[Clinicopathological features and prognosis in patients with presacral recurrent rectal cancer].

Objective: To investigate the clinicopathological features and prognostic factors in patients with presacral recurrent rectal cancer (PRRC). Methods: PRRC was defined as recurrence of rectal cancer after radical surgery involving posteriorly the presacral soft tissue, the sacrum/coccyx, and/or sacral nerve root. The diagnosis is confirmed with clinical symptoms (pain of pelvis/back/lower limb, bloody stools, increased frequency of defecation, and abnormal secretions), physical examination of perineal or pelvic masses, radiological findings, colonoscopy with histopathological biopsy, and the evaluation by multi-disciplinary team (MDT). Inclusion criteria: (1) primary rectal cancer undergoing radical surgery without distant metastasis; (2) PRRC was diagnosed; (3) complete inpatient, outpatient and follow-up data. According to the above criteria, clinical data of 72 patients with PRRC in Peking University People's Hospital from January 2008 to December 2017 were retrospectively analyzed. The clinicopathological features and follow-up data were summarized. Cox proportional hazard models was used to analyze the prognostic factors of PRRC. Results: Among 72 patients, 45 were male and 27 were female with a male-to-female ratio of 1.7:1.0. The median age at recurrence was 58 (34 to 83) years and the median interval from surgery to recurrence was 2.0 (0.2 to 17.0) years. The main symptom was pain in 48.6% (35/72) of patients. In addition, gastrointestinal symptoms were found in 25.0% (18/72) of patients. The presacral recurrent sites were presacral fascia in 36 (50.0%) patients, lower sacrum (S3~S5 or coccyx) in 25 (34.7%) patients, and higher sacrum (S1~S2) in 11 (15.3%) patients. Forty-seven (65.3%) patients underwent radical surgery (abdominal resection, abdominoperineal resection, sacrectomy, abdominosacral resection), 12 (16.7%) underwent non-radical surgery (colostomy, cytoreductive surgery), and 13 (18.1%) did not undergo any surgery but only receive palliative chemoradiotherapy and nutritional support treatment. Thirty-three (45.8%) patients received radiotherapy and/or chemotherapy (oxaliplatin, 5-fluorouracil, capecitabine, irinotecan, etc.). All the patients received follow-up, and the median follow-up time was 19 (2 to 72) months. The median overall survival time was 14 (1 to 65) months. The 1- and 3-year overall survival rates were 67.1% and 32.0%, respectively. Univariate analysis showed that age at recurrence (P=0.031) and radical resection (P<0.001) were associated with prognosis. Multivariate analysis demonstrated that radical resection was independent factor of good prognosis (RR=0.140, 95%CI: 0.061-0.322, P<0.001). Conclusions: Patients tend to develop presacral recurrent rectal cancer within 2 years after primary surgery. The main symptom is pain. Patients undergoing radical resection have a relatively good prognosis.

[Comparison of clinical efficacy among different surgical methods for presacral recurrent rectal cancer].

