The hemostatic system is assumed to be similar in children and adults and reference ranges established for adults are commonly used to evaluate children suspected of having congenital or acquired hemostatic problems. However, we know that the hemostatic system is not fully mature by 6 months of age and comprehensive studies of healthy older children have not been published. Therefore, we conducted a prospective cohort study of the hemostatic system in healthy children having minor, elective day surgery. After obtaining informed consent, a 3-mL blood sample was obtained at the time routine preoperative blood work was drawn. The plasma was fractioned and stored at -70 degrees C for batch assaying. We measured the concentration of 33 components of the hemostatic system (functional and immunologic assays) and the bleeding time (automated pediatric device) in 246 children aged 1 to 16 inclusive (a minimum of four subjects at each age). Eleven components of hemostasis (fibrinogen, prekallikrein, high-molecular weight kininogen, factors VIII and XIII, antithrombin III [ATIII], heparin cofactor II [HCII], alpha 1-antitrypsin [alpha 1AT], protein S, plasminogen, alpha 2-antiplasmin [alpha 2AP]) had mean values and ranges of normal that were similar to adults. Mean values of seven coagulants (II, V, VII, IX, X, XI, XII) were significantly lower than adult values and varied with age. Values for three inhibitors, alpha 2-macroglobulin (alpha 2M), protein C, and protein C1-inhibitor (C1-Inh) also differed from adults. Alpha 2M and C1-Inh inhibitor levels were elevated throughout childhood, whereas protein C levels were low, with a lower limit of normal of 0.40 U/mL until the age of 11. Finally, the upper limit of normal for the bleeding time was longer in children during the first 10 years of life, but decreased to adult values in the teenage years. In summary, there are important physiologic differences in the hemostatic system in children compared with adults. The decreased levels of several critical coagulants and increased levels of alpha 2M may contribute in part to the lower risk of thrombotic events in childhood. Age-matched controls should be used for evaluation of the hemostatic system in children with suspected congenital or acquired defects.

The usefulness of isoniazid (INH), a key component of short-course chemotherapy of tuberculosis, is threatened by the emergence of drug-resistant strains of Mycobacterium tuberculosis with mutations in the katG gene. It is shown here that the most commonly occurring KatG mutation, where Ser 315 is replaced by Thr (S315T), is associated with clinically significant levels of INH resistance. In contrast to another resistant isolate, in which Pro replaces Thr 275, the S315T mutant produces active catalase-peroxidase and is virulent in the mouse model of the disease, indicating that a significant loss of bacterial fitness does not result from this frequent mutation. The implications of this finding for the transmission and reactivation of multidrug-resistant strains of M. tuberculosis are severe.

MPF, a protein kinase complex consisting of cyclin and p34cdc2 subunits, promotes the G2 to M phase transition in eukaryotic cells. The pathway of activation and inactivation of MPF is not well understood, although there is strong evidence that removal of phosphate from a tyrosine residue on p34cdc2 is part of the activation process. INH was originally identified as an activity that could inhibit the posttranslational activation of a latent form of MPF, called pre-MPF, in immature (G2 phase-arrested) Xenopus oocytes. We have purified INH and demonstrated that it is a form of protein phosphatase 2A. Both INH and the catalytic subunit of protein phosphatase 2A can directly inactivate an isolated p34cdc2-cyclin complex. Both cyclin and p34cdc2 become dephosphorylated; the rate of inactivation closely parallels the removal of phosphate from a specific site on p34cdc2. We propose that INH opposes MPF activation by reversing this critical phosphorylation.

A 5.5 kb DNA fragment carrying the functions necessary for the specific secretion of the extracellular metalloproteases B and C produced by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi has been sequenced. The fragment contains four transcribed and translated genes: inh, which codes for a protease inhibitor and is not required for protease secretion, and prtD, prtE and prtF, which share significant homology with the hlyB, hlyD and tolC genes required for alpha-haemolysin secretion in Escherichia coli. Mutations in any of the three prt genes abolish protease secretion. The prtD and prtE products (60 and 50 kd) contain at least one hydrophobic segment and the prtF gene product contains a signal sequence.

