Biomedical subjects

L F Wu

Publications and source records attributed to L F Wu.

At least 37 records · Page 2Linked to original sources

PubMed1996

Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants.

The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), the sugar-specific enzymes II, are energized by sequential phosphoryl transfer from phosphoenolpyruvate to (i) enzyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA domains of the permeases, and (iv) the enzyme IIBC domains of the permeases which transport and phosphorylate their sugar substrates. A number of site-specific mutants of HPr were examined by using kinetic approaches. Most of the mutations exerted minimal effects on the kinetic parameters characterizing reactions involving phosphoryl transfer from phospho-HPr to various sugars. However, when the well-conserved aspartyl 69 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosamine, and beta-glucosides decreased markedly without changing the maximal reaction rates. The same mutation reduced the spontaneous rate of phosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent glutamyl residue 70 in HPr was changed to a lysyl residue, the Vmax values of the reactions catalyzed by the enzymes II were reduced, but the Km values remained unaltered. Changing this residue to alanine exerted little effect. Site-specific alterations in the C terminus of the beta-glucoside enzyme II which reduced the maximal reaction rate of phosphoryl transfer about 20-fold did not alter the relative kinetic parameters because of the aforementioned mutations in HPr. Published three-dimensional structural analyses of HPr and the complex of HPr with the glucose-specific enzyme IIA (IIAGlc) (homologous to the beta-glucoside and N-acetylglucosamine enzyme IIA domains) have revealed that residues 69 and 70 in HPr are distant from the active phosphorylation site and the IIAGlc binding interface in HPr. The results reported therefore suggest that residues D-69 and E-70 in HPr play important roles in controlling conformational aspects of HPr that influence (i) autophosphohydrolysis, (ii) the interaction of this protein with the sugar permeases of the bacterial phosphotransferase system, and (iii) catalysis of phosphoryl transfer to the IIA domains in these permeases.

Acetylglucosamine↗

PubMed1995

Purification and characterization of the periplasmic nickel-binding protein NikA of Escherichia coli K12.

The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system specific for nickel. In this report, we describe the overproduction of the periplasmic nickel-binding protein NikA by cloning the nikA gene into an overexpression vector, pRE1. NikA was purified free of nickel to near homogeneity from the periplasm by hydrophobic and ion-exchange chromatography. N-terminal amino acid sequencing confirmed that the leader peptide of NikA had been removed. The nickel-binding properties of the protein has been studied by monitoring the quenching of intrinsic protein fluorescence. NikA binds one atom of nickel/molecule of protein with a dissociation constant (Kd) of less than 0.1 microM. Other metals (cobalt, copper, iron) are bound at least 10-fold less tightly. The high specificity for Ni2+ is also demonstrated by high-performance immobilized-metal-ion affinity chromatography. Biosynthesis of NikA occurred only under anaerobic conditions and was dependent on the general anaerobic regulator FNR. It was repressed by the presence of 250 microM Ni2+ in the growth medium and was not affected by either 30 mM formate or 100 mM nitrate. Anaerobically grown wild-type strain MC4100 contains about 23,000 molecules of NikA/cell. In addition to the effect on nickel transport, nikA mutation affects also the nickel sensing in Tar-dependent repellent chemotaxis.

Bacterial Proteins↗

PubMed1995

Plasticity of complex terminals in lamina II in partially deafferented spinal cord: the cat spared root preparation.

Projections to the dorsal horn change in adult mammals in response to complete or partial deafferentation. The number of synaptic terminals remains constant after complete lumbosacral deafferentation, indicating replacement of lost dorsal root terminals by newly formed terminals from spared intrinsic systems. The density of a spared central projection of a dorsal root is increased in dorsal horn after partial deafferentation, consistent with sprouting by the axons in the spared root. In this study, we have used electron microscopy to study morphological changes in a specific class of terminals in the dorsal horn induced by partial deafferentation. Complex terminals (CTs) in the dorsal horn originate exclusively from dorsal roots and are readily distinguished morphologically. The CTs and the postsynaptic densities (PSDs) associated with CTs were measured in lamina II at L5 and L6 in cats subjected to unilateral spared root (L6) dorsal rhizotomies and compared to CTs in the control side. Acutely following partial deafferentation, the number of CT profiles decreased. At more chronic survivals, the number of CT profiles were restored to normal levels, and both the number and the length of PSDs were increased. The changes in CTs and PSDs suggest sprouting and synaptogenesis by the spared dorsal root fibers that produce changes in the postsynaptic neuron. Spared root deafferentation thus elicits compensatory changes in presynaptic terminals of the spared root and also in their postsynaptic target neurons.

