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The characteristics of monolithically integrated light-emitting diodes (LEDs), photodetectors (PDs), and waveguides on a GaN-on-Si wafer are investigated. The InGaN/GaN multi-quantum wells, which are responsible for blue light emission in LEDs, are also used for photodetection in PDs. Despite the Stokes shift, a spectral overlap of ∼25  nm between the emission and absorption spectra provides the PDs with sufficient photosensitivity to signals from the emitter while remaining insensitive to ambient lighting. Optical interconnects in the form of linear or bent suspended waveguides bridging the LEDs and PDs are formed by selective detachment of etched GaN mesas from the Si substrate. Additionally, the PDs can be detached from the substrate and remounted on an elevated platform, owing to the flexibility of the thin-film waveguide. The 150  μm×150  μm LEDs and PDs exhibit rapid response on nanosecond time scales, which is attributed to fast radiative recombinations as well as minimized resistive-capacitive (RC) delays, enabling transmission of pseudorandom binary sequence (PRBS) data signals at rates of 250 Mb/s with an opening in the eye diagram. Together with multichannel transmission free of crosstalk, the ability of the planar and three-dimensional monolithic photonic systems to handle visible-light communication (VLC) applications is demonstrated.

Immunotherapeutic approaches, particularly programmed death 1/programmed death ligand 1 (PD-1/PD-L1) blockade, have improved the treatment of non-small-cell lung cancer (NSCLC), supporting the premise that evasion of immune destruction is of importance for NSCLC progression. However, the signals responsible for upregulation of PD-L1 in NSCLC cells and whether they are integrated with the regulation of other immune-related genes are not known. Mucin 1 (MUC1) is aberrantly overexpressed in NSCLC, activates the nuclear factor-κB (NF-κB) p65→ZEB1 pathway and confers a poor prognosis. The present studies demonstrate that MUC1-C activates PD-L1 expression in NSCLC cells. We show that MUC1-C increases NF-κB p65 occupancy on the CD274/PD-L1promoter and thereby drives CD274transcription. Moreover, we demonstrate that MUC1-C-induced activation of NF-κB→ZEB1 signaling represses the TLR9(toll-like receptor 9), IFNG, MCP-1(monocyte chemoattractant protein-1) and GM-CSFgenes, and that this signature is associated with decreases in overall survival. In concert with these results, targeting MUC1-C in NSCLC tumors suppresses PD-L1 and induces these effectors of innate and adaptive immunity. These findings support a previously unrecognized central role for MUC1-C in integrating PD-L1 activation with suppression of immune effectors and poor clinical outcome.

The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (P<0.05). FOXP1 knockdown in ABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL patients (n=150), reduced HLA-DRA (<90% frequency) expression correlated with inferior overall survival (P=0.0003) and progression-free survival (P=0.0012) and with non-GCB subtype stratified by the Hans, Choi or Visco–Young algorithms (all P<0.01). In non-GCB DLBCL cases with <90% HLA-DRA, there was an inverse correlation with the frequency (P=0.0456) and intensity (P=0.0349) of FOXP1 expression. We propose that FOXP1 represents a novel regulator of genes targeted by the class II MHC transactivator CIITA (MHC II and CD74) and therapeutically targeting the FOXP1 pathway may improve antigen presentation and immune surveillance in high-risk DLBCL patients.

We demonstrate all-optical ultrahigh-speed swept-source optical coherence tomography (OCT) based on amplified optical time-stretch (AOT). Such an inertia-free wavelength-swept mechanism, via group velocity dispersion, enables us to realize OCT with an A-scan rate well above MHz. More importantly, the key significance of AOT-OCT is its simultaneous broadband Raman amplification during the time-stretch process–greatly enhancing the detection sensitivity compared with prior attempts to apply optical time-stretch to OCT. Here, we report on an AOT-OCT system which is operated at an A-scan rate of 7.14 MHz, a superior roll-off performance (>2  mm/dB), a record-high sensitivity of time-stretch-based OCT (>80  dB) with a broadband gain bandwidth of 80 nm, which results in an axial resolution of ∼15  μm. Our AOT-OCT system is thus able to, for the first time to the best of our knowledge, perform time-stretch-based OCT of biological tissue in vivo. It represents a major step forward in utilizing AOT as an alternative for achieving practical MHz OCT, without any long-term mechanical stability concerns as in typical swept-source OCT or bypassing the speed limitation of the image sensor employed in spectral-domain OCT.

