Upregulation of inflammatory gene transcripts in periosteum of chronic migraineurs: Implications for extracranial origin of headache.

OBJECTIVE: Chronic migraine (CM) is often associated with chronic tenderness of pericranial muscles. A distinct increase in muscle tenderness prior to onset of occipital headache that eventually progresses into a full-blown migraine attack is common. This experience raises the possibility that some CM attacks originate outside the cranium. The objective of this study was to determine whether there are extracranial pathophysiologies in these headaches. METHODS: We biopsied and measured the expression of gene transcripts (mRNA) encoding proteins that play roles in immune and inflammatory responses in affected (ie, where the head hurts) calvarial periosteum of (1) patients whose CMs are associated with muscle tenderness and (2) patients with no history of headache. RESULTS: Expression of proinflammatory genes (eg, CCL8, TLR2) in the calvarial periosteum significantly increased in CM patients attesting to muscle tenderness, whereas expression of genes that suppress inflammation and immune cell differentiation (eg, IL10RA, CSF1R) decreased. INTERPRETATION: Because the upregulated genes were linked to activation of white blood cells, production of cytokines, and inhibition of NF-κB, and the downregulated genes were linked to prevention of macrophage activation and cell lysis, we suggest that the molecular environment surrounding periosteal pain fibers is inflamed and in turn activates trigeminovascular nociceptors that reach the affected periosteum through suture branches of intracranial meningeal nociceptors and/or somatic branches of the occipital nerve. This study provides the first set of evidence for localized extracranial pathophysiology in CM. Ann Neurol 2016;79:1000-1013.

Perturbing the general base residue Glu166 in the active site of class A ß-lactamase leads to enhanced carbapenem binding and acylation.

Most class A ß-lactamases cannot hydrolyze carbapenem antibiotics effectively. The molecular mechanism of this catalytic inefficiency has been attributed to the unique stereochemistry of carbapenems, including their 6-α-hydroxyethyl side chain and the transition between two tautomeric states when bound at the active site. Previous studies have shown that the 6-α-hydroxyethyl side chain of carbapenems can interfere with catalysis by forming hydrogen bonds with the deacylation water molecule to reduce its nucleophilicity. Here our studies of a class A noncarbapenemase PenP demonstrate that substituting the general base residue Glu166 with Ser or other residues leads to a significant enhancement of the acylation kinetics by ∼100-500 times toward carbapenems like meropenem. The structures of PenP and Glu166Ser both in apo form and in complex with meropenem reveal that Glu166 is critical for the formation of a hydrogen bonding network within the active site that locks Asn170 in an orientation to impose steric clash with the 6-α-hydroxyethyl side chain of meropenem. The Glu166Ser substitution weakens this network and enables Asn170 to adopt an alternative conformation to avoid steric clash and accommodate faster acylation kinetics. Furthermore, the weakened hydrogen bonding network caused by the Glu166Ser substitution allows the 6-α-hydroxyethyl moiety to adopt a catalytically favorable orientation as seen in class A carbapenemases. In summary, our data identify a previously unreported role of the universally conserved general base residue Glu166 in impeding the proper binding of carbapenems by restricting their 6-α-hydroxyethyl group.

Protective effect of an intrinsic antioxidant, HMH (5-hydroxy-1-methylhydantoin; NZ-419), against cellular damage of kidney tubules.

HMH (5-hydroxy-1-methylhydantoin; NZ-419) is a mammalian creatinine metabolite and an intrinsic antioxidant. HMH prevents the progression of chronic kidney disease in rats when a sufficient amount is taken orally. We assessed whether intrinsic and higher levels of HMH could protect tubular epithelial cells, LLC-PK(1) cells, against known cellular damage caused by xenobiotics, such as cisplatin and cephaloridine, or by hypoxia/reoxygenation treatment. Both cell damage and peroxidation, monitored as the leakage of lactate dehydrogenase (LDH) and malondialdehyde (MDA), respectively, from cells into the media, were inhibited by HMH in a concentration-dependent manner. The minimum effective concentration of HMH (2.5 µM) seemed to be too low for HMH to only be a direct hydroxyl radical scavenger. Additional antioxidant effect(s) inhibiting reactive oxygen species generation and/or modulating signal transduction pathways were suggested. The possibility that intrinsic HMH could be a protectant for the kidney was indicated. At the same time, for sufficient inhibition, higher concentrations than intrinsic HMH concentrations may be necessary. Patterns of efficacies of HMH on LDH and MDA against different kinds of cellular damage were compared with our reported data on those of corresponding, naturally occurring antioxidants. A common and specific inhibitory mechanism as well as common target(s) in kidney injuries were indicated.