Animals↗

PubMed1995

A family of homologous substrate-binding proteins with a broad range of substrate specificity and dissimilar biological functions.

The uptake of peptides is accomplished mainly by a family of homologous oligopeptide or dipeptide transporters in bacteria. Computer-aided sequence analyses expand members of the oligopeptide-binding protein family to nickel and heme permeases and other proteins, including an enzyme hyaluronate synthase. They are involved in human pathogenicity, bacterial virulence, substrate-sensing, bacterial conjugation and bacterial metabolic reactions distinct from nutrient uptake. These homologous proteins are found in both purple bacteria and Gram-positive bacteria, indicating the presence of a common ancestor before the appearance of the two eubacterial phyla. Nevertheless, the pheromone-binding proteins, involved in bacterial conjugation, and the hyaluronate synthase are present only in the low G-C Gram-positive eubacteria subdivision, which suggests that these proteins diverged from the common ancestor after the appearance of this subdivision.

Amino Acid Sequence↗

PubMed1994

Crystallization and preliminary X-ray diffraction study of the nickel-binding protein NikA of Escherichia coli.

NikA, a periplasmic nickel-binding protein involved in nickel-repellent chemotaxis has been crystallized. The crystals are hexagonal with space group P6(2) (or its enantiomorph) with a = 160.3 A and c = 138.4 A and they diffract to at least 3.0 A resolution in the laboratory. NikA presents sequence homology with several periplasmic solute-binding proteins from Gram-negative bacteria.

ATP-Binding Cassette Transporters↗

PubMed1994

Antagonistic effect of nickel on the fermentative growth of Escherichia coli K-12 and comparison of nickel and cobalt toxicity on the aerobic and anaerobic growth.

The facultative anaerobic enterobacterium Escherichia coli requires the activity of nickel-containing hydrogenase for its anaerobic growth. Deficiency of the specific nickel transport system led to a hydrogenase-minus phenotype and slowed down the fermentative growth in the nik mutant. Addition of 300 microM nickel to the growth medium could restore the hydrogenase activity. This restoration resulted in the recovery of anaerobic growth. A further increase of nickel concentration inhibited growth. Thus nickel shows an antagonistic effect on the anaerobic growth of E. coli. To study the mechanism of nickel toxicity, two classes of nickel-resistant mutants were isolated. The nkr mutant was obtained by selecting colonies grown on nickel-containing minimal plate. It acquired simultaneously the resistance to cobalt. A nonspecific magnesium transport mutant corA was isolated on cobalt-containing plate. The corA mutant was also resistant to nickel. When analyzing the influence of nickel and cobalt on the bacterial growth, we obtained two interesting observations. First, anaerobic growth was less sensitive than aerobic growth to cobalt toxicity. In contrast, nickel toxicity did not vary from the growth conditions. Second, cobalt seems to abolish the growth, while nickel appears to slow down the growth rate under the condition used.

Aerobiosis↗

PubMed1994

[A 4-year retrospective study of neonatal outcome on preterm premature rupture of the membranes].

During a four year period, 60 patients with premature rupture of membranes (PROM) met the inclusion criteria of having a single living fetus with gestational age between 25 to 36 weeks and more than 24 hours between PROM and delivery were admitted in Karolinska Hospital, Sweden. These cases were reviewed retrospectively. Five neonates died postnatally and the total survival rate was 91.7%. Three of them had major malformations and one died of hyaline membrane disease with 29 weeks of gestational age. In only one case the immediate cause of death was due to infection. The present protocol of expectant treatment for PROM in this hospital tends to be a minimum of unnecessary intervention for obtaining a high survival rate.

Female↗

PubMed1994

[Puerperal infection of ureaplasma urealyticum].