Nanostructures of the tin oxide, indium oxide and tin-doped indium oxide have been fabricated on silicon by chemical vapor deposition from a mixture of metal oxide nanoparticles and single-wall carbon nanotubes (SWCNT). Different ratios of the metal oxide to SWCNT have been used. It was found that the morphology of the nanostructures depends on the substrate temperature. The morphology, growth direction and optical properties have been studied by scanning electron microscopy, transmission electron microscopy and photoluminescence spectroscopy.

The effectiveness of energy‐dissipative tuned mass dampers (TMD) on structural dynamic responses have been studied extensively since its development in the 1970s. Its applications have recently been extended to the use of active multiple tuned mass dampers. However, the process of a damper in improving a structure's ability to dissipate the earthquake's input energy has never been investigated. In this research, the objective lies in studying the process of structural energy transfer and the ability for the structure to dissipate energy. Numerical simulations are performed to study the energy responses of structures with and without TMD installed. The effectiveness of TMD in reduction of energy responses is also studied by using energy spectra and in a case study of a six‐storey building. Results show that TMD is more effective in reducing the energy responses for structures with a moderate to long period of vibration than those for short period structures. In addition, TMD is very effective in reducing both the maximum kinetic energy and strain energy of the structure, while it is also effective in reducing the amount of damping energy that must be dissipated through the structure by providing an alternating route of damping energy dissipation through the TMD. Copyright © 2004 John Wiley & Sons, Ltd.

Background: This study aimed to evaluate clinically and manometrically the anorectal function of patients with imperforate anus after repair with laparoscopically assisted anorectoplasty (LAR), as compared with the function of patients after undergoing the conventional method, posterior sagittal anorectoplasty (PSARP). Methods: The defecation status and anorectal manometry of patients with high or intermediate type imperforate anus repaired with LAR ( n = 9) and age-matched patients repaired with PSARP ( n = 13) were assessed and compared during the first year of postoperative follow-up evaluation. The defecation status was classified by the frequency of bowel openings (<1, 1–4, and >5 times per day). Manometric assessment was performed by an open-tip hydraulic capillary infusion system. The presence of the rectoanal relaxation reflex was determined, and the resting sphincteric pressure and resting rectal pressure were measured. Results: Seven of nine LAR patients had an “acceptable” frequency of one to four bowel openings per day, in contrast to 7 of 13 PSARP patients. The difference in the presentation of daily stooling is not significant ( p > 0.05). A positive RAR was detected in 88.9% (8/9) of the LAR patients, and in only 30.8% (4/13) of the PSARP patients ( p < 0.01). The presence of a rectoanal relaxation reflex also significantly correlated with an acceptable frequency of bowel opening (1–4 times per day) in both LAR and PSARP patients ( p < 0.05). Moreover, a rectoanal relaxation reflex was detected significantly earlier in LAR than in PSARP patients (4.9 ± 1.2 vs 10.1 ± 2.5 months; postoperatively p < 0.0001). Both the LAR and PSARP patients had a similar resting sphincteric pressure (21.5 ± 4.7 vs 25.4 ± 6.2 cm H 2O; p > 0.05). By contrast, the resting rectal pressure was significantly lower in LAR than in PSARP patients (7.7 ± 1.5 vs 11.5 ± 1.3 cmH 2O; p < 0.05). Conclusions: In the early postoperative stage, patients repaired with LAR had more favorable findings in anorectal manometry than patients repaired with PSARP. Long-term follow-up studies to confirm a superior defecation continence achieved with LAR are warranted.