OmpA is the principal nonspecific slow porin of Acinetobacter baumannii.

Acinetobacter species show high levels of intrinsic resistance to many antibiotics. The major protein species in the outer membrane of Acinetobacter baumannii does not belong to the high-permeability trimeric porin family, which includes Escherichia coli OmpF/OmpC, and instead is a close homolog of E. coli OmpA and Pseudomonas aeruginosa OprF. We characterized the pore-forming function of this OmpA homolog, OmpA(Ab), by a reconstitution assay. OmpA(Ab) produced very low pore-forming activity, about 70-fold lower than that of OmpF and an activity similar to that of E. coli OmpA and P. aeruginosa OprF. The pore size of the OmpA(Ab) channel was similar to that of OprF, i.e., about 2 nm in diameter. The low permeability of OmpA(Ab) is not due to the inactivation of this protein during purification, because the permeability of the whole A. baumannii outer membrane was also very low. Furthermore, the outer membrane permeability to cephalothin and cephaloridine, measured in intact cells, was about 100-fold lower than that of E. coli K-12. The permeability of cephalothin and cephaloridine in A. baumannii was decreased 2- to 3-fold when the ompA(Ab) gene was deleted. These results show that OmpA(Ab) is the major nonspecific channel in A. baumannii. The low permeability of this porin, together with the presence of constitutive ß-lactamases and multidrug efflux pumps, such as AdeABC and AdeIJK, appears to be essential for the high levels of intrinsic resistance to a number of antibiotics.

Quantitative structure activity relationship for inhibition of human organic cation/carnitine transporter.

Organic cation/carnitine transporter (OCTN2; SLC22A5) is an important transporter for L-carnitine homeostasis, but can be inhibited by drugs, which may cause L-carnitine deficiency and possibly other OCTN2-mediated drug-drug interactions. One objective was to develop a quantitative structure-activity relationship (QSAR) of OCTN2 inhibitors, in order to predict and identify other potential OCTN2 inhibitors and infer potential clinical interactions. A second objective was to assess two high renal clearance drugs that interact with OCTN2 in vitro (cetirizine and cephaloridine) for possible OCTN2-mediated drug-drug interactions. Using previously generated in vitro data of 22 drugs, a 3D quantitative pharmacophore model and a Bayesian machine learning model were developed. The four pharmacophore features include two hydrophobic groups, one hydrogen-bond acceptor, and one positive ionizable center. The Bayesian machine learning model was developed using simple interpretable descriptors and function class fingerprints of maximum diameter 6 (FCFP_6). An external test set of 27 molecules, including 15 newly identified OCTN2 inhibitors, and a literature test set of 22 molecules were used to validate both models. The computational models afforded good capability to identify structurally diverse OCTN2 inhibitors, providing a valuable tool to predict new inhibitors efficiently. Inhibition results confirmed our previously observed association between rhabdomyolysis and C(max)/K(i) ratio. The two high renal clearance drugs cetirizine and cephaloridine were found not to be OCTN2 substrates, and their diminished elimination by other drugs is concluded not to be mediated by OCTN2.

Kidney injury molecule-1 expression in rat proximal tubule after treatment with segment-specific nephrotoxicants: a tool for early screening of potential kidney toxicity.

