[Application of the two-step sentinel lymph node biopsy with double-tracer in early staged endometrial cancer].

Objective: To explore the feasibility and clinical value of sentinel lymph node (SLN) biopsy through cervix-uterine combined two-step injection with two tracers in patients with early stage endometrial cancer. Methods: From July 2019 to April 2021, a total of 73 patients, aged (54.2±3.3) year, who were preoperatively diagnosed as stage Ⅰ-Ⅱ endometrial cancer (including 56 low-risk patients and 17 medium-high risk patients) in Affiliated Hospital of Qingdao University were selected. According to the different sites of tracer injection, the patients were randomly divided into three groups: cervical injection group (25 cases): 1 ml of nano-carbon was used to inject at 3 and 9 o'clock in the cervix; uterine injection group (21 cases): the magnetic resonance imaging examination was performed to determine the location of the lesion, and 4 ml of methylene blue was injected into the uterine body at 2 sites where the lesion was located; combined injection group (27 cases): cervical injection of nano-carbon (1 ml) combined with uterine injection of methylene blue (4 ml). The SLN in all patients were identified under laparoscopy, removed, and followed by frozen pathological examination. Pathological ultra-staging was performed if the postoperative pathological outcome of SLN was negative. The total detection rate of SLN, bilateral pelvic SLN detection rate, sensitivity, negative predictive value, and location of SLN in each group were calculated and compared. Results: (1) In 73 patients with endometrial cancer, the overall detection rate of SLN was 88% (64/73), the detection rate of bilateral pelvic SLN was 67% (49/73), and the detection rate of para-aortic SLN was 49% (36/73). The overall detection rate of SLN (71%, 15/21) and bilateral pelvic SLN (43%, 9/21) in the intrauterine injection group were significantly lower than those in the cervical injection group [92% (23/25), 76% (19/25), respectively] and the combined injection group [96% (26/27), 78% (21/27), respectively; all P<0.05]; the detection rate of para-aortic SLN in the cervical injection group (28%, 7/25) was significantly lower than those in the intrauterine injection group and combined injection group [52% (11/21) and 67% (18/27), respectively; both P<0.05]. Among 73 cases with endometrial cancer, 9 had lymph node metastasis confirmed by postoperative pathological examination, 8 of them had lymph node metastasis detected by SLN and 1 had no lymph node metastasis detected by SLN, with a total sensitivity of 89% and a negative predictive value of 98%. The sensitivity and negative predictive value of cervical injection group and combined injection group were 100%, while the sensitivity and negative predictive value of intrauterine injection group were 67% and 95%. Among 56 low-risk patients, only one patient with lymph node metastasis was confirmed by postoperative pathology by SLN detection, and the metastasis rate was 2% (1/56), and the sensitivity and negative predictive value were 100%. Lymph node metastasis was confirmed in 8 of 17 patients (8/17) with a sensitivity of 88% and a negative predictive value of 90%. (2) A total of 459 SLN were detected in 73 endometrial cancer patients, with the highest proportion of external iliac (33.3%, 153/459).The obturator foramen was 25.3% (116/459), para-aortic 19.6% (90/459), iliac 12.0% (55/459), and presacral 9.8% (45/459). The proportion of para-aortic SLN in the cervical injection group was 12.4% (21/169), which were significantly lower than that in the intrauterine injection group and the combined injection group [27.4% (26/95) and 22.1% (43/195), respectively; both P<0.05]. (3) Pathological super-staging results: among 64 patients with negative SLN routine paraffin pathology, 4 cases of lymph node micro-metastases and 1 case of isolated tumor cell metastasis were detected, and the SLN micro-metastases rate was 8% (5/64), including 2 cases of low-risk patients and 3 cases of medium-high risk patients. Conclusions: SLN biopsy has high sensitivity and negative predictive value in patients with early endometrial cancer and could be used as an alternative to systematic lymph node dissection in low-risk patients. The SLN mapping through cervical-uterine combined injection could further improve the detection rate effectively and avoid the missed detection of positive para-aortic lymph node, especially for high-risk patients or patients with fundal tumor involvement.

Quantum circuits with many photons on a programmable nanophotonic chip.