Objectives: To assess the prevalence of sleep‐­disordered breathing (SDB) and its associated symptoms in a group of commercial bus drivers in Hong Kong. Methods: Two hundred and sixteen of 410 bus drivers from three different shifts were interviewed with the Sleep & Health Questionnaire (SHQ) and the Epworth sleepiness scale (ESS) at a Hong Kong bus depot. Seventeen subjects from each shift were then randomly selected for at‐home sleep study using the Mesam IV device (Madaus Medizin−Elektronik, Freiburg, Germany). Results: There were 207 men and nine women (mean age 42.4 ± 7.5 years; body mass index (BMI) 25.4 ± 4.5 kg/m2; ESS 5.3 ± 4.2). From the SHQ it was discovered that: (i) daytime sleepiness was reported by 87 subjects (40%), (ii) snoring ≥ 3 times per week was reported by 80 subjects (37%), (iii) witnessed apnoea was reported by 17 subjects (7.9%) and (iv) 29 subjects (13.4%) reported having fallen asleep during driving. Among the 51 subjects who underwent the at‐home sleep study: (i) 31 subjects (61%) had respiratory disturbance index (RDI) ≥ 5 per hour of sleep, (ii) 21 subjects (41%) had RDI ≥ 10 per hour of sleep, (iii) 12 subjects (24%) had RDI ≥ 15 per hour of sleep and (iv) 35 subjects (68.6%) snored objectively ≥ 10% of the night. Ten subjects (20%) had RDI ≥ 5 and sleepiness at work, while five subjects (9.8%) had RDI ≥ 5 and ESS > 10. No significant differences were noted in the SHQ responses, ESS, objective snoring or RDI among the three groups. Multiple regression analysis showed that BMI and witnessed apnoea were the only positive independent predictors of RDI. Conclusions: This study showed a high prevalence of objective snoring and SDB in a group of commercial bus drivers. Neither self‐reported sleepiness nor the ESS could identify subjects with SDB. (Intern Med J 2002; 32: 149−157)

The wetting and wicking behavior of linen treated with low-temperature oxygen and argon plasma is presented. Wetting and wicking abilities of plasma treated linen are investigated using contact angles and upward and downward water wicking methods. The downward wicking method is more suitable for distinguishing the effects of plasma treatment under various conditions.

Background: Venous access for chemotherapy patients at the Singapore General Hospital has conventionally been via an externalised (Hickman) catheter. A peripherally implanted permanent subcutaneous central venous access device (P.A.S.Port) was introduced in 1996. Aims: We review its use, complications and cost competitiveness with other chemotherapy delivery systems. Methods: A retrospective study of clinical records was conducted with follow up interviews of patients and nursing staff. Results: Twelve patients had the P.A.S.Port inserted for administration of chemotherapy as an outpatient procedure. Immediate complications were pain (8%) and bruising (8%). One port-related infection (8%) one week following insertion was reported and one patient complained of interference with elbow movement as a late complication. Cost analysis was favourable for P.A.S.Port over an externalised Hickman catheter or chest-placed port at one year. Patient and nursing staff acceptance was high. Conclusion: P.A.S.Port insertion is a safe outpatient procedure and is an alternative for permanent venous access.

Low temperature plasma treatments have been applied to linen fabrics prior to enzyme treatments. The effects of using oxygen and argon plasma on the bulk fibre structure, fabric properties, surface morphology and properties have been investigated experimentally by a range of methods, before and after the enzyme treatment. The results detailed in this paper indicate that the effectiveness of enzyme treatment can be enhanced by the means of plasma pre‐treatment.

Three enzyme preparations (crude cellulase, laccase, and proteinase) were evaluated for their potential to improve the papermaking properties of mechanical pulp. After treating a long fibre-rich fraction of the pulp with enzyme, the fibres were recombined with untreated fines for handsheet making and testing. None of the enzymes altered the retention of fines or the consolidation of the furnish mix during handsheet formation. All three enzymes increased tensile stiffness index, which is a measure of the initial resistance of the handsheets to strain. Only the laccase preparation, an enzyme that modifies pulp lignin, consistently increased fibre bonding to enhance other strength properties of the handsheets.