Primary airway epithelial cells grown in air-liquid interface differentiate into cultures that resemble native epithelium morphologically, express ion transport similar to those in vivo, and secrete cytokines in response to stimuli. Comparisons of cultures derived from normal and cystic fibrosis (CF) individuals are difficult to interpret due to genetic differences besides CFTR. The recently discovered CFTR inhibitor, CFTR(inh)-172, was used to create a CF model with its own control to test if loss of CFTR-Cl(-) conductance alone was sufficient to initiate the CF inflammatory response. Continuous inhibition of CFTR-Cl(-) conductance for 3-5 days resulted in significant increase in IL-8 secretion at basal (P = 0.006) and in response to 10(9) Pseudomonas (P = 0.0001), a fourfold decrease in Smad3 expression (P = 0.02), a threefold increase in RhoA expression, and increased NF-kappaB nuclear translocation upon TNF-alpha/IL-1beta stimulation (P < 0.000001). CFTR inhibition by CFTR(inh)-172 over this period does not increase epithelial sodium channel activity, so lack of Cl(-) conductance alone can mimic the inflammatory CF phenotype. CFTR(inh)-172 does not affect IL-8, IL-6, or granulocyte/macrophage colony-stimulating factor secretion in two CF phenotype immortalized cell lines: 9/HTEo(-) pCEP-R and 16HBE14o(-) AS, or IL-8 secretion in primary CF cells, and inhibitor withdrawal abolishes the increased response, so CFTR(inh)-172 effects on cytokines are not direct. Five-day treatment with CFTR(inh)-172 does not affect cells deleteriously as evidenced by lactate dehydrogenase, trypan blue, ciliary activity, electron micrograph histology, and inhibition reversibility. Our results support the hypothesis that lack of CFTR activity is responsible for the onset of the inflammatory cascade in the CF lung.

Isoniazid (INH), a frontline antitubercular drug, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. Here, we report that the INH-NAD adduct is a slow, tight-binding competitive inhibitor of InhA. Demonstration that the adduct binds to WT InhA by a two-step enzyme inhibition mechanism, with initial, weak binding (K(-1) = 16 +/- 11 nM) followed by slow conversion to a final inhibited complex (EI*) with overall Ki = 0.75 +/- 0.08 nM, reconciles existing contradictory values for the inhibitory potency of INH-NAD for InhA. The first order rate constant for conversion of the initial EI complex to EI* (k2 = 0.13 +/- 0.01 min(-1)) is similar to the maximum rate constant observed for InhA inhibition in reaction mixtures containing InhA, INH, NADH, and the INH-activating enzyme KatG (catalase/peroxidase from M. tuberculosis), consistent with an inhibition mechanism in which the adduct forms in solution rather than on the enzyme. Importantly, three mutations that correlate with INH resistance, I21V, I47T, and S94A, have little impact on the inhibition constants. Thus, drug resistance does not result simply from a reduction in affinity of INH-NAD for pure InhA. Instead, we hypothesize that protein-protein interactions within the FASII complex are critical to the mechanism of INH action. Finally, for M161V, an InhA mutation that correlates with resistance to the common biocide triclosan in Mycobacterium smegmatis, binding to form the initial EI complex is significantly weakened, explaining why this mutant inactivates more slowly than WT InhA when incubated with INH, NADH, and KatG.