Growing interest in quantum computing for practical applications has led to a surge in the availability of programmable machines for executing quantum algorithms1,2. Present-day photonic quantum computers3-7 have been limited either to non-deterministic operation, low photon numbers and rates, or fixed random gate sequences. Here we introduce a full-stack hardware-software system for executing many-photon quantum circuit operations using integrated nanophotonics: a programmable chip, operating at room temperature and interfaced with a fully automated control system. The system enables remote users to execute quantum algorithms that require up to eight modes of strongly squeezed vacuum initialized as two-mode squeezed states in single temporal modes, a fully general and programmable four-mode interferometer, and photon number-resolving readout on all outputs. Detection of multi-photon events with photon numbers and rates exceeding any previous programmable quantum optical demonstration is made possible by strong squeezing and high sampling rates. We verify the non-classicality of the device output, and use the platform to carry out proof-of-principle demonstrations of three quantum algorithms: Gaussian boson sampling, molecular vibronic spectra and graph similarity8. These demonstrations validate the platform as a launchpad for scaling photonic technologies for quantum information processing.

Abnormal expression of Rap2A as a prognostic marker for human breast cancer.

OBJECTIVE: The aim of this study was to explore the expression of GTPase protein Ras-related protein Rap-2a (Rap2A) in breast cancer (BC). Furthermore, the associations of Rap2A with clinicopathological parameters of BC patients were investigated. PATIENTS AND METHODS: quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to examine Rap2A expression in BC tissues and cells. The association between Rap2A expression and clinicopathological characteristics was analyzed by Chi-square test. Low expression of Rap2A in BC cells was conducted by transfection of small interfering RNA (siRNA). Subsequently, colony formation assay and transwell assay were used to detect the proliferation and invasion abilities of BC cells, respectively. RESULTS: Rap2A was highly expressed in both BC tissues and cells (p<0.05). Further analysis showed that tumor size, clinical stage, and distant metastasis were correlated with Rap2A expression (p<0.05). Besides, inhibition of Rap2A significantly decreased the proliferation and invasion abilities of BC cells (p<0.05). CONCLUSIONS: Rap2A acted as a promotor in the development of BC. Our findings suggested that Rap2A might be a new potential therapeutic target marker for BC treatment.

[Clinical analysis of surgical treatment of post-infarction left ventricular aneurysm: a series of 254 patients].

Objective: To examine the efficacy of two surgical procedures on post-infarction left ventricular aneurysm. Methods: The clinic data of 254 patients with post-infarction left ventricular aneurysm, who underwent surgical ventricular reconstruction between January 1997 and December 2019 in Department of Cardiovascular Surgery, Nanjing Hospital Affiliated to Nanjing Medical University was analyzed retrospectively. There were 183 males and 71 females aged from 31 to 81 years, with a median age of 64.6 years. Based on the size of the ventricular aneurysm, there were 73 patient received linear reconstruction (linear group) and 181 patients received endoventricular patch plasty technique (patch plasty group). Ejection fraction, left ventricular systolic and end diastolic volume and left ventricular systolic and end diastolic volume index were recorded preoperatively, 2-week, 3-month, 1-year and 5-year after operation. The survival curves were plotted with Kaplan-Meier method and the survival rates were compared by Log-rank test. Results: All patients underwent surgery with a mean cardiopulmonay bypass duration of (92±32) minutes (44 to 196 minutes) and aortic cross clamp duration of (67±22) minutes (33 to 152 minutes).There were 9 perioperative deaths with a mortality rate of 3.5%. Angina pectoris of other cases are relief and heart function improved greatly. Five years after operation, the percentage of cardiac function (New Yord Heart Association) class Ⅲ to Ⅳ patients decreased from 96.1%(244/254) to 9.9%(16/161). There was no significant difference in survival rate between linear group and patch plasty group at 1-, 3-, 5-years postoperatively (96%, 91%, 77% vs. 96%, 90%, 79%, P=0.562). Ejection fraction increased from (39±10)% (range: 22% to 50%) preoperatively to (46±6)% (range: 39% to 54%) 1-year postoperatively in the linear group, while increased from (38±13)% (range: 26% to 51%) preoperatively to (50±6)% (range: 39% to 55%) in the patch plasty group. Conclusions: Left ventricular reconstruction is quite effective for patients with post-infarction left ventricular aneurysm. The choice of operative approaches is determined by the size and range of ventricular aneurysm. Both linear reconstruction and endoventricular patch plasty technique can got similarly surgical outcomes with near and late curative effect.

[Scar management strategy in children].