A series of 23 patients with carcinoma of the renal pelvis is presented. The different presentations are described and discussed, and the difficulties in establishing a preoperative diagnosis are considered. There were 3 squamous cell carcinomas in the series, all of them associated with renal stone and urinary tract infection.

Summary Transmission of a Canadian arctic isolate of Northway virus has been demonstrated after incubation of arcticAedes communis mosquitoes at 13° C for 27 days after intrathoracic injection of 300 plaque forming units of virus. Replication has also been demonstrated after intrathoracic injection of domesticA. aegypti mosquitoes of this virus. Virions of Northway virus, 84–92 nm diameter were morphologically typical of a bunyavirus after propagation in salivary glands ofA. communis or in tissue cultures of baby hamster kidney (BHK-21) cells. An Ontario isolate of St. Louis encephalitis was transmitted by bites ofA. communis after 27 days incubation at 13° C after oral ingestion of 3 or 30 mouse LD50 virus. This mosquito species transmitted virus after 13 to 76 days incubation at 13° C following intrathoracic injection of 3 mouse LD50 or higher virus doses.

Epstein-Barr virus (EBV) receptors (EBV/C3d receptors) were detected, using the monoclonal antibody HB5, on 23 ectocervical and 5 endocervical biopsies of the uterine cervix. Elevated IgA liters against the viral capsid antigen and early antigen of EBV were also found in the cervical secretions from cervical carcinoma patients (83%), compared with samples from patients with cervical intraepithelial neoplasia (75%), herpes simplex virus-infected patients (0%), and gynecologic patients with nonmalignant conditions (0%). EBV DNA was present in 63% of cervical carcinoma biopsies detected by in situ hybridization. These observations suggest a positive association between EBV and carcinoma of the cervix. Copyright 1993, 1999 Academic Press

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

A general method was developed for the isolation of Salmonella thyphimurium LT2 Mu d1–8 (Aprlac) operon fusions in a gene displacing a Tn10 insertion. Random Mu d1–8 fusion pools were prepared to grow phage P22 lysates which transduced chlC::Tn10 to AprTets on fusaric acidampicillin plates. Among these AprTets potential chlC::Mu d1–8 fusions, a simple spot test identified the fusions that were closely linked to the Tn10 insertion in chlC. Out of 68 AprTets isolates 7 chlC::Mu d1–8 fusions with a nitrate-induced Lac+ phenotype were obtained. When oxrA::Tn10 was transduced into these chlC::Mu d1–8 fusions, they became Lac- even in the presence of nitrate, confirming that they were chlC::Mu d1–8 fusions.

The use of antisense RNA for the inhibition of gene expression and viral pathogenesis appears promising for gene therapeutic use. We have designed a family of transducing viral vectors, based upon the noncytopathic adeno-associated virus (AAV), that encode antisense RNA targeting critical early events in the replicative cycle of targeted viruses, including human immunodeficiency virus-1 (HIV-1) and herpes simplex virus-1 (HSV-1). AAV vectors efficiently transduce cells of different types and integrate into chromosomal DNA in a stable fashion. Genes inserted into AAV vectors may be precisely designed to direct the transcription of short antisense RNA molecules since there is no transcriptional interference from the base vector. Cells expressing AAV-encoded antisense RNA show a significant reduction in the production of the cognate virus. Thus, AAV vectors may become powerful analytic and therapeutic tools for the stable introduction of transgenes including antisense genes into cells.Copyright 1993, 1999 Academic Press

Two patients are described who developed acute leukemia during a 7- to 20-year quiescent phase of inflammatory bowel disease. The possibility of an association of leukemia to the diagnostic radiation received or a susceptibility to malignancy other than adenocarcinoma in patients with inflammatory bowel disease is suggested.

