The synthesis of the subunits of the C1 complex (C1q, C1s, C1r), and its regulator C1 inhibitor (C1-Inh) by human monocytes has been previously established. However, surface expression of these molecules by monocytes has not been shown. Using flow cytometry and antigen-capture enzyme-linked immunosorbent assay, we show here for the first time that, in addition to C1q, peripheral blood monocytes, and the monocyte-derived U937 cells express C1s and C1r, as well as Factor B and C1-Inh on their surface. C1s and C1r immunoprecipitated with C1q, suggesting that at least some of the C1q on these cells is part of the C1 complex. Furthermore, the C1 complex on U937 cells was able to trigger complement activation via the classical pathway. The presence of C1-Inh may ensure that an unwarranted autoactivation of the C1 complex does not take place. Since C1-Inh closely monitors the activation of the C1 complex in a sterile or infectious inflammatory environment, further elucidation of the role of C1 complex is crucial to dissect its function in monocyte, dendritic cell, and T cell activities, and its implications in host defense and tolerance.

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG.

BACKGROUND

A non-invasive test, which could predict the presence of sperm during a testicular sperm extraction (TESE) procedure in men with non-obstructive azoospermia (NOA), would be of profound clinical importance. Inhibin B (Inh-B) and anti-Müllerian hormone (AMH) have been proposed as direct markers of Sertoli cell function and indirect markers of spermatogenesis.

METHODS

A search was conducted in the electronic databases MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials from inception through June 2009. Thirty-six different studies reported data on the predictive value of one or more index markers (serum Inh-B: 32 studies, seminal Inh-B: 5 studies, serum AMH: 2 studies, seminal AMH: 4 studies) and were included in the systematic review. Nine studies, which had serum Inh-B as index marker, met the predefined criteria and were included in the meta-analysis.

RESULTS

Serum Inh-B demonstrated a sensitivity of 0.65 (95% confidence interval [CI]: 0.56-0.74) and a specificity of 0.83 (CI: 0.64-0.93) for the prediction of the presence of sperm in TESE. When the pre-test probability of 41% was incorporated in a Fagan's nomogram, resulted in a positive post-test probability of 73% and a negative post-test probability of 23% for the presence of sperm in TESE.

CONCLUSIONS

Serum Inh-B cannot serve as a stand-alone marker of persistent spermatogenesis in men with NOA. Although limited, evidence on serum AMH and serum/seminal AMH do not support their diagnostic value in men with NOA.

Interferons-alpha, -beta and -gamma (IFNs-alpha, -beta and -gamma) stimulated the synthesis of the second complement component (C2), Factor B (B) and C1 inhibitor (C1-inh) by human monocytes in vitro. The degree of increase of the secretion rates of C2, B and C1-inh was dose-dependent and proportional to increases in the abundances of their respective mRNAs. IFN-gamma was the most effective at stimulating monocyte C1-inh synthesis, whereas IFN-alpha and IFN-beta were marginally more effective at stimulating monocyte C2 and B synthesis. Kinetic studies showed that the effect of the IFNs was rapid, with maximum stimulation occurring within 1-2 h for all three proteins. After the removal of IFNs from cultures the C1-inh mRNA abundance remained elevated for over 24 h in IFN-gamma-treated monocytes but returned to control levels within 8 h in IFN-alpha-treated and IFN-beta-treated monocytes. The abundances of C2 mRNA and B mRNA also returned to basal values within 8 h after removal of any of the three cytokines from the cultures. Both IFN-alpha and IFN-beta acted synergistically with IFN-gamma to stimulate synthesis of C1-inh and B. This synergistic effect only occurred when the cytokines were present in the cultures simultaneously. The effects of IFN-gamma plus IFN-alpha or IFN-beta on C2 synthesis appeared to be additive rather than synergistic. IFN-gamma inhibited synthesis of C3 by monocytes, but IFN-alpha and IFN-beta had no effect on the synthesis of this protein. Furthermore, none of the three cytokines had any effect on the expression of actin mRNA in monocytes.

We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca(2+) influx, (iii) promote Ca(2+)-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca(2+)-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1β, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-β without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1β, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.