Children are a special group, which have unique physiological characteristics and are still in the period of physical and mental growth and development, thus the prevention and treatment of scar in children are different from that in adults. Scar management in children is a complex and multifaceted system engineering. The grade of scar in children needs to be adjusted according to the age, period, and severity. Corresponding method needs to be chosen for the treatment of scar in children according to the classification of the scar. The compliance of children is poor, and the key to scar treatment is the persistence and cooperation of the parents, so doctors should strengthen propaganda and education to the parents of children with scar. For children with scar, individualized and comprehensive treatment should be used according to the characteristics of children to achieve good results.

[Application effects of enhanced computed tomography and three-dimensional reconstruction technology in reconstruction of pediatric post-burn scars with expanded flaps].

Objective: To explore the application effects of enhanced computed tomography (CT) and three-dimensional reconstruction technology in the reconstruction of pediatric post-burn scars with expanded flaps. Methods: From May 2016 to March 2019, 19 children with hypertrophic scars after thermal injuries were admitted to our unit, including 10 boys and 9 girls, aged from 4 years and 5 months to 15 years and 11 months. The area of scars ranged from 5 cm×4 cm to 23 cm×9 cm. One or more skin and soft tissue expanders with suitable volume and shape were implanted into the normal skin area around scar of children. Three to six months later, enhanced CT and three-dimensional reconstruction were performed before the second stage operation to obtain three-dimensional images of the vascular branches in the donor site for expanded flaps to be cut, so as to determine the course and distribution of the vascular branches and guide the design of expanded flaps. According to the design scheme, the resection of scar, removal of expanders, and excision and transfer of flaps were performed to repair the wounds after scar resection. The area of flaps ranged from 6 cm×4 cm to 25 cm×10 cm. The donor site was closed directly. The number of flaps was counted. The anatomical structure, vascular distribution, and adverse reactions during enhanced CT and three-dimensional reconstruction of site for expanded flaps to be cut, the survival of expanded flaps and the follow-up after the second-stage operation were observed. Results: A total of 48 expanded flaps were designed and excised in 19 children. The anatomical structure of the site for expanded flaps to be cut and the adjacent spatial position relationship were visually observed through the three-dimensional reconstruction after enhanced CT, and no adverse reactions were observed. Arterial branch blood supply or venous return was observed in 29 sites for expanded flaps to be cut. All the expanded flaps survived well without blood supply disorder after the second stage operation. The children were followed up for 6 months to 1 year and 6 months after the second stage operation. The appearance of the flaps was natural, and the color and thickness of the flaps were similar to those of the surrounding normal skin, except for one child with obvious linear scar. Conclusions: Enhanced CT and three-dimensional reconstruction can assist the vascular assessment of the expended flaps, which is helpful for rational design of the flap excision and transfer protocol to improve the survival rate of flaps. Thus, it has certain clinical application value in the reconstruction of post-burn scar in children with expanded flaps.

Euxanthone exhibits anti-proliferative and anti-invasive activities in hepatocellular carcinoma by inducing pyroptosis: preliminary results.

OBJECTIVE: Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths. Euxanthone, a xanthone compound extracted from Polygala caudata, possesses a variety of pharmacological activities. This work aimed to explore whether euxanthone exhibits anti-cancer activities in HCC. PATIENTS AND METHODS: Tissue specimen was collected and the mRNA and protein expression of marker proteins were detected by qRT-PCR and Western blot, respectively. The viable cell number was assessed by CCK-8 assay. TUNEL assay was utilized to determine cell death. Cell migration was measured by Wound healing. Cell invasion was measured by transwell assay. Xenograft model was established to evaluate the efficacy of euxanthone in vivo. RESULTS: We demonstrated that HCC tissue and HCC cell line presented with lower expression of caspase-1, IL-1ß, and IL-18. Euxanthone markedly suppressed the proliferation of HCC cells and induced cell death. In addition, euxanthone inhibited cell migration and invasion. Meanwhile, our results showed that euxanthone promoted cell death in a caspase-2 dependent manner. In vivo data also showed that euxanthone suppressed tumor growth and promoted pyroptotic cell death. CONCLUSIONS: We showed that that euxanthone may be a candidate of anticancer agent and provide clinical benefits for patients with HCC.

Doublets versus single-agent therapy as first-line therapy for elderly patients with advanced non-small cell lung cancer? A systematic review of randomised controlled trials.

