Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor.

The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome's central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA.

Alterations in the ß flap and ß' dock domains of the RNA polymerase abolish NusA-mediated feedback regulation of the metY-nusA-infB operon.

The RimM protein in Escherichia coli is important for the in vivo maturation of 30S ribosomal subunits and a ΔrimM mutant grows poorly due to assembly and translational defects. These deficiencies are suppressed partially by mutations that increase the synthesis of another assembly protein, RbfA, encoded by the metY-nusA-infB operon. Among these suppressors are mutations in nusA that impair the NusA-mediated negative-feedback regulation at internal intrinsic transcriptional terminators of the metY-nusA-infB operon. We describe here the isolation of two new mutations, one in rpoB and one in rpoC (encoding the ß and ß' subunits of the RNA polymerase, respectively), that increase the synthesis of RbfA by preventing NusA from stimulating termination at the internal intrinsic transcriptional terminators of the metY-nusA-infB operon. The rpoB2063 mutation changed the isoleucine in position 905 of the ß flap-tip helix to a serine, while the rpoC2064 mutation duplicated positions 415 to 416 (valine-isoleucine) at the base of the ß' dock domain. These findings support previously published in vitro results, which have suggested that the ß flap-tip helix and ß' dock domain at either side of the RNA exit tunnel mediate the binding to NusA during transcriptional pausing and termination.

Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine.

The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.

Optimising the signal peptide for glycosyl phosphatidylinositol modification of human acetylcholinesterase using mutational analysis and peptide-quantitative structure-activity relationships.

Glycosyl phosphatidylinositol (GPI)-modified proteins have a C-terminal signal peptide (GPIsp) that mediates the addition of a GPI-anchor to an amino acid residue at the cleavage and modification site (omega-site). Within the GPIsp, a stretch of hydrophilic amino acid residues are found which constitutes the spacer region that separates the omega-site residue from a hydrophobic C-terminus. Deletions and insertions into the spacer region of human acetylcholinesterase (AChE) show that the length of this spacer region is very important for efficient GPI-modification. Surprisingly, the natural length of the spacer region in human AChE was not optimal for the highest degree of GPI modification. The importance of the two adjacent residues downstream of the omega-site, the omega+1 and omega+2 residues, was investigated by peptide-quantitative structure-activity relationships (Peptide-QSAR). A model was made that predicts the efficiency of the GPI modification when these residues are substituted with others, and suggests important features for these residues. The most preferred omega+1 and omega+2 residues, predicted by the model, in combination with an ideal spacer length resulted in an optimised GPIsp. This mutant protein is more efficiently GPI-modified than any mutant AChE tested thus far.

The GTPase activity of the Escherichia coli Ffh protein is important for normal growth.

The Escherichia coli (E. coli) Ffh protein is homologous to the 54kDa subunit of the eukaryotic signal recognition particle. We have examined an intrinsic GTPase activity of this protein and have created mutations in one sequence motif (GXXXXGK) of the putative GTP binding site. When glycine-112 was changed to valine (Ffh-G112V), Vmax was reduced to only 4% of the wildtype level. On the other hand, when glutamine-109 was altered to glycine (Ffh-Q109G), the major effect was a 50-fold increase in Km. These results show that the residues Q-109 and G-112 are essential for the binding and hydrolysis of GTP and that they are part of a catalytic site structurally related to that of many other GTPase proteins. Expression of the mutant protein Ffh-G112V in E. coli was highly toxic in the presence of the wildtype protein. In contrast, genetic complementation experiments showed that a viable strain could be constructed where the Ffh-Q109G mutant protein replaced wildtype Ffh. However, expression of the mutant protein had a negative effect on growth rate at 30 degrees C and resulted in elongated cells. These results demonstrate that the GTPase activity of the Ffh protein is required for proper function of the protein in vivo.

Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli.