Dose-response expression of kidney injury molecule-1 (KIM-1) gene in kidney cortex and its correlation with morphology and traditional biomarkers of nephrotoxicity (plasma creatinine and blood urea nitrogen, BUN) or segment-specific marker of proximal tubule injury (kidney glutamine synthetase, GSK) were studied in male rats treated with proximal tubule segment-specific nephrotoxicants. These included hexachloro-1:3-butadiene (HCBD, S(3) segment-specific), potassium dichromate (chromate, S(1)-S(2) segment-specific), and cephaloridine (Cph, S(2) segment-specific). Rats were treated with a single intraperitoneal (ip) injection of HCBD 25, 50, and 100 mg/kg, subcutaneous (sc) injection of chromate 8, 12.5, and 25 mg/kg; or ip injection of Cph 250, 500, and 1,000 mg/kg. KIM-1 gene showed a dose-dependent up-regulation induced by all segment-specific nephrotoxicants. Interestingly, magnitude of the up-regulation reflected the severity of microscopic tubular changes (degeneration, necrosis, and regeneration). Even low-severity microscopic observations were evidenced by significant gene expression changes. Furthermore, KIM-1 showed significant up-regulation even in the absence of morphological changes. In contrast, traditional and specific markers demonstrated low sensitivity or specificity. In conclusion, this study suggested KIM-1 as a sensitive molecular marker of different levels of tubular injury, and it is likely to represent a potential tool for early screening of nephrotoxicants.

Penicillin sulfone inhibitors of class D beta-lactamases.

OXA beta-lactamases are largely responsible for beta-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the beta-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D beta-lactamases. To overcome this challenge, we explored the abilities of beta-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D beta-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low K(i) values for the OXA-1 beta-lactamase (0.70 +/- 0.14 --> 1.60 +/- 0.30 microM) and demonstrated significant K(i) improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low K(i)s for the OXA-10, -14, -17, and -24/40 beta-lactamases (0.20 +/- 0.04 --> 17 +/- 4 microM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [t(n)] = 0) and t(n)s ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA beta-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 beta strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type beta-lactamases.

Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli.

Multidrug efflux transporters, especially those that belong to the resistance-nodulation-division (RND) family, often show very broad substrate specificity and play a major role both in the intrinsic antibiotic resistance and, with increased levels of expression, in the elevated resistance of Gram-negative bacteria. However, it has not been possible to determine the kinetic behavior of these important pumps so far. This is partly because these pumps form a tripartite complex traversing both the cytoplasmic and outer membranes, with an outer membrane channel and a periplasmic adaptor protein, and it is uncertain if the behavior of an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli containing the intact multiprotein complex AcrB-AcrA-TolC, and measure the kinetic constants for various cephalosporins, by assessing the periplasmic concentration of the drug from their rate of hydrolysis by periplasmic beta-lactamase and the rate of efflux as the difference between the influx rate and the hydrolysis rate. Nitrocefin efflux showed a K(m) of about 5 microM with little sign of cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, however, kinetics showed strong positive cooperativity, which is consistent with the rotating catalysis model of this trimeric pump. For the very hydrophilic cefazolin there was little sign of efflux.

Carnitine/organic cation transporter OCTN2 (Slc22a5) is responsible for renal secretion of cephaloridine in mice.

Carnitine/organic cation transporter (OCTN) 2 (SLC22A5) plays a pivotal role in renal tubular reabsorption of carnitine, a vitamin-like compound, on apical membranes of proximal tubules, but its role in relation to therapeutic drugs remains to be clarified. The purpose of the present study was to elucidate the involvement of OCTN2 in renal disposition of a beta-lactam antibiotic, cephaloridine (CER), based on experiments with juvenile visceral steatosis (jvs) mice, which have a functional deficiency of the octn2 gene. Renal clearance of CER during constant intravenous infusion in wild-type mice was much higher than could be accounted for by glomerular filtration, but was decreased by increasing the infusion rate with minimal change in kidney-to-plasma concentration ratio, suggesting the existence of saturable transport mechanism(s) across the apical membranes. The plasma concentration profile and kidney-to-plasma concentration ratio after intravenous injection in jvs mice were higher than those in wild-type mice, whereas renal clearance in jvs mice was much lower than that in wild-type mice and could be accounted for by glomerular filtration. Uptake of CER by mouse OCTN2 was shown in Xenopus laevis oocytes expressing mouse OCTN2. The CER transport by OCTN2 exhibited saturation with K(m) of approximately 3 mM, which is similar to the renal CER concentration exhibiting saturation in renal clearance in vivo. The OCTN2-mediated CER transport was inhibited by carnitine and independent of Na(+) replacement in the medium. These results show OCTN2 on apical membranes of proximal tubules plays a major role in renal secretion of CER in mice.