Serological diagnosis and follow-up of paracoccidioidomycosis (PCM) patients have relied mainly on the detection of antibody responses by using techniques such as complement fixation (CF) and immunodiffusion. We recently described a novel <em>inh</em>ibition enzyme-linked immunosorbent assay (<em>inh</em>-ELISA) which proved to be useful in the diagnosis of PCM via the detection of an 87-kDa determinant in patient sera (<em>B</em>. L. Gomez, J. I. Figueroa, A. J. Hamilton, <em>B</em>. Ortiz, M. A. Robledo, R. J. Hay, and A. Restrepo, J. Clin. Microbiol. 35:3278-3283, 1997). This test has now been assessed as a means of following up PCM patients. A total of 24 PCM patients, classified according to their clinical presentation (6 with the acute form of the disease, of whom two had AIDS, 12 with the multifocal form of the disease, and 6 with the unifocal form of the disease), were studied. The four human immunodeficiency virus-negative patients with acute PCM showed a statistically significant decrease in circulating antigen levels after the start of antifungal therapy. Antigen levels in this group became negative by our criteria (</=2.3 microgram/ml) before week 20 and remained so in three of four of these patients. In contrast, the two AIDS patients who also presented with the acute form of PCM showed no statistically significant decrease in circulating antigen levels even after 68 weeks of therapy. Taken together as a group, the patients with the multifocal form showed a statistically significant decrease in antigenemia after 28 weeks of therapy. In addition, five of six patients with the unifocal form became antigen negative by week 40. Antigen level decrease mirrored clinical cure in the majority of patients in all clinical groups; in contrast, measurement of anti-PCM antibodies via the CF test showed wide fluctuations in titers during the follow-up period. The <em>inh</em>-ELISA for the detection of the 87-kDa Paracoccidioides brasiliensis determinant would appear to be a valuable additional tool in the follow-up of PCM patients.

Using mini-Tn5CmR::gusA, a transposon that allows transcriptional fusions to a promoterless beta-glucuronidase gene, a mutant of Erwinia carotovora subsp. carotovora SCC3193 deficient in extracellular protease production and soft-rot pathogenicity in plants was isolated. The mutant, designated SCC6004, produced normal levels of pectate lyase, polygalacturonase and cellulase. The region of the transposon insertion was partially sequenced to permit the design of specific oligonucleotide primers to amplify a 2.7 kb Clal fragment from E. carotovora subsp. carotovora SCC3193. The DNA sequence of the cloned fragment contained two complete and one partial ORFs. One of the complete ORFs (ORF1) was designated prtW and encodes a secreted protease. The deduced amino acid sequence of PrtW showed a high overall identify of 60-66% to the previously described Erwinia chrysanthemi proteases, but no homology to other proteases isolated from different E. carotovora strains. Downstream from ORF1, a further complete ORF (ORF2) and a partial ORF (ORF3) were found, with deduced peptide sequences that have significant similarity to the Inh and PrtD proteins, respectively, from E. chrysanthemi, which are involved in protease secretion. Gene fusion to the gusA reporter was employed to charaterize the regulation of prtW. The prtW gene was found to be strongly induced in the presence of plant extracts. The mutant exhibited reduced virulence, suggesting that PrtW enhances the ability of strain SCC3193 to macerate plant tissue.

