[Characteristics of the white matter involvement sites on MRI in patients with multiple gliomas].

The retrospective study included 122 cases of multiple glioma and 183 cases of single glioma. Of these, there were 74 males and 48 female with multiple gliomas, aged 18 to 83 (53±13) years, and 104 males and 79 females with single gliomas, aged 10 to 84 (51±14) years. A standard spatial-based lesion analysis method was used for constructing a spatially distributed frequency heatmap of multiple gliomas, to observe the characteristics of their white matter invasion sites. The spatial distribution was more frequent in the subventricular zone, corpus callosum and cingulate gyrus in the multiple glioma group compared to the single glioma group (P<0.001).The white matter areas of multiple gliomas were more extensively involved, with more frequent involvement of the conjoined fibers (corpus callosum, P<0.05) and contact fibers (cingulate, dome, 0.05

[Interleukin-1 receptor associated kinase 4 deficiency: a case report and literature review].

Objective: To summarize the clinical characteristics of children with interleukin-1 receptor associated kinase 4 (IRAK4) deficiency. Methods: The clinical data of a child with IRAK4 deficiency who was admitted to the Department of Neurology of Shenzhen Children's Hospital for several times from June 2019 to August 2020 were retrospectively analyzed. Related literature up to January 2021 with the key words "IRAK4 gene variation", and "interleukin-1 receptor-associated kinase 4 deficiency" in PubMed, CNKI, Wanfang, and CQVIP databases were searched. The clinical characteristics of this disease were summarized and analyzed. Results: The boy was 6 years of age and had recurrent respiratory tract infections. He was improved after antibiotic treatment. His clinical manifestation included Streptococcus pneumoniae meningoencephalitis, multiple sclerosis, invasive discitis and inflammatory bone destruction. Family-based whole exome sequencing showed that the boy had a homozygous frameshift variation in the IRAK4 gene, NM_016123.3:C.540del (p.Phe180leufs*26), and both parents were heterozygous. A total of 23 cases were reported in ten English articles. Together with this case, there were 24 cases, including 13 males and 11 females. The age of onset was 8 days to 7 years. The main manifestations were recurrent invasive bacterial infection, including 11 cases with Streptococcus pneumoniae meningitis, 9 cases with Streptococcus pneumoniae and (or) Staphylococcus aureus septicemia, 1 case with Pseudomonas aeruginosa meningitis, 1 case of salmonella infection, and 1 case with Staphylococcus aureus skin abscess. Only 1 case had recurrent virus infection. There were 2 patients with autoimmune diseases, 1 with autoimmune encephalitis and the other one with juvenile idiopathic arthritis. Among the 24 cases, 10 died (9 in infancy). Most of the surviving children were diagnosed early and received antibiotics preventively and intravenous immunoglobulin (IVIG). Their susceptibility to infection decreased year by year, and could be close to normal children at the age of 14 years. Among the 24 cases, 21 cases had homozygous variation of IRAK4 gene and 3 cases had complex heterozygous variation. There were 15 kinds of variation, including 9 kinds of frameshift variation, 4 kinds of nonsense variation and 2 kinds of missense variation. One candidate variation hotspot was c.877 c>T (3 cases). Conclusions: IRAK4 deficiency mainly manifest as recurrent and invasive bacterial infection, with Streptococcus pneumoniae meningitis or septicemia being the most common. A few patients are complicated with autoimmune diseases. The mortality rate is high in infancy, early diagnosis and treatment can avoid severe illness or death.

[Clinical evaluation of the effect of reducing tooth sensitivity caused by in office bleaching using dentifrices].