Objective: To investigate the efficacy and prognosis of three surgical methods for presacral recurrent rectal cancer (PRRC). Methods: A retrospective cohort study was carried out. Case inclusion criteria: (1) primary rectal cancer without distant metastasis and undergoing radical surgery; (2) patients undergoing radical surgery after the diagnosis of PRRC; (3) complete inpatient, outpatient and follow-up data. Clinical data of 47 patients meeting the above criteria who underwent operation at the Department of Gastrointestinal Surgery, The Peking University People's Hospital from January 2008 to December 2017 were reviewed and analyzed retrospectively. Of the 47 patients, 31 were male and 16 were female; the mean age was 57 years old; 9 (19.1%) were low differentiation or signet ring cell carcinoma, 38 (80.9%) were medium differentiation; 19 (40.4%) received neoadjuvant therapy. According to operative procedure, 22 patients were in the abdominal/abdominoperineal resection group, 15 in the sacrectomy group and 10 in the abdominosacral resection group. The operative data, postoperative data and prognosis were compared among the three groups. Survival curve was conducted using the Kaplan-Meier method, and log-rank test was used to compare survival difference among three groups. Results: There were no significant differences in baseline data among three groups (all P>0.05). All the 47 patients completed the radical resection successfully. The mean operation time was (4.7±2.1) hours, the median intraoperative blood loss was 600 ml, and the median postoperative hospitalization time was 17 days. Fifteen cases (31.9%) had perioperative complications, of which 3 cases were grade III-IV. There was no perioperative death. The mean operative time was (7.4±1.6) hours in the abdominosacral resection group, (4.9±1.6) hours in the abdominal/abdominoperineal resection group, and (3.0±1.1) hours in the sacroectomy group, with a significant difference (F=25.071, P<0.001). There were no significant differences in intraoperative blood loss, postoperative hospitalization days and perioperative complications among the three groups (all P>0.05). The median follow-up period of all the patients was 24 months, 12 cases (25.5%) developed postoperative dysfunction. The incidence of postoperative dysfunction in the abdominosacral resection group was 5/10, which was higher than 4/15 in the sacrectomy group and 3/22 (13.6%) in the abdominoperineal resection group with statistically significant difference (χ(2)=9.307, P=0.010). The 1-year and 3-year overall survival rates were 86.1% and 40.2% respectively. The 1-year overall survival rates were 86.0%, 86.7% and 83.3%, and the 3-year overall survival rates were 33.2%, 40.0% and 62.5% in the abdominal/abdominoperineal resection group, sacrectomy group and abdominosacral resection group, respectively, whose difference was not statistically significant (χ(2)=0.222, P=0.895). Conclusions: Abdominal/abdominoperineal resection, sacrectomy and abdominosacral resection are all effective for PRRC. Intraoperative function protection should be concerned for patients undergoing abdominosacral resection.

[Comparative study of functional prognosis of transanal total mesorectal excision and conventional total mesorectal excision based on propensity score matching].

Objective: To compare the postoperative functional prognosis of transanal mesorectal excision (taTME) and conventional total mesorectal excision (TME) in rectal cancer. Methods: Totally 49 patients underwent taTME and 478 patients underwent conventional TME at Department of Gastroenterological Surgery, Peking University People's Hospital from January 2015 to December 2019 were retrospectively collected. Propensity score matching method was used to perform 1 versus 1 matching between the taTME and conventional TME groups, and 36 pairs of patients were successfully matched. After matching, the median age of patients in taTME group and conventional TME group was 60.5 (16.0) years and 60.5 (13.0) years (M(Q(R))), respectively, and the proportion of male patients was 66.7% (24/36) and 55.6% (20/36) , respectively. EORTC QLQ-C30 scale was used to assess quality of life, low anterior resection syndrome (LARS) scale and Wexner constipation score were used to evaluate anal function, international prostate symptom score (IPSS) was used to evaluate urinary function,international index of erectile function (IIEF) -5 and female sexual function index (FSFI) score were used to evaluate male and female sexual function, respectively, and generalized anxiety disorder (GAD-7) and patient health questionnaire (PHQ-9) scale were used to evaluate psych function. The t test, Mann-Whitney U test, χ(2) test, and Fisher exact test were used for comparison between groups, and Wilcoxon rank sum test or McNemar test was used for comparison between paired data. Results: There were no significant differences in surgery time, postoperative hospital stays, conversion rate, morbidity rate, surgery cost, and numbers of lymph node yield between the two groups (all P>0.05). Compared with the conventional TME group, the intraoperative blood loss in the taTME group was significantly higher (100 (100) ml vs. 80 (50) ml, U=424.5, P=0.010), the prophylactic stoma rate was significantly higher (96.9%(31/36) vs. 63.6%(21/36), χ(2)=11.218, P<0.01), the total hospitalization cost was significantly lower (74 297.7 (16 746.4) CNY vs. 91 781.3 (26 228.4) CNY, U=413.0, P=0.008). There were no significant differences in anal and urinary function between the two groups (LARS scalescore: Z=-0.513, P=0.608, Wexner constipation score: Z=-0.992, P=0.321, IPSS: Z=-1.807, P=0.071). In terms of psych function, significant difference in GAD-7 scale was seen between the two groups (Z=-2.311, P=0.021), patients with generalized anxiety disorder accounting for 26.7% (8/30) and 46.9% (15/32), respectively. Conclusions: Compared with conventional TME surgery, taTME has a significantly increased blood loss and prophylactic stoma rate. There are no significant difference in the incidence of postoperative anal, urinary, and sexual dysfunction between taTME and conventinal TME. taTME can alleviate the financial burden and general anxiety disorder to a certain extent.