L-type fatty acid binding protein transgenic mouse as a novel tool to explore cytotoxicity to renal proximal tubules.

A novel biomarker of renal dysfunction, liver-type fatty acid binding protein (L-FABP), which is expressed in human proximal tubules, binds to lipid peroxidation products during renal injury and is excreted into the urine. Here, we examined the usefulness of human L-FABP transgenic (Tg) mice as a tool to explore nephrotoxicity, employing two model drugs, cephaloridine and cisplatin, which are taken up by renal tubules via organic anion and cation transporters, respectively. Urinary excretion of L-FABP increased after administration of cephaloridine in most of the Tg mice, whereas glomerular filtration markers such as blood-urea-nitrogen (BUN) and plasma creatinine (CRE) were almost unchanged. Thus, L-FABP is a highly sensitive detector of the nephrotoxicity of cephaloridine. Urinary excretion of L-FABP in the Tg mice also increased after administration of cisplatin, and this increase was reduced by coadministration of cimetidine. Both BUN and CRE also increased after the cisplatin treatment, but these parameters were minimally affected by coadministration of cimetidine, suggesting that cimetidine reduces cisplatin-induced renal tubular toxicity with only a minimal effect on the glomerulus. These results indicate that the L-FABP Tg mouse should be a useful drug screening system to evaluate specifically the toxicity of transporter substrates to renal tubules.

L-Carnitine suppresses oleic acid-induced membrane permeability transition of mitochondria.

Membrane permeability transition (MPT) of mitochondria has an important role in apoptosis of various cells. The classic type of MPT is characterized by increased Ca(2+) transport, membrane depolarization, swelling, and sensitivity to cyclosporin A. In this study, we investigated whether L-carnitine suppresses oleic acid-induced MPT using isolated mitochondria from rat liver. Oleic acid-induced MPT in isolated mitochondria, inhibited endogenous respiration, caused membrane depolarization, and increased large amplitude swelling, and cytochrome c (Cyt. c) release from mitochondria. L-Carnitine was indispensable to beta-oxidation of oleic acid in the mitochondria, and this reaction required ATP and coenzyme A (CoA). In the presence of ATP and CoA, L-carnitine stimulated oleic acid oxidation and suppressed the oleic acid-induced depolarization, swelling, and Cyt. c release. L-Carnitine also contributed to maintaining mitochondrial function, which was decreased by the generation of free fatty acids with the passage of time after isolation. These results suggest that L-carnitine acts to maintain mitochondrial function and suppresses oleic acid-mediated MPT through acceleration of beta-oxidation.

Transcriptomic analysis of nephrotoxicity induced by cephaloridine, a representative cephalosporin antibiotic.

Cephaloridine (CER) is a classical beta-lactam antibiotic that has long served as a model drug for the study of cephalosporin antibiotic-induced acute tubular necrosis. In the present study, we analyzed gene expression profiles in the kidney of rats given subtoxic and toxic doses of CER to identify gene expression alterations closely associated with CER-induced nephrotoxicity. Male Fischer 344 rats were intravenously injected with CER at three different dose levels (150, 300, and 600 mg/kg) and sacrificed after 24 h. Only the high dose (600 mg/kg) caused mild proximal tubular necrosis and slight renal dysfunction. Microarray analysis identified hundreds of genes differentially expressed in the renal cortex following CER exposure, which could be classified into two main groups that were deregulated in dose-dependent and high dose-specific manners. The genes upregulated dose dependently mainly included those involved in detoxification and antioxidant defense, which was considered to be associated with CER-induced oxidative stress. In contrast, the genes showing high dose-specific (lesion-specific) induction included a number of genes related to cell proliferation, which appeared to reflect a compensatory response to CER injury. Of the genes modulated in both manners, we found many genes reported to be associated with renal toxicity by other nephrotoxicants. We could also predict potential transcription regulators responsible for the observed gene expression alterations, such as Nrf2 and the E2F family. Among the candidate gene biomarkers, kidney injury molecule 1 was markedly upregulated at the mildly toxic dose, suggesting that this gene can be used as an early and sensitive indicator for cephalosporin nephrotoxicity. In conclusion, our transcriptomic data revealed several characteristic expression patterns of genes associated with specific cellular processes, including oxidative stress response and proliferative response, upon exposure to CER, which may enhance our understanding of the molecular mechanisms behind cephalosporin antibiotic-induced nephrotoxicity.