OBJECTIVE: To evaluate tooth sensitivity and the efficacy of in-office bleaching when using different desensitizing dentifrices. METHODS: In total, 150 eligible individuals were recruited and randomized by computer-generated block randomization into three groups: potassium nitrate group (n=50), stannous fluoride group (n=50), and placebo group (n=50). Participants were asked to use a desensitizing dentifrice and toothbrush 15 days prior to and after the in-office bleaching. Each patient received a one-session in-office bleaching with 35% hydrogen peroxide whitening gel. The in-office bleaching included three 15 min operations, totally 45 min. Tooth sensitivity was evaluated by 100 mm visual analog scale (VAS) scores immediately 0 d and 1 d, 2 d, 7 d, 14 d, and 30 d after in-office bleaching. In total, 48 (96%), 45 (90%), and 46 (92%) individuals in the potassium nitrate, stannous fluoride, and placebo groups, respectively, completed the follow-up observations. RESULTS: Data were analyzed using analysis of covariance (ANOVA). For tooth sensitivity produced by in-office bleaching, the mean VAS values of the three groups were analyzed. At 0 d immediately after surgery, the mean VAS of the potassium nitrate group was 39.22±15.08, which was lower than that of the stannous fluoride group (47.18±12.59) and the placebo group (52.53±14.05), and the difference was statistically significant (P<0.05). The results of the stannous fluoride group and the placebo group were similar, and the difference was not statistically significant (P>0.05). On 1 day postoperatively, the mean VAS of the potassium nitrate group was 38.27±16.52, which was lower than that of the stannous fluoride group (44.69±14.92) and the placebo group (44.45±13.54), P<0.05. The results of the stannous fluoride group and the placebo group were similar, and the difference was not statistically significant (P>0.05). The mean values of VAS were similar 2 d, 7 d, 14 d and 30 d after operation, and the difference was not statistically significant (P>0.05). CONCLUSION: Compared with the stannous fluoride group and the placebo group, the use of potassium nitrate desensitizing toothpaste 15 days before surgery can effectively alleviate the tooth sensitivity during and after in-office bleaching.

[Course and prognosis analysis of intratympanic dexamethasone in the treatment of sudden sensorineural hearing loss].

Objective:To compare the recovery of different times of intratympanic dexamethasone in the treatment of sudden sensorineural hearing loss(SSNHL) and investigate the related prognostic factors. Method: Ninety patients with SSNHL were randomly divided into group A(3 times, every 2 days) and group B(5 times, every 2 days) according to the times of intratympanic dexamethasone. All patients were treated with blood flow promoting agents and neural nutrients. After 2 weeks of treatment, the recovery was compared. Based on treatment outcomes, patients were divided into two groups: recovery and no recovery, we evaluated the possible prognostic factors. Result:In the group A, the threshold improvement was(20.2±16.7) dB HL and the overall recovery rate was 78.7%(complete recovery rate, marked recovery rate and recovery rate were 17.0%, 21.3% and 40.4%, respectively), in the group B, the threshold improvement was(20.4±17.4) dB HL and the overall recovery rate was 81.4%, the complete recovery rate, marked recovery rate and recovery rate were 20.9%, 32.6% and 27.9%, respectively, the differences of recovery variables between the 2 groups were not statistically significant. After 2 weeks of treatment, 71 patients got overall recovery(78.9%), 19 patients(21.1%) resulted in no recovery, Interval between onset of symptoms and therapy of recovery group was shorter than that in no recovery group ï¼»(5.6±3.6）days vs. （7.7±4.0）days, P<0.05ï¼½, the presence rate of vertigo in recovery group was lower than that in no recovery(14.1% vs. 68.4%, P<0.01). Gender, ear side, age, concurrence of tinnitus, initial hearing threshold, times of intratympanic dexamethasone and audiogram have no influence on the prognosis of SSNHL. Conclusion:SSNHL patients treated with 3 times and 5 times of intratympanic dexamethasone got similar results. Earlier treatment and absence of vertigo lead to favorable hearing results.

[Effects of bioactive glass on reducing the hypersensitivity after full crown preparation].

