Plant Physiol, 1994 May, 105(1), 269 - 77 Expression of lipoxygenase in wounded tubers of Solanum tuberosum L; Geerts A et al.; A lipoxygenase cDNA clone from Solanum tuberosum L . was analyzed to study the role of lipoxygenases in potato development and wound response . Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant lipoxygenases . Expression of the cDNA sequences in Escherichia coli and subsequent analysis of bacterial protein extracts showed lipoxygenase activity using linoleic, linolenic, or arachidonic acid as substrates . Transcripts encoding the potato lipoxygenase were most abundant in tuber tissue, lower in roots, and hardly detectable in leaves, petioles, and stems . The induction of lipoxygenase expression in tubers by wounding was dependent on various parameters . Whereas lipoxygenase transcript levels increased in discs from stored tubers incubated under aerobic conditions, tubers taken from a growing plant did not accumulate lipoxygenase transcripts in response to wounding . Incubation of tuber discs in buffer did not lead to an increase in lipoxygenase RNA levels; however, methyl jasmonate stimulated lipoxygenase expression after 24 h in stored tubers . Proteinase inhibitor II mRNAs decreased in stored tubers as well as in discs from growing tubers. Free Radic Biol Med, 1994 May, 16(5), 555 - 9 Paraquat diaphorases in Escherichia coli; Liochev SI et al.; Extracts of E . coli contain at least three easily separable NAD(P)H:paraquat diaphorases . One of these is identified as thioredoxin reductase, which accounts for most of the PQ++ diaphorase in a thioredoxin reductase overproducer but is only 25% of this activity in a wild type . NADP+, but not NAD+, inhibited the diaphorase activity of thioredoxin reductase . All of the soluble PQ++ diaphorases of E . coli are stable during fractionation by HPLC and none depend upon the cooperative action of components separable by this technique . GSSG reductase is inhibited by PQ++ and is not, to any significant degree, a contributor to the diaphorase activity of E . coli. Free Radic Biol Med, 1994 May, 16(5), 539 - 45 Reactive oxygen inducing vasoconstriction in the isolated perfused rat liver; Ikai I et al.; The effect of reactive oxygen generation on intact livers was studied . Production of reactive oxygen species in perfused livers isolated from normal and endotoxin-treated rats was measured using chemically enhanced chemiluminescence . The resting state chemiluminescence of the livers increased on endotoxin administration and was maximal about 6 h after treatment . Chemiluminescence from the livers was further stimulated severalfold by inclusion of phorbol myristate acetate in the perfusion medium, reaching maximum intensity 3 h after endotoxin treatment . Oxygen consumption by the endotoxin-treated liver showed a transient increase followed by a significant decrease on phorbol myristate acetate stimulation, which was inhibited by dexamethasone . These results are consistent with the occurrence of a respiratory burst followed by oxygen-radical-species-induced vasoconstriction in the intact perfused liver . The evaluation of reactive oxygen species by resident and accumulated macrophages in the intact liver is made possible by these studies, and related effects on the liver could be conveniently and quantitatively followed using this model. Microbiology, 1994 May, 140 ( Pt 5), 1035 - 43 Molecular analysis of the operon which encodes the RNA polymerase sigma factor sigma 54 of Escherichia coli; Jones DH et al.; The rpoN gene (encoding the sigma factor sigma 54) of Escherichia coli was cloned and its nucleotide sequence determined . Promoter probe analysis confirmed the presence of a promoter in a 350 bp fragment covering the start of rpoN . The likely promoter was identified . The nucleotide sequence of the region extending 2.1 kb downstream of rpoN was also determined . This region contained four open reading frames encoding potential polypeptides of 10750, 17959, 32492 and 9810 Da; maxicell and T7 promoter studies showed that four polypeptides of similar molecular masses were expressed from this region . The amino acid sequence of the 17959 Da polypeptide showed homology to the enzyme IIA domains of several proteins of the bacterial sugar phosphotransferase system (PTS), and the 9810 Da polypeptide showed homology to the HPr proteins of the bacterial PTS . The proteins encoded downstream of rpoN are known to negatively regulate sigma 54 activity . The homologies therefore suggest that this effect on sigma 54 may be mediated by sequential protein phosphorylation and suggest that there is a link between signal transduction and transcription of sigma 54-dependent genes. Microbiology, 1994 May, 140 ( Pt 5), 1027 - 34 The high-spin cytochrome o' component of the cytochrome bo-type quinol oxidase in membranes from Escherichia coli: formation of the primary oxygenated species at low temperatures is characterized by a slow 'on' rate and low dissociation constant; Poole RK et al.; Cytochromes b and o in membrane vesicles from aerobically grown Escherichia coli were readily reduced by succinate; one cytochrome, which we propose should be called cytochrome o', reacted with CO in the Fe(II) state to give a photodissociable CO adduct . The photodissociation spectrum (photolysed minus pre-photolysis) at sub-zero temperatures had a relatively high gamma/alpha absorbance ratio, indicating a high-spin haem, which, in the reduced state, probably contributes little to the sharp alpha absorbance of the oxidase complex in membranes . Reaction with oxygen of the unliganded high-spin haem between -132 degrees C and -95 degrees C following photolytic activation gave a product that is identified as the oxygenated form, being spectrally similar to, but not identical with, the CO adduct . In membranes, the forward velocity constant at -95 degrees C was 61 M-1s-1, and the dissociation constant was 1.6 x 10(-5) M O2, as it is in intact cells . These data clearly distinguish the oxygen-trapping strategy of the cytochrome o' in this oxidase from that of cytochrome a3 and also suggest that the presence of the soluble flavohaemoglobin (Hmp) in intact cells is without effect on such measurements of the primary oxygen reaction . In view of recent findings that this oxidase complex contains predominantly one mole of haem O and one of haem B, a revised nomenclature for the oxidase complex is proposed, namely, cytochrome bo'. Biol Reprod, 1994 May, 50(5), 1108 - 12 Lipopolysaccharide-induced fetal death: the role of tumor-necrosis factor alpha; Silver RM et al.; Lipopolysaccharide (LPS) administration has been known to cause murine fetal death for over 50 years, but the responsible mechanism(s) remains unclear . We used the LPS-hyporesponsive murine strain, C3H/HeJ, to 1) establish whether LPS-induced fetal death is due to a maternal or fetal response to LPS and 2) to investigate the involvement of tumor necrosis factor alpha (TNF alpha) in fetal death caused by LPS . C3H/HeJ (LPS-hyporesponsive) or C3H/HeN (LPS-responsive) females were mated with C57B1/6 or C3H/HeN males (both LPS-responsive) . Administration of 10 micrograms LPS caused fetal death in C3H/HeN mothers . However, up to 1000 micrograms LPS did not result in the death of LPS-responsive fetuses when administered to C3H/HeJ mothers . Systemic administration of TNF alpha was able to cause fetal death in both C3H/HeN and C3H/HeJ strains of mice . Pretreatment of pregnant C3H/HeN mice with anti-TNF alpha antibodies significantly reduced fetal death caused by LPS administration . The administration of a sublethal dose of TNF alpha plus 10 micrograms LPS to pregnant C3H/HeJ mice restored abortifacient activity . These results indicate that LPS-induced fetal death is due to a maternal response as opposed to a direct fetal response to LPS and that TNF alpha appears to be an important mediator of fetal death caused by LPS. DNA Cell Biol, 1994 May, 13(5), 521 - 30 Characterization of a Max:DNA complex by cross-linking to photoactive oligonucleotides; Mullen MA et al.; The structure of the Max:DNA complex was investigated by cross-linking Max to a series of photoactive oligonucleotides . A single photoactive aryl azide was introduced into oligonucleotides at defined positions downstream from the specific CACGTG binding site . Purified Max homodimers bound to and were cross-linked to oligonucleotides containing a photoactive group either 2, 5, 8, or 11 bp downstream from the binding site . Further analysis revealed that an amino-terminal chymotryptic peptide of Max was cross-linked to the oligonucleotide containing a photoactive probe 11 bp downstream from the specific binding site . This result is consistent with the recent crystal structure of the Max:DNA complex (Ferre-D'Amare et al., 1993) and further suggests that amino acid residues near the amino-terminus of Max are in close proximity to a region of DNA that is separated from the core binding site by one turn of the double helix. Arzneimittelforschung, 1994 May, 44(5), 636 - 41 Comparative in vitro investigations of the influence of mofebutazone, phenylbutazone and diclofenac on phagocytosis and respiratory burst of human peripheral blood leucocytes; Neumuller J et al.; The phagocytosis and the release of oxidative products generated by the respiratory burst have been studied under the influence of the non-steroidal anti-inflammatories (NSAID) phenylbutazone (PB), mofebutazone (monophenylbutazone, MPB) and diclofenac (DF) using phagocytes of the peripheral blood from healthy human donors and patients with soft tissue rheumatism . The measurement of phagocytosis by flow cytometry (FC) was carried out in order to investigate the uptake of FITC-labelled bacteria (E . coli), separately by monocytes (MON) and polymorphonuclear leucocytes (PMN) . In addition the luminol-dependent chemiluminescence (CL) was measured using opsonized Zymosan on leucocytes of the peripheral blood that were purified by lysis of erythrocytes . In FC, PMNs and MONs could be identified by gating in the whole blood in which erythrocytes had been lysed . The NSAID were added to the in vitro tests for 30 min in concentrations of 10(-3) mol/l, 10(-4) mol/l, 10(-5) mol/l, 10(-6) mol/l, and 10(-8) mol/l . Using the FC phagocytosis test it was found that PB and MPB decreased the percentage of phagocytosing PMNs as well as MONs while DF increased it slightly in contrast to the controls . However, the fluorescence intensity of the phagocytes, which indicates the amount of ingested bacteria, was found to be unchanged . PB effected a significant concentration-dependent inhibition of CL in all of the concentrations used, with the exception of 10(-8) mol/l . MPB resulted in a borderline inhibition at 10(-3) mol/l although the measurement of every individual proband showed a concentration dependency . DF inhibited the luminol-dependent CL significantly only at a concentration of 10(-3) mol/l. Plant J, 1994 May, 5(5), 613 - 23 Sequencing, processing, and localization of the petunia CMS-associated mitochondrial protein; Nivison HT et al.; The petunia mitochondrial fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS . Pcf transcripts are modified by editing at 11 sites . Codon usage and nearest neighbor analysis suggest that the urfS region is not derived originally from a plant mitochondrial coding region . Although the gene contains an open reading frame coding for a 43 kDa protein, a 25 kDa gene product has previously been identified (Nivison and Hanson, 1989) . N-terminal sequencing revealed that the 25 kDa protein is encoded within the urfS portion of pcf and that its actual molecular mass is 19.5 kDa . Through pulse-chase labeling of protein in isolated mitochondria, the 25 kDa protein was found to be processed from a 43 kDa precursor protein representing the entire pcf gene sequence . Antibodies to synthetic peptides encoded by the atp9 and coxII portions of pcf recognized petunia ATP9 or COXII but no other mitochondrial proteins on immunoblots . Controlled proteolysis experiments showed that both the 43 kDa precursor and the 25 kDa protein are soluble or loosely associated with membranes . Thus, the 25 kDa protein appears to be the only pcf-encoded protein that accumulates in mitochondria. J Biomol NMR, 1994 May, 4(3), 433 - 54 1H, 15N and 13C resonance assignments for the first three zinc fingers of transcription factor IIIA; Liao X et al.; The first three zinc fingers (ZF1-3) of transcription factor IIIA (TFIIIA) from Xenopus have been shown to contribute the majority of the binding energy to the intact TFIIIA-DNA interaction {Liao et al . (1992) J . Mol . Biol., 223, 857-871} . We have expressed a 92-amino acid polypeptide containing the three N-terminal zinc fingers of TFIIIA . This three-fingered polypeptide has been isotopically labeled with 15N and 13C in E . coli and purified to homogeneity . Assignment of backbone 1H, 15N, aliphatic 1H and 13C and aromatic 1H and 13C resonances of delta NZF1-3 has been obtained using a combination of single-, double- and triple-resonance multidimensional NMR experiments . The secondary structures for each finger have been determined from NOE connectivities, 3JNH alpha values and chemical shifts . The results show that each finger folds into a canonical beta-sheet-helix zinc finger structural motif, while the linkers adopt an extended structure . The helix between the two histidine ligands in ZF3 is distorted by zinc coordination, to accommodate the presence of four intervening amino acids instead of three as in ZF1 and ZF2. J Biomol NMR, 1994 May, 4(3), 411 - 32 Effect of disulfide bridge formation on the NMR spectrum of a protein: studies on