The trmD operon of Escherichia coli encodes the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and a 21,000 Mr protein of unknown function. Here we demonstrate that, in contrast to the expression of other ribosomal protein operons, the amount of trmD operon mRNA and the rate of synthesis of the proteins encoded by the operon respond to increased gene dosage. The steady-state level of the mRNA was about 18 times higher, and the relative rate of synthesis of the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and the 21,000 Mr protein was 15, 9, 25 and 23 times higher, respectively, in plasmid-containing cells than in plasmid-free cells. Overproduced tRNA(m1G37)methyltransferase and 21,000 Mr protein were as stable as E. coli total protein, whereas the two ribosomal proteins were degraded to a large extent. The steady-state amount of S16 and L19 in the plasmid-containing cells exceeded that in plasmid-free cells by threefold and twofold, respectively. No significant effect on the synthesis of the trmD operon proteins from the chromosomally located genes was observed when parts of the operon were expressed on different plasmids. Taken together, these results suggest that the expression of the trmD operon is not subject to transcriptional or translational feedback regulation, and demonstrate that not all ribosomal protein operons are regulated in the same manner. We propose that ribosomal protein operons that do not encode proteins that bind directly to rRNA are not under autogenous control. Metabolic regulation at the transcriptional level and protein degradation are plausible mechanisms for the control of expression of such operons.

The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation.

The transcriptional promoting activity of DmpR is under the strict control of its aromatic effector ligands that are bound by its regulatory N-terminal domain. The positive control function of DmpR resides within the central C-domain that is highly conserved among activators of sigma(54)-RNA polymerase. The C-domain mediates ATP hydrolysis and interaction with sigma(54)-RNA polymerase that are essential for open-complex formation and thus initiation of transcription. Wild-type and loss-of-function derivatives of DmpR, which are defective in distinct steps in nucleotide catalysis, were used to address the consequences of nucleotide binding and hydrolysis with respect to the multimeric state of DmpR and its ability to promote in vitro transcription. Here, we show that DmpR derivatives deleted of the regulatory N-terminal domain undergo an aromatic-effector independent ATP-binding triggered multimerisation as detected by cross-linking. In the intact protein, however, aromatic effector activation is required before ATP-binding can trigger an apparent dimer-to-hexamer switch in subunit conformation. The data suggest a model in which the N-terminal domain controls the transcriptional promoting property of DmpR by constraining ATP-mediated changes in its oligomeric state. The results are discussed in the light of recent mechanistic insights from the AAA(+) superfamily of ATPases that utilise nucleotide hydrolysis to restructure their substrates.

Importance of mRNA folding and start codon accessibility in the expression of genes in a ribosomal protein operon of Escherichia coli.

The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kilodalton protein (21K) of unknown function, the tRNA(m1G37)methyltransferase (TrmD), and r-protein L19, in that order. The synthesis of the 21K and TrmD proteins is 12 and 40-fold lower, respectively, than that of the two r-proteins, although the corresponding parts of the mRNA are equally abundant. This translational control of expression of at least the 21K protein gene (21K), is mediated by a negative control element located between codons 18 and 50 of 21K. Here, we present evidence for a model in which mRNA sequences up to around 100 nucleotides downstream from the start codon of 21K fold back and base-pair to the 21K translation initiation region, thereby decreasing the translation initiation frequency. Mutations in the internal negative control element of 21K that would prevent the formation of the proposed mRNA secondary structure over both the Shine-Dalgarno (SD) sequence and the start codon increased expression up to about 20-fold, whereas mutations that would disrupt the base-pairing with the SD-sequence had only relatively small effects on expression. In addition, the expression increased 12-fold when the stop codon of the preceding gene, rpsP, was moved next to the SD-sequence of 21K allowing the ribosomes to unfold the postulated mRNA secondary structure. The expression increased up to 150-fold when that stop codon change was combined with the internal negative control element base-substitutions that derepressed translation about 20-fold. The negative control element of 21K does not seem to be responsible for the low expression of the trmD gene located downstream. However, a similar negative control element native to trmD can explain at least partly the low expression of trmD. Possibly, the two mRNA secondary structures function to decouple translation of 21K and trmD from that of the respective upstream cistron in order to achieve their independent regulation.

The novel NADPH oxidase 4 inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice.