The aims of this study were: 1) to describe, in relation to the date of final menses, the average hormone levels of women in the years before and after this date and to determine the extent to which these average levels were dependent on age and body mass index (BMI); and 2) to determine the degree of tracking in residual hormone levels [i.e., the extent to which individuals above (below) the mean for their age or time relative to final menstrual period (FMP) and BMI remain above (below) the mean as time progresses]. Serial levels of serum FSH, circulating estradiol (E2), and the dimeric inhibins (INH) A and B were measured annually in 150 women who experienced a natural menopause during 6 years of follow-up. Means of the log-transformed hormonal levels were analyzed as a double-logistic function of time relative to FMP, as well as age and BMI and correlations between repeated hormonal levels, were measured. Mean FSH levels started to increase from about 2 years before the FMP, increased most rapidly about 10 months before the FMP, and had virtually plateaued by 2 years after the FMP. FSH levels were, on average, 3% greater for each year of age and 2% lower for each kg/m2 of BMI. After adjusting for time relative to the FMP, logFSH showed modest tracking. Age-adjusted values of logFSH were moderately correlated across time, and much of this tracking was explained by the actual timing of a woman's FMP. Mean E2 levels started to decrease about 2 years before the FMP, decreased most rapidly around the time of the FMP, and had virtually plateaued by 2 years after the FMP. E2 levels were lower, on average, by about 9% per year of age, and residual values showed weak tracking. Levels of both INHA and INHB decreased, on average, in the years before the FMP and were undetectable (INHA, < 10 pg/mL; INHB, < 25 pg/mL) in the majority of women by the time of the FMP and in almost all women by 4 years post-FMP. Significant negative correlations between log serum FSH and log E2 (r = -0.73) and log INHA (r = -0.41) and log INHB (r = -0.36) were observed. It is concluded that substantial changes in reproductive hormone levels occur within 1-2 yr on each side of the FMP, that falling concentrations of E2 and the INH contribute to the rising concentrations of FSH, and that there is no single reliable hormonal marker of menopausal status for an individual woman.

The purpose of the present investigation was to purify a urine-derived tumor necrosis factor alpha inhibitor (TNF alpha INH) and to characterize its mechanism of action. For the purification procedure, urine was concentrated and TNF alpha INH purified by ion-exchange chromatographies, gel filtration, TNF alpha affinity column, and reverse-phase chromatography. The TNF alpha INH migrates with an apparent Mr of approximately 33,000 when estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under both reducing and nonreducing conditions. Elution of TNF alpha INH activity from the gel yields also a approximately 33,000-Da inhibitory fraction. Besides inhibiting TNF alpha-induced cytotoxicity in L929 cells in the presence of actinomycin D, the TNF alpha INH impeded in a dose-dependent manner prostaglandin E2 production and expression of cell-associated interleukin-1 by human dermal fibroblasts. Therefore, TNF alpha INH is active on both actinomycin D-treated and untreated cells. In contrast to TNF alpha, TNF beta-induced cytotoxicity was only slightly affected by the inhibitor. This specificity was confirmed by the fact that it affected neither interleukin-1 alpha nor interleukin-1 beta biologic activities. The mechanism of action of TNF alpha INH involves blocking of 125I-TNF alpha binding to the promonocytic cell line U937. Moreover, preincubation of 125I-TNF alpha with TNF alpha INH increased binding inhibition, suggesting an interaction between TNF alpha and the inhibitor.

Escherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1-INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn), cleaves C1-INH to produce (unique) approximately 60-65 kDa fragments. StcE does not digest other serine protease inhibitors, extracellular matrix proteins or universal protease targets. We also observed that StcE causes the aggregation of cultured human T cells but not macrophage-like cells or B cells. Substitution of aspartic acid for glutamic acid at StcE position 435 within the consensus metalloprotease active site ablates its abilities to digest C1-INH and to aggregate T cells. StcE is secreted by the etp type II secretion pathway encoded on pO157, and extracellular StcE levels are positively regulated by the LEE-encoded regulator, Ler. StcE antigen and activity were detected in the faeces of a child with an E. coli O157:H7 infection, demonstrating the expression of StcE during human disease. Cleavage of C1-INH by StcE could plausibly cause localized pro-inflammatory and coagulation responses resulting in tissue damage, intestinal oedema and thrombotic abnormalities.