OBJECTIVE: To evaluate the efficacy of the 45S5 bioactive glass on the reduction of hypersensitivity of teeth prepared for full coverage crowns. METHODS: In the study, 31 prepared abutment teeth with vital pulp from 18 patients satisfied the inclusion criteria and exclusion criteria. The patients were informed consent and recruited into the study. This was a before and after self-control, single blind clinical trial. The severity of tooth hypersensitivity at baseline was evaluated, after application of the placebo, which was the powder of zinc oxide, and after application of the 45S5 bioactive glass on the same tooth. Tooth hypersensitivity was evaluated by Yeaple probe and visual analogue scale (VAS) when the tooth was stimulated by a fixed value of mechanical pressure and a blast of cold air respectively. And the dentist gave the subjective score, and the Schiff score at the same time. The three methods of measurement were repeated at baseline, after application of placebo and after application of the powder of 45S5 bioactive glass. Student's t test and nonparametric test were used for statistical analysis. RESULTS: The indexes measured at baseline, after the application of the placebo and after the application of the 45S5 bioactive glass were as follows, respectively: Tactile value (29.03±9.44) g, (29.68±9.48) g and (44.19±11.19) g, VAS value (50.79±22.92) mm, (46.63±22.06) mm and (30.90±20.30) mm, Schiff score 2.13±0.67, 1.97±0.66 and 1.42±0.56. After the application of bioactive glass, the values of Tactile, the VAS, and the Schiff score were statistically different from those at baseline or after application of the placebo (P<0.05). The data measured at baseline and after application of the placebo were of no statistical difference (P>0.05). Statistical differences between the Schiff scores of the groups were associated with gender, tooth position, primary or secondary tooth preparation. CONCLUSION: RESULTS of the clinical trial showed that the powders of 45S5 bioactive glass were significantly effective on reducing the hypersensitivity of teeth with vital pulp after the full crown preparation.

[Clinical value of prenatal MRI in the diagnosis of fetal simple expansion of lateral ventricle and follow-up after birth].

Objective: To explore the value of prenatal MRI in the diagnosis of fetal simple expansion of lateral ventricle (ventriculomegaly) , and follow up the nervous system development status after birth. Methods: Simple expansion of the lateral ventricle fetus by prenatal MRI examination were collected in Huzhou Maternal and Child Care Hospital from May 2013 to June 2015, 126 cases of live births in expansion group, 50 normal cases were recruited in the same period as the control group. In expansion group, fetal subgroup analysis was done: (1) unilateral or bilateral lateral ventricle expasion: one group was 98 cases was lateral ventricle expansion (77.8%, 98/126), expansion of bilateral ventricle group was 28 cases (22.2%, 28/126). (2) Prenatal MRI in the diagnosis of the lateral ventricle of expansion: expansion of the lateral ventricle width was greater than 10.0 mm, if both sides were expanding, the expand width was the heavier one side, divided into 3 subgroups: ①Expansion in group A (lateral ventricle width 10.0-12.0 mm) were 88 cases (69.8%, 88/126). ②Expansion in group B (lateral ventricle width 12.1-15.0 mm) were 29 cases (23.0%, 29/126). ③Expansion of group C (lateral ventricle width> 15.0 mm) were 9 cases (7.12%, 9/126). All 176 cases were followed up after birth at the 3rd, 6th, 12th, 18th month (corrected age was used for premature babies), and Gesell developmental schedules (GDS) were used to evaluate the neurobehavioral development. Results: (1) The MRI results after birth: 21 cases were followed up by MRI after birth. In group A, 11 cases had MRI and 9 were normal (the ventricular width <10.0 mm after birth), the other 2 cases were stable (the ventricular width measured first time after birth was ≥10.0 mm, but the difference was within 2.0 mm from the MRI before birth). In group B, 4 cases had MRI, 1 was normal, 1 was stable, and 2 cases were getting better (the ventricular width measured first time after birth was ≥10.0 mm, but the width decreased more than 2.0 mm from the MRI before birth). In group C, 6 cases had MRI. 3 cases were getting better and 3 cases were stable. (2) Overall GDS results: expansion group after the birth of the 3rd, 6th, 12th, 18th month GDS evaluation results compared with control group, respectively, the differences were not statistically significant (all P>0.05). (3) The GDS results among the subgroups: in each evaluation after birth, there were no statistically significant differences between group A and the control group (all P>0.05). The GDS results of group B at the 3rd and 6th month were lower than those of the control group (P<0.05); while there were no statistically significant differences between the 2 goups at the 12th and 18th month (P>0.05). And for group C, statistically significant differences were found compared to the control group at each follow-up time (all P<0.05). (4) GDS results at different times after birth in the expansion group: there was no statistically significant difference between the results at the 3rd and 6th month (P>0.05). But when the result at the 3rd month was compared to the results of the 12th or 18th month, the differences were statistically significant (P<0.05). GDS result of 6th months after birth compared with 12th and 18th months, respectively, there were no statistically significant differences (P>0.05). There was no statistically significant difference between the results at the 12th and 18th month (P>0.05). (5) The GDS results in unilateral and bilateral ventricle expansion: at the 18th month, among the 98 unilateral cases, 86 (87.8%, 86/98) had normal GDS results(>85 scores); 8 (8.2%, 8/98) had borderline results (75-85 scores); 4 (4.1%, 4/98) had delayed results (<75 scores). Among the 28 bilateral cases, 23 (82.1%, 23/28) had normal GDS results; 3 (10.7%, 3/28) had borderline results; 2 (7.1%, 2/28) had delayed results. There was no statistically significant difference (P>0.05). Conclusions: Among the simple expansion of lateral ventricle, those whose ventricular width are ≤12.0 mm may not need clinical treatment. If the width is between 12.1 to 15.0 mm, closely follow-up and targeted rehabilitation training after birth are recommended. When the width is more than 15.0 mm, the risk of the central nervous system function delay is significantly increased, and early intervention might improve the prognosis.