In vitro gene expression analysis of nephrotoxic drugs in rat primary renal cortical tubular cells.

Rat primary renal cortical tubular cells were exposed to seven test substances, some with, and some without, known direct renal tubular cell toxicity. Cells were exposed to the substances at either one-third or one-tenth of the TC50 for cytotoxicity for 6 h or 24 h, so as not to induce cytotoxicity but to cause some transcriptional changes. Transcriptional profiles were investigated by using the Affymetrix Rat Toxicology U34 arrays, containing probes for more than 850 genes and ESTs. Four direct toxicants, cisplatin (CDDP), its less nephrotoxic analogue carboplatin (CBDCA), cephaloridine and gentamicin, were grouped together in a hierarchical clustering. In addition, the four direct toxicants affected more than 32 transcripts at their subcytotoxic concentrations at either 6 h or 24 h exposure. On the other hand, diclofenac, cyclosporine A and zinc, which are not considered to be directly toxic to tubules, affected less than 12 transcripts. Decreased Map3k12 and increased Hmox1 were commonly observed among the four direct toxicants, which appeared to be responses to cellular damage. Two platinum complexes, CDDP and CBDCA, induced similar changes, regardless of exposure duration or concentration. The types of transcriptional changes observed in this study were consistent with previously reported in vivo data, although there were some differences. These observations suggest that an in vitro gene expression analysis approach using GeneChip is feasible for screening for direct tubular toxicity of drugs and may help to clarify the underlying mechanisms of tubular toxicity.

Safe use of selected cephalosporins in penicillin-allergic patients: a meta-analysis.

BACKGROUND: Recent analysis of clinical data and a clearer understanding of the role of chemical structure in the development of cross-reactivity indicate that the increased risk of an allergic reaction to a cephalosporin in penicillin-allergic patients is smaller than previously postulated. METHOD: Medline and EMBASE databases were searched with the keywords: cephalosporin, penicillin, allergy, and cross-sensitivity for the years 1960 through 2005. Among 219 articles retrieved, 9 served as source material for this evidence-based meta-analysis. RESULTS: A significant increase in allergic reactions to cephalothin (odds ratio [OR] = 2.5; 95% confidence interval [CI] = 1.1 to 5.5), cephaloridine (OR = 8.7; CI = 5.9 to 12.8), and cephalexin (OR = 5.8; CI = 3.6 to 9.2), and all first generation cephalosporins plus cefamandole (OR = 4.8; CI = 3.7 to 6.2) were observed in penicillin allergic patients; no increase was observed with second generation cephalosporins (OR = 1.1; CI, 0.6 to 2.1) or third generation cephalosporins (OR = 0.5; CI = 0.2 to 1.1). Clinical challenges, skin testing, and monoclonal antibody studies point to the paramount importance of similarities in side chain structure to predict cross-allergy between cephalosporins and penicillins. CONCLUSION: First-generation cephalosporins have cross-allergy with penicillins, but cross-allergy is negligible with second- and third-generation cephalosporins. Particular emphasis should be placed on the role of chemical structure in determining the risk of cross-reactivity between specific agents.

The aminolysis of N-aroyl beta-lactams occurs by a concerted mechanism.