Inhibins (INH) are dimeric glycoproteins, composed of an alpha-subunit (INH-alpha) and one of two possible beta-subunits (INH-betaA or -betaB). Aims of this study were to determine the frequency and tissue distribution of INH-alpha, -betaA and -betaB in breast cancer tissue. Paraffin-fixed ductal carcinoma in situ (DCIS; n=7), invasive ductal carcinomas without lymph node metastases (IDC; n=8), infiltrating ductal carcinomas with their lymph node metastases (IDC/LN; n=8), primary ductal carcinomas with their subsequent recurrence (n=7) were analyzed by immunohistochemical means with monoclonal antibodies against inhibin-alpha, -betaA and -betaB subunits. INH-alpha was observed in DCIS (5/7), while its expression was significantly higher in DCIS than IDC (1/7; p<0.05) and IDC/LN (0/8; p<0.005) and recurrent breast cancer tissue (0/7; p<0.005). The INH-betaA subunit was also demonstrated in all DCIS cases with a significantly higher intensity compared to IDC (p<0.05), IDC/LN (p<0.01) and primary carcinoma with subsequent recurrence (p<0.05). INH-betaA expression was significant higher in primary tumors with subsequent recurrence compared to IDC/LN (p<0.05). The metastatic lymph nodes expressed the lowest inhibin-betaA compared to all other groups (p<0.01). INH-betaB was also demonstrated in all mammary carcinoma tissues, but without any statistical differences. The differential expression of INH-alpha in DCIS might suggest a function as a tumor suppressor in breast tissue, suggesting a useful marker for recognizing patients with subsequent risk of developing invasive ductal cancer. The higher INH-betaA expression in DCIS than invasive cancer suggests an important role in mammary carcinogenesis. Interestingly, primary breast tumor with a subsequent recurrence expressed a higher intensity of the inhibin-betaA subunit, suggesting an important role in metastatic pathogenesis, and utilization as a tumor marker. The immunoreactivity of inhibin-betaA was significantly higher in DCIS than invasive ductal carcinomas, suggesting an important role in mammary carcinogenesis. The metastatic lymph nodes expressed lower INH-betaA and -betaB than the primary tumor, which might be the cause of less differentiated and aggressive tumor cells within the primary tumor. Therefore, inhibin/activin subunits might be useful prognostic markers for breast cancer.

The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl(-) channel in the apical membrane of epithelial cells for cAMP-dependent Cl(-) secretion. Here we report on the synthesis and screening of a small library of 6-phenylpyrrolo[2,3-b]pyrazines (named RP derivatives) evaluated as activators of wild-type CFTR, G551D-CFTR, and F508del-CFTR Cl(-) channels. Iodide efflux and whole-cell patch-clamp recordings analysis identified RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine] as a submicromolar activator of wild-type (WT)-CFTR [human airway epithelial Calu-3 and WT-CFTR-Chinese hamster ovary (CHO) cells], G551D-CFTR (G551D-CFTR-CHO cells), and F508del-CFTR (in temperature-corrected human airway epithelial F508del/F508del CF15 cells). The structural analog RP108 [7-n-butyl-6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine], contrary to RP107, was a less potent activator only at micromolar concentrations. RP107 and RP108 did not have any effect on the cellular cAMP level. Activation was potentiated by low concentration of forskolin and inhibited by glibenclamide and CFTR(inh)-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-thiazolidinone]but not by calixarene or DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid). Finally, we found significant stimulation of short circuit current (I(sc)) by RP107 (EC(50) = 89 nM) and RP108 (EC(50) = 103 microM) on colon of Cftr(+)(/)(+) but not of Cftr(-/-) mice mounted in Ussing chamber. Stimulation of I(sc) was inhibited by glibenclamide but not affected by DIDS. These results show that RP107 stimulates wild-type CFTR and mutated CFTR, with submicromolar affinity by a cAMP-independent mechanism. Our preliminary structure-activity relationship study identified 4-hydroxyphenyl and 7-n-butyl as determinants required for activation of CFTR. The potency of these agents indicates that compounds in this class may be of therapeutic benefit in CFTR-related diseases, including cystic fibrosis.

BACKGROUND

Anoctamin 1 (ANO1 or TMEM16A) Ca(2+)-gated Cl(-) channels of nociceptor neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl(-) concentrations ([Cl(-)]i) sensory neuronal Cl(-) channels are excitatory. The ability of sensory neuronal ANO1 to trigger action potentials and subsequent nocifensive (pain) responses were examined by direct activation with an N-aroylaminothiazole. ANO1 channels are also activated by intracellular Ca(2+) ([Ca(2+)]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. Thus, sensory neuronal ANO1 can facilitate TRPV1 triggering of action potentials, resulting in enhanced nociception. This was investigated by reducing ANO1 facilitation of TRPV1 effects with: (1) T16A[inh]-A01 ANO1-inhibitor reagent at physiological [Cl(-)]i and (2) by lowering sensory neuronal [Cl(-)]i to switch ANO1 to be inhibitory.