oxidized and reduced Escherichia coli thioredoxin; Chandrasekhar K et al.; As a prelude to complete structure calculations of both the oxidized and reduced forms of Escherichia coli thioredoxin (M(r) 11,700), we have analyzed the NMR data obtained for the two proteins under identical conditions . The complete aliphatic 13C assignments for both oxidized and reduced thioredoxin are reported . Correlations previously noted between 13C chemical shifts and secondary structure are confirmed in this work, and significant differences are observed in the C beta and C gamma shifts between cis- and trans-proline, consistent with previous work that identifies this as a simple and unambiguous method of identifying cis-proline residues in proteins . Reduction of the disulfide bond in the active-site Cys32-Gly-Pro-Cys35 sequence causes changes in the 1H, 15N and 13C chemical shifts of residues close to the active site, some of them quite far distant in the amino acid sequence . Coupling constants, both backbone and side chain, show some differences between the two proteins, and the NOE connectivities and chemical shifts are consistent with small changes in the positions of several side chains, including the two tryptophan rings (Trp28 and Trp31) . These results show that, consistent with the biochemical behavior of thioredoxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein. J Biomol NMR, 1994 May, 4(3), 385 - 410 Evaluation of an algorithm for the automated sequential assignment of protein backbone resonances: a demonstration of the connectivity tracing assignment tools (CONTRAST) software package; Olson JB Jr et al.; The peptide sequential assignment algorithm presented here was implemented as a macro within the CONnectivity TRacing ASsignment Tools (CONTRAST) computer software package . The algorithm provides a semi- or fully automated global means of sequentially assigning the NMR backbone resonances of proteins . The program's performance is demonstrated here by its analysis of realistic computer-generated data for IIIGlc, a 168-residue signal-transducing protein of Escherichia coli {Pelton et al . (1991) Biochemistry, 30, 10043-10057} . Missing experimental data (19 resonances) were generated so that a complete assignment set could be tested . The algorithm produces sequential assignments from appropriate peak lists of nD NMR data . It quantifies the ambiguity of each assignment and provides ranked alternatives . A 'best first' approach, in which high-scoring local assignments are made before and in preference to lower scoring assignments, is shown to be superior (in terms of the current set of CONTRAST scoring routines) to approaches such as simulated annealing that seek to maximize the combined scores of the individual assignments . The robustness of the algorithm was tested by evaluating the effects of imposed frequency imprecision (scatter), added false signals (noise), missing peaks (incomplete data), and variation in user-defined tolerances on the performance of the algorithm. J Biomol NMR, 1994 May, 4(3), 349 - 66 1H, 15N and 13C resonance assignments, secondary structure, and the conformation of substrate in the binary folate complex of Escherichia coli dihydrofolate reductase; Falzone CJ et al.; By using fully 15N- and 15N/13C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific 1H and 15N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the 13C alpha resonances in the binary folate complex . These assignments were made through a variety of three-dimensional proton-detected 15N and 13C experiments . A smaller but significant subset of side-chain 1H and 13C assignments were also determined . In this complex, only one 15N or 13C resonance was detected per 15N or 13C protein nucleus, which indicated a single conformation . Proton-detected 13C experiments were also performed with unlabeled DHFR, complexed with 13C-7/13C-9 folate to probe for multiple conformations of the substrate in its binary complex . As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7 . These results are consistent with an earlier report based on 1H NMR data {Falzone, C.J . et al . (1990) Biochemistry, 29, 9667-9677} and suggest that the E . coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex . This feature of the E . coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date. Plant Mol Biol, 1994 May, 25(2), 167 - 77 The 24 kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning, in vivo expression and import pathway of a protein with unusual properties; Fischer K et al.; The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane . Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template . This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence . The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da . It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues . Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca . 25 kDa) is due to its abnormal amino acid composition . Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane . Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP. FEMS Microbiol Lett, 1994 May 1, 118(1-2), 57 - 63 Identification, isolation and sequencing of the recA gene of Streptomyces lividans TK24; Nussbaumer B et al.; An internal fragment of the recA gene of Streptomyces cattleya was amplified by the polymerase chain reaction (PCR) employing degenerate oligonucleotide primers . Using this fragment as a hybridization probe, a recA homologous gene could be shown in each tested Streptomyces strain . A 4.4 kb BamHI fragment which carried the complete recA gene was isolated from Streptomyces lividans TK24 . Sequence analysis suggested that the coding region of the recA gene consists of 1122 bp . The highest similarity (approximately 78%) could be detected to the recA genes of Mycobacterium tuberculosis and Mycobacterium leprae . After fusion with an E . coli promoter the S . lividans recA gene could partially complement an Escherichia coli recA mutant. FEMS Microbiol Lett, 1994 May 1, 118(1-2), 51 - 6 Cloning and sequence analysis of a recA-like gene from Streptomyces venezuelae ISP5230; Yao W et al.; When genomic DNA fragments from Streptomyces venezuelae ISP5230 were probed at moderate stringency with recA from Mycobacterium tuberculosis, a 2.0-kb SmaI fragment was identified . The fragment was isolated by cloning a BamHI digest of S . venezuelae DNA in pHJL400 and screening the plasmids in Escherichia coli by Southern hybridization using a sib-selection technique . Sequencing the hybridizing region located an open reading frame encoding 377 amino acids . Its deduced amino acid sequence resembled that of recA genes from other bacteria . The cloned S . venezuelae gene conferred partial resistance to ethyl methanesulfonate when expressed in E . coli from the lacZ promoter. FEMS Microbiol Lett, 1994 May 1, 118(1-2), 167 - 73 Identification of an Escherichia coli periplasmic acid phosphatase containing of a 27 kDa-polypeptide component; Rossolini GM et al.; An acid phosphatase containing a 27-kDa polypeptide component has been identified in Escherichia coli by means of a zymogram technique . The enzyme is secreted in the periplasmic space and is able to hydrolyze several organic phosphate esters, but not diesters, showing preferential activity on p-nitrophenyl phosphate and other phenolic phosphate esters . Production of the enzyme apparently occurs only in cells growing on carbon sources other than glucose. Biophys Chem, 1994 May, 50(1-2), 47 - 61 De novo design and creation of a stable artificial protein; Tanaka T et al.; Protein de novo design has been performed, as an exercise of the inverse folding problem . A beta/alpha-barrel protein was designed and synthesized using the Escherichia coli expression system for the structural characterization . A tertiary model with a two-fold symmetry was built, based upon the geometrical parameters extracted from X-ray crystal structures of several beta/alpha-barrel proteins . Amino acid frequencies at each position on the alpha- and beta-structures were investigated, and an amino acid sequence with 201 residues was designed . The associated gene was chemically synthesized and the fusion protein with human growth hormone was expressed in Escherichia coli . The purified protein after being cleaved and refolded was found to be stable and globular with the large amount of secondary structures . However, it has similar characteristics to the molten globules of natural proteins, with loose packing of side-chains . The approach for the tight packing is discussed. Dev Comp Immunol, 1994 May-Jun, 18(3), 193 - 200 Mutation "white pupae" in the integument of Ceratitis capitata affects both defense and melanogenesis; Charalambidis ND et al.; Studying defense and melanogenesis processes in the cuticle of the "white pupae" (wp) and "dark pupae" (dp) mutant strains of Ceratitis capitata, we showed that both processes function equally well only in the cuticle of dp mutants, as in the wild-type cuticle . The cuticle of wp mutants lacks the ability to form Escherichia coli aggregates and to melanize in vivo . However, in this mutant, tyrosinase and dopachrome conversion factor activities, as well as melanin content and nonself-recognition proteins are expressed as in the wild strain . The present results indicate that the inability of wp mutant cuticle to immobilize E . coli seems to be due to lack of suitable site(s) on nonself-recognition proteins for adduct formation with tyrosine derivatives by the action of tyrosinase, and the inability to melanize, very probably due to deficiency of tyrosine derivatives (tanning precursors). Res Microbiol, 1994 May, 145(4), 333 - 40 Easy-to-perform modified Elek test to identify Shiga-like toxin-producing diarrhoeogenic Escherichia coli; Germani Y et al.; We determined whether Shiga-like toxin I (SLT-I) -producing diarrhoeogenic Escherichia coli could be detected by a modified Elek tests . The test (SLT Elek test) is based on the principle of the Elek test and the Ouchterlony double-gel diffusion . The development of the SLT Elek test was preceded by a preliminary study; the purpose of the later was to establish whether a simplified purification procedure of SLT-I (involving bacterial sonic extract, "Affi-Gel Blue" chromatography and anion- and cation-exchange liquid chromatography) could be employed in the preparation of rabbit antisera to SLT-I . SLT-I-specific antisera were obtained after adsorption of sera with bacterial sonic extract from non-toxigenic E . coli . A total of 135 strains of E . colo were tested by the SLT Elek test (100 SLT-I-negative and 35 SLT-I-positive) . The results of the SLT Elek test and the Vero cell test correlated well: 30 strains gave positive results and 100 strains gave negative results in both tests . Only 5 strains gave discrepant results: they were weakly positive in the Vero cell assay, whereas 3 gave borderline reactions and 2 were negative in the SLT Elek test . Positive predictive value was 1, negative predictive value was 0.98; the SLT Elek test was 91% sensitive and 100% specific. Res Microbiol, 1994 May, 145(4), 309 - 25 Ufr/s variation in Escherichia coli K12: a reversible double-mutation or alternate chromosome expression in non-complementing diploids? Gratia JP. A novel form of bacterial variation found in an FhuA- mutant of Escherichia coli K12 was characterized by the alternation of (1) simultaneous resistance to lipopolysaccharide-specific phage U3 and to FhuA-specific agents (Ufr phenotype); and (2) a return to the sensitivity pattern of the initial strain (Ufs) . In Ufr cells, loss of the U3 receptor permitted C21 adsorption without modifying the sensitivity to other tested phages or colicins . Genetic analysis revealed that Ufr variants were altered at two distinct loci . Ufr bacteria, though derived from a strain F- devoid of classical gene transfer mechanisms, were transiently able to promote mating between themselves and, to some extent, with other bacteria, including Rec- . Heterogenic matings resulted in the formation of persistent heterozygotes segregating Ufr- and Ufs-like bacteria . Pedigree analysis and subcloning of heterozygotic isolates indicate that they were diploids, as was the initial Ufr strain . Functional genetic complementation between these two genomes was only transient and the alternative forms were likely to result from the expression of a single chromosome of the heterozygotes . Mutation occurred in either form without causing any change in the alternative form. Mikrobiologiia, 1994 May-Jun, 63(3), 450 - 6 {Effect of over-synthesis of cloned gene products on methanol metabolism in the recombinant methylotrophic yeast Hansenula polymorpha}; Gorlatova NV et al.; As is shown expression homologous (dihydroxyacetone kinase) and heterologous (HBsAg, beta-galactosidase) genes in methylotrophic yeasts Hansenula polymorpha DL1 negatively affects on the growth parameters of a host strain . The reducing of specific growth rate (mu max) and yield of biomass per the unit of a consumed substrate (Yx/s) were found in all recombinant strains grown on methanol . Overproduction of dihydroxyacetone kinase and beta-galactosidase in recombinant H . polymorpha was accompanied by two-fold increasing of the activity of alcohol oxidase, which is the first enzyme of methanol oxidation . Otherwise, the activity of formaldehyde dehydrogenase two-fold decreased in the recombinant strain overproducing HBsAg compared with the host strain . It is suggested that the over-synthesis of foreign proteins requiring an additional energetic and metabolic expenses might reduce the growth parameters and the activities of some enzymes of methanol metabolism in recombinant methylotrophic yeast H . polymorpha. J Struct Biol, 1994 May-Jun, 112(3), 216 - 30 Transmission electron microscopy of GroEL, GroES, and the symmetrical GroEL/ES complex; Harris JR et al.; Two new 2-D crystal forms of the Escherichia coli chaperone GroEL (cpn60) 2 x 7-mer have been produced using the negative staining-carbon film (NS-CF) technique . These 2-D crystals, which contain the cylindrical GroEL in side-on and end-on orientations, both possess p21 symmetry, with two molecules in the respective unit cells . The crystallographically averaged images correlate well with those obtained by other authors from single particle analysis of GroEL and our own previous crystallographic analysis . 