The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, inhA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates inhA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C(26:0)), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of InhA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the inhA gene of Mycobacterium smegmatis that rendered InhA inactive at 42 degrees C. Thermal inactivation of InhA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C(16:0)) and a concomitant increase of tetracosanoic acid (C(24:0)) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C(16:0), and a concomitant accumulation of C(26:0). Moreover, the InhA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that InhA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with InhA being a primary target of INH.

Urine of some febrile patients exhibits a TNF-alpha inhibitory activity (TNF-alpha INH), sensitive to heat and trypsin, with an apparent mol wt of 40-60 X 10(3) and a pI range of 5.5-6.1. As for the Il-1 INH, the TNF INH activity involves a competitive mechanism of action suggesting the existence of a family of negative feedback-regulating molecules interfering with cytokines actions.

Angioedema is an underestimated clinical problem. Many cases are nonallergic reactions, e.g. bradykinin-induced angioedema caused by genetic defects and angiotensin-converting enzyme (ACE) inhibitors. This difference is crucial for successful therapy, in particular when complete emergency care is not available. Five important forms of nonallergic angioedema can be distinguished: hereditary (HAE), acquired (AAE), renin-angiotensin-aldosterone system (RAAS)-blocker-induced (RAE), pseudoallergic angioedema (PAE) and idiopathic angioedema (IAE). Some angioedema are present in the larynx and may cause death. A vast majority of nonallergic angioedema are RAE, particularly those caused by ACE inhibitors. It appears important to emphasize that in patients with complete intolerance to RAAS-blockers, cessation of RAAS-blockers is likely to be associated with increased cardiovascular risk. Currently, there is no published algorithm for diagnosis and treatment. Angioedema is usually treated by a conservative clinical approach using artificial ventilation, glucocorticoids and antihistamines. Today, a plasma pool C1-esterase inhibitor (C1-INH) concentrate is the therapy of choice in HAE. The current pharmacotherapy of nonallergic angioedema is not satisfactory, thus requiring the identification of effective agents in clinical trials. Recently, several new drugs were developed: a recombinant C1-INH, a kallikrein inhibitor (ecallantide) and a specific bradykinin-B2-receptor antagonist (icatibant). According to currently available reports, these drugs may improve the treatment of kinin-induced angioedema.

There are three major structural classes of mycolic acids in the cell envelope of Mycobacterium tuberculosis (MTB): alpha-, methoxy- and ketomycolate. The two oxygen-containing classes are biosynthetically related through a common alpha-methyl hydroxymycolate intermediate. BCG strains that fail to produce methoxymycolate and instead produce only keto- and alpha-mycolic acids show apparent defects in the O-methyltransferase MMAS-3. Overproduction of MMAS-3 from MTB resulted in a complete replacement of ketomycolate by methoxymycolate in both BCG and MTB. In vitro growth of these recombinant strains lacking ketomycolate was impaired at reduced temperatures but appeared to be normal at 37 degrees C. Glucose uptake was significantly decreased in such strains, but uptake of chenodeoxycholate and glycine was unaffected. Although sensitivity to INH remained unchanged, these cells were found to be hypersensitive to ampicillin and rifampicin. Infectivity of BCG and H37Rv wild type or MMAS-3 overproducers in THP-1 cells was somewhat affected, but the ability of the strains lacking ketomycolate to grow within this macrophage-like cell line was severely compromised. In vivo labelling of mycolic acids during growth of H37Rv within THP-1 cells revealed a substantial increase in ketomycolate and alphamycolate synthesized by intracellularly grown mycobacteria. These results establish a critical role for mycolate composition in proper cell wall function during the growth of MTB in vivo.