Inducible nitric oxide synthase expression.

This unit contains three protocols that can be used to determine iNOS expression in mouse macrophage-like cells, RAW 264.7, by measuring end product, protein, and mRNA. A bacterial product, lipopolysaccharide (LPS), stimulates iNOS expression in RAW 264.7 cells.

Induction of MRP1 and gamma-glutamylcysteine synthetase gene expression by interleukin 1beta is mediated by nitric oxide-related signalings in human colorectal cancer cells.

Treatment of human colorectal cancer cells HT29 with interleukin 1beta (IL-1beta) induces expression of the multidrug resistance protein (MRP1) gene encoding the ATP-dependent glutathione S-conjugate export (GS-X) pump and the gamma-glutamylcysteine synthetase (gamma-GCSh) gene encoding heavy (catalytic) subunit of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the biosynthesis of glutathione (GSH). The induction can be suppressed by N(G)-methyl-L-arginine, a specific inhibitor of nitric oxide synthase (NOS). These results suggest that IL-1beta-mediated MRP1 and gamma-GCSh induction involve nitric oxide (NO) -related signaling. Further supports to the involvement of NO in the induction of MRP1 and gamma-GCSh expression are made by the following observations. (i) Expression of MRP1 and gamma-GCSh genes were induced by treating the cells with NO donors, i.e., S-nitro-N-acetyl-D,L-penicillamide (SNAP) and S-nitroso-L-glutathione, in a concentration-dependent manner. (ii) Ectopic expression of inducible NOS (iNOS) activity by transfecting expressible recombinant iNOS cDNA encoding functional iNOS but not the nonfunctional version resulted in elevated expression of MRP1 and gamma-GCSh. We also demonstrated that HT-29 cells treated with either 1L-1beta or SNAP induced ceramide production, and addition of C2 or C6 ceramides into cultured HT-29 cells resulted in induction of gamma-GCSh but not MRP1 expression. Collectively, our results demonstrate that induction of MRP1 and gamma-GCSh by IL-1beta is regulated, at least in part, by an NO-related signaling, and induction of gamma-GCSh is by NO-related ceramide signaling.

Alkyl hydroperoxide reductase subunit C (AhpC) protects bacterial and human cells against reactive nitrogen intermediates.

In Salmonella typhimurium, ahpC encodes subunit C of alkyl hydroperoxide reductase, an enzyme that reduces organic peroxides. Here, we asked if ahpC could protect cells from reactive nitrogen intermediates (RNI). Salmonella disrupted in ahpC became hypersusceptible to RNI. ahpC from either Mycobacterium tuberculosis or S. typhimurium fully complemented the defect. Unlike protection against cumene hydroperoxide, protection afforded by ahpC against RNI was independent of the reducing flavoprotein, AhpF. Mycobacterial ahpC protected human cells from necrosis and apoptosis caused by RNI delivered exogenously or produced endogenously by transfected nitric oxide synthase. Resistance to RNI appears to be a physiologic function of ahpC. ahpC is the most widely distributed gene known that protects cells directly from RNI, and provides an enzymatic defense against an element of antitubercular immunity.

Novel triterpenoids suppress inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse macrophages.