Fingerprinting of RNA by arbitrarily primed PCR (RAP) can be used to identify conditionally expressed genes in prokaryotes. Differential gene expression in Salmonella typhimurium LT2 in response to peroxide treatment was examined as a system in which to demonstrate this strategy. This treatment models the induction of bacterial protective proteins that may occur when mammalian phagocytes use peroxide to fight S. typhimurium infection. To identify genes inducible by hydrogen peroxide stress, total RNA from peroxide-treated and untreated bacterial cultures were RAP fingerprinted with six different arbitrarily selected primers. A 435-base RAP product that was differentially amplified by RAP using the reverse sequencing primer was cloned and sequenced. Northern blot analysis confirmed that the RNA corresponding to this clone, RSP435, was induced when bacteria were treated with hydrogen peroxide. The RNA was not induced in an oxyR1 mutant that constitutively expressed a subset of hydrogen peroxide-inducible genes. Using pulsed-field gel electrophoresis and dot blot hybridization to an array of induced Mud-P22 integrations, the gene corresponding to RSP435 was mapped to two places, one between 19 and 21.5 min and one between 56 and 57 min. Thus, two similar or identical stress-inducible genes were found in different parts of the genome. Identification, cloning, and mapping of the conditionally expressed RSP435 cDNA were performed entirely by physical means, demonstrating that the strategy should complement genetic methods for many prokaryotic or archaebacterial systems and should be applicable to organisms in which genetic methods are difficult to perform or have not yet been developed.

A polylinker with rare restriction sites was introduced into a mini-Tn5 derivative. These sites include M.XbaI-DpnI (TCTAGATCTAGA), which is rare in most bacterial genomes, SwaI (ATTTAAAT) and PacI (TTAATTAA), which are rare in G+C-rich genomes, NotI (GCGGCCGC) and SfiI (GGCCN5GGCC), which are rare in A+T-rich genomes, and BlnI (CCTAGG), SpeI (ACTAGT), and XbaI (TCTAGA), which are rare in the genomes of many gram-negative bacteria. This Tn5(pfm) (pulsed-field mapping) transposon carries resistance to chloramphenicol and kanamycin to allow selection in a wide variety of background genomes. This Tn5(pfm) was integrated randomly into the Salmonella typhimurium and Serratia marcescens genomes. Integration of the new rare SwaI, PacI, BlnI, SpeI, and XbaI sites was assayed by restriction digestion and pulsed-field gel electrophoresis. Tn5(pfm) constructs could be valuable tools for pulsed-field mapping of gram-negative bacterial genomes by assisting in the production of physical maps and restriction fragment catalogs. For the first applications of a Tn5(pfm), we bisected five of the six largest BlnI fragments in the S. typhimurium genome, bisected the linearized 90-kb pSLT plasmid, and used Tn5(pfm) and Tn10 to trisect the largest BlnI fragment.

A semi-implicit finite element scheme is proposed for two-dimensional tidal flow computations. In the scheme, each term of the governing equations, rather than each dependent variable, is expanded in terms of the unknown nodal values and it helps to reduce computer execution time. The friction terms are represented semi-implicitly to improve stability, but this requires no additional computational effort. Test cases where analytic solutions have been obtained for the shallow water equations are employed to test the proposed scheme and the test results show that the scheme is efficient and stable. An numerical experiment is also included to compare the proposed scheme with another finite element scheme employing Serendipity-type Hermitian cubic basis functions. A numerical model of an actual bay is constructed based on the proposed scheme and computed tidal flows bear close resemblance to flows measured in field survey.

In this paper, we propose a high-level state-based dependency description and structuring formalism for flexible manufacturing systems (FMS). This extended cellular automaton model (ECAM) is designed to capture the behaviour and performance of a large complex concurrent system in an easier and more concise way than is possible with other contemporary FMS models. Concurrency problems of FMS can be clearly identified by the model.