Although Cl-inhibitor (Cl-INH) and alpha(2)-macroglobulin (alpha(2)M) have been reported as the major inhibitors of plasma kallikrein in normal plasma, there is little quantitative support for this conclusion. Thus, we studied the inactivation of purified kallikrein in normal plasma, as well as in plasma congenitally deficient in Cl-INH, or artificially depleted of alpha(2)M by chemical modification of the inhibitor with methylamine. Under pseudo-first-order conditions, the inactivation rate constant of kallikrein in normal plasma was 0.60 min(-1). This rate constant was reduced to 0.35, 0.30, and 0.06 min(-1), in plasma deficient respectively in Cl-INH, alpha(2)M, or both inhibitors. Thus Cl-INH (42%) and alpha(2)M (50%) were found to be the major inhibitors of kallikrein in normal plasma. Moreover all the other protease inhibitors present in normal plasma contributed only for 8% to the inactivation of the enzyme. To confirm these kinetic results, (125)I-kallikrein (M(r) 85,000) was completely inactivated by various plasma samples, and the resulting mixtures were analyzed by gel filtration on Sepharose 6B CL for the appearance of (125)I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (M(r) 370,000) with Cl-INH, while 48% formed a complex (M(r) 850,000) with alpha(2)M. After inactivation by Cl-INH-deficient plasma, >90% of the active (125)I-kallikrein was associated with alpha(2)M. A similar proportion of the label was associated with Cl-INH in plasma deficient in alpha(2)M. After inactivation by plasma deficient in both Cl-INH and alpha(2)M, (125)I-kallikrein was found to form a complex of M(r) 185,000. This latter complex, which may involve antithrombin III, alpha(1)-protease inhibitor, and/or alpha(1)-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either Cl-INH or alpha(2)M, even after the addition of heparin (2 U/ml). These observations demonstrate that Cl-INH and alpha(2)M are the only significant inhibitors of kallikrein in normal plasma confirming previous predictions based on experiments in purified systems. Moreover, in the absence of either Cl-INH or alpha(2)M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.

Tuberculosis (TB) treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis (Mtb) to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4) inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH). Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment.

We demonstrate in vitro expression of complement components, i.e. C3, factor H (FH), factor B (FB), C4, C1-inhibitor (C1-inh), C1q, C5, C6, C7 and C9, by four human neuroblastoma cell lines IMR32, SKNSH, SH-SY5Y and KELLY. Activating proteins C4, C9 and C1q, and regulatory proteins FH and C1-inh were produced constitutively by the four cell lines. C3, C6 and FB were mainly produced by SKNSH and SH-SY5Y. Western blot experiments showed that secreted proteins were structurally similar to their serum counterparts. An additional polypeptide of 43 kDa with FH immunoreactivity was detected, which could correspond to the N-terminal truncated form found in plasma. Regulation of complement expression by inflammatory cytokines, lipopolysaccharide and dexamethasone was tested in vitro. These factors had no significant effects on activating synthesis of components C3, FB and C4, but expression of regulating components C1-inh and FH was strongly increased particularly by IFN-gamma and tumor necrosis factor-alpha. The rate of synthesis of complement components was dependent on the differentiation of neuroblastoma cells. This effect of differentiation was also observed on normal rat neurons. Rat cerebellar granule cells constitutively expressed mRNA for C4 and C1q, but expression of C3 mRNA was induced by differentiation. This study shows that neurons could be another local source of complement in the brain, besides astrocytes and microglia. Human neuroblastoma cell lines can constitute an interesting model to analyze complement biosynthesis by human neurons. Local complement expression by neurons in vivo may be implicated in some physio-pathological processes.

Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three DeltaF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Omega.cm(2). In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1beta, TNF-alpha, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.

INH, a type 2A protein phosphatase (PP2A), negatively regulates entry into M phase and the cyclin B-dependent activation of cdc2 in Xenopus extracts. INH appears to be central to the mechanism of the trigger for mitotic initiation, as it prevents the premature activation of cdc2. We first show that INH is a conventional form of PP2A with a B alpha regulatory subunit. We next explore the mechanism by which it inhibits cdc2 activation by examining the effect of purified PP2A on the reaction pathways controlling cdc2 activity. Our results suggest that although PP2A inhibits the switch in tyrosine kinase and tyrosine phosphatase activities accompanying mitosis, this switch is a consequence of the inhibition of some other rate-limiting event. In the preactivation phase, PP2A inhibits the pathway leading to T161 phosphorylation, suggesting that this activity may be one of the rate-limiting events for transition. However, our results also suggest that the accumulation of active cdc2/cyclin complexes during the lag is only one of the events required for triggering entry into mitosis.