We have synthesized more than 80 novel triterpenoids, all derivatives of oleanolic and ursolic acid, as potential anti-inflammatory and chemopreventive agents. These triterpenoids have been tested for their ability to suppress the de novo formation of two enzymes, inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2), using IFN-gamma-stimulated primary mouse macrophages or lipopolysaccharide (LPS)-activated RAW 264.7 macrophages as assay systems. Two synthetic oleananes, 3,12-dioxoolean-1-en-28-oic acid (TP-69) and 3,11-dioxoolean-1,12-dien-28-oic acid (TP-72), were highly active inhibitors of de novo formation of both iNOS and COX-2. Both TP-69 and TP-72 blocked the increase in iNOS or COX-2 mRNA induced by IFN-gamma or LPS. In addition, TP-72 suppressed NF-KB activation in primary macrophages treated with the combination of IFN-gamma and LPS or IFN-gamma and tumor necrosis factor. The 3-alpha(axial)-epimer of ursolic acid suppressed de novo formation of COX-2, in contrast to naturally occurring 3-beta(equatorial)-ursolic acid. Inhibitory effects of TP-69 or TP-72 on iNOS formation were not blocked by the glucocorticoid receptor antagonist RU-486, indicating that these triterpenoids do not act through the glucocorticoid receptor, nor does TP-72 act as an iNOS or COX-2 enzyme inhibitor when added to RAW cells in which synthesis of these two enzymes in response to LPS has already been induced. It may be possible to develop triterpenoids as useful agents for chemoprevention of cancer or other chronic diseases with an inflammatory component.

Nitric oxide and macrophage function.

At the interface between the innate and adaptive immune systems lies the high-output isoform of nitric oxide synthase (NOS2 or iNOS). This remarkable molecular machine requires at least 17 binding reactions to assemble a functional dimer. Sustained catalysis results from the ability of NOS2 to attach calmodulin without dependence on elevated Ca2+. Expression of NOS2 in macrophages is controlled by cytokines and microbial products, primarily by transcriptional induction. NOS2 has been documented in macrophages from human, horse, cow, goat, sheep, rat, mouse, and chicken. Human NOS2 is most readily observed in monocytes or macrophages from patients with infectious or inflammatory diseases. Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, fungi, protozoa, helminths, and tumor cells. The antimicrobial and cytotoxic actions of NO are enhanced by other macrophage products such as acid, glutathione, cysteine, hydrogen peroxide, or superoxide. Although the high-output NO pathway probably evolved to protect the host from infection, suppressive effects on lymphocyte proliferation and damage to other normal host cells confer upon NOS2 the same protective/destructive duality inherent in every other major component of the immune response.

Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimer's disease.

In Alzheimer's disease (AD), affected neurons accumulate beta amyloid protein, components of which can induce mouse microglia to express the high-output isoform of nitric oxide synthase (NOS2) in vitro. Products of NOS2 can be neurotoxic. In mice, NOS2 is normally suppressed by transforming growth factor beta 1 (TGF-beta 1). Expression of TGF-beta 1 is decreased in brains from AD patients, a situation that might be permissive for accumulation of NOS2. Accordingly, we investigated the expression of NOS2 in patients with AD, using three monospecific antibodies: a previously described polyclonal and two new monoclonal antibodies. Neurofibrillary tangle-bearing neurons and neuropil threads contained NOS2 in brains from each of 11 AD patients ranging in age from 47 to 81 years. NOS2 was undetectable in brains from 6 control subjects aged 23-72 years, but was expressed in small amounts in 3 control subjects aged 77-87 years. Thus, human neurons can express NOS2 in vivo. The high-output pathway of NO production may contribute to pathogenesis in AD.

Inducible nitric oxide synthase requires both the canonical calmodulin-binding domain and additional sequences in order to bind calmodulin and produce nitric oxide in the absence of free Ca2+.