RESULTS

ANO1 effects on action potential firing of mouse dorsal root ganglia (DRG) neurons in vitro and mouse nocifensive behaviors in vivo were examined with an N-aroylaminothiazole ANO1-activator (E-act), a TRPV1-activator (capsaicin) and an ANO1-inhibitor (T16A[inh]-A01). At physiological [Cl(-)]i (40 mM), E-act (10 µM) increased current sizes (in voltage-clamp) and action potential firing (in current-clamp) recorded in DRG neurons using whole-cell electrophysiology. To not disrupt TRPV1 carried-Ca(2+) activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was measured by perforated-patch (Amphotericin-B) current-clamp technique. Subsequently, at physiological [Cl(-)]i, capsaicin (15 µM)-induced action potential firing was diminished by co-application with T16A[inh]-A01 (20 µM). Under conditions of low [Cl(-)]i (10 mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-act (5 mM) or capsaicin (50 µM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3 mM).

CONCLUSIONS

An ANO1-activator (E-act) induced [Cl(-)]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicin-triggering of action potentials and capsaicin-induced nocifensive behaviors. These results indicate ANO1 channels are involved with TRPV1 actions in sensory neurons and inhibition of ANO1 could be a novel means of inducing analgesia.

Activation of complement by beta-amyloid (A beta) contributes to the pathology of Alzheimer's disease (AD). Here, we show that C1-Inhibitor (C1-Inh) protects cultured rat hippocampal cells against beta-amyloid induced complement lysis indicating a classical pathway-mediated activation mechanism. We report on screening of compound libraries to identify compounds that inhibit C1q binding to beta-amyloid. Characterization of these compounds revealed that C1q possessed distinct binding sites for beta-amyloid and antibodies. One selected compound protected cultured hippocampal cells against complement-dependent beta-amyloid toxicity. These results provide evidence that complement has the potential to damage hippocampal cells, and C1q is a relevant target to suspend this deleterious mechanism in Alzheimer's disease.

Radioimmunoassays (RIAs) for the detection of C-1-inhibitor (C-1-Inh) complexed to either kallikrein or activated Hageman factor (factor XIIa) are described. Kallikrein-C-1-Inh or factor XIIa-C-1-Inh complexes were bound to Sepharose to which monospecific antibodies against (pre)kallikrein or factor XII, respectively, were coupled. Bound complexes were subsequently detected by an incubation with affinity purified 125I-labeled antibodies against C-1-Inh. These RIAs were used to detect activation of the contact system of coagulation in vitro and in vivo. Addition of dextran sulfate (DXS) (20 micrograms/ml) to fresh plasma resulted at 37 degrees C in the rapid generation of amidolytic kallikrein activity, which was maximal after 1 to 2 min of incubation and subsequently decreased within a few minutes. The generation of kallikrein activity coincided with the appearance of both kallikrein-C-1-Inh and factor XIIa-C-1-Inh complexes. However, in contrast to kallikrein activity, both types of complexes remained detectable in the incubation mixtures during the incubation period. Experiments with purified kallikrein. C-1-Inh and partly purified beta-factor XIIa, and activation experiments in plasmas deficient in either factor XII or prekallikrein, demonstrated the specificity of both RIAs. The minimal amount of DXS that resulted in the generation of measurable amounts of both types of complexes in plasma was 2-3 micrograms per ml. Similar experiments with kaolin showed that with limiting amounts of activator (1-2 mg/ml), only kallikrein-C-1-Inh complexes were detected in plasma. When larger amounts of kaolin were added to plasma, factor XIIa-C-1-Inh complexes were additionally detected in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Proenzymic C1r was purified from human plasma in a two-step technique involving indirect affinity chromatography on Sepharose Ig anti-C1s. The capacity of C1r to monomerize at pH 5.0 and to redimerize at neutral pH was used for selective elution of C1r. The yield in purified C1r was 39% from plasma; no trace of contaminating serine proteases was detected from [3H]diisopropyl phosphorofluoridate labelling of C1r. 2. C14 was able to undergo a two-way autoactivation: an intramolecular catalytic process catalysed by proenzymic C1r itself and an intermolecular reaction catalysed by activated C1r formed in the process of the reaction. DFP (5mM) and C1 Inh at a C1 Inh/C1r ratio of 1:1 were effective on the solely intermolecular activation, leading to partial inhibition of the autoactivation from proenzymic C1r: C1r formed during the activation was titrated by the inhibitors. Calcium, high ionic strength or acid pH decreased C1r activation. The pH effect was characterized by a slowed-down reaction below pH 6.0 and no net influence at values as high as 10.5. The two types of activation developed similarly as a function of pH. 3. Peripheral iodination of C1r revealed differences in label distribution between proenzymic (A chain moiety 48%, B chain moiety 52%) and activated C1r (A chain 20%, B chain 80%). Two different conformational states of C1r were also suggested by 125I-labelling at different temperatures.