A new synthesis of the magnetic protein magnetoferritin is reported. Addition of increments of Fe(II) to anaerobic solutions of the demetalated protein, apoferritin, at pH = 8.6 and 65 °C, followed by stoichiometric amounts of the oxidant trimethylamine-N-oxide (Me3NO), results in the formation of a dispersed magnetic bioinorganic nanocomposite. By limiting the Fe:protein ratio to not more than ~140 atoms/protein molecule and the Fe(II):Me3NO ratio to 3:2 in each increment, ferrimagnetic nanocrystals of magnetite/maghemite (Fe3O4)/(γ-Fe2O3) are synthesized in the 8 nm diameter protein cage. Controlling the number of stepwise cycles of the Fe(II)/oxidant additions produces biomimetic proteins with different iron loadings (100, 260, 530, 1000, 2040, and 3150 Fe atoms/protein molecule) and concomitant changes in the size of the inorganic nanocores. Magnetoferritins prepared with less than 1000 Fe atoms/molecule were discrete nanocomposites with protein-encapsulated magnetic cores. Samples with higher iron loadings were aggregated on the TEM grid and showed a progressive increase in the number of cores with dimensions greater than the protein cavity. The temperature-dependent magnetic properties of magnetoferritins with different Fe loadings were studied by SQUID magnetometry. An approximately linear dependence of the superparamagnetic blocking temperature with iron loading was determined.

A commercial xylanase from Trichoderma longibrachiatum was used to treat the fractions of Douglas-fir kraft pulp of different fibre length. Enzymatic prebleaching was followed by chelation and peroxide bleaching. An evaluation of both optical and physical properties of the distinct fractions was conducted. A difference in susceptibility of the fractions of different fibre length to xylanase prebleaching was observed. The bleach-boosting effect observed with all fractions appeared to be related to the high-molecular-mass UV-absorbing material solubilized during enzyme treatments. Xylanase treatments resulted in beneficial effects to handsheet density and roughness as well as to some of the strength properties. However, the response to the xylanase treatments exhibited by all fractions of different fibre length was not uniform, indicating that fiber composition played a key role in the effectiveness of xylanase treatments.

Two thermophilic xylanases (xylanase II from Thielavia terrestris 255B and the 32-kDa xylanase from Thermoascus crustaceus 235E) were studied to determine if they had different and complementary modes of action when they hydrolysed various types of xylans. Partial amino acid sequencing showed that these two enzymes belonged to different families of ß-1,4-glycanases. Xylanase II achieved faster solubilization of insoluble xylan whereas the 32-kDa xylanase was more effective in producing xylose and short xylooligomers. An assessment of the combined hydrolytic action of the two xylanases did not reveal any co-operative action. The sugars released when the two thermophilic xylanases were used together were almost identical to those released when the 32-kDa xylanase acted alone. The two xylanases were able to remove about 12% of the xylan remaining in an aspen kraft pulp. This indicated that either one of these thermophilic enzymes may be useful for enhancing the bleaching of kraft pulps.

Using a genomic approach, we have identified a new Salmonella pathogenicity island, SPI-4, which is the fourth Salmonella pathogenicity island to be identified. SPI-4 was located at 92 min on the chromosome map and was flanked by the ssb and soxSR loci. The DNA sequence covering the entire SPI-4 and both boundaries was determined. The size of SPI-4 was about 25 kb and it contains 18 putative open reading frames (ORFs). Three of these ORFs encode proteins that have significant homology with proteins involved in toxin secretion. Another five ORFs encode proteins that have significant homology with hypothetical proteins from Synechocystis sp. strain PCC6803 or Acinetobacter calcoaceticus. The rest of the ORFs encode novel proteins, one of which has five membrane-spanning domains. SPI-4 is likely to carry a type I secretion system involved in toxin secretion. Furthermore, a previously identified locus (ims98), which is required for intramacrophage survival, was also mapped within the SPI-4 region. These findings suggested that SPI-4 is needed for intramacrophage survival.

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.15 +/- 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret lambdamax at 396 nm with a shoulder at 460 nm and contained 0. 28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 +/- 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.