In type 2 diabetes, it has been proposed that pancreatic beta-cell dysfunction is promoted by oxidative stress caused by NADPH oxidase (NOX) overactivity. Five different NOX enzymes (NOX1-5) have been characterized, among which NOX1 and NOX2 have been proposed to negatively affect beta-cells, but the putative role of NOX4 in type 2 diabetes-associated beta-cell dysfunction and glucose intolerance is largely unknown. Therefore, we presently investigated the importance of NOX4 for high-fat diet or HFD-induced glucose intolerance using male C57BL/6 mice using the new NOX4 inhibitor GLX351322, which has relative NOX4 selectivity over NOX2. In HFD-treated male C57BL/6 mice a two-week treatment with GLX351322 counteracted non-fasting hyperglycemia and impaired glucose tolerance. This effect occurred without any change in peripheral insulin sensitivity. To ascertain that NOX4 also plays a role for the function of human beta-cells, we observed that glucose- and sodium palmitate-induced insulin release from human islets in vitro was increased in response to NOX4 inhibitors. In long-term experiments (1-3 days), high-glucose-induced human islet cell reactive oxygen species (ROS) production and death were prevented by GLX351322. We propose that while short-term NOX4-generated ROS production is a physiological requirement for beta-cell function, persistent NOX4 activity, for example, during conditions of high-fat feeding, promotes ROS-mediated beta-cell dysfunction. Thus, selective NOX inhibition may be a therapeutic strategy in type 2 diabetes.

Active center differences between cathepsins L and B: the S1 binding region.

The substrate peptide bond cleaved by cathepsins B and L is determined not by the amino acid contributing the carboxyl group to this bond as in the case of serine proteases but rather by the presence of a neighboring amino acid with a large hydrophobic side chain. From a study of the inhibitory potency in a series, Cbz-Phe-X-CHN2, in which Phe promotes binding at S2 (terminology of [(1968) Biochem. Biophys. Res. Commun. 32, 898-902]) while the amino acid X probes S1, it is shown that this region of cathepsin L also has the ability to accommodate large hydrophobic side chains. In this respect cathepsin L differs from cathepsin B. Thus Cbz-Phe-Tyr(O-t-Bu)CHN2 inactivates cathepsin L with a rate 2.5 x 10(4) greater than that for cathepsin B.

Interactions of papaya proteinase IV with inhibitors.

Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by 'papain' reported previously. PPIV is slowly bound by human alpha 2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues.

Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine.

Thrombin is the key enzyme in the blood coagulation system, and inhibitors of its proteolytic activity are of therapeutic interest since they are potential anticoagulants. The most potent inhibitor of the benzamidine type is N alpha-[(2-naphthylsulfonyl)glycyl]-4-amidinophenylalanylpiperid ide (NAPAP). However, NAPAP and other benzamidine derivatives do not show favorable pharmacological properties; above all, they have very low systemic bioavailability after oral administration. The goal of designing new compounds was to obtain potent inhibitors with improved pharmacokinetic properties. Piperazide derivatives of 3-amidinophenylalanine as the key building block were synthesized. The piperazine moiety opened the possibility to introduce quite different substituents on the second nitrogen using common synthetic procedures. Some of the newly synthesized compounds are potent inhibitors of thrombin and offer an approach to study structure-function relationships for inhibition of thrombin and related enzymes and for the improvement of their pharmacokinetic properties.

Tumor associated macrophages in human prostate cancer: relation to clinicopathological variables and survival.

Tumor associated macrophages (TAM) influence diverse processes such as angiogenesis, tumor cell proliferation, and metastasis during tumor progression. In a variety of tumor types, the amount of TAM has been associated with prognosis, but their role in prostate cancer has not been elucidated. The purpose of this study was to investigate the role of TAM in a series of 85 cases of prostatic carcinoma, diagnosed at transurethral resection of the prostate between 1975-1983, using immunohistochemistry and morphometrical techniques. Macrophage density was assessed as the maximum number of TAM per field in the three most macrophage dense areas (TAMmax) and as the average volume density of TAM in an estimate of the whole resected tumor. Furthermore, the individual cell profile area of TAM was assessed with an image analyzer. Macrophage variables were thereafter related to histological grade, tumor stage, metastasis as well as to vascular density, tumor cell proliferation and survival. Patients with a volume density of TAM in the fourth quartile had a shorter median cancer specific survival time than patients in the first to third quartile (3.3 vs. 5.9 years, p=0.005). Furthermore, an increased macrophage cell profile area was related to poor clinical outcome (4.6 vs. 5.9 years, p=0.039) whereas TAMmax gave no prognostic information. In a multivariate analysis, metastasis and the volume density of macrophages gave independent prognostic information (p=0.0008, p=0.010). However, when excluding metastasis from the analysis, only Gleason score was an independent predictor of cancer specific survival (p= 0.005). The volume density of TAM, the macrophage cell profile area and TAMmax increased with increasing Gleason score (p=0.001, p=0.0001, p=0.0001 respectively). A correlation was found between the volume density of TAM and tumor cell proliferation (rs=0.44, p=0.001) and an increased macrophage cell profile area was associated to microvessel density (rs=0.42, p=0.0001). Together these results suggest that both the functional state (as reflected by cell size), number and location of the macrophages are of importance for their influence on prostate tumors, but macrophage quantification is not a strong independent prognostic factor.