Angioedema due to an acquired deficiency in the inhibitor of the first component of human complement (CI-INH) is a rare syndrome that is usually identified as acquired angioedema (AAE). The clinical features of C1-INH deficiency, which may also be of genetic origin (hereditary angioedema, HAE), include subcutaneous, non-pruritic swelling, involvement of the upper respiratory tract, and abdominal pain due to partial obstruction of the gastrointestinal tract. Unlike those with HAE, AAE patients have no family history of angioedema and are characterised by the late onset of symptoms and various responses to treatment due to the hypercatabolism of C1-INH. The reduction in C1-INH function leads to activation of the classical complement pathway and complement consumption, as well as activation of the contact system leading to the generation of the vasoactive peptide bradykinin, increased vascular permeability, and angioedema. AAE is frequently associated with lymphoproliferative diseases ranging from monoclonal gammopathies of uncertain significance (MGUS) to non-Hodgkin's lymphoma (NHL) and/or anti-C1-INH inactivating autoantibodies. The coexistence of true B cell malignancy, non-malignant B cell proliferation and pathogenic autoimmune responses suggests that AAE patients are all affected by altered B cell proliferation control although their clinical evolution may vary.

Previous in vitro and in vivo studies from our laboratory showed that progesterone (P(4)), corticosterone (B), and testosterone (T) increase intracellular content and release of FSH in the anterior pituitary. Activin (Act) and inhibin (Inh) are structurally related proteins with antagonistic actions, as Act stimulates and Inh inhibits FSH secretion from the anterior pituitary. Together with follistatin (FS), a protein that bioneutralizes Act, they form an autocrine-paracrine loop in the anterior pituitary that tightly regulates FSH secretion. The objective of the present study was to test the hypothesis that P(4), B, and T modulate this autocrine-paracrine loop to favor increased FSH secretion. If Act were to mediate steroid-induced FSH release, FS would be expected to block these effects. To test this interaction, cell cultures were prepared from anterior pituitaries of male and female rats, and treated with Act, B, P(4), or T in the absence or presence of FS. Act, B, P(4), and T increased FSH release; FS suppressed both basal and Act- and steroid-stimulated FSH release to approximately 50% below basal levels. Cell cultures from anterior pituitary of female rats were used to compare the interaction of incremental concentrations of FS on dose-related Act- and P(4)-stimulated FSH release. With increasing concentrations of Act, the FS-induced suppression of FSH release was attenuated and eventually abolished; in contrast, maximally stimulatory concentrations of P(4) did not fully overcome the FS-induced suppression of FSH release. The effects of P(4), B, and Act in the presence and absence of estradiol on steady-state mRNA levels of FSHbeta, Actbeta(B), and FS were determined in primary pituitary cell cultures from metestrous female rats by reverse transcription-polymerase chain reaction. Whereas Act, P(4), B increased FSHbeta mRNA levels, only Act raised the level of FS mRNA, and neither steroid increased Actbeta(B) mRNA. The results support the hypothesis that endogenous Act is a common mediator of the action of P(4), B, and T in the rat primary anterior pituitary cell culture. We conclude that the stimulation of FSH release and intracellular content in the gonadotroph by P(4), B, and T is mediated, in part, by Act and involves modulation of a tightly regulated Act/FS autocrine-paracrine loop.