Using indirect agglutination test (IAT), ELISA and metabolic inhibition test (MI), serum specific IgM antibodies to ureaplasma urealyticum (Un) was investigated in 160 women of late pregnancy during the period of 1991-1992. 81 cases (50.6%) were found positive. 65 out of 81 positive mothers had umbilical blood samples examined and placenta tissues cultured. Specific IgM was detected in the umbilical blood of 7 women (10.8%), and ureaplasma urealyticum was isolated from 6 placenta (9.2%). Among Un IgM positive women, 28 (34.6%) were complicated with puerperal fever, and 30 newborns suffered from neonatal pneumonia and fever, which were significantly higher than that in the Un IgM negative group (P < 0.05). This study indicated that ureaplasma urealyticum might be one of the important pathogens for puerperal and neonatal infection.

Adult↗

PubMed1993

The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel.

The complete nucleotide sequence of the Escherichia coli nik locus, which has been suggested to encode the specific transport system for nickel, has been determined. It was found to contain five overlapping open reading frames that form a single transcription unit. Deduced amino acid sequence of the nik operon shows that its five gene products, NikA to NikE, are highly homologous to components of oligopeptide- and dipeptide-binding protein-dependent transport systems from several Gram-negative and Gram-positive species. NikA represents the periplasmic binding protein, NikB and NikC are similar to integral membrane components of periplasmic permeases, and NikD and NikE possess typical ATP-binding domains that suggest their energy coupling role to the transport process. Insertion mutations in nik genes totally abolished the nickel-containing hydrogenase activity under nickel limitation and markedly altered the rate of nickel transport. Taken together, these data support the notion that the nik operon encodes a typical periplasmic binding-protein-dependent transport system for nickel.

Adenosine Triphosphate↗

PubMed1993

Microbial hydrogenases: primary structure, classification, signatures and phylogeny.

Thirty sequenced microbial hydrogenases are classified into six classes according to sequence homologies, metal content and physiological function. The first class contains nine H2-uptake membrane-bound NiFe-hydrogenases from eight aerobic, facultative anaerobic and anaerobic bacteria. The second comprises four periplasmic and two membrane-bound H2-uptake NiFe(Se)-hydrogenases from sulphate-reducing bacteria. The third consists of four periplasmic Fe-hydrogenases from strict anaerobic bacteria. The fourth contains eight methyl-viologen- (MV), factor F420- (F420) or NAD-reducing soluble hydrogenases from methanobacteria and Alcaligenes eutrophusH16. The fifth is the H2-producing labile hydrogenase isoenzyme 3 of Escherichia coli. The sixth class contains two soluble tritium-exchange hydrogenases of cyanobacteria. The results of sequence comparison reveal that the 30 hydrogenases have evolved from at least three different ancestors. While those of class I, II, IV and V hydrogenases are homologous, i.e. sharing the same evolutionary origin, both class III and VI hydrogenases are neither related to each other nor to the other classes. Sequence comparison scores, hierarchical cluster structures and phylogenetic trees show that class II falls into two distinct clusters composed of NiFe- and NiFeSe-hydrogenases, respectively. These results also reveal that class IV comprises three distinct clusters: MV-reducing, F420-reducing and NAD-reducing hydrogenases. Specific signatures of the six classes of hydrogenases as well as some subclusters have been detected. Analyses of motif compositions indicate that all hydrogenases, except those of class VI, must contain some common motifs probably participating in the formation of hydrogen activation domains and electron transfer domains. The regions of hydrogen activation domains are highly conserved and can be divided into two categories. One corresponds to the 'nickel active center' of NiFe(Se)-hydrogenases. It consists of two possible specific nickel-binding motifs, RxCGxCxxxH and DPCxxCxxH, located at the N- and C-termini of so-called large subunits in the dimeric hydrogenases, respectively. The other is the H-cluster of the Fe-hydrogenases. It might comprise three motifs on the C-terminal half of the large subunits. However, the motifs corresponding to the putative electron transfer domains, as well as their polypeptides chains, are poorly or even not at all conserved. They are present essentially on the small subunits in NiFe-hydrogenases. Some of these motifs resemble the typical ferredoxin-like Fe-S cluster binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

Osmotic repression of anaerobic metabolic systems in Escherichia coli.