Role of transforming growth factor-beta1 in prostate cancer.

TGF-beta1 is an important regulator of the normal and malignant prostate. In the non-malignant prostate, TGF-beta1 stimulates cell differentiation, inhibits epithelial cell proliferation, and induces epithelial cell death. TGF-beta1 is secreted into semen where it is an important immunosuppressive factor. Prostate cancer cells express high levels of TGF-beta1, which seems to enhance prostate cancer growth and metastasis by stimulating angiogenesis and by inhibiting immune responses directed against tumour cells. Prostate cancer cells frequently lose their TGF-beta receptors and acquire resistance to the anti-proliferative and pro-apoptotic effects of TGF-beta1. Accordingly, high expression of TGF-beta1 and loss of TGF-beta receptor expression have been associated with a particularly bad prognosis in human prostate cancer patients. TGF-beta1 also seems to be a mediator of castration-induced apoptosis in androgen dependent normal and malignant prostate epithelial cells. The ability of some prostate tumours to avoid castration-induced apoptosis may not, however, be simply due to loss of TGF-beta receptor type I or type II expression in the tumour cells. It may also be related to an inability of these cells to up-regulate TGF-beta receptor levels in response to castration or possibly due to defects downstream of the receptors. Short-term therapy-induced changes in the TGF-beta system in prostate tumours can probably be used to predict the long-term response to androgen ablation treatment. Further investigations into the TGF-beta system in the prostate are needed, however, to elucidate how alterations in this system affect the behaviour of prostate tumours, and whether this system can be manipulated for therapeutical purposes.

DNA recovery and PCR quantification of catechol 2,3-dioxygenase genes from different soil types.

With the objective of monitoring xenobiotic degrading bacteria in soil, a method for rapid extraction of DNA from soil, amenable to amplification by PCR, was developed. The method was based on lysis by freeze-thawing and subsequent addition of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide and proteinase K. The extraction method required 2 h and was tested on six different soils differing in organic content, water holding capacity and pH, including ones from which DNA extraction is difficult. DNA yields from the soils ranged from 6.1 to 54.0 micrograms of DNA per g soil. The efficiency and reproducibility of the DNA extraction method were evaluated by competitive PCR. The organic content in the soils was a major factor affecting the amount of obtained DNA amenable for amplification by PCR. A PCR primer-pair was designed on the basis of the known nucleotide sequences of several catechol 2,3-dioxygenase genes. The specificity of the primer-pair was demonstrated on different sequenced catechol 2,3-dioxygenase genes and on site-specific bacterial isolates from polycyclic aromatic hydrocarbon (PAH)-contaminated soil. The concentration of catechol 2,3-dioxygenase DNA in PAH-contaminated sediment undergoing an ex situ compost process was quantified by competitive PCR over a period of 16 weeks. The concentration of PAHs and catechol 2,3-dioxygenase DNA in the soil samples, was found to correlate.

The interaction between impulsivity and neighborhood context on offending: the effects of impulsivity are stronger in poorer neighborhoods.

This research blends 2 traditions of theorizing on the causes of crime, one focused on the role of individual differences and the other focused on structural and contextual variables. Two related studies examined the relations among impulsivity, neighborhood context, and juvenile offending. The first, cross-sectional study uses a large sample of 13-year-old inner-city boys, whereas the second, longitudinal study offers a conceptual replication using 17-year-old inner-city boys who are a subset of the original sample. Across both studies, results indicate that the effects of impulsivity on juvenile offending are stronger in poorer neighborhoods. Furthermore, nonimpulsive boys in poor neighborhoods were at no greater risk for delinquency than nonimpulsive boys in better-off neighborhoods.