All three mammalian isoforms of nitric oxide synthase (NOS) must bind calmodulin (CaM) for enzymatic activity. Only NOS2 (the inducible isoform, iNOS) does so at the low levels of free Ca2+ in resting cells and when almost all Ca2+ is chelated in cell-free preparations. To test directly whether the predicted CaM-binding region of mouse NOS2 accounts for its Ca2+ independence, we prepared chimeric NOS's in which mouse NOS2 residues 503-532 were reciprocally exchanged with the corresponding residues 725-754 of rat NOS1 (neuronal NOS). Unlike either parent, both chimeras required an intermediate level of free Ca2+ to bind CaM and generate NO. In cell lysates, the concentration of Ca2+ necessary for half-maximal activity (EC50) was approximately 0 for NOS2, 200-300 n for NOS1, and 7-10 n for the chimeras. Results were similar when the region exchanged was enlarged by 7-8 residues toward the amino terminus. In contrast, when the carboxyl-terminal half of NOS2 (residues 454-1144) was replaced with that of NOS1 (residues 675-1429), the resulting chimera resembled NOS1 (EC50, 200-300 n free Ca2+). Truncation analysis suggested that NOS2 residues within the sequence 484-726 were required for Ca2+-independent CaM-binding. Thus, both the canonical CaM-binding domain and additional residues within the region 484-726 are necessary for NOS2's ability to bind CaM and produce NO when Ca2+ levels approach zero.

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes.

Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis.

The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% of bronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2.

Inducible nitric oxide synthase: identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase.

Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death.

Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells.

Previous studies from our laboratory demonstrated an inverse relationship between the expression level of inducible nitric oxide synthase (iNOS) and the metastatic potential of murine K-1735 melanoma cells. The purpose of this study was to provide direct evidence that the expression of iNOS suppresses metastatic potential of melanoma cells. Highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containing 3 mM NG-methyl-L-arginine (NMA). Positive transfectants were identified by Southern and Northern blot analyses and homogeneous staining with a specific anti-iNOS monoclonal antibody. Semiconfluent cultures of C4.P (parental), C4.Neo.3 (control transfection), C4.S2.3 (inactive iNOS), and C4.L8.5 (functional iNOS) were harvested, and viable cells were injected intravenously into syngeneic C3H/HeN mice and allogeneic BALB/c nude mice. C4.P, C4.Neo.3, and C4.S2.3 cells were highly metastatic whereas C4.L8.5 cells were not metastatic. Experiments with [125I]dUrd-labeled tumor cells demonstrated that the initial arrest in the lung microvasculature did not differ among the lines, but that C4.L8.5 cells died by 48-72 h after injection. Enhanced survival of all K-1735 C4 cells (including C4.L8.5) was found in mice given twice daily injections of 20 mg NMA. The C4.L8.5 cells produced slow growing subcutaneous tumors in nude mice, whereas the other three lines produced fast growing tumors. In vitro studies confirmed that in the absence of NMA the expression of iNOS in C4.L8.5 cells induced apoptosis. Collectively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis.

Vesicle membrane association of nitric oxide synthase in primary mouse macrophages.

The isoform of nitric oxide synthase (NOS) whose activity is independent of elevated Ca2+ and exogenous calmodulin (iNOS; NOS type II) is inducible in a wide variety of cells and plays a major role in pathophysiology. The notion that iNOS is predominantly cytosolic is based on studies of a transformed cell line; almost nothing is known about the subcellular localization of iNOS in primary cells. Accordingly, we undertook a combined immunoelectron microscopic and biochemical analysis of iNOS in primary mouse macrophages. Approximately one-half of their iNOS activity and protein could be sedimented from 1 M KCl at 100,000 x g. The morphologic counterpart of particulate iNOS was a population of 50 to 80 nm vesicles that did not correspond to lysosomes nor peroxisomes. Vesicular iNOS arose from cytosolic iNOS by undergoing a post-translational modification that increased its apparent molecular mass by 4.5 kDa and promoted its salt-, detergent-, acid- and urea-resistant association with membranes, in the absence of detectable alternative splicing, myristoylation, palmitoylation, acetylation, glycosylation, or COOH-terminal truncation. Although primary macrophage iNOS underwent phosphorylation, ubiquitinylation, and binding of calmodulin tightly enough to resist boiling in SDS, these modifications did not allow us to distinguish between the cytosolic and particulate variants. The apparently novel iNOS-positive vesicles may translocate to phagosomes containing appropriately opsonized particles.

