissues than for membranes from less hardy tissue. Addition of partially purified soluble phospholipase D to the reaction system containing membranes caused only a slight stimulation of the degradation of endogenous phospholipids. Phospholipid degradation in vivo during freezing of less hardy tissue may be catalyzed mainly by the bound enzyme. Disintegration of the tonoplast, however, besides releasing soluble phospholipase D into the cytosol, would release organic acids (lowering the pH) and free Ca²⁺. Both factors would stimulate drastically the membrane-bound phospholipase D, causing degradation of membrane phospholipids.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed C NMR

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the ¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting ¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴-10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However, ¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl ¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, ¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.