Regulation of FSH secretion in the male involves a complex balance between stimulation by GnRH from the hypothalamus, inhibitory feedback by sex steroids (T and E2) and inhibin B (Inh B) from the gonads, and autocrine/paracrine modulation by activin and follistatin within the pituitary. The aim of the present study was to delineate the feedback control of FSH in the human male with specific reference to the relative roles of sex steroids vs. Inh B. Two experimental human models were used: 1) normal (NL) men subjected to acute sex steroid withdrawal (-T, -E2, + Inh B), and 2) functional castrate males (-T, -E2, -Inh B). Nine NL men (age range, 25-45 yr) and three castrate males (age range, 23-47 yr) were studied. The NL men underwent acute sex steroid suppression using high dose ketoconazole (1-g loading dose, followed by 400 mg, orally, four times daily for 150 h). Gonadotropin secretion was characterized by frequent blood sampling every 10 min for 12 h at baseline and on d 3 and 6 of sex steroid ablation. In the three castrate subjects, blood sampling was performed every 5 min for 24 h 8 wk after discontinuing androgen replacement therapy. In the NL men, treatment with ketoconazole resulted in a decline to castrate levels in T (451 +/- 20 to 38 +/- 7 ng/dl; P < 0.0005) and E2 (39 +/- 4 to 15 +/- 2 pg/ml; P < 0.005) and a modest, but significant, decline in Inh B levels, which remained within the normal range (183 +/- 19 to 136 +/- 13 pg/ml; P < 0.005). This suppression of sex steroids was associated with a more marked increase in mean LH (9.5 +/- 0.9 to 24.9 +/- 2.0 IU/liter; P < 0.0001) than FSH levels (5.1 +/- 0.7 to 10.0 +/- 1.5 IU/liter; P < 0.005), with the latter not exceeding the normal adult male range. The castrate subjects had a mean T level of 66 +/- 8 ng/dl, an E2 level of 20 +/- 1 pg/ml, and undetectable Inh B levels. Despite a similar sex steroid milieu, the mean FSH levels observed in NL men after acute sex steroid ablation were approximately 6-fold lower than those seen in the castrate subjects (10.0 +/- 1.5 vs. 59.5 +/- 17.7 IU/liter; P < 0.0005). In contrast, mean LH levels in the NL men were less than 3-fold lower than those in castrate subjects (24.9 +/- 2.0 vs. 66.8 +/- 20.1 IU/liter; P < 0.005). From this human model of acute sex steroid withdrawal, we conclude that Inh B is likely to be the major feedback regulator of FSH secretion in the human male.

Biological tenderness differences between longissimus muscles (LM) from Bos indicus and Bos taurus breeds were evaluated. Steers and heifers of Hereford x Angus (H x A, n = 10), 3/8 Sahiwal x H, A or H x A (3/8 SAH, n = 6) and 5/8 Sahiwal x H, A or H x A (5/8 SAH, n = 11) crosses were utilized. Muscle temperature and pH were monitored every 3 h for the first 12 h and at 24 h. Samples were obtained within 1 h and at 24 h postmortem from the LM for determination of calcium-dependent protease (CDP) -I and -II and CDP inhibitor (INH) activities. At 1 and 14 d postmortem, LM samples were removed for determining cathepsin B and B + L activity, soluble and total collagen, sarcomere length, muscle-fiber histochemistry, shear force and sensory-panel traits. Data were analyzed using least squares procedures with fixed effects of breed cross, sex and their interaction. No significant breed cross effects were observed for carcass traits or rates of pH and temperature decline. Steaks from H x A had lower (P less than .05) shear-force values and higher (P less than .05) sensory scores for tenderness at 1 and 14 d postmortem than steaks from 3/8 and 5/8 SAH. Correspondingly, 5/8 SAH had lower (P less than .05) myofibril fragmentation indices than H x A at 1, 3, 7 and 14 d postmortem. Breed cross effects were not significant for sarcomere length, fiber types, soluble and total collagen, cathepsin B and B + L specific activity, CDP-I and -II activity and INH activity within 1 h postmortem. However, INH total activity/100 g of muscle was greater (P less than .01) at 24 h postmortem for 5/8 SAH (208.8 +/- 14.8) and 3/8 SAH (195.6 +/- 19.3) than for H x A (136.3 +/- 14.9). For H x A, SDS-PAGE revealed that by d 1 desmin had been subjected to proteolysis, and by d 14 desmin could not be detected, but a 30,000-dalton component was clearly evident. However, in 5/8 SAH, desmin remained visible at d 14 without a 30,000-dalton component appearing. This reduced protein hydrolysis may account for less tender meat in SAH; INH apparently influences this process.