Macrophages derived from C3H/HeJ (Lpsd) mice respond to bacterial lipopolysaccharide by activating NF-kappa B.

The effects of bacterial lipopolysaccharide (LPS) on macrophage gene expression are mediated in part by its ability to induce activation of transcription factor NF-kappa B. We compared the ability of LPS-treated macrophages from Lpsn (LPS-responsive) C3H/HeN and Lpsd (LPS-hyporesponsive) C3H/HeJ mice to mobilize NF-kappa B by electrophoretic mobility shift assays with oligonucleotide probes containing a unique NF-kappa B sequence from the promoter of inducible nitric oxide synthase (iNOS). In response to ng/ml concentrations of LPS, this probe bound proteins that appeared rapidly in the nuclei of thioglycollate-elicited macrophages and bone marrow-derived macrophage cell lines from both Lpsn and Lpsd mice. Only in macrophages from Lpsn mice, however, was LPS able to induce iNOS or tumor necrosis factor alpha. NF-kappa B-containing DNA-protein complexes from Lpsd macrophages were formed in lesser amounts than from Lpsn macrophages but shared the same composition, insofar as they displayed the same electrophoretic mobilities and content of heterodimers of p50/RelA (p65) and p50/c-rel. Two conclusions emerge from these findings: (1) NF-kappa B activity alone is not sufficient for induction of certain LPS-responsive genes and (2) An LPS-response pathway involving activation of NF-kappa B is preserved in Lpsd mice. The inability of cells from Lpsd mice to induce gene expression in response to LPS thus cannot be attributed to inability to activate NF-kappa B.

Carboxyl terminus of inducible nitric oxide synthase. Contribution to NADPH binding and enzymatic activity.

Cloning of a nitric oxide synthase (NOS) from RAW 264.7 mouse macrophages (Xie, Q.-w., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T., and Nathan, C. (1992) Science 256, 225-228) yielded two sets of cDNA: one with a longer coding region of 1144 amino acids, whose sequence matched that of the purified protein, and another with a shorter coding region of 1122 amino acids, in which the last 10 carboxyl-terminal amino acids differed completely from those of the long form. We have now found that the short form lacks NOS activity. To determine the basis of this defect, we prepared recombinant chimeric, deletional, and point mutants of the long and short NOS variants, monitored their expression by immunoblot, and tested their enzymatic activity. By itself, lack of the 22-carboxyl-terminal residues of the long form NOS was scarcely consequential. Mutation of Phe1122, the only aromatic residue within one of the longest conserved regions shared by all NOSs of reported sequence, reduced enzymatic activity by 41%. Deletion of 23 carboxyl-terminal amino acids (including Phe1122) reduced activity by 71%. Further loss of Ile1121, another completely conserved residue, reduced activity by 95%, and with the deletion of the rest of the conserved region, NOS activity was undetectable. Normal dimerization and binding of heme and calmodulin by the short variants militated against distortions of tertiary structure affecting the amino-terminal half or middle portion of the protein. In contrast, the short variants were deficient in binding to NADPH, as predicted by a model of tertiary structure based on that of spinach ferredoxin-NADP+ reductase. This is the first demonstration that the carboxyl terminus of NOS is a functionally critical region.

The presence of an active S-adenosylmethionine decarboxylase gene increases the growth defect observed in Saccharomyces cerevisiae mutants unable to synthesize putrescine, spermidine, and spermine.

Saccharomyces cerevisiae spe1 delta SPE2 mutants (lacking ornithine decarboxylase) and spe1 delta spe2 delta mutants (lacking both ornithine decarboxylase and S-adenosylmethionine decarboxylase) are equally unable to synthesize putrescine, spermidine, and spermine and require spermidine or spermine for growth in amine-free media. The cessation of growth, however, occurs more rapidly in spe1 delta SPE2 cells than in SPE1 spe2 delta or spe1 delta spe2 delta cells. Since spe1 delta SPE2 cells can synthesize decarboxylated adenosylmethionine (dcAdoMet), these data indicate that dcAdoMet may be toxic to amine-deficient cells.