The influence of the osmolarity of the growth medium on anaerobic fermentation and nitrate respiratory pathways was analyzed. The levels of several enzymes, including formate dehydrogenase, hydrogenase, and nitrate reductase, plus a nickel uptake system were examined, as was the expression of the corresponding structural and regulatory genes. While some functions appear to be only moderately affected by an increase in osmolarity, others were found to vary considerably. An increase in the osmolarity of the medium inhibits both fermentation and anaerobic respiratory pathways, though in a more dramatic fashion for the former. fnr expression is affected by osmolarity, but the repression of anaerobic gene expression was shown to be independent of FNR regulatory protein, at least for hyd-17 and fdhF. This repression could be mediated by the intracellular concentration of potassium and is reversed by glycine betaine.

Anaerobiosis↗

PubMed1992

Functional interactions between proteins of the phosphoenolpyruvate:sugar phosphotransferase systems of Bacillus subtilis and Escherichia coli.

Proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus subtilis were overexpressed, purified to near homogeneity, and characterized. The proteins isolated include Enzyme I, HPr, the glucose-specific IIA domain of the glucose-specific Enzyme II (IIAglc), and the mannitol-specific IIA protein, IIAmtl. Site specific mutant proteins of IIAglc and HPr were also overexpressed and purified, and their properties were compared with those of the wild type proteins. These proteins and their phosphorylated derivatives were characterized with respect to their immunological cross-reactivities employing the Western blot technique and in terms of their migratory behavior during sodium dodecyl sulfate-gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. The interactions between homologous and heterologous Enzymes I and HPrs, between homologous and heterologous HPrs and the IIAglc proteins, and between homologous and heterologous IIAglc proteins and IIBCscr of B. subtilis as well as IICBglc of Escherichia coli were defined and compared kinetically. The mutant HPrs and IIAglc proteins were also characterized kinetically as PTS phosphocarrier proteins and/or as inhibitors of the phosphotransferase reactions of the PTS. These studies revealed that complexation of IIAglc with the mutant form of HPr in which serine 46 was replaced by aspartate (S46D) did not increase the rate of phosphoryl transfer from phospho Enzyme I to S46D HPr more than when IIAmtl was complexed to S46D HPr. These findings do not support a role for HPr(Ser-P) in the preferential utilization of one PTS carbohydrate relative to another. Functional analyses in E. coli established that IIAglc of B. subtilis can replace IIAglc of E. coli with respect both to sugar transport and to regulation of non-PTS permeases, catabolic enzymes, and adenylate cyclase. Site-specific mutations in histidyl residues 68 and 83 (H68A and H83A) inactivated IIAglc of B. subtilis with respect to phosphoryl transfer and its various regulatory roles.

Amino Acid Sequence↗

PubMed1992

[Treatment of infantile hepatitis syndrome with injection of yin zhi huang].

The infantile hepatitis syndrome is defined as a liver disease which occurs during the infantile period with the clinical manifestation of jaundice, hepatomegaly and hepatic dysfunction. The cause of disease is complex and the treatment is difficult. From March 1987 to June 1988, 55 patients (treatment group) were treated with injection of yin zhi huang, and 15 cases (control group) with injection of Inosine, vitamin C and glucose. The course of treatment lasted 15 days. At the beginning and the end of the treatment, serum bilirubin level, sALT level, duodenal juice bilirubin level, liver size and T cell subsets in peripheral blood had been detected separately. The results showed that among the treatment group, serum bilirubin level was significantly lower at the end of the treatment than that of the control group. The sALT, and hepatomegaly were also reduced or lessened, while the duodenal juice bilirubin level was increased. But there was no difference in T cell subsets between the two groups.

CD4-CD8 Ratio↗

PubMed1991

The hydC region contains a multi-cistronic operon (nik) involved in nickel transport in Escherichia coli.