Tuberculosis (TB) is notoriously difficult to cure, requiring administration of multiple antibiotics for 6 mo or longer. Conventional anti-TB drugs inhibit biosynthetic processes involved in cell growth and division, such as DNA replication, RNA transcription, protein translation, and cell wall biogenesis. Although highly effective against bacteria cultured in vitro under optimal growth conditions, these antibiotics are less effective against bacteria grown in vivo in the tissues of a mammalian host. The factors that contribute to the antibiotic tolerance of bacteria grown in vivo are unknown, although altered metabolism and sluggish growth are hypothesized to play a role. To address this question, we identified mutations in Mycobacterium tuberculosis that impaired or enhanced persistence in mice treated with isoniazid (INH), a front-line anti-TB drug. Disruption of cydC, encoding a putative ATP-binding cassette transporter subunit, accelerated bacterial clearance in INH-treated mice without affecting growth or survival in untreated mice. Conversely, transposon insertions within the rv0096-rv0101 gene cluster attenuated bacterial growth and survival in untreated mice but paradoxically prevented INH-mediated killing of bacteria in treated mice. These contrasting phenotypes were dependent on the interaction of the bacteria with the tissue environment because both mutants responded normally to INH when grown in macrophages ex vivo or in axenic cultures in vitro. Our findings have important implications because persistence-impairing mutations would be missed by conventional genetic screens to identify candidate drug targets. Conversely, persistence-enhancing mutations would be missed by standard diagnostic methods, which are performed on bacteria grown in vitro, to detect drug resistance.

Complement activation products C1q, C4c/d, and C3c/d in amyloid plaques in Alzheimer's disease probably result from direct binding and activation of C1 by amyloid beta peptides. RT-PCR and in situ hybridization studies have shown that several complement factors are produced in the brain parenchyma. In the present study, cytokines that can be detected in amyloid plaques (i.e., interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-alpha) were found to differentially stimulate the expression of C1 subcomponents, C1-Inhibitor (C1-Inh), C4, and C3, by astrocyte and microglial cell cultures derived from postmortem adult, human brain specimens and by neuroblastoma cell lines in culture. C1r and C1s were secreted at low levels by astrocytes and neuroblastoma cell lines. Exposure of cells to IL-1 alpha, IL-1 beta, TNF-alpha and to a far lesser extent IL-6, markedly upregulated C1r, C1s, and C3 production. C4 synthesis increased in response to interferon (IFN)-gamma and IL-6, whereas that of C1-Inh could be stimulated only by IFN-gamma. Thus, C1-Inh production is refractory to stimulation by plaque-associated cytokines, whereas these cytokines do stimulate C1r, C1s, and also C4 and C3 secretion by astrocytes and neuronal cells in culture. In contrast to the amyloid plaque associated cytokines IL-1 beta, IL-1 alpha, and TNF-alpha, the amyloid peptide A beta 1-42 itself did not stimulate C1r and C1s synthesis by astrocytes, microglial cells, or neuroblastoma cell lines. Microglial cells were the only cell type that constitutively expressed C1q. The ability of C1q to reassociate with newly formed C1r and C1s upon activation of C1 and subsequent inactivation by C1-Inh, may enable ongoing complement activation at sites of amyloid deposition, especially when C1-Inh is consumed and not replaced.