2-D crystallization of the smaller chaperone GroES (cpn10) 7-mer has also been achieved using the NS-CF technique . Crystallographically averaged images of GroES single particle images indicate considerable variation in molecular shape, which is most likely due to varying molecular orientation on the carbon support film . The quaternary structure of GroES does, nevertheless, approximate to a ring-like shape . The complex formed by GroEL and GroES in the presence of ATP at room temperature has been shown to possess a symmetrical hollow ellipsoidal conformation . This symmetrical complex forms in the presence of a 2:1 or greater molar ratio of GroES:GroEL . At lower molar ratios linear chains of GroEL form, apparently linked by GroES in a 1:1 manner, which provide supportive evidence for the ability of both ends of the GroEL cylinder to interact with GroES . The apparent discrepancy between our data and that of other groups who have described an asymmetrical "bullet-shaped" (holo-chaperone) GroEL/ES complex is discussed in detail. Minerva Chir, 1994 May, 49(5), 429 - 31 {The conservative treatment of infected vascular prostheses in surgery to revascularize the lower limbs}; Celoria G et al.; Infected vascular grafts are associated with very high rates of limb loss and mortality . "Classic" treatment has invariably included graft excision . Recent reports have suggested that a more conservative approach may be indicated in selected cases, leaving the graft in place and using an aggressive local treatment associated with appropriate intravenous antibiotics . The authors report their experience with two patients with infected prosthetic vascular grafts in the groin . They both had purulent drainage from the groin wound, with the graft exposed close to the femoral anastomosis . They were both treated successfully without graft removal, and both graft maintained patency, with a follow-up of 22 and 19 months. J Biochem (Tokyo), 1994 May, 115(5), 958 - 64 Hot spots for sulfhydryl inactivation of Cys mutants in the widely conserved sequence motifs of the metal-tetracycline/H+ antiporter of Escherichia coli; Yamaguchi A et al.; Unique hot spots for sulfhydryl inactivation of Cys mutants of the metal-tetracycline/H+ antiporter (TetA) were found at the fourth positions of the dual conserved sequence motifs, GKXXDRXGRR and GRXXXKXGEK {Yamaguchi, A., Kimura, T., Someya, Y., & Sawai, T . (1993) J . Biol . Chem . 268, 6496-6504} . The fourth positions of these motifs are occupied by Ser65 and Ala269, respectively . N-Ethylmaleimide (NEM) rapidly bound to and inactivated the S65C and A269C mutants . M64C and I268C showed low reactivity to NEM, probably due to the partial cripticity of these residues . In contrast, NEM rapidly bound to T270C but did not inactivate it . NEM completely inactivated S65C, whereas a small sulfhydryl reagent, methyl methanethiosulfonate (MMTS), caused only 40% inactivation . {14C}NEM binding to S65C was inhibited by tetracycline . These observations indicated that position 65 is located close to the substrate-protein interaction site and that the inactivation by sulfhydryl reagents comprises volume-dependent steric hindrance . The S65M mutant, having a side chain analogous to one of the thiomethyl cysteines of MMTS-modified S65C, showed about 30% of the Vmax value for the wild-type tetracycline transport, while the S65F mutant had completely lost the activity, confirming the idea of volume-dependent steric hindrance at position 65 . On the other hand, the A269C mutant was greatly inactivated by both NEM and MMTS . The degree of the inactivation by MMTS (90%) was higher than that of NEM (80%) . The synergetic inactivation observed for the S65C/A269C double mutant suggested different inactivation mechanisms for A269C and S65C.(ABSTRACT TRUNCATED AT 250 WORDS) J Biochem (Tokyo), 1994 May, 115(5), 1021 - 6 Archae-opsin expressed in Escherichia coli and its conversion to purple pigment in vitro; Sugiyama Y et al.; Archae-opsin-1 (aO-1) has been expressed efficiently as a fusion protein with 13 heterologous amino acids at the amino terminus of the mature aO-1 in Escherichia coli under the control of T7 promoter . The E . coli-expressed aO-1, designated as aO-1002, which was located in the membrane fraction, was extracted with 8 M urea and partially purified by gel filtration chromatography in the presence of SDS . When all-trans retinal was added, aO-1002 in dimyristoylphosphatidylcholine and detergent-mixed micelles was converted to a purple pigment with lambda max at 558 nm at 20 degrees C via a 435/460 nm intermediate . Conversion of the intermediate to purple pigment was the rate-limiting step and proceeded as a two-state transition, because an isosbestic point was seen at 485 nm . Similar spectral changes were also observed in the regeneration process of hydroxylamine-bleached claret membranes and aO-1 isolated from claret membranes . Thus, the polypeptide of aO-1002 is considered to fold and form a retinal binding pocket in phospholipid and detergent micelles similarly to aO-1 isolated from the halobacterial membranes . Purple pigment showed a light-driven proton-pumping activity when reconstituted into phosphatidylcholine liposomes. Biochem Mol Biol Int, 1994 May, 33(2), 377 - 84 Altered conformation and increased strand breaks in neuronal and astroglial DNA of aging rat brain; Bhaskar MS et al.; Melting temperatures (Tm) of the DNA isolated from young, adult, and old rat brain neurons and astrocytes were recorded under different conditions . There was a rise in Tm and decrease in hyperchromicity in the old when compared to the young and adult . Single and double strand breaks were assessed by using nick translation type incubation of DNA with E . coli Pol I and addition of nucleotides at the terminal 3'-OH by calf thymus terminal deoxynucleotidyl transferase . Results show that DNA from old brain cells is more compact in conformation . However, there is also an increase in the number of single and double strand breaks with age in both neuronal and astroglial DNA. Appl Biochem Biotechnol, 1994 May-Jun, 47(2-3), 175 - 89; discussion 189-90 Phage display of enzymes and in vitro selection for catalytic activity; Soumillion P et al.; Despite recent progress, our understanding of enzymes remains limited: the prediction of the changes that should be introduced to alter their properties or catalytic activities in an expected direction remains difficult . An alternative to rational design is selection of mutants endowed with the anticipated properties from a large collection of possible solutions generated by random mutagenesis . We describe here a new technique of in vitro selection of genes on the basis of the catalytic activity of the encoded enzymes . The gene coding for the enzyme to be engineered is cloned into the genome of a filamentous phage, whereas the enzyme itself is displayed on its surface, creating a phage enzyme . A bifunctional organic label containing a suicide inhibitor of the enzyme and a ligand with high affinity for an immobilized receptor are constructed . On incubation of a mixture of phage enzymes, those phages showing an activity on the inhibitor under the conditions of the experiment are labeled . These phages can be recovered by affinity chromatography . The design of the label and the factors controlling the selectivity of the selection are analyzed . The advantages of the technique and its scope in terms of the enzymes that can be engineered are discussed. J Biolumin Chemilumin, 1994 May-Jun, 9(3), 113 - 22 Low-light imaging technology in the life sciences; Hooper CE et al.; Photon imaging is an increasingly important technique for the measurement and analysis of chemiluminescence and bioluminescence . New high-performance low-light level imaging systems have recently become available for the life sciences . These systems use advances in camcera design and digital image processing and are now being used for a wide range of luminescence applications . They offer good sensitivity for photon detection and large dynamic range, and are suitable for quantitative analysis . This is achieved using a range of software techniques including image arithmetic, histogramming or summing regions of interest, feature extraction and multiple image processing for kinetics or assay screening . Improvements in image-processing hardware and software have increased the usefulness of these systems in the biosciences . Low-light imaging is a rapid and non-invasive method for the sensitive detection and analysis of luminescent assays . As such it offers a powerful and sensitive tool for investigating processes, both at the cellular level (luc and lux reporter genes, intracellular signalling) and for measurement of macro samples (immunoassays, gels and blots, tissue sections). Somat Cell Mol Genet, 1994 May, 20(3), 233 - 42 Removal of cyclobutane pyrimidine dimers from a UV-irradiated shuttle vector introduced into human cells; Ganesan AK et al.; A shuttle vector (pZH-1) carrying the E . coli lacZ gene under control of the SV40 early promoter was irradiated with UV and introduced into repair-proficient or repair-deficient human cell lines . The expression of irradiated lacZ compared to unirradiated lacZ was greater in repair-proficient cells (HT-1080) than in repair-deficient cells (XP12RO-SV40) belonging to xeroderma pigmentosum complementation group A . To ascertain whether the expression of lacZ in the repair-proficient cells was correlated with the removal of cyclobutane pyrimidine dimers (CPDs), we purified DNA from the recipient cells and used the CPD-specific enzyme T4 endonuclease V to measure the frequency of CPDs remaining in the plasmid as a whole and in two restriction fragments derived from it . We found that removal of CPDs occurred in both fragments in the repair-proficient cells but not in the repair-deficient cells . Our results provide the first direct evidence for the removal of CPDs from UV irradiated plasmids introduced into human cells and support the notion that expression of the UV-damaged lacZ gene in repair-proficient human cells reflects the removal of transcription blocking lesions from the gene. Mutagenesis, 1994 May, 9(3), 245 - 51 Induction of the SOS response and mutations by reactive oxygen-generating compounds in various Escherichia coli mutants defective in the mutM, mutY or soxRS loci; Kato T et al.; Derivatives of E . coli WP2s (uvrA trpE) defective in 7,8-dihydro-8-oxoguanine (8-OG) DNA glycosylase activity (mutM), MutY glycosylase activity on an A:8-OG mispair (mutY), and/or an adaptive response to oxidative stress by superoxide (soxRS) were constructed to compare the mutability to various reactive oxygen-generating compounds . Induction of Trp+ reversion was assayed both in the presence and absence of plasmid pKM101 . Phenazine methosulfate and phenazine ethosulfate showed mutagenic activity at a relatively low dose in soxRS mutants . In comparison to the parent strain WP2s, however, the introduction of mutM, mutY, or soxRS mutations, in any combination, did not make the strain hypersensitive in terms of mutability (i.e . mutation induction at relatively low doses) to hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or phenylhydrazine . Mutagenicity of formaldehyde was detected only in the pKM101-carrying strains . On the other hand, bleomycin, menadione, plumbagin, paraquat, and diquat were not mutagenic to any strain, with or without pKM101 . The SOS-response inducing activity was measured by monitoring the expression of a umu'-'lacZ fusion gene, carried on the plasmid pSK1002 . The induction of the SOS response by hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, formaldehyde, phenylhydrazine, and bleomycin was of almost the same magnitude between the parent strain and a mutM or soxRS mutant . Phenazine methosulfate and phenazine ethosulfate induced the SOS response only in the soxRS derivatives . No induction was detected by treatment with redox-cycling compounds, such as menadione, plumbagin, paraquat, or diquat. Mutagenesis, 1994 May, 9(3), 183 - 5 Measurement of gene mutation in vivo using Muta Mouse and positive selection for lacZ- phage; Dean SW et al.; The use of transgenic animals for in vivo mutagenicity studies following rescue of the bacterial lacZ transgene has been hindered by the sheer scale of the experimental work involved . We describe here a new positive selection protocol which is based upon a modified E . coli bacterial host . This new system is potentially capable of generating mutation data much faster and more cheaply than previous methods. Mol Microbiol, 1994 May, 12(4), 631 - 8 Novel growth rate control of dam gene expression in Escherichia coli; Rasmussen LJ et al.; Transcription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters . Single-copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence . Promoter P2 is a typical sigma 70 promoter with 18 bp spacing between the -10 and -35 hexamers . Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response . Beta-galactosidase specific activity from a single-copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein . Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feedback and enhanced transcription by Fis protein . We devised a procedure for selection of mutant cells in which dam gene expression was unregulated . One such mutant (cde-4), obtained by miniTn10 insertion, showed the same level of beta-galactosidase activity at all growth rates tested . In contrast, growth rate-dependent expression of the rrnB gene was unaffected by cde-4 confirming the different modes of regulation . The cde-4::miniTn10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene. Mol Microbiol, 1994 May, 12(4), 621 - 9 Control of the LexA regulon by pH: evidence for a reversible inactivation of the LexA repressor during the growth cycle of Escherichia coli; Dri AM et al.; The LexA repressor controls the expression of several genes, including lexA, recA, and sfiA, which are induced when exponentially growing bacteria are exposed to DNA-damaging agents . Induction of this so-called SOS response takes place while LexA is cleaved in a reaction that requires the RecA protein and damaged DNA . We have shown that large fluctuations in the cellular concentration of the LexA repressor and in the rate of transcription of the sfiA gene also occur spontaneously during bacterial growth in complex medium such as LB . The possibility that changes in external or internal pH may explain these fluctuations has been explored . A consistent pattern was established whereby conditions leading to either increased or decreased pH were associated with altered expression of the lexA and sfiA genes . These data can be explained by a model in which the LexA repressor exists in either of two forms in equilibrium: a form favoured at homeostatic internal pH, which has a low affinity for the operators of LexA-controlled genes; and a form accumulated in response to a transient decrease in internal pH, which has a high affinity for operators. Mol Microbiol, 1994 May, 12(4), 579 - 86 NarK is a nitrite-extrusion system involved in anaerobic nitrate respiration by Escherichia coli; Rowe JJ et al.; Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration . A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter . The longest-lived radioactive isotope of nitrogen, 13N-nitrate (half-life = 9.96 min) and the nitrite-sensitive fluorophore N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide have now been used to define the function of NarK . At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange . This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase. Mol Microbiol, 1994 May, 12(4), 571 - 8 Role of rpoS (katF) in oxyR-independent regulation of hydroperoxidase I in Escherichia coli; Ivanova A et al.; We present evidence showing that rpoS (katF) is a regulator of katG gene transcription in an oxyR-independent manner . Mutation of the rpoS gene in several different Escherichia coli strains caused a significant reduction in catalase HPI activity . In rpoS-delta oxyR double mutants, the level of HPI was considerably lower compared to the delta oxyR parent strain, and was restored when transformed with an rpoS+ plasmid . Overproduction of HPI in oxyR- suppressor strains was greatly diminished after inactivation of the rpoS gene and was accompanied by a substantial increase in sensitivity to menadione . Beta-galactosidase expression from a katG::lacZ promoter was lower in rpoS strains compared to rpoS+ isogenic parents . Several delta oxyR strains had detectable levels of katG transcription that was significantly diminished after rpoS gene inactivation. J Antimicrob Chemother, 1994 May, 33 Suppl A, 93 - 7 Bacteriuria of pregnancy--an update on significance, diagnosis and management; Bint AJ et al.; Bacteriuria of pregnancy is a common condition which is thought to be associated with serious sequelae in mother and fetus . A programme of screening, treatment and follow-up is likely to be cost-effective, but will depend on local prevalences of bacteriuria and pyelonephritis . If treatment is undertaken, nitrofurantoin and cephalexin are both safe and effective and have resistance rates of below 10% . Amoxycillin is also safe, but resistance rates in Escherichia coli are about 40% . A 7 day course of treatment remains advisable. Exp Eye Res, 1994 May, 58(5), 573 - 84 Expression of recombinant bovine gamma B-, gamma C- and gamma D-crystallins and correlation with native proteins; Hay RE et al.; Despite the use of bovine gamma-crystallins in numerous biophysical and chemical studies, characterization of these proteins at the molecular level is incomplete . Bovine lenses have at least six gamma-crystallin protein fractions currently assigned as gamma s/I, gamma A/IVb, gamma B/II, gamma C/IIIb, gamma D/IIIa and gamma E/IVa . A lack of primary sequence data for corresponding gamma-crystallin genes and proteins, however, has made these assignments tenuous . To clarify these assignments, we have over-expressed recombinant bovine gamma-crystallin proteins in Escherichia coli using complementary DNAs corresponding to gamma B-, gamma C-, and gamma D-crystallin genes . The recombinant crystallins were characterized by chromatographic and spectroscopic comparisons with native bovine crystallin fractions gamma II, gamma IIIa and gamma IIIb . The elution of recombinant gamma B and native gamma II proteins was identical on cation-exchange chromatography as expected; however, recombinant gamma C coeluted with gamma IIIa and recombinant gamma D co-eluted with gamma IIIb . Sequential Edman degradation through the first 29 residues of the native gamma IIIa and gamma IIIb polypeptides confirmed the colinearity of their sequences with those predicted from the gamma C- and gamma D-crystallin genes, respectively . Absorption and UV circular dichroism (CD) spectra of the recombinant proteins were almost identical to those of their native counterparts, indicating that the secondary and tertiary structures of the recombinant proteins were characteristic for gamma-crystallins . Based on these data the bovine gamma-crystallins proteins and genes are correlated as follows: II/gamma B, IIIa/gamma C and IIIb/gamma D . The assignment of gamma IIIb (previously characterized as having a low critical temperature for phase separation) with gamma D rather than gamma C proves an exception to the hypothesis that the gamma ABC-crystallin group is more resistant to phase separation than the gamma DEF group . These corrected assignments should provide a more substantial base for investigations of residues responsible for phase separation and other biophysical characteristics . Additionally, expression of recombinant gamma-crystallins with structures similar to native proteins may prove to be useful in probing specific structure-function relationships of the gamma-crystallins. Electrophoresis, 1994 May, 15(5), 647 - 53 Purification of human recombinant superoxide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes; Wenisch E et al.; Human recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (pIs) in the pH range 4.8 to 5.1, the pI 4.80 form representing the minor component . Due to the fact that this enzyme is expressed in E . coli, N-terminal acetylation or glycosylation should be ruled out . When purified by small-scale preparative isoelectric focusing in immobilized pH gradient gels, it was found that, upon subsequent analysis, the pI 5.07 form would band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nearly the same amounts, whereas the lowest pI component would always generate both the intermediate and upper forms . Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, slightly higher than that of the starting material . Metal analysis indicated that all three forms contained the same metal/protein ratio, approaching the value Cu2Zn2-SOD, as reported in the literature . Circular dichroism spectra of the pI 4.80 and 5.07 forms showed the same profile in the 190-240 nm range, but marked differences in the 250-350 nm region . Treatment with EDTA produces 1-2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher pI components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms . It is concluded that these three isoforms could represent interconvertible species, the highest pI component representing the most stable conformer. Chin Med J (Engl), 1994 May, 107(5), 338 - 41 Detection of genes for heat-stable enterotoxin in Escherichia coli by biotinylated ST-DNA probes; Zhu QY et al.; Reference strains of enterotoxigenic Escherichia coli (ETEC), non-enterotoxigenic Escherichia coli (non-ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), and other enteropathogenic bacteria were used to prove the reliability of BIO-ST-DNA probe hybridization . In addition, 417 strains of E . coli isolated from children with diarrheal diseases in Shanxi Children's Hospital were examined for BIO-ST-DNA probe hybridization . In the test, BIO-ST-DNA hybridization was compared with suckling mouse assay in identifying ST-ETEC . The results obtained by both methods showed no significant difference . It was found that identification of ST-ETEC using hybridization is a simple, sensitive and more practical method. Bioorg Med Chem, 1994 May, 2(5), 331 - 8 Functionalized 3,5-dihydroxybenzoates as potent novel inhibitors of EPSP synthase; Miller MJ et al.; Aromatic analogues of the EPSP synthase enzyme substrate (S3P), reaction intermediate (1), and product (EPSP) were synthesized from 3,5-dihydroxybenzoic acid and were evaluated as inhibitors of E . coli EPSP synthase . These simple, synthetically accessible aromatic analogues are highly effective competitive inhibitors versus S3P with an apparent Ki for the tetrahedral intermediate analogue 4 of 160 +/- 40 nM . This demonstrates that a simple benzene ring is a quite suitable substitute for the complex shikimate ring in the design of EPSP synthase inhibitors. J Am Soc Nephrol, 1994 May, 4(11), 1890 - 5 The effect of uremia on tumor necrosis factor-alpha release after an in vitro whole-blood endotoxin challenge; Oettinger CW et al.; Uremia has been associated with immunologic aberrations, including anergy, increased susceptibility to infections, and reduced phagocytic activity of polymorphonuclear leukocytes . In this study, cytokine release in uremic and nonuremic blood after in vitro endotoxin stimulation was studied . Blood from nonuremic controls, chronic renal failure patients not on dialysis, and chronic hemodialysis patients predialysis and postdialysis was spiked with 10 ng/mL of Escherichia coli endotoxin and incubated for 2 and 26 h . Plasma tumor necrosis factor-alpha (TNF alpha) concentrations were determined by ELISA after each incubation period . To further study which uremic blood component may be responsible for enhanced release of TNF alpha, plasma and cellular components of chronic renal failure patients and controls were switched and then given an in vitro endotoxin stimulation (1 ng/mL) . It was found that (1) TNF alpha release is enhanced by uremia and is exacerbated with progressive declines in renal function, (2) enhanced TNF alpha release is related to a blood cellular phenomenon induced by uremia, and (3) enhanced TNF alpha release in hemodialysis patients is associated with a prolonged stimulation and/or reduced plasma elimination of TNF alpha. J Invest Surg, 1994 May-Jun, 7(3), 175 - 86 Role of topical phospholipids in the prophylaxis of silicone elastomer-associated infection in the abdominal cavity; Guo W et al.; The present study evaluated the influence of phospholipids (phosphatidyl choline and phosphatidyl inositol) on the prevention of abdominal biomaterial-associated infection . Phospholipid-impregnated silicone elastomer (SE) fragments were either intraperitoneally implanted in rats or immersed in serum for 0, 4, and 14 days, and 3 x 10(9) cfu of 3H-labeled, live Escherichia coli were added in the peritoneal cavity or in vitro incubation medium . Three hours after incubation, the adherence of bacteria significantly decreased to phospholipid-impregnated SE fragments, which had been immersed/implanted for 0 and 4 days . However, the number of adhering bacteria did not differ between the impregnated and unimpregnated SE fragments after 14 days of immersion/implantation . A significantly lower number of adhering bacteria was noted on all unimpregnated SE fragments when phospholipid was supplemented in the peritoneal cavity or in vivo medium, compared with fragments with no supplement . The rate of bacterial DNA synthesis decreased significantly after incubation with phospholipid 2 h or more . Phospholipids did not further influence peritoneal morphology . Thus topical administration of phospholipids by impregnation to the surface of SE fragments or supplement in the incubation medium prevented bacterial adherence onto the SE fragments . This implies that the use of phospholipids might be a mode of preventing biomaterial-associated infections. AIDS Res Hum Retroviruses, 1994 May, 10(5), 595 - 600 Identification and characterization of the Bel 3 protein of human foamy virus; Weissenberger J et al.; The human foamy virus (HFV) is a complex retrovirus that contains several regulatory and auxiliary bel genes besides the gag, pol, and env genes . In contrast to the gene products of bel 1 and bel 2/bet that were identified previously, the Bel 3 protein has not been described to date . Here we report the identification of Bel 3 in HFV-infected cells by immunoprecipitation, indirect immunofluorescence, and expression cloning under the control of a strong heterologous promoter . Bel 3 was immunoprecipitated with an antiserum directed against a bacterially expressed and purified form of recombinant Bel 3 antigen . Bel 3 was found to be expressed in low amounts in the cytoplasm of HFV-infected cells and to migrate with an apparent molecular mass of 19.4 kDa on electrophoresis in SDS-polyacrylamide gels, consistent with the calculated value of 18.2 kDa . Radioimmunoprecipitation of HFV-infected cell lysates with the hyperimmune serum against Bel 3 revealed at least two additional immunoreactive bands of 15.5 and 10.6 kDa . The results indicate that Bel 3 was labile, because it was partially degraded even at early time points after infection . On transfection and expression in transfected COS cells, recombinant Bel 3 was immunoprecipitated and migrated in three polypeptide bands of 18.7, 14.8, and 9.3 kDa under denaturing conditions . In the absence of reducing agents, the bacterially expressed and purified recombinant Bel 3 protein of 16.1 kDa can form homodimers of 30 kDa. Protein Eng, 1994 May, 7(5), 689 - 96 Site-directed mutagenesis of a calcium binding site modifies specifically the different biochemical properties of annexin I; Trave G et al.; All the functions of annexins in vitro as well as in vivo are mediated and probably regulated by calcium . We have used recombinant annexin I, synthesized by Escherichia coli, and we have performed site-directed mutagenesis . We have mutated the endonexin fold of domain 2 that binds calcium . Mutations were performed in this domain of the molecule because it perfectly matches the calcium binding consensus sequence . The two glycines of this fold were mutated into glutamic acid . The helix content and the stability of the mutants are identical to those of the wild-type, suggesting that the mutations did not drastically affect the structure of the protein . The two mutants showed modified calcium binding affinities . However, the calcium binding affinity of the G131E mutant was far more altered than that of the G129E mutant . Furthermore, other biochemical properties of these mutants were modified to different extents . The binding to phospholipid was not seriously affected, whereas the self-association was lost by the G131E mutant . In the same way, liposome aggregation is conserved, but modified, while the calcium affinity measured by equilibrium dialysis is dramatically altered. Mol Microbiol, 1994 May, 12(3), 423 - 32 Post-transcriptional regulation of the groEL1 gene of Streptomyces albus; Servant P et al.; Thermally induced expression of the heat-shock gene groEL is subject to post-transcriptional regulation in Streptomyces albus . When S . albus cells were shifted from 30 degrees C to 41 degrees C, synthesis of three GroEL-like proteins was induced from two genes transcribed from associated promoters P1 and P2 . Surprisingly, analyses of transcriptional fusions of these promoters with various reporter genes indicated constitutive expression independent of heat shock . In contrast, neo expression was thermally inducible as a GroEL1-APH translational fusion protein . Furthermore, expression of the groEL1-neo gene was heat inducible even after the groEL1 promoter region was replaced by a heterologous non-heat-inducible promoter such as the Escherichia coli lac promoter . Finally, synthesis of GroE proteins, as well as the GroEL-APH fusion protein, was heat inducible when their transcription was inhibited by rifampicin . Post-transcriptional regulatory signals needed for heat-induced GroEL1 synthesis were mapped within of the groEL1 structural gene. Mol Microbiol, 1994 May, 12(3), 387 - 401 CooC and CooD are required for assembly of CS1 pili; Froehlich BJ et al.; Many strains of enterotoxigenic Escherichia coli (ETEC) isolated from patients with diarrhoeal disease exhibit CS1 pili on their surfaces . These appendages, which are thought to be important for colonization of the upper intestine, are composed largely of multiple identical protein subunits encoded by cooA . We have sequenced the DNA directly downstream of cooA and identified two open reading frames, cooC and cooD, transcribed in the same direction as cooB and cooA . Following cooD is DNA homologous to an insertion sequence, so cooB, A, C and D appear to encode all the information needed for E . coli K-12 to synthesize CS1 pili . Complementation analysis of mutants cloned in E . coli K-12 and constructed in an ETEC-derived strain indicates that cooC and cooD are not required for stability of the major CS1 pilin protein or for its transport to the periplasm, but, like cooB, both are needed for assembly of cooA into pili. FEMS Microbiol Lett, 1994 May 1, 118(1-2), 83 - 8 Morphological appearances of K88ab fimbriae and optical diffraction analysis of K88 paracrystalline structures; Simons BL et al.; K88ab fimbriae are filamentous protein structures at the surface of certain enterotoxigenic Escherichia coli strains . Electron microscopy analysis of K88ab fimbriae showed that these structures have different morphological appearances dependent on the medium in which cells expressing these fimbriae or in which purified fimbriae were suspended . Thin and curled structures, thin and flexible fimbriae, a wider and rigid form of the fimbriae, and, in addition, paracrystalline structures were detected . Optical diffraction analysis of the paracrystalline structures indicated a helical conformation of K88ab fimbriae. J Bacteriol, 1994 May, 176(10), 2938 - 45 Effects of transcriptional start site sequence and position on nucleotide-sensitive selection of alternative start sites at the pyrC promoter in Escherichia coli; Liu J et al.; In Escherichia coli, expression of the pyrC gene is regulated primarily by a translational control mechanism based on nucleotide-sensitive selection of transcriptional start sites at the pyrC promoter . When intracellular levels of CTP are high, pyrC transcripts are initiated predominantly with CTP at a site 7 bases downstream of the Pribnow box . These transcripts form a stable hairpin at their 5' ends that blocks ribosome binding . When the CTP level is low and the GTP level is high, conditions found in pyrimidine-limited cells, transcripts are initiated primarily with GTP at a site 9 bases downstream of the Pribnow box . These shorter transcripts are unable to form a hairpin at their 5' ends and are readily translated . In this study, we examined the effects of nucleotide sequence and position on the selection of transcriptional start sites at the pyrC promoter . We characterized promoter mutations that systematically alter the sequence at position 7 or 9 downstream of the Pribnow box or vary the spacing between the Pribnow box and wild-type transcriptional initiation region . The results reveal preferences for particular initiating nucleotides (ATP > or = GTP > UTP >> CTP) and for starting positions downstream of the Pribnow box (7 >> 6 and 8 > 9 > 10) . The results indicate that optimal nucleotide-sensitive start site switching at the wild-type pyrC promoter is the result of competition between the preferred start site (position 7) that uses the poorest initiating nucleotide (CTP) and a weak start site (position 9) that uses a good initiating nucleotide (GTP) . The sequence of the pyrC promoter also minimizes the synthesis of untranslatable transcripts and provides for maximum stability of the regulatory transcript hairpin . In addition, the results show that the effects of the mutations on pyrC expression and regulation are consistent with the current model for translational control . Possible effects of preferences for initiating nucleotides and start sites on the expression and regulation of other genes are discussed. J Bacteriol, 1994 May, 176(10), 2814 - 21 Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action; Jiang P et al.; The effects of inhibition of Escherichia coli phospholipid synthesis on the accumulation of intermediates of the fatty acid synthetic pathway have been previously investigated with conflicting results . We report construction of an E . coli strain that allows valid {14C}acetate labeling of fatty acids under these conditions . In this strain, acetate is a specific precursor of fatty acid synthesis and the intracellular acetate pools are not altered by blockage of phospholipid synthesis . By use of this strain, we show that significant pools of fatty acid synthetic intermediates and free fatty acids accumulate during inhibition of phospholipid synthesis and that the rate of synthesis of these intermediates is 10 to 20% of the rate at which fatty acids are synthesized during normal growth . Free fatty acids of abnormal chain length (e.g., cis-13-eicosenoic acid) were found to accumulate in glycerol-starved cultures . Analysis of extracts of {35S}methionine-labeled cells showed that glycerol starvation resulted in the accumulation of several long-chain acyl-acyl carrier protein (ACP) species, with the major species being ACP acylated with cis-13-eicosenoic acid . Upon the restoration of phospholipid biosynthesis, the abnormally long-chain acyl-ACPs decreased, consistent with transfer of the acyl groups to phospholipid . The introduction of multicopy plasmids that greatly overproduced either E . coli thioesterase I or E . coli thioesterase II fully relieved the inhibition of fatty acid synthesis seen upon glycerol starvation, whereas overexpression of ACP had no effect . Thioesterase I overproduction also resulted in disappearance of the long-chain acyl-ACP species . The release of inhibition by thiosterase overproduction, together with the correlation between the inhibition of fatty acid synthesis and the presence of abnormally long-chain acyl-ACPs, suggests with that these acyl-ACP species may act as feedback inhibitors of a key fatty acid synthetic enzyme(s). EMBO J, 1994 May 1, 13(9), 2089 - 96 Relaxation of replication control in chaperone-independent initiator mutants of plasmid P1; Mukhopadhyay G et al.; Escherichia coli chaperones DnaJ, DnaK and GrpE increase P1 plasmid initiator binding to the origin by promoting initiator folding . The binding allows initiation and also promotes pairing of origins which is believed to control initiation frequency . Chaperone-independent DNA binding mutants are often defective in replication control . We show here that these mutants have increased rates of association for DNA binding and defects in origin pairing . The increases in association rates were found to be due either to increased protein folding into active forms or to increases in the association rate constant, kon . Since the dissociation rate constants for DNA release with these mutants are not changed, it is unlikely that the DNA binding domain is affected . The pairing domain may thus control replication and modulate DNA binding . The role of the pairing domain in DNA binding can be significant in vivo as the selection for chaperone-independent binding favors pairing-defective mutants. Am J Med Sci, 1994 May, 307(5), 335 - 9 Pigeon and dove eggwhite protect mice against renal infection due to P fimbriated Escherichia coli; Johnson JR et al.; Pigeon and dove eggwhite exhibit high level P1 antigenic activity and are potent and specific inhibitors of adherence mediated by P fimbriae of uropathogenic Escherichia coli . To evaluate pigeon and dove eggwhite as P fimbrial receptor analogues in the prevention of ascending renal infection, mice were challenged with a P fimbriated E . coli urosepsis isolate suspended in saline alone or in saline plus various inhibitors of adherence, including D-mannose, globoside, and chicken, dove, and pigeon eggwhite . D-mannose inhibited mannose-sensitive adherence but not P fimbrial adherence, and failed to prevent renal infection . Globoside and chicken eggwhite also failed to inhibit P fimbrial adherence; chicken eggwhite had little and globoside had no impact on renal infection . In contrast, dove and pigeon eggwhite eliminated P fimbrial adherence and significantly reduced the incidence and intensity of renal infection . These findings suggest that pigeon and dove eggwhite provide P1-antigen-specific protection against ascending renal infection in mice due to P fimbriated uropathogenic E . coli. J Bacteriol, 1994 May, 176(9), 2513 - 6 Identification of the Shine-Dalgarno sequence required for expression and translational control of the pyrC gene in Escherichia coli K-12; Liu J et al.; Expression of the pyrC gene in Escherichia coli K-12 is regulated by a translational control mechanism in which CTP (and perhaps GTP) pool sizes determine the selection of alternative transcriptional start sites at the pyrC promoter . High CTP levels cause transcription to start primarily at a site that directs the synthesis of untranslatable pyrC transcripts . These transcripts form a hairpin at their 5' ends that blocks ribosome binding to the Shine-Dalgarno (SD) sequence . The pyrC ribosome binding site is unusual in that it contains two potential SD sequences, designated SD1 and SD2, which are located 11 and 4 nucleotides upstream of the translational initiation codon, respectively . In this study, we examined the functions of these two SD sequences in translational initiation . Mutations that inactivate either SD1 or SD2 were constructed and incorporated separately into a pyrC::lacZ protein fusion . The effects of the mutations on pyrC::lacZ expression, regulation, and transcript levels were determined . The results indicate that SD1 is the only functional pyrC SD sequence . The SD2 mutation did cause a small reduction in expression, but this effect appeared to be due to a decrease in transcript stability . In addition, we constructed a mutation that introduces a long spacer region between