Emission properties and CW laser operation of Pr:YLF in the 910 nm spectral range.

The polarized emission spectra for the 3P0→1G4 emission transition of the Pr3+ ion around 910 nm in the Pr3+:LiYF4 (Pr:YLF) laser crystal were registered and calibrated in unit of cross sections for the first time. Continuous-wave (CW) laser operation is demonstrated at 915 nm in π polarization by pumping the crystal with an optically pumped semiconductor laser (OPSL) at 479.2 nm. An output power of 218 mW is thus obtained with a laser slope efficiency of about 24% for an output coupler (OC) transmission of 1.9%. CW laser operation is also demonstrated at 907 nm in σ polarization by using a thin plate oriented at Brewster angle. An output power of about 89 mW with a slope efficiency of about 10% is then obtained for an OC transmission of 0.8%.The round-trip cavity losses are estimated for different experimental cavity configurations to be about 1% and the typical beam quality M2 factors measured in the transverse x and y directions are found equal to about 1.07 and 1.04, respectively. Finally, we also report on a double laser wavelength operation by using an OC with a transmission of about 0.05%, such effect resulting from joint-etalon effects inside the cavity.

Inertia sensor-based guidance system for upperlimb posture correction.

Stroke rehabilitation is labor-intensive and time-consuming. To assist patients and therapists alike, we propose a wearable system that measures orientation and corrects arm posture using vibrotactile actuators. The system evaluates user posture with respect to a reference and gives feedback in the form of vibration patterns. Users correct their arm posture, one DOF at a time, by following a protocol starting from the shoulder up to the forearm. Five users evaluated the proposed system by replicating ten different postures. Experimental results demonstrated system robustness and showed that some postures were easier to mimic depending on their naturalness.

Raman fiber laser harmonically mode-locked by exploiting the intermodal beating of CW multimode pump source.

We report here the first demonstration of a harmonic mode-locked Raman fiber laser using the intermodal beating of a continuous-wave (CW) multiple-longitudinal-mode pump laser. By matching the Raman-cavity round-trip frequency with the intermodal-beating one of a 1064 nm CW pump source, harmonic mode-locking in phosphosilicate Raman fiber laser is stably initiated at the first-order Stokes of 1239.5 nm, and generates rectangular-shape nanosecond pulses with the pulse energy up to 4.25 nJ. Using the new type of mode-locking, the harmonic order can be discretely tuned from 78 th- to 693 rd-order, and the cavity supermode is suppressed up to 51.1 dB with the signal-to-noise ratio of more than 65 dB.