N-aroyl beta-lactams are imides with exo- and endocyclic acyl centres which react with amines in aqueous solution to give the ring opened beta-lactam aminolysis product. Unlike the strongly base catalysed aminolysis of beta-lactam antiobiotics, such as penicillins and cephaloridines, the rate law for the aminolysis of N-aroyl beta-lactams is dominated by a term with a first-order dependence on amine concentration in its free base form, indicative of an uncatalysed aminolysis reaction. The second-order rate constants for this uncatalysed aminolysis of N-p-methoxybenzoyl beta-lactam with a series of substituted amines generates a Brønsted betanuc value of +0.90. This is indicative of a large development of positive effective charge on the amine nucleophile in the transition state. Similarly, the rate constants for the reaction of 2-cyanoethylamine with substituted N-aroyl beta-lactams gives a Brønsted betalg value of -1.03 for different amide leaving groups and is indicative of considerable change in effective charge on the leaving group in the transition state. These observations are compatible with either a late transition state for the formation of the tetrahedral intermediate of a stepwise mechanism or a concerted mechanism with simultaneous bond formation and fission in which the amide leaving group is expelled as an anion. Amide anion expulsion is also indicated by an insignificant solvent kinetic isotope effect, kH2ORNH2/kD2ORNH2, of 1.01 for the aminolysis of N-benzoyl beta-lactam with 2-methoxyethylamine. The Brønsted betalg value decreases from -1.03 to -0.71 as the amine nucleophile is changed from 2-cyanoethylamine to propylamine. The Brønsted betanuc value is more invariant although it changes from +0.90 to +0.85 on changing the amide leaving group from p-methoxy to p-chloro substituted. The sensitivity of the Brønsted betanuc and betalg values to the nucleofugality of the amide leaving group and the nucleophilicity of the amine nucleophiles, respectively, indicate coupled bond formation and bond fission processes.

Protective effect of serum thymic factor, FTS, on cephaloridine-induced nephrotoxicity in rats.

Serum thymic factor (FTS), a thymic peptide hormone, has been reported to increase superoxide disumutase (SOD) levels in senescence-accelerated mice. In the present study, we examined the effect of FTS on cephaloridine (CER)-induced nephrotoxicity in vivo and in vitro. We previously reported that CER led to extracellular signal-regulated protein kinase (ERK) activation in the rat kidney. So, we also investigated whether FTS has an effect on ERK activation induced by CER. Treatment of male Sprague-Dawley rats with intravenous CER (1.2 g/kg) for 24 h markedly increased BUN and plasma creatinine levels and urinary excretion of glucose and protein, decreased creatinine clearance and also led to marked pathological changes in the proximal tubules, as revealed by electron micrographs. An increase in phosphorylated ERK (pERK) was detected in the nuclear fraction prepared from the rat kidney cortex 24 h after CER injection. Pretreatment of rats with FTS (50 microg/kg, i.v.) attenuated the CER-induced renal dysfunction and pathological damage. FTS also suppressed CER-induced ERK activation in the kidney. In vitro treatment of the established cell line, LLC-PK1 cells, with FTS significantly ameliorated CER-induced cell injury, as measured by lactate dehydrogenase (LDH) leakage. Our results, taken together with our previous report that MEK inhibitors ameliorated CER-induced renal cell injury and ERK activation induced by CER, suggest that FTS participates in protection from CER-induced nephrotoxicity by suppressing ERK activation induced by CER.

The growth rate of Mycobacterium smegmatis depends on sufficient porin-mediated influx of nutrients.

Mycobacteria have a unique outer membrane (OM) that is thicker than any other known biological membrane. Nutrients cross this permeability barrier by diffusion through porins. MspA is the major porin of Mycobacterium smegmatis. In this study we showed that three paralogues of MspA, namely MspB, MspC and MspD are also porins. However, only the mspA and mspC genes were expressed in the wild-type strain. None of the single deletion mutants displayed a significant OM permeability defect except for the mspA mutant. Deletion of the mspA gene caused activation of transcription of mspB and/or mspD in three independent strains by unknown chromosomal mutations. It is concluded that mspB and mspD provide backup porins for M. smegmatis. This also indicated that a minimal porin-mediated OM permeability is essential for survival of M. smegmatis. Electron microscopy in combination with quantitative image analysis of protein gels revealed that the number of pores per cell dropped from 2400 to 800 and 150 for the DeltamspA and DeltamspA DeltamspC mutant (ML10) respectively. The very low number of pores correlated well with the at least 20-fold lower channel activity of detergent extracts of the ML10 strain and its 15- and 75-fold lower permeability to nutrient molecules such as serine and glucose respectively. The amount of Msp porin and the OM permeability of the triple porin mutant lacking mspA, mspC and mspD was not altered. The growth rate of M. smegmatis dropped drastically with its porin-mediated OM permeability in contrast to porin mutants of Escherichia coli. These results show that porin-mediated influx of nutrients is a major determinant of the growth rate of M. smegmatis.