A fast and simple turbidimetric method for the determination of sulfate in sulfate-reducing bacterial cultures.

A standard turbidimetric assay for the determination of sulfate in water was modified with the objective of achieving a quick and simple method for monitoring the decrease of sulfate in cultures of sulfate-reducing bacteria. The effects of sulfate concentration, mixing time and the ratio of sample to conditioning reagent were optimized using a central composite face-centered response surface model design. The results suggested that a mixing time of 30 s resulted in smaller absorbance variance, the variance in absorbance measurements tended to increase with concentration of sulfate and that the ratio between the amount of conditioning reagent and sample had no significant influence on the absorbance variance. The modified assay thus developed is simple and quick, and covers a comparatively large sulfate concentration range (0-5 mM) compared to the standard turbidimetric assay.

Structure of a Natural Microbial Community in a Nitroaromatic Contaminated Groundwater Is Altered during Biodegradation of Extrinsic, but Not Intrinsic Substrates.

A BSTRACTThis study demonstrates microbial community changes over time in a nitroaromatic-contaminated groundwater upon amendment with hydrocarbons previously unknown to the microbial community (extrinsic) and hydrocarbons previously known to the microbial community (intrinsic). Sealed flasks, shaken and incubated at 25 degrees C, containing contaminated groundwater and salts were amended twice with extrinsic hydrocarbons including phenol, benzoic acid, and naphthalene, and intrinsic hydrocarbons including 2,4-dinitrotoluene (2,4-DNT) and para-nitrotoluene ( p-NT). Microbial growth, biodegradation, and community structure changes measured by random amplified polymorphic DNA (RAPD) and quantitative PCR (qPCR) targeting catechol-2,3-dioxygenase (C23O) genes were monitored over time. All amended substrates were biodegraded after both substrate amendments except for 2,4-DNT, which was only partially degraded after the second amendment. Unique microbial communities were developed in flasks amended with phenol, benzoic acid, and naphthalene. However, in the flasks amended with intrinsic hydrocarbons the microbial community remained similar to the unamended control flasks. The relative amount of C23O genes detected by qPCR correlated with the biodegradation of phenol and naphthalene but not with 2,4-DNT. The results showed that a selection for microorganisms capable of catabolizing extrinsic hydrocarbons naturally and initially present in the nitroaromatic-contaminated groundwater occurred. However, growth-linked biodegradation of added intrinsic hydrocarbons was not selective.

Synthetic urokinase inhibitors as potential anti-invasive drugs.

In carcinogenesis, proteinases play an important role in metastasis of solid malignant tumors. Aside from several matrix metalloproteinases and cathepsins, the urokinase-plasmin system seems to be one of the main players within the cell surface-associated proteolytic activity, facilitating tumor cell migration and invasion. Upon binding to a specific receptor, the enzymatic activity of urokinase is focused to the cell surface. The significance of the plasminogen activator system in malignancy is underlined by the finding of elevated levels of urokinase and its receptor in tumor tissues. Therefore, the possible use of urokinase inhibitors in the therapeutic treatment of cancer and metastasis has been the subject of intensive investigations. This review covers synthetic inhibitors of urokinase described up to December 2000, although the number of lead structures is still relatively small.

Novel plasma kallikrein inhibitors of the benzamidine type.

Besides guanidino compounds and amines structurally related to arginine and lysine, compounds with an amidino moiety are inhibitors of trypsin-like enzymes. However, in most cases ordinary benzamidine derivatives are not selective inhibitors. Relatively selective inhibitors of some enzymes were found among amino acids containing a benzamidine moiety at the side chain. For example, derivatives of phenyl-alpha-aminobutyric acid with one or two amidino moieties such as the 4'-amidinoanilide of N alpha-[4-amidinophenylsulfonyl]-phenyl-alpha- aminobutyric acid are potent and selective inhibitors of plasma kallikrein (Ki = 0.15 microM). Using N alpha-arylsulfonylated 3-amidinophenylalanine as the key building block, novel inhibitors of plasma kallikrein were developed. Several esters and amides of the nipecotic acid derivative were synthesized which inhibit plasma kallikrein competitively with Ki values between 0.1 and 1.0 microM. The compounds prolonged aPTT in micromolar concentration, indicating an inhibition of the intrinsic coagulation pathway.