the hairpin at the 5' end of the pyrC transcript and a new pyrC SD sequence . As predicted by the model for translational control, this mutation caused constitutive expression of a pyrC::lacZ protein fusion. Antibiot Khimioter, 1994 May, 39(5), 3 - 11 {Actinomycete plasmid and integrative vectors based on DNA of the temperate Phi C31 actinophage, determining limitation of lytic development of phage Phi C31, not dependent on repressor}; Sladkova IA et al.; Bireplicon plasmids were constructed . The plasmids consist of DNAs of the streptomycete plasmid pIJ702, the Escherichia coli plasmid pUC19 and the phi C31 actinophage genome fragment encoding the function of the site-specific integration into the chromosome . Part of the plasmids transformed Streptomyces lividans TK64 to thiostreptone resistance . The DNA transforming activity depended on the mutual orientations of the blocks used for the construction and probably depended on the structural stability of the plasmids in S . lividans . The integrative vectors consisting of the pUC19 plasmid DNA and the phage genome fragment with the integrative function efficiently transformed S . lividans . No phage plagues were detected with the standard procedure of integrants' infection by phi C31 phage, despite the absence of the phi C31 phage repressor gene in the integrated DNA . The phi C31 phage mutants including clear and virulent ones were not capable of lytic growth on the integrants . The region determining the limitation of the phi C31 phage lytic development was localized by the deletion analysis of the bireplicon plasmids . As a result actinomycete monoreplicon plasmids were formed . The region is the 1.3 kb phage fragment whose right end maps at 0.2 kb preceding the right end of the phi C31 phage genome linear map. Zhonghua Yu Fang Yi Xue Za Zhi, 1994 May, 28(3), 147 - 50 {Inhibitory effects of fifteen kinds of Chinese herbal drugs, vegetables and chemicals on SOS response}; Jin ZC et al.; Effects of 15 kinds of herbal drugs, vegetables and chemicals on lex-dependent sfi-SOS response were determined by micropersistent and/or pulse models induced by 4-Nitroquinoline-N-oxide (4NQO) and Mitomycin C (MMC) in Escherichia coli(E . coli) PQ37 and PQ35, respectively . Results showed the water extract of Rhizoma Polygonati (RP), Fructus Chebulae (FC), Radix Polygoni Multiflori (RPM), Fructus Ligustri Lucidi (FLL), Bulbus Fritillariae Thunbergii (BFT), shell of water chestnut with a pedicle, Chinese chives juice, and solutions of 5-Fluorouracil, Tannic acid and garlicin could inhibit SOS responses with a dose-response relationship and suggested the inhibitory effects took place both inside and outside E . coli cells . Water extract of FC, FLL, BFT, shell of water chestnut with a pedicle, Chinese chives juice and solution of 5-Fluorouracil and Tannic acid could intracellularly inhibit SOS responses induced by MMC in E . coli PQ35, and acetone extract of Grifola Frondosa (GF) could extracellularly inhibit SOS responses in E . coli PQ37 and intracellularly in PQ35 induced by 4NQO or MMC . Water extract of raw hawthorn . Radix Angelicae Duhuricae (RAD), Radix Ophiopogonis (RO), and 5-Fluorodeoxyuridine could extracellularly inhibit SOS responses induced by 4NQO in E coli PQ37 . The possible mechanisms of intracellular inhibition and antidamage repair were discussed in the paper. Biochem Cell Biol, 1994 May-Jun, 72(5-6), 175 - 81 Effect of pH and nonphysiological salt concentrations on human immunodeficiency virus-1 protease dimerization; Tyagi SC et al.; Human immunodeficiency virus-1 (HIV-1) protease is catalytically active as a dimer of identical subunits that associate through noncovalent interactions . To investigate the forces stabilizing HIV-1 protease in its active form, we have studied the effects of pH and salts on structure and function of the enzyme . Enzymatic activity was measured by following the hydrolysis of a fluorogenic substrate . Dissociation of the dimer into its subunits was monitored by gel filtration, while conformational changes in the enzyme were probed by measurements of intrinsic tryptophan fluorescence . Mg2+ ions were capable of dissociating the dimeric enzyme with a concomitant red shift and increase in quantum yield of the tryptophan fluorescence, indicating increased accessibility of tryptophan to the aqueous environment . These structural changes also were associated with a loss of catalytic activity which was insensitive to substrate concentration, consistent with noncompetitive inhibition . Both structural and functional changes could be attributed to binding of Mg2+ ions to a site with an apparent dissociation constant of approximately 2 M . In contrast, increasing concentrations of Na ions up to 5 M were without effect . Increasing pH had similar effects on HIV-1 protease as increasing Mg2+ ions concentration, with concomitant dissociation into subunits, increase in quantum yield and red shift in tryptophan fluorescence, and loss in catalytic activity . The apparent pKa for these structural and functional transitions was 6.95 +/- 0.08 . This value is consistent with that of an aspartic acid residue with an anomalously high pKa, which has been implicated in the catalytic activity of HIV-1 protease. Microb Pathog, 1994 May, 16(5), 337 - 48 Cloning and expression of hemolysin genes from Treponema denticola strains ATCC 35404 (TD-4) and human clinical isolate GM-1 in Escherichia coli; Karunakaran T et al.; The oral spirochete, Treponema denticola is a putative etiologic agent in adult periodontitis, and acute necrotizing ulcerative gingivitis . In vitro, the oral treponeme produces several factors including proteases, hemolysins, hemin-binding proteins, which could potentially be involved in the virulence of this spirochete . Our laboratory has been investigating the pathobiology of T . denticola, and has demonstrated the production of several hemolysins by T . denticola . In this report two hemolysin genes from T . denticola strains ATCC 35404 (TD-4) and GM-1 were isolated by screening genomic DNA libraries of T . denticola on sheep blood agar plates . Physical maps of the insert fragments were not identical . Southern blot analyses suggested some degree of homology in the nucleotide sequence . Maxicell analyses of {35S}-methionine-labeled polypeptides from the recombinant plasmids have suggested the synthesis of an approximately 62.5 kDa polypeptide . Biochemical characterization of the T . denticola hemolysin genes indicated the activity to be inhibited by Mg2+, Ca2+ and Zn2+ but not by EDTA . Dithiothreitol and glutathione moderately enhanced the hemolytic activity of the recombinant plasmids . Iron partially inhibited the hemolytic activities . Addition of 2-2' bipyridyl moderately enhanced the activities, possibly by iron limitation . These results suggest the isolation of an identical hemolysin gene from T . denticola strains TD-4 and GM-1. Vet Q, 1994 May, 16 Suppl 2, S117 - 21 Laboratory and clinical evaluation of a chromogenic endotoxin assay for horses with acute intestinal disorders; Steverink PJ et al.; In this study the laboratory and clinical performance of a chromogenic endotoxin assay for equine plasma was evaluated . The assay was sensitive (detection limit 3 ng LPS/L plasma), reproducible (within and between-assay CV at 50 ng LPS/L E . coli O111:B4 LPS standard addition was 5% and 7.5%, respectively), and not substantially affected by enhancement or inhibition phenomena (recovery of an in vitro spike was 75-125% in 80% of the samples) . LPS added to whole blood was rapidly inactivated upon incubation at 37 degrees C but not at 0 degrees C . A recently developed blood collection tube for LPS testing was found suitable, i.e . LPS-free and providing non-contaminated samples . In 48 horses suffering from acute abdominal diseases requiring surgical treatment, LPS levels were significantly higher in platelet-rich plasma (PRP) than in platelet-poor plasma (PPP), and the proportional difference was related to the PRP platelet count (r = 0.52, p < 0.001, mean difference 48%, range 8-77%) . LPS levels were also significantly higher in horses that died or were euthanized than in surviving horses (mean 16.5 and 7.1 ng/L PRP, respectively, p < 0.05) . We conclude that LPS can be measured in equine plasma with picogram sensitivity and recommend the use of PRP instead of PPP for clinical LPS testing . For clinical use a decision limit for endotoxaemia of 5 ng LPS/L PRP appeared to be inadequate . Analysis at a higher cut-off level for endotoxaemia and the evaluation of clinical, pathological, and laboratory parameters would be more meaningful. Biosci Biotechnol Biochem, 1994 May, 58(5), 895 - 9 High level secretion of calf chymosin using a glucoamylase-prochymosin fusion gene in Aspergillus oryzae; Tsuchiya K et al.; A recombinant chymosin was secreted at high levels using fusion genes with A . oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system . Two portions of the A . oryzae glucoamylase, one with almost the entire glucoamylase (GA1-603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1-511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA . Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing . The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture . Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture. Biotechnol Prog, 1994 May-Jun, 10(3), 314 - 9 Clonal variation in the Spodoptera frugiperda IPLB-SF21-AE insect cell population; Pasumarthy MK et al.; Clones have been isolated from the heterogeneous Spodoptera frugiperda IPLB-SF21-AE insect cell population . Five of these clones, in addition to the parent cell line and the SF9 cell line (another clonal isolate of the parent cell line), have been compared in regards to morphology, growth, budded virus synthesis, and recombinant protein synthesis . No significant differences in cell morphology were found among these cell lines . There was, however, a significant difference in the average cell size, with diameters ranging from 9.30 +/- 0.184 to 11.11 +/- 0.22 microns and from 9.17 +/- 0.05 to 11.25 +/- 0.24 microns for cells growing in Excell 401 serum-free medium in spinner flask cultures and in TNM-FH medium supplemented with 10% FBS in tissue flask cultures, respectively . While no significant differences in the growth rates were found in TNM-FH medium containing 10% calf serum, significant differences were found in Excell 401 serum-free medium, with population doubling times ranging from 38.5 +/- 6.6 to 64.5 +/- 6.4 h in spinner flask studies . Significant differences in expression levels of Escherichia coli beta-galactosidase (beta-gal) were also found in both 12-well plates and spinner flasks . In the 12-well plate studies, the peak levels of beta-galactosidase obtained by these cell lines ranged from 0.332 +/- 0.091 to 0.805 +/- 0.117 mg/10(6) cells and from 0.580 +/- 0.130 to 1.458 +/- 0.132 mg/10(6) cells in Excell 401 and Hyclone Hy-Q serum-free media, respectively . In the spinner flask studies, peak expression levels ranged from 0.128 +/- 0.053 to 0.573 +/- 0.215 mg/10(6) cells in Excell 401 serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS) Biotechnol Prog, 1994 May-Jun, 10(3), 237 - 45 Characterization and polyelectrolyte precipitation of beta-galactosidase containing genetic fusions of charged polypeptides; Niederauer MQ et al.; Genetically engineered versions of beta-galactosidase were constructed through the addition of charged polypeptide fusion tails for the purpose of enhancing polyelectrolyte precipitation . Negatively charged aspartic acid tails and positively charged poly(arginine) tails were added to beta-galactosidase from Escherichia coli . These fusion proteins were all shown to possess specific activity equal to that of the native enzyme . Gel permeation and ion-exchange chromatography provided evidence concerning the integrity of the tails as well as their altered charge characteristics . All enzymes containing charged tails displayed enhanced polyelectrolyte precipitation over the native enzyme . An optimal number of charged residues, beyond which no further enhancement of precipitation was observed, was found to be approximately 10 residues for each type of tail . No interference from nucleic acids was observed in the precipitation of positively tailed beta-galactosidase. Biotechnology (N Y), 1994 May, 12(5), 517 - 20 Efficient sampling of protein sequence space for multiple mutants; Caren R et al.; We describe here a method capable of generating a very large population of multiple mutants, the size of which is primarily limited by volume constraints . This method, referred to as recombination-enhanced mutagenesis, combines the power of in vitro mutagenesis with the high frequencies of in vivo recombination that can be achieved using single-stranded transduction systems . The recombination frequency between two mutations separated by as little as 19 amino acids is 0.02; this frequency approaches a value of 0.1 for mutations separated by more than 38 amino acids . Up to 10(8) independent recombinants were generated in 1 ml of an E . coli culture, and this number scales linearly (or better) with increasing volume . To prove the method's effectiveness, we applied it to the problem of reverting multiple mutants of mouse dihydrofolate reductase, which could not be reverted using mutagenesis alone . Thus, given an appropriate screen or selection scheme, recombination-enhanced mutagenesis is well-suited for addressing a range of combinatorially complex problems, such as antigen recognition, enzyme catalysis, protein folding, and transport/transduction across biomembranes. Shock, 1994 May, 1(5), 359 - 61 