BlnI or AvrII (5'-CCTAGG) sites are very rare in the Salmonella typhimurium LT2 genome. BlnI was used to construct a physical map which was correlated with the genetic map by using three methods. First, Tn10 carries BlnI sites, and the extra restriction sites produced by 34 genetically mapped Tn10 insertions were physically mapped by using pulsed-field gel electrophoresis. Second, six genetically mapped Mud-P22 prophage insertions were used to assign BlnI fragments. Integration of Mud-P22 introduces 30 kb of DNA that can easily be detected by a "shift up" in all but the largest BlnI fragments. Finally, induced Mud-P22 insertions package more than 100 kb of genomic DNA adjacent to one side of the insertion. Some of the smaller BlnI fragments were localized by hybridization to a dot blot array of 52 lysates from induced Mud-P22 insertions. Of the 10 BlnI sites mapped, 6 probably occur in or near the 16S rRNA genes at about 55, 71, 83, 86, 88.5, and 89.5 min. There is one BlnI site in the 90-kb pSLT plasmid. Two additional BlnI fragments of about 7 and 4 kb have not been localized. The size of the genome was estimated as 4.78 Mb (+/- 0.1 Mb) excluding pSLT but including prophages Fels-1 and Fels-2. One BlnI fragment that maps between 55 and 59 min showed a 40-kb reduction in size in a strain cured of the approximately 40-kb Fels-2 prophage.

MexA-MexB-OprM is an efflux system in Pseudomonas aeruginosa. OprM overproduced from the cloned gene was able to complement OprM-deficient mutants but did not alter the resistance of a wild-type P. aeruginosa strain to the different antimicrobial agents tested. This suggests that OprM cannot function by itself to efflux antibiotics, including beta-lactams targeted to the periplasm.

The v-abl oncogene of Abelson murine leukemia virus encodes a deregulated form of the cellular nonreceptor tyrosine kinase. v-Abl activates c-myc transcription, and c-Myc is an essential downstream component in the v-Abl transformation program. To explore the mechanism by which v-Abl activates c-myc transcription, a cotransfection assay was developed. We show that transactivation of a c-myc promoter by v-Abl requires the SH1 (tyrosine kinase) and SH2 domains of v-Abl; the C-terminal domains are not required for transactivation. The assay also identified the E2F site in the c-myc promoter as a v-Abl-responsive element. In addition, multimerized E2F sites were shown to be sufficient to confer v-Abl-dependent activation on a minimal promoter. This is the first identification of a v-Abl response element for transcriptional activation. v-Abl tyrosine kinase-dependent changes in proteins binding the c-myc E2F site were also demonstrated, including induction of a complex containing DP1, p107, cyclin A, and cdk2. Identification of v-Abl-dependent changes in E2F-binding proteins provides an important link between v-Abl, transcription, cell cycle regulation, and control of cellular growth.

Pyruvate formate-lyase (EC 2.3.1.54), a key enzyme in the anaerobic metabolism of Salmonella typhimurium, catalyzes the conversion of pyruvate to acetyl coenzyme A and formate. pfl::Mu dA operon fusions were isolated for the study of transcriptional regulation. pfl was transcribed both aerobically and anaerobically, but the activity increased about sixfold under anaerobic conditions. The addition of pyruvate, formate, and acetate in nutrient broth did not have any effect on the anaerobic expression of pfl. However, the addition of pyruvate to minimal glucose medium increased the anaerobic expression of pfl. The expression of pfl varied in different growth media. Anaerobic expression of pfl was lower when the culture was grown in minimal glucose medium than when it was grown in nutrient broth. When Casamino Acids (Difco Laboratories, Detroit, Mich.) were added to minimal glucose medium, the expression of pfl increased proportionally with the amount of Casamino Acids added. The transcription of pfl was positively controlled by the oxrA gene product and was affected by both the cya and crp mutations. However, mutations in genes affecting the cyclic AMP-cyclic AMP receptor protein complex or oxrA could not completely abolish the anaerobic derepression of pfl. In merodiploid strains, pfl::Mu dA/F' pfl+, the beta-galactosidase activities were decreased. The mutations gyrA, oxrC, and oxrE, which affected anaerobic metabolism, did not affect anaerobic expression of pfl.