Haemolytic activities of the classical and alternative complement pathways, and levels of C1, C4, C3, factor B and C1 inhibitor (C1-INH) were measured in 85 serum samples from 46 patients with chronic lymphocytic leukaemia (CLL). Significantly decreased mean C1 and C4 levels were found, and the haemolytic activities of these components were low or low normal in more than 50% of the sera tested. In 15 sera from 5 patients a complement profile characteristic of acquired C1-IHN deficiency was observed. These results indicate that the depression of the activity of the classical complement pathway is a frequently occurring feature in CLL.

CH50 and the concentrations of C3, C4, C1 INH and factor B have been measured in sera from 34 control subjects and 178 patients with various hepatobiliary diseases, including primary biliary cirrhosis (PBC), chronic active hepatitis (CAH), cryptogenic cirrhosis (CC), alcoholic liver disease (ALD), Wilson's disease (WD), large duct biliary obstruction (LDBO) and viral hepatitis (VH). CH50 was decreased in CAH and CC. C3 was increased in PBC, LDBO and VH and decreased in CAH and CC. C4 was decreased in PBC, CAH, ALD and WD. C1 INH was increased in PBC, CAH, ALD, LDBO and VH. Factor B was increased in LDBO and VH and decreased in CC. In none of the patient groups was the mean C4 level increased or the mean C1 INH level decreased. All 5 indices of serum complement were lower in ascitic than nonascitic patients. Data on serum complement were similar in HBsAg positive and negative VH. Discriminant analysis facilitated separation of all the patient groups on the basis of complement data, except PBC and VA. Analysis of data using a within-group correlation matrix revealed a significant negative correlation between C4, the most discriminating variable of serum complement in CAH, and gamma-globulin concentration in CAH. The possible contribution of factors such as activation of complement, impaired hepatic synthesis of complement components, an acute phase response and cholestasis to altered serum complement profiles in different hepatobiliary diseases is discussed.

Ethanol and aqueous extracts of the different parts of Piper sarmentosum were analysed by HPLC for marker compounds to standardise these extracts. The standardised extracts were investigated for antioxidant activity (beta-carotene linoleate model and DPPH model), anti-TB activity (microplate tetrazolium assay), and estimation of total phenolic and amide contents. The extracts of the different parts exhibited different antioxidant activity, phenolic and amide contents (p < 0.01). The ethanol extracts exhibited better antioxidant activity as compared to the aqueous extracts. The leaf ethanol extract was further investigated for dose response relationship and its EC(50) was found to be 38 microg mL(-1). All the extracts have exhibited anti-TB activity with MIC/MBC 12.5 microg mL(-1). The leaf methanol extract was fractionated and the ethyl acetate fraction exhibited anti-TB activity with MIC/MBC 3.12 microg mL(-1) while MIC/MBC of isoniazid (INH) was found to be 0.5 microg mL(-1). A positive correlation was found between antioxidant activity and total polyphenols, flavonoids and amides, in the beta-carotene linoleate model (p = 0.05) and in the DPPH model (p = 0.01). The analytical method was found to have linearity >0.9922, coefficient of variance <5% and accuracy 95.5 +/- 5 to 96.9 +/- 5. This plant possesses promising antioxidant as well as anti-TB properties.