Protective effect of a protein kinase inhibitor on cellular injury induced by cephaloridine in the porcine kidney cell line LLC-PK(1).

We investigated the effects of a protein kinase C inhibitor and a tyrosine kinase inhibitor on the cellular injury induced by cephaloridine in an established renal epithelial cell line, LLC-PK(1). Cephaloridine increased the leakage of lactate dehydrogenase (LDH) from LLC-PK(1) cells into the medium and also caused an increase in the level of lipid peroxide (index of oxidative stress) in the cells. Treatment of the cells with a hydroxyl radical scavenger, dimethylthiourea (DMTU), inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK(1) cells exposed to cephaloridine. A protein kinase C inhibitor, H-7, and tyrosine kinase inhibitors, genistein and lavendustinA, inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK(1) cells exposed to cephaloridine. These results suggest that a signaling pathway which involves protein kinase C and tyrosine kinase plays a role in the generation of reactive oxygen species in LLC-PK(1) cells damaged by cephaloridine.

Peptidoglycan precursor pools associated with MraY and FtsW deficiencies or antibiotic treatments.

Blocking peptidoglycan synthesis in Escherichia coli with moenomycin or vancomycin led to the accumulation of UDP-MurNAc-pentapeptide and of its immediate upstream precursors, whereas with cephaloridine or penicillin G the pool of UDP-MurNAc-pentapeptide decreased. With MraY and FtsW deficiencies the decrease of UDP-MurNAc-pentapeptide was accompanied by an increase of the upstream nucleotide precursors and the appearance of UDP-MurNAc-tetrapeptide.

Identification of novel targets of cephaloridine in rabbit renal proximal tubules synthesizing glutamine from alanine.

Cephaloridine, which accumulates in the renal proximal tubule, is a model compound used for studying the toxicity of antibiotics towards this nephron segment. Several studies have demonstrated that cephaloridine alters renal intermediary and energy metabolism, but the mechanism by which this compound interferes with renal metabolic pathways remains incompletely understood. In an attempt to improve our knowledge in this field, we have studied the influence of cephaloridine on the synthesis of glutamine, which represents a key metabolic process involving several important enzymatic steps in the rabbit kidney. For this, suspensions of rabbit renal proximal tubules were incubated for 90 and 180 min in the presence of 5 mM alanine, an important glutamine precursor, both in the absence and the presence of 10 mM cephaloridine. Glutamate accumulation and glutamine synthesis were found to be inhibited by cephaloridine after 90 and 180 min of incubation, and cephaloridine accumulation in the renal proximal cells occurred in a time-dependent manner. The renal proximal tubule activities of alanine aminotransferase and glutamate dehydrogenase, which initiates alanine removal and releases the ammonia needed for glutamine synthesis, respectively, were inhibited to a significant degree and in a concentration-dependent manner by cephaloridine concentrations in the range found to accumulate in the renal proximal cells. Citrate synthase and glutamine synthetase activities were also inhibited by cephaloridine, but to a much lesser extent. The above enzymatic activities were not found to be inhibited when they were measured after successive dilutions of renal proximal tubules incubated for 180 min in the presence of 5 mM alanine and 10 mM cephaloridine. When microdissected segments (S1-S3) of rabbit renal proximal tubules were incubated for 180 min with 5 mM alanine with and without 5 and 10 mM cephaloridine, glutamate accumulation and glutamine synthesis were also inhibited in the three renal proximal segments studied; the latter cephaloridine-induced inhibitions observed were concentration-dependent except for glutamine in the S3 segment. These results are consistent with the view that cephaloridine accumulates and is toxic along the entire rabbit renal proximal tubule. They also demonstrate that cephaloridine interferes in a concentration-dependent and reversible manner mainly with alanine aminotransferase and glutamate dehydrogenase, which are therefore newly-identified targets of the toxic effects of cephaloridine in the rabbit renal proximal tubule.