We have cloned the multigenic hydC locus of Escherichia coli on a 7.1-kb BamHI-EcoRI fragment. Since its gene products are likely to be involved in the specific nickel transport [Wu et al., Mol. Microbiol. 3 (1989) 1709-1718], we propose a new gene designation, nik, to replace hydC. In vivo gene expression studies and complementation analysis support the notion that the nik locus is a multi-cistronic operon consisting of two to five genes. The first two genes, arranged in the order nikA and nikB, encode two proteins of 59 and 27.5 kDa, respectively. The downstream genes direct the biosynthesis of three polypeptides of 30, 28 and 25.5 kDa. Southern-blot analysis showed that mutant HYD720 carries a chromosomal deletion of 5.1-kb covering both nikA and nikB, whereas a 0.8-kb deletion in mutant HYD790 includes the sole nikB region. The cloned nik operon and the deletion mutants will aid greatly in the further molecular characterization of the multicomponent-specific transport of nickel in E. coli.

Bacterial Proteins↗

PubMed1991

Insertion of the mannitol permease into the membrane of Escherichia coli. Possible involvement of an N-terminal amphiphilic sequence.

The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches. Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro. The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses. Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage. Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed. In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase. The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence. The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm. This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment. Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation. The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis. The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly.... The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaline Phosphatase↗

PubMed1991

Evolution of the MIP family of integral membrane transport proteins.

Six integral membrane proteins of bacterial, animal, and plant origin, which are believed to function in solute transport, share sequence identity and are grouped together as members of the MIP family. These include the Escherichia coli glycerol facilitator, the major intrinsic protein from bovine lens fibre junction membranes, a plant tonoplast membrane protein, a soybean protein from the peribacteroid membrane, and a Drosophila neurogenic protein. These proteins, each of which appears to consist of six transmembrane helical segments per subunit, apparently arose by internal duplication of a three-transmembrane segment. Phylogenetic 'trees' interrelating these proteins and segments are presented.

Amino Acid Sequence↗

PubMed1991

Nucleotide sequence of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinases of Escherichia coli.

The fruK gene encoding fructose-1-phosphate kinase (FruK), located within the fructose (fru)-catabolic operon of Rhodobacter capsulatus, was sequenced. FruK of R. capsulatus (316 amino acids; molecular weight = 31,232) is the same size as and is homologous to FruK of Escherichia coli, phosphofructokinase B (PfkB) of E. coli, phosphotagatokinase of Staphylococcus aureus, and ribokinase of E. coli. These proteins therefore make up a family of homologous proteins, termed the PfkB family. A phylogenetic tree for this new family was constructed. Sequence comparisons plus chemical inactivation studies suggested the lack of involvement of specific residues in catalysis. Although the Rhodobacter FruK differed markedly from the other enzymes within the PfkB family with respect to amino acid composition, these enzymes exhibited similar predicted secondary structural features. A large internal segment of the Rhodobacter FruK was found to be similar in sequence to the domain bearing the sugar bisphosphate-binding region of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase of plants and bacteria. Proteins of the PfkB family did not exhibit statistically significant sequence identity with PfkA of E. coli. PfkA, however, is homologous to other prokaryotic and eukaryotic ATP- and PPi-dependent Pfks (the PfkA family). These eukaryotic, ATP-dependent enzymes each consist of a homotetramer (mammalian) or a heterooctamer (yeasts), with each subunit containing an internal duplication of the size of the entire PfkA protein of E. coli. In some of these enzymes, additional domains are present. A phylogenetic tree was constructed for the PfkA family and revealed that the bacterial enzymes closely resemble the N-terminal domains of the eukaryotic enzyme subunits whereas the C-terminal domains have diverged more extensively. The PPi-dependent Pfk of potato is only distantly related to the ATP-dependent enzymes. On the basis of their similar functions, sizes, predicted secondary structures, and sequences, we suggest that the PfkA and PfkB families share a common evolutionary origin.

Amino Acid Sequence↗

PubMed1990

Evolution of permease diversity and energy-coupling mechanisms with special reference to the bacterial phosphotransferase system.

Different classes of apparently unrelated permeases couple different forms of energy to solute transport. While the energy coupling mechanisms utilized by the different permease classes are clearly distinct, it is proposed, based on structural comparisons, that many of these permeases possess transmembrane, hydrophobic domains which are evolutionarily related. Carriers may have arisen from transmembrane pore-forming proteins, and the protein constituents or domains which are specifically responsible for energy coupling may have had distinct origins. Thus, complex permeases may possess mosaic structures. This suggestion is substantiated by recent findings regarding the evolutionary origins of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). Mechanistic implications of this proposal are presented.

Biological Evolution↗