There is an urgent need for the development of new therapeutic strategies for Alzheimer's disease (AD). The dual-specificity tyrosine phosphorylation-regulated kinase-1A (Dyrk1a) is a protein kinase that phosphorylates the amyloid precursor protein (APP) and tau and thus represents a link between two key proteins involved in AD pathogenesis. Furthermore, Dyrk1a is upregulated in postmortem human brains, and high levels of Dyrk1a are associated with mental retardation. Here, we sought to determine the effects of Dyrk1 inhibition on AD-like pathology developed by 3xTg-AD mice, a widely used animal model of AD. We dosed 10-month-old 3xTg-AD and nontransgenic (NonTg) mice with a Dyrk1 inhibitor (Dyrk1-inh) or vehicle for eight weeks. During the last three weeks of treatment, we tested the mice in a battery of behavioral tests. The brains were then analyzed for the pathological markers of AD. We found that chronic Dyrk1 inhibition reversed cognitive deficits in 3xTg-AD mice. These effects were associated with a reduction in amyloid-β (Aβ) and tau pathology. Mechanistically, Dyrk1 inhibition reduced APP and insoluble tau phosphorylation. The reduction in APP phosphorylation increased its turnover and decreased Aβ levels. These results suggest that targeting Dyrk1 could represent a new viable therapeutic approach for AD.

In this study we examined the mechanisms of resistance to rifampin (RMP) and isoniazid (INH) in 352 clinical isolates of Mycobacterium tuberculosis from Sevilla, Spain, using three different molecular methods: 1) PCR-single strand polymorphism analysis; 2) the commercial system Inno-LiPA RTB for RMP resistance; and 3) sequence analysis. Resistance to RMP was found in 21 strains, where the following mutations in the rpoB gene were detected: Ser531->>Leu (n = 14 strains); His526->>Asp (n = 3), Asn518->>Ser (n = 1), Gln513->>Leu (n = 1) and a nine nucleotide deletion (n = 1). Resistance to INH occurred in 29 strains, with mutations observed in: a) katG gene: Ser315->>Thr (n = 12), Ile304->>Val (n = 1), and a partial deletion (n = 4); b) regulatory region of the inhA gene: nucleotide substitution C209T (n = 3). No mutation was found in the ahpC promoter.

It is currently believed that isoniazid (INH) is oxidised inside Mycobacterium tuberculosis to generate, by covalent attachment to the nicotinamide ring of NAD(H) (beta-nicotinamide adenine dinucleotide), a strong inhibitor of InhA, an enzyme essential for mycolic acid biosynthesis. This work was carried out to characterise the InhA inhibitors (named INH-NAD(H) adducts) which are generated, in the presence of the nicotinamide coenzyme NAD+, by oxidation of INH with manganese(III) pyrophosphate, a nonenzymatic and efficient oxidant used to mimic INH activation by the catalase-peroxidase KatG inside M. tuberculosis. The oxidation process is almost complete in less than 15 minutes (in comparison to the slow activation obtained in the KatG-dependent process (2.5 hours) or in the nonenzymatic O2/Mn(II)-dependent activation (5 hours)). The alkylation of NAD+ by the postulated isonicotinoyl radical generates, in solution, a family of INH-NAD(H) adducts. Analyses with liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) and experiments performed with 18O- and 2H-labelled substrates allowed us to propose two open and four hemiamidal cyclised dihydropyridine structures as the main forms present in solution; these result from the combination of the isonicotinoyl radical and the nicotinamide part of NAD+. A small amount of a secondary oxidation product was also detected. Structural data on the forms present in solution should help in the design of inhibitors of enzymes involved in the biosynthesis of mycolic acids to act as potential antituberculosis drugs.

The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP(4) receptor has been described [2-5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP(4) receptor or β adrenergic receptor (βAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either βAR or the EP(4) receptor were also studied. In oocytes co-expressing ClC-2 and βAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the β adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP(4) did not respond to the presence of 0.1, 1, or 10 μM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and βAR did not respond to hyperpolarization, acidic pH, or 1 μM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTR(inh)172. Co-expression of CFTR and EP(4) resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTR(inh)172. The EC(50) for lubiprostone mediated CFTR activation was ~10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP(4) receptor in oocytes.