Expression of N-methyl-D-aspartate receptor and its effect on nitric oxide production of rat alveolar macrophages.

We early show that glutamate (Glu) mediate hyperoxia-induced newborn rat lung injury through N-methyl-D-aspartate receptor (NMDAR). In this study, we search for evidence of NMDAR expression on newborn rat alveolar macrophages (AMs) and the difference between newborn and adult rat AMs, and the possible effect on nitric oxide (NO) production of AMs by exogenous NMDA. The protein of NMDAR was showed by immunocytochemistry, and the mRNA was examined by RT-PCR and real-time PCR. The results show that: (i) both newborn and adult rat AMs express NMDAR1 and the four NMDAR2 subtypes and newborn rat AMs are higher expression. (ii) NMDA administration increase NO production, inducible nitric oxide synthase (iNOS) activity and iNOS mRNA expression of AMs. (iii) NMDAR activation elevates NO secretion of AMs, which suggests that AM may be one of the key cellular origin of the elevated NO secretion in hyperoxia-induced lung injury.

Fine-tuning the synthesis of ZnO nanostructures by an alcohol thermal process.

A simple and efficient alcohol thermal technique was applied to control the growth of the dimensions and morphology of ZnO nanostructures under mild conditions, where surfactant was not necessary. The size of ZnO nanocrystals increased with growth temperature and they transformed into nanorods with different aspect ratios through tuning the reaction time. The length of nanorods increased significantly with the reaction time, but their thickness only slightly increased. The as-prepared ZnO nanocrystals were monocrystalline and the growth orientation of ZnO nanorods was [001]. Photoluminescence measurements showed a blue shift in violet emission with a reduction in crystal size and revealed the quantum confinement effect. Electron irradiation induced structural damage was observed in the ZnO nanorods synthesized at 120 degrees C. Experimental results proved that the possible growth mechanism of ZnO nanostructures was oriented attachment.

The Agrobacterium tumefaciens rnd homolog is required for TraR-mediated quorum-dependent activation of Ti plasmid tra gene expression.

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-L-homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation of tra genes on pTiC58DeltaaccR. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd of Escherichia coli, an RNase known to be involved in tRNA processing. The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2, surround A. tumefaciens rnd, but none of these genes exerted a detectable effect on the expression of the tra reporter. In the mutant, traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of tra, but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect in tra gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.

Construction of a derivative of Agrobacterium tumefaciens C58 that does not mutate to tetracycline resistance.

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

Quorum-sensing signal binding results in dimerization of TraR and its release from membranes into the cytoplasm.

Promoter binding by TraR and LuxR, the activators of two bacterial quorum-sensing systems, requires their cognate acyl-homoserine lactone (acyl-HSL) signals, but the role the signal plays in activating these transcription factors is not known. Soluble active TraR, when purified from cells grown with the acyl-HSL, contained bound signal and was solely in dimer form. However, genetic and cross-linking studies showed that TraR is almost exclusively in monomer form in cells grown without signal. Adding signal resulted in dimerization of the protein in a concentration-dependent manner. In the absence of signal, monomer TraR localized to the inner membrane while growth with the acyl-HSL resulted in the appearance of dimer TraR in the cytoplasmic compartment. Affinity chromatography indicated that the N-terminus of TraR from cells grown without signal is hidden. Analysis of heterodimers formed between TraR and its deletion mutants localized the dimerization domain to a region between residues 49 and 156. We conclude that binding signal drives dimerization of TraR and its release from membranes into the cytoplasm.

The antiactivator TraM interferes with the autoinducer-dependent binding of TraR to DNA by interacting with the C-terminal region of the quorum-sensing activator.