Drug resistance in Mycobacterium tuberculosis has become a serious global health threat, which is now complicated by the emergence of extensively drug-resistant strains. New drugs that are active against drug-resistant tuberculosis (TB) are needed. We chose to search for new inhibitors of the enoyl-acyl carrier protein (ACP) reductase InhA, the target of the first-line TB drug isoniazid (also known as isonicotinoic acid hydrazide [INH]). A subset of a chemical library, composed of 300 compounds inhibiting Plasmodium falciparum enoyl reductase, was tested against M. tuberculosis. Four compounds were found to inhibit M. tuberculosis growth with MICs ranging from 1 μM to 10 μM. Testing of these compounds against M. tuberculosis in vitro revealed that only two compounds (CD39 and CD117) were bactericidal against drug-susceptible and drug-resistant M. tuberculosis. These two compounds were also bactericidal against M. tuberculosis incubated under anaerobic conditions. Furthermore, CD39 and CD117 exhibited increased bactericidal activity when used in combination with INH or rifampin, but CD39 was shown to be toxic to eukaryotic cells. The compounds inhibit InhA as well the fatty acid synthase type I, and CD117 was found to also inhibit tuberculostearic acid synthesis. This study provides the TB drug development community with two chemical scaffolds that are suitable for structure-activity relationship study to improve on their cytotoxicities and bactericidal activities in vitro and in vivo.

The type 1 protein phosphatase (PP1) has been reported to be overactivated in the failing heart, leading to a depression in cardiac function. We investigated whether in vivo PP1 inhibition by myocardial gene transfer of inhibitor-2 (INH-2), an endogenous PP1 inhibitor, alleviates heart failure (HF) progression in the cardiomyopathic (CM) hamster, a well-established HF model. Adenoviral INH-2 gene delivery improved % fractional shortening of the left ventricle (LV) accompanied by reduced chamber size at 1 wk. In vivo myocardial INH-2 gene delivery induced an increase in cytosolic PP1 catalytic subunit alpha (PP1Calpha) without inducing the corresponding increase in cytosolic PP1 activity. On the other hand, INH-2 delivery induced a decrease in microsomal PP1Calpha, resulting in a preferential decrease in microsomal PP1 activity, thereby increasing in phospholamban phosphorylation at Ser16. INH-2 gene transfer alleviated brain natriuretic peptide expression, presumably reflecting improved cardiac function. Moreover, adeno-associated virus-mediated INH-2 gene delivery significantly extended the survival time for 3 mo. These results indicate that increased PP1 activity is an exacerbating factor during progression of genetic cardiomyopathy and modulation of PP1 activity by INH-2 provides a potential new treatment for HF without activating protein kinase A signaling in cardiomyocytes.

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in alpha-hydroxypyridinemethanesulfonic acid increased (14)CO(2) incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of (14)CO(2) into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of (14)C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of (14)CO(2) into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O(2)/CO(2) ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O(2), but these compounds increased the O(2) inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.

Previous studies have shown that urine of febrile patients contains a tumor necrosis factor alpha inhibiting activity (TNF-alpha Inh) when tested in a cytotoxicity assay using the tumor necrosis factor alpha (TNF-alpha)-susceptible cell line L929. In the present study, we investigated the relationship between the TNF-alpha Inh and a potential soluble form of the receptor, as the former has been shown to block TNF-alpha activities by binding to the ligand. We demonstrate that human TNF-alpha is affected to a greater extent than is murine TNF-alpha. This species specificity of the inhibitor correlates with the binding studies of TNF receptor interactions already reported. We raised a polyclonal antibody to TNF-alpha Inh that neutralizes its activity and does not recognize TNF-alpha. Solubilized cross-linked 125I-labeled TNF-alpha receptor complex could be immunoprecipitated by using either anti-TNF-alpha or anti-TNF-alpha Inh antibody, suggesting immunological cross-reactivity between the receptor and the inhibitor. By using fluorescein isothiocyanate-coupled TNF-alpha, it was possible to visualize by fluorescence-activated cell sorter analysis the TNF-alpha receptor on phytohemagglutinin/interleukin 2-activated T cells. A similar increase of immunofluorescence intensity of the activated T cells was observed by using anti-TNF-alpha Inh antibody revealed with a fluorescein isothiocyanate-coupled goat anti-rabbit IgG1 conjugate, suggesting that the TNF-alpha Inh is also expressed as a membrane protein. Taken together, our results suggest that the TNF-alpha Inh originally described might be a soluble form of the TNF receptor itself.