Lack of alteration in cardiac beta-adrenoceptor site concentration in rats treated with an adenylate cyclase-stimulating dose of endotoxin; Gardey C et al.; In previous experiments on male rats we showed that 2 mg kg-1, intravenously, lipopolysaccharide from Escherichia coli, a sublethal nonhypotensive dose, induced circulating cardiodepressant activity, maximal alterations in steroid hormonal response, and myocardial adenylate cyclase hyperactivity 4 h later . The purpose of the present study was to investigate a possible relation between this adenylate cyclase hyperactivity and alterations in total ventricular beta-adrenoceptors . To this end, ventricular beta-adrenoceptor concentration and affinity were measured under the same conditions . Binding assays were performed using {3H}dihydroalprenolol . No significant change was observed in both parameters. Shock, 1994 May, 1(5), 347 - 53 Effects of NW-nitro-L-arginine and dexamethasone on early events following lipopolysaccharide injection: observations in the hamster cheek pouch microcirculation; Bouskela E et al.; The effects of NW-nitro-L-arginine (L-NAG) and dexamethasone in the microcirculatory changes observed in early stages of endotoxemia was investigated in male hamsters treated with Escherichia coli lipopolysaccharide (LPS) . The cheek pouch was studied in vivo by means of intravital microscopy and mean arterial and venous pressures, mean arteriolar internal diameter, spontaneous arteriolar vasomotion, microvascular blood flow, macromolecular permeability, leukocyte adhesion, and mean survival time were evaluated in animals treated with either LPS alone or the combination of LPS with L-NAG, an inhibitor of both the constitutive and inducible NO synthases (NOs) . The intravenous injection of LPS (100 mg/kg) elicited a significant reduction in mean arterial blood pressure (MABP) and arteriolar blood flow . The observed arterioles dilated and the spontaneous vasomotion ceased . The combination LPS + L-NAG, both given intravenously, prevented the reduction of MABP and the vasodilation but did not help either the reduction of arteriolar blood flow or the cessation of vasomotion . In order to separate the effect of the two NOs, a group of hamsters was pretreated with dexamethasone (10 mg/kg, also intravenously) which inhibits the induction of the inducible NO synthase (iNOs) . In this group, the hypotension, vasodilation, and cessation of vasomotion were prevented but the decrease in arteriolar blood flow was not affected . The mean survival time was significantly decreased by the combination of LPS + L-NAG (35 +/- 6 h) and significantly increased by the pretreatment with dexamethasone (92 +/- 5 h) compared to LPS alone (56 +/- 7 h).(ABSTRACT TRUNCATED AT 250 WORDS) Exp Appl Acarol, 1994 May, 18(5), 301 - 8 Transient expression of a Drosophila melanogaster hsp70 promoter/lacZ construct injected into larvae of two species of predatory mites (Acari: Phytoseiidae); Presnail JK et al.; The Drosophila melanogaster heat shock 70 promoter (hsp70) was used to regulate expression of the Escherichia coli beta-galactosidase gene (lacZ) in transiently-transformed predatory mite larvae . A construct containing the hsp70 promoter upstream of the D . melanogaster alcohol dehydrogenase (adh) translational start site and Escherichia coli lacZ gene fusion (adh/lacZ) was injected into larvae of Metaseiulus occidentalis and Amblyseius finlandicus . LacZ expression was compared to expression of a similar construct lacking any upstream regulatory sequence . Expression from the hsp70 promoter was strong and heat shock-dependent in both species . The Drosophila hsp70 promoter therefore appears useful for regulating expression of exogenous DNA in both phytoseiid species and may be broadly applicable in the Phytoseiidae . Furthermore, the lacZ gene is a useful gene for analysis of expression in both species . Larval microinjection provides a method of assessing transient expression and of examining native regulatory sequences in these two phytoseiids and will likely be useful in other phytoseiid mites with only minor modifications. Fiziol Zh, 1994 May-Aug, 40(3-4), 101 - 3 {Interleukin-1 production by exudate and bone marrow macrophages in experimental acute infectious peritonitis}; Dygai AM et al.; The model of acute infectious peritonitis in mice has been used to show that inflammation is attended by a marked phasic increase in interleukin-1 (IL-1) production by macrophages of exudate and bone marrow . The increased IL-1 production by macrophages of exudate proceeds earlier than that by macrophages of the bone marrow . This indicates that activation of the bone marrow macrophages may be a result of the effect of OL-1 and other haemopoietic factors released by macrophages on the inflammatory focus. Gene Ther, 1994 May, 1(3), 185 - 91 Folate receptor mediated DNA delivery into tumor cells: potosomal disruption results in enhanced gene expression; Gottschalk S et al.; We have used a particular folate receptor, which is overexpressed in tumor cells, for targeted DNA delivery into these cell types . This folate receptor internalizes folate through caveolae by a process named potocytosis, which is distinct from endocytosis, through clathrin-coated pits . When folate conjugated to poly-L-lysine was used to deliver the E . coli beta-galactosidase gene into tumor cells overexpressing the folate receptor, only low levels of beta-galactosidase activity were detectable . When a replication-defective adenovirus was coincubated with the DNA/folate complexes, 20 to 30% of the cells stained blue with X-gal and a 1000-fold increase of beta-galactosidase activity was observed . Thus, for high efficient DNA delivery and gene expression via the caveolae system, a potosomal disruption agent is needed . Furthermore, folate-mediated DNA delivery is restricted to tumor cells that highly overexpress the folate receptor, which will permit future development of tumor cell-specific delivery of toxic genes for cancer gene therapy. Mol Microbiol, 1994 May, 12(3), 375 - 85 Synthesis of ribosomal proteins during growth of Streptomyces coelicolor; Blanco G et al.; Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied . Proteins being synthesized were pulse-labelled with {35S}-methionine, separated by two-dimensional polyacrylamide gel electrophoresis, and quantified using the BioImage computer software . Most of the ribosomal proteins were synthesized throughout the life cycle . Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase . These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coli beta-galactosidase . The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHI fragment that was cloned and sequenced . The linkage and order of the genes coincide with other L10-L7/L12 operons . However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E . coli and other bacteria . Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHI fragment. Mol Cell Biol, 1994 May, 14(5), 2975 - 84 Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene; Charest H et al.; Leishmania protozoans are the causative agents of leishmaniasis, a major parasitic disease in humans . During their life cycle, Leishmania protozoans exist as flagellated promastigotes in the sand fly vector and as nonmotile amastigotes in the mammalian hosts . The promastigote-to-amastigote transformation occurs in the phagolysosomal compartment of the macrophage cell and is a critical step for the establishment of the infection . To study this cytodifferentiation process, we differentially screened an amastigote cDNA library with life cycle stage-specific cDNA probes and isolated seven cDNAs representing amastigote-specific transcripts . Five of these were closely related (A2 series) and recognized, by Northern (RNA) blot analyses, a 3.5-kb transcript in amastigotes and in amastigote-infected macrophages . Expression of the amastigote-specific A2 gene was induced in promastigotes when they were transferred from culture medium at 26 degrees C and pH 7.4 to medium at 37 degrees C and pH 4.5, conditions which mimic the macrophage phagolysosomal environment . A2 genes are clustered in tandem arrays, and a 6-kb fragment corresponding to a unit of the cluster was cloned and partially sequenced . An open reading frame found within the A2-transcribed region potentially encoded a 22-kDa protein containing repetitive sequences . The recombinant A2 protein produced in Escherichia coli cells was specifically recognized by immune serum from a patient with visceral leishmaniasis . The A2 protein repetitive element has strong homology with an S antigen of Plasmodium falciparum, the protozoan parasite responsible for malaria . Both the A2 protein of Leishmania donovani and the S antigen of P . falciparum are stage specific and developmentally expressed in mammalian hosts. Circ Shock, 1994 May, 43(1), 18 - 25 Modulation of pyrogen-induced upregulation of endothelial cell adhesion molecules (CAMs) by interleukin-4: transcriptional mechanisms and CAM-shedding; Kapiotis S et al.; The pyrogens interleukin 1 (IL-1), tumor necrosis factor (TNF), and bacterial lipopolysaccharides (LPS) are known to increase endothelial cell (EC) adhesiveness for leukocytes by stimulating surface expression of various adhesion molecules . IL-4, a product of activated T-cells, was shown to affect pyrogen-mediated regulation of EC adhesion molecule surface expression . In the present study, we investigated the effect of IL-4 on pyrogen-induced upregulation of the cell adhesion molecules (CAMs) ICAM-1 (intercellular cell adhesion molecule-1), ELAM-1 (endothelial leucocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) in cultured human umbilical vein EC (HUVEC) . Surface expression of adhesion molecules was quantified by flow cytometry, HUVEC mRNA content was estimated by Northern blot analysis, and ICAM-1 antigen in conditioned media was measured by ELISA . Incubation of HUVEC with IL-1 (100 U/ml), TNF (500 U/ml), and LPS (10 micrograms/ml) caused significant increase in ICAM-1, ELAM-1, and VCAM-1 surface expression; IL-1 caused about an eightfold increase in ICAM-1 expression, about a 13-fold increase in ELAM-1 surface expression, and about a fourfold increase in VCAM-1 expression . Coincubation of pyrogens with IL-4 (500 U/ml) differentially influenced their proadhesive effects on the HUVEC surface . In the presence of IL-4, IL-1-induced ICAM-1 upregulation was reduced, ELAM-1 upregulation was not significantly influenced by IL-4, and induction of VCAM-1 was enhanced by IL-4.(ABSTRACT TRUNCATED AT 250 WORDS) J Biochem (Tokyo), 1994 May, 115(5), 814 - 9 Modification of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and its derivative ND 28 with polyethylene glycol; Yamasaki M et al.; Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been obtained from genetically engineered Escherichia coli as an unglycosylated protein . Both native glycosylated hG-CSF and rhG-CSF are rapidly cleared from the circulation, which may limit their effectiveness for clinical use . To improve this biological property, rhG-CSF and its derivative ND 28, which has a higher specific activity than does rhG-CSF, were modified with polyethylene glycol (PEG) . Modified rhG-CSF and ND 28 in which 1 to 3 mol of PEG were bound, were purified by two-step chromatography and characterized by several methods . The results of their physicochemical characterization suggest that PEG-modification does not appreciably change the conformation of rhG-CSF and ND 28 . As a result of the whole characterization, the PEG-modification of rhG-CSF and ND 28 enhanced the stability of rhG-CSF and ND 28 and decreased the plasma clearance rate, which led to more effective hemopoiesis. Biol Pharm Bull, 1994 May, 17(5), 564 - 7 Measurement of staphylokinase by enzyme-linked immunosorbent assay using monoclonal antibodies; Mike A et al.; Hybridoma clones producing monoclonal antibodies specific for staphylokinase were isolated . A competitive assay revealed that the monoclonal antibodies studied could be divided into at least two groups . Representatives of these groups, AS22 and B3E6, recognized quite different epitopes on staphylokinase . This finding led us to develop an assay system for the quantitative analysis of staphylokinase by enzyme-linked immunosorbent assay using AS22 as the capturing antibody and biotinylated B3E6 as the "detector" . The lower limit of sensitivity of the assay was 20 pg of staphylokinase per ml . The assay exhibited good reproducibility, with values of 5.8 and 3.8% for the intra- and inter-assay coefficients of variation, respectively . Staphylokinase could be assayed in the presence of human plasma when the plasma was diluted more than 320-fold, and the measurement was unaffected by the presence of physiological concentrations of human plasminogen . Hence, this assay was considered useful for the detection and quantification of staphylokinase in clinical samples. Biochem Mol Biol Int, 1994 May, 33(1), 187 - 94 cDNA and protein sequences coding for the precursor of phospholipase A2 from Taiwan cobra, Naja naja atra; Pan FM et al.; The cDNA sequence encoding phospholipase A2 (PLA2) was determined by analysis of polymerase-chain-reaction (PCR) product amplified from total cDNA mixture which had been constructed from the poly(A)+RNA of venom glands obtained from Taiwan cobras . Two oligonucleotide segments corresponding to the 5'- and 3'-noncoding regions of sea-snake PLA2 gene were used as primers for PCR-amplified reaction . Plasmids of transformed E . coli strain JM109 containing amplified PLA2 cDNA were purified and prepared for nucleotide sequencing by dideoxynucleotide chain-termination method . Sequencing more than five clones containing about 0.5 kb DNA inserts revealed two isoforms with complete reading frames of 468 base pairs each covering a precursor for phospholipase A2 with a deduced mature protein sequence of 119 amino acids and a 27 amino-acid signal peptide . These two enzymes of Group I PLA2 differ in six nucleotide residues at the gene level and three amino acids along the whole polypeptide chain, each consisting of 14 cysteine residues similar to all reported PLA2 of different snake venoms . The signal peptides and hydropathy profiles of Group I PLA2 reported here are distinctly different from those of Group II PLA2 in viperid snakes. J Virol Methods, 1994 May, 47(3), 331 - 43 Use of TrpE fusion protein to identify antigenic domains within the BIV envelope protein; Chen P et al.; Nine different recombinant clones spanning various regions of the bovine immunodeficiency-like virus (BIV) envelope gene open reading frame were generated . These clones span the entire external glycoprotein as well as the transmembrane glycoprotein region . These proteins were expressed as fusions to the TrpE protein in E . coli . The levels of recombinant protein expressed varied, some clones expressed enough protein that can be detected in a Coomassie blue-stained gel, whereas other proteins could only be detected by Western blot analyses . A recombinant env protein representing the extracellular domain of the env protein was detected by BIV-infected bovine sera . In addition, a 134 amino acid peptide which may represent a major immunoreactive epitope was identified . This peptide is located at the amino terminus of the transmembrane glycoprotein and was specifically recognized by all BIV-infected calf sera tested . The identification of this epitope and the use of recombinant envelope protein will enable us to develop a more effective screening test to study the epidemiology of BIV infection. Can J Microbiol, 1994 May, 40(5), 341 - 4 Survey of cytotoxin production among Escherichia coli strains characterized as enteropathogenic (EPEC) by serotyping and presence of EPEC adherence factor (EAF) sequences; Guth BE et al.; A total of 108 Escherichia coli strains characterized as enteropathogenic (EPEC) by serotyping and the presence of EPEC adherence factor (EAF) sequences were examined for cytotoxin production by cell line assays and colony hybridization with Shiga-like toxin (SLT) probes . Cytolethal distending toxin (CLDT) production was found in three (2.8%) strains belonging to serotype O86:H34, while one O111ab:NM strain hybridized with a SLT-II probe but did not express any cytotoxic activity . All four strains showed localized adherence to HeLa cells and hybridized to an E . coli attaching-effacing gene (eae) probe . The CLDT-producing strains had multiple plasmids and some were present in all strains, including a plasmid of approximately 54 MDa that hybridized with the EAF probe. J Immunother Emphasis Tumor Immunol, 1994 May, 15(4), 292 - 302 A phase I trial of intravenous interleukin-6 in patients with advanced cancer; Weber J et al.; Eighteen patients were treated with escalating doses of recombinant, Escherichia coli-derived human interleukin-6 (IL-6) intravenously every 8 h . Therapy was given for two cycles of 7 days each separated by a week off therapy . Fevers and chills were observed in most patients . Mild renal and liver function abnormalities were noted at higher doses of IL-6 . Dose-limiting toxicity was reached at 30 micrograms/kg i.v . every 8 h due to reversible neurotoxicity, but significant rapidly reversible anemia and hyperglycemia were seen at lower doses . Platelet counts, white blood cell counts, and acute phase reactant levels were substantially elevated . No antitumor responses were seen . A maximum tolerated dose of 10 micrograms/kg i.v . every 8 h for two 7-day cycles is recommended for future phase II trials. Biophys J, 1994 May, 66(5), 1388 - 97 Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior; Nekolla S et al.; LamB, a sugar-specific channel of Escherichia coli outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated using fast Fourier transformation . The current noise through the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz . The spectral density of the noise of the open LamB channels was a quadratic function of the applied voltage . Its magnitude was not correlated to the number of channels in the lipid bilayer membrane . Upon addition of sugars to the aqueous phase the current decreased in a dose-dependent manner . Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the sugars with the binding site inside the channel . The frequency dependence of the spectral density was of Lorentzian type, although the power of its frequency dependence was not identical to -2 . Analysis of the power density spectra using a previously proposed simple model (Benz, R., A . Schmid, and G . H . Vos-Scheperkeuter . 1987 . J . Membr . Biol . 100: 12-29), allowed the evaluation of the on- and the off-rate constants for the maltopentaose binding to the binding site inside the LamB channels . This means also that the maltopentaose flux through the LamB channel could be estimated by assuming a simple one-site, two-barrier model for the sugar transport from the results of the noise analysis. Br J Pharmacol, 1994 May, 112(1), 289 - 91 The effect of nitric oxide synthase inhibition on the plasma fibrinolytic system in septic shock in rats; Korbut R et al.; 1 . We have investigated the effect of pretreatment of rats with nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on the E . coli lipopolysaccharide (LPS)-induced changes in the plasma fibrinolytic system, platelet count, fibrinogen level, as well as in gross and microscopic pathophysiological changes indicative of disseminated intravascular coagulation (DIC) in rats . 2 . E . coli LPS (6 mg kg-1, i.p.) produced a decrease in the levels of plasma fibrinogen and a drop in the blood platelet count 6 h after administration . The decrease in fibrinogen but not the drop in platelet count was reversed by pretreatment with L-NAME (30 mg kg-1, i.p., 24 h and 15 min before administration of LPS) . 3 . Pretreatment with L-NAME antagonized the LPS-induced activation of fibrinolysis as measured by changes in the euglobulin clot lysis time (ECLT) and enhanced the LPS-induced rise in the plasma level of plasminogen activator inhibitor (PAI) . In animals pretreated with L-NAME there was also a marked reduction in the histological changes indicative of DIC . 4 . We propose that L-NAME can act as a protective agent in LPS-induced DIC, and this protection is due to an increased generation of PAI following inhibition of NO synthase. Microbiology, 1994 May, 140 ( Pt 5), 1119 - 24 Frequencies of lipopolysaccharide core types in Escherichia coli strains from bacteraemic patients; Appelmelk BJ et al.; We have investigated the distribution of the various core types (R1, R2, R3, R4 and K-12) in 138 Escherichia coli isolates obtained from positive blood cultures . Rabbit antisera, raised against five rough strains expressing the respective core types, were made monospecific by extensive absorption . The reactivity of the antisera was tested in ELISA with bacterial cells that had been autoclaved for full exposure of core epitopes . One hundred and thirty strains could be typed directly, while eight strains required prior digestion with proteinase K for removal of cross-reactions . Ninety-four of the strains (68%) expressed the R1 type, and 9 (6.5%), 12 (8.7%), 7 (5.1%) and 3 (2.2%) strains expressed the R2, R3, R4 and K-12 core types, respectively . An R1R4 mixed core type, hitherto not yet described, was found in 13 (9.4%) strains . Results obtained with polyclonal antisera were in agreement with those obtained with monoclonal antibodies to the R1, R2 and R3 core types . Core typing may serve as an additional serological marker next to conventional typing of O-, H- and K-antigens. Rinsho Byori, 1994 May, 42(5), 460 - 6 {Molecular cloning, expression and epitope mapping of nuclear antigen}; Yamamoto K; The antinuclear antibody is a major characteristic phenomenon in systemic type of autoimmune diseases . There is a good correlation between symptoms or diseases and the types of antinuclear antibodies in these patients . Therefore, the study of the mechanisms of antinuclear antibody production is thought to be important in the elucidation of the pathogenesis of such autoimmune diseases . The conventional methods to detect antinuclear antibodies have been useful to date in clinical studies . However, these methods are not sufficient for more precise investigations . Thus, we have applied the recently developed molecular biology to this field . cDNAs which encode nuclear antigens have been cloned and their protein products were expressed as recombinant fusion protein in E . coli . By this procedure, we could establish simple and reproducible method to detects antinuclear antibodies . Furthermore, epitope mapping of nuclear antigens could be performed using deletion mutants generated by enzymatic manipulations of the cDNAs . These epitope studies are facilitating a more precise understanding of the mechanisms of antinuclear antibody production. FEMS Microbiol Lett, 1994 May 1, 118(1-2), 23 - 30 Characterization of Mip proteins of Legionella pneumophila; Ludwig B et al.; The Mip ('macrophage infectivity potentiator') protein of Legionella pneumophila has been shown to be an essential virulence factor, exhibiting peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506 . The cloning and sequencing of mip genes from three different L . pneumophila strains revealed a single amino acid substitution which did not affect the isomerase property of the enzyme . Mip proteins isolated from two wild-type L . pneumophila strains and from two corresponding Escherichia coli K-12 recombinant clones derived from these strains exhibited identical enzymatic properties and the precursor proteins are processed at identical cleavage sites . The mature Mip proteins exist in an oligomeric form . Site-directed mutagenesis demonstrated that a substitution of an Asp residue at position 142 by a Leu residue affects PPIase activity of Mip. J Bacteriol, 1994 May, 176(10), 2807 - 13 The RNA chain elongation rate in Escherichia coli depends on the growth rate; Vogel U et al.; We determined the rates of mRNA and protein chain elongation on the lacZ gene during exponential growth on different carbon sources . The RNA chain elongation rate was calculated from measurements of the time elapsing between induction of lacZ expression and detection of specific hybridization with a probe near the 3' end of the mRNA . The elongation rate for the transcripts decreased 40% when the growth rate decreased by a factor of 4, and it always correlated with the rate of translation elongation . A similar growth rate dependency was seen for transcription on the infB gene and on a part of the rrnB gene fused to a synthetic, inducible promoter . However, the untranslated RNA chain specified by the rrnB gene was elongated nearly twice as fast as the two mRNA species encoded by infB and lacZ. Leukemia, 1994 May, 8(5), 780 - 5 Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside; Flasshove M et al.; Deficiency of deoxycytidine kinase (dCK) activity represents one possible cause of resistance to cytosine arabinoside (ara-C) . Mutations of the dCK gene have recently been shown to be responsible for dCK deficiency and increased resistance in vitro . In order to define the relevance of this mechanism in vivo, we analyzed the dCK gene in 16 adult patients with relapsed/refractory acute myeloid leukemia (AML) and clinical resistance to standard-dose and/or high-dose ara-C . Southern blot analysis using genomic DNA from peripheral blood or bone marrow samples containing > or = 70% leukemic blasts and agarose gel electrophoresis of cDNA obtained by RT-PCR did not reveal gross rearrangements of the dCK gene . Sequencing of the dCK coding region showed point mutations in seven patients . Besides two silent mutations (or RFLPs) in codon 42 and 86, base pair mutations resulting in amino acid replacements were found in five patients affecting codon 20, 93, 98, 99, and 154, respectively . dCK cDNA clones from three patients with > or = 50% of sequenced clones revealing the specific base pair alteration were bacterially expressed in E . coli and analyzed for dCK activity . Normal enzyme activity was found in two patients (codon 20 and 98), and a complete loss of activity in one patient (codon 99) . We conclude that structural alteration of the coding region of the dCK gene represents one possible mechanism for ara-C resistance in vivo, but, considering the frequency of this event, other mechanisms may play a more important role for clinical resistance to ara-C in patients with AML. Mutat Res, 1994 May 1, 307(1), 53 - 9 Enzyme-dependent pausing during in vitro replication of O4-methylthymine in a defined oligonucleotide sequence; Menichini P et al.; We had previously reported that an oligonucleotide containing a site-specifically incorporated O4-methylthymine (m4T) was replicated under kinetic conditions by the Klenow fragment of E . coli DNA polymerase I (Kf) (Dosanjh et al., 1993) . Using other polymerases for complete replication, but with limiting enzyme, a pause site before the m4T was observed . In order to investigate whether such a pause could be due to enzyme dissociation or stalling, trapping experiments were designed to aid in differentiating the two mechanisms . Rather than the generally used heparin or sheared DNA trap, these experiments utilized as the acceptor the same oligonucleotid