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via the transcriptional activator TraR and the acyl-homoserine lactone Agrobacterium autoinducer (AAI). Unique to this system, the activity of TraR is negatively modulated by an antiactivator called TraM. Analyses from yeast two-hybrid studies suggest that TraM directly interacts with the activator, but the conditions under which these components interact and the region of TraR responsible for this interaction are not known. Induction of traM in a strain in which TraR was activating transcription of a reporter system led to rapid cessation of gene expression. As assessed by a genetic assay that measures AAI-dependent DNA binding, TraM inhibited TraR function before and after the transcription factor had bound to its DNA recognition site. Consistent with this observation, in gel retardation assays, purified TraM abolished the DNA binding activity of TraR in a concentration-dependent manner. Such inhibition occurred independent of the order of addition of the reactants. As assessed by far Western analyses TraM interacts with TraR by directly binding the activator. TraM in its native form interacted with native TraR and also with heat-treated TraR but only when SDS was included with the denatured protein. TraM interacted with TraR on blots prepared with total lysates of cells grown in the presence and absence of AAI. Far Western analysis of N- and C-terminal deletion mutants localized a domain of TraR contributing to TraM binding to the C-terminal portion of the activator protein. Random mutagenesis by hydroxylamine treatment and error-prone polymerase chain reaction identified several residues in this region of TraR important for interacting with TraM as well as for transcriptional activation or/and DNA binding. We conclude that TraM inhibits TraR by binding to the activator at a domain within or close to the helix-turn-helix motif located at the C terminus of the protein.

[Modulation of epidermal growth factor on the synthesis of pulmonary surfactant and its mechanism].

AIM: To study the effect of EGF at physiological level on PS synthesis on cultured lung explants without serum. METHODS: The total phospholipids and major phospholipid components in lung tissues were determined. The amount of 3H-choline incorporation into PC was measured. RESULTS: 1. 10(-9) mol/L EGF enhanced 3H-choline incorporation into PC at 8 h and reached the maximum at 16 h. 2. The synthesis of total phospholipids, PC and PG increased from lung tissues exposed to 10(-9) mol/L EGF, but membrane characteristic phospholipid showed no change. 3. TP 47, a PTK inhibitor and H7, a PKC inhibitor could block EGF-induced PS synthesis. The release of LDH from lung explants could not be changed by EGF, TP 47 and H7. CONCLUSION: EGF at physiological level could enhance the adult rat PS synthesis, and its modulated mechanisms involved PTK and PKC pathways.

[The influence of serotonin on cochlear microcirculation].

OBJECTIVE: The present study was undertaken to investigate the influence of serotonin (5-HT) on cochlear microcirculation. METHOD: 2 microliters 0.5% 5-HT were applied to round window membrane in anaesthetized guinea pigs. The three parameters as blood flow (CBF), number of blood cell moving (CMBC) and blood velocity (Veloc) of cochlear microcirculation were measured with laser doppler flowmeter. The mean arterial pressure (MABP) was recorded simultaneously. RESULT: 5-HT induced a biphasic response of reduction in cochlear blood flow. In the first phase, cochlear microcirculation showed a transient abruptly reduction. The CBF, CMBC and Veloc reached to 86.8%, 90.2% and 96.3% of the baseline respectively. The second phase reduced slowly. The CBF and CMBC decreased to 89.7% and 89.6% by average throughout the experiment period respectively. The Veloc and MABP had no significant changes. CONCLUSION: 5-HT could decrease the cochlear, blood flow and result in cochlear microcirculation dysfunction.

[Fibronectin or RGD peptide promotes nitric oxide synthesis of rabbit bronchial epithelial cells].

To find out whether the extracellular matrix component fibronectin plays a regulatory and protective role in bronchial epithelial cells, the present study was undertaken to detect the NO released from primary cultured rabbit bronchial epithelial cells (BEC) and the activity of NO synthesase (NOS) in cells. Stress with ozone was taken as the positive control, and the effects of fibronectin (Fn) or its specific sequence Arg-Gly-Asp (RGD peptide) was observed. The results showed: (1) Ozone stress enhanced NO release from BEC, and treatment with Fn also promoted the NO production. The effect of Fn could be blocked by calmodulin inhibitor W(7). (2) Both Fn and RGD showed a dose-dependent promotion on NO release. (3) The NOS activity was significantly elevated in Fn treated group and the effect of Fn was abolished by W(7). A dose-response relation in NOS activity was observed in either Fn treated or RGD treated group. It is concluded that the binding of fibronectin or its specific sequence RGD peptide with integrins of BEC plays a role in upregulating the NOS activity and the NO release, and calmodulin may take part in the pathway of signal transduction.

Modulating quorum sensing by antiactivation: TraM interacts with TraR to inhibit activation of Ti plasmid conjugal transfer genes.

Conjugal transfer of the Ti plasmid pTiC58 is regulated by a quorum-sensing system involving the transcriptional activator TraR and the acyl homoserine lactone autoinducer N-(3-oxo-octanoyl)-L-homoserine lactone (AAI). Activation of tra gene expression by TraR and AAI is inhibited by TraM, an 11 kDa protein also coded for by the Ti plasmid. Previous studies suggested that TraM interferes with TraR activity by directly interacting with the activator protein. Using the yeast two-hybrid system, constructs of Saccharomyces cerevisiae containing a fusion of traR to the B42 domain of the prey plasmid pJG4.5 and a fusion of traM to the lexA gene of the bait plasmid pEG202 produced beta-galactosidase and grew on medium lacking leucine, both phenotypes indicative of an interaction between the two proteins. Early termination mutants and substitution mutants mapping to the C-terminus of TraM were isolated by screening for alleles unable to interfere with TraR activity in Agrobacterium tumefaciens. These mutants all failed to interact with the TraR fusion in the two-hybrid system. An N-terminal deletion mutant of TraM lacking the first 27 residues weakly interacted with TraR in the two-hybrid system whereas deletions of 48 amino acids or more abolished the interaction. As assessed by Western blot analysis, the mutant fusion proteins were produced at levels indistinguishable from that of the wild-type TraM in the yeast tester strain. Mutants of TraR that were not inhibited by TraM in A. tumefaciens were isolated and fell into two classes. In the first, the mutation resulted in increased expression of wild-type TraR. In the second, a proline residue at position 176 was changed to serine (P176 --> S) or to leucine (P176 --> L). The P176 --> S mutant interacted with wild-type TraM, but at a detectably lower level, in the two-hybrid assay. Mutants of TraR with N-terminal deletions as large as 105 amino acids interfered with the ability of TraM to inhibit wild-type TraR in A. tumefaciens. Two-hybrid assays indicated that these mutants, as well as a C-terminal 49 residue fragment of TraR, can interact with TraM. We conclude that TraM and TraR interact in vivo and that this interaction is responsible for inhibition of TraR-mediated activation. We also conclude that the two proteins interact with each other through domains located at their respective C-termini.

Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity.

TraR, a member of the LuxR family of quorum-sensing transcription factors, is responsible for the population density-dependent regulation of Ti plasmid conjugal transfer. The protein requires as coinducer an acyl-homoserine lactone signal molecule called AAI (Agrobacterium autoinducer) that is produced by the bacteria themselves. TraR only activates its target genes, making it difficult to determine whether interaction with AAI is required for binding DNA or for initiating transcription. To assess this, we converted TraR into a repressor by placing a copy of the tra box, an 18-bp inverted repeat believed to be the recognition site for this protein, over the -10 region of a promoter driving expression of lacZ. Repression of this promoter by TraR depended on AAI or, at higher concentrations, VAI, the closely related signal of Vibrio fischeri. C-terminal deletions as short as 2 aa and N-terminal deletions as short as 4 aa in TraR abolished both repressor and activator functions. The C-terminal mutants were strongly dominant over TraR, suggesting that they can form heteromultimers with the wild-type activator. Mutants of TraR with substitutions at Asp-10 and Gly-123 failed to activate a positively controlled reporter but continued to repress the chimeric promoter in an AAI-dependent manner. We conclude that TraR recognizes the tra box as its binding site, that binding of TraR to this site depends on AAI, and that the N-terminal half of the protein contains one or more domains that are required for activation but not for multimerization, for interaction with the acyl-homoserine lactone, or for DNA binding.