INTRODUCTION: Although the standard treatment for advanced non-small cell lung cancer (NSCLC) patients is platinum-based doublet chemotherapy, single-agent therapy is still preferred in elderly patients. Comparison of the efficacy of various combinations of doublets with single-agent chemotherapy is somehow contradictory. This study conducted a systematic review to evaluate the efficacy and tolerability of the third-generation agent-based doublets vs. single-agent chemotherapy in elderly NSCLC patients. METHODS: Electronic (PubMed, EMBASE and Cochrane Library database) and manual searches were conducted to collect data from published, randomised, phase 2 and 3 trials which compared doublets with a third-generation single-agent chemotherapy in elderly patients. Pooled relative risks (RRs) were calculated for the incidences of overall response rate (ORR), 1-year survival rate (1-y SR), and grade 3/4 toxicities. RESULTS: Seven eligible trials (2219 patients) were selected from 1170 studies that were initially identified. A significant difference in ORR favouring doublets over single agents was observed [RR, 1.59; 95% confidence interval (95% CI), 1.36-1.86; p < 0.0001] with a slightly, but not significantly improved 1-y SR (RR, 1.19; 95% CI, 0.98-1.45, p = 0.007). Subgroup analysis suggested that platinum (RR, 1.94; 95% CI, 1.47-2.55, p < 0.0001) or non-platinum- (RR, 1.45; 95% CI, 1.20-1.75, p < 0.0001) based doublets could improve ORR, and the grade 3/4 thrombocytopaenia (RR, 6.64; 95% CI, 1.78-24.86, p = 0.005) and anaemia (RR, 2.86; 95% CI, 1.62-5.05, p < 0.0001) were preferred to occur in platinum-based doublets. CONCLUSIONS: Doublets appear to be more effective and tolerable than single-agent therapy for treating elderly advanced NSCLC patients, and therefore could be considered as a treatment option for elderly populations with good physical status.

A new meroditerpenoid from Mayodendron igeum.

A new meroditerpenoid, igeumone (1), together with 18 known compounds (2-19), were isolated from ethanolic extract of the bark of Mayodendron igeum. Their structures were determined by analysis of spectral data or comparison with authentic samples. X-ray crystallographic analysis was employed to unambiguously determine the structure of 1.

Pectinase production by Aspergillus niger using wastewater in solid state fermentation for eliciting plant disease resistance.

An elicitor of plant disease resistance, pectinase, was produced by solid state fermentation with Aspergillus niger. Sugar beet pulp was used as carbon source and the wastewater from monosodium glutamate production was used as nitrogen and water source. The composition of the fermentation medium was: 11 ml concentrated wastewater (containing NH3-N 38.2 mg/ml), sugar beet pulp 10 g, Na2HPO4.12H2O 0.2 g, KH2PO4 0.04 g in a 500 ml Erlenmeyer flask. The fermentation temperature was 30 degrees C and the relative humidity of the air was 75-90%. The maximum production of pectinase was reached after 96 h cultivation. The crude pectinase extracted from the fermented materials could elicit disease resistance in cucumber and tomato seedlings.

Chromosomal polymorphism of mandarin vole, Microtus mandarinus (Rodentia).

The mitotic and meiotic chromosomes of mandarin vole, Microtus mandarinus Milne-Edwards, from Shandong Province of China were analyzed by conventional, G- and C-banding and Silver-staining techniques. We detected chromosomal polymorphism in the vole, exhibiting diploid chromosome numbers 2n = 48-50 and variable morphology of the 1st pair, one medium sized telocentric pair and the X chromosomes. Four types of karyotypes were revealed in the population. According to banding analysis, there were pericentric inversion, Robertsonian fusion and translocation in M. mandarinus karyotype evolution. The X displayed two different morphologies, which could be explained by prericentric inversion and a telocentric autosome translocation.

Evidence for a novel GTPase priming step in the SRP protein targeting pathway.

Protein targeting by the signal recognition particle (SRP) pathway requires the interaction of two homologous GTPases that reciprocally regulate each other's GTPase activity, the SRP signal peptide- binding subunit (SRP54) and the SRP receptor alpha-subunit (SRalpha). The GTPase domain of both proteins abuts a unique 'N domain' that appears to facilitate external ligand binding. To examine the relationship between the unusual regulation and unique architecture of the SRP pathway GTPases, we mutated an invariant glycine in Escherichia coli SRP54 and SRalpha orthologs ('Ffh' and 'FtsY', respectively) that resides at the N-GTPase domain interface. A G257A mutation in Ffh produced a lethal phenotype. The mutation did not significantly affect Ffh function, but severely reduced interaction with FtsY. Likewise, mutation of FtsY Gly455 produced growth defects and inhibited interaction with Ffh. The data suggest that Ffh and FtsY interact only in a 'primed' conformation which requires interdomain communication. Based on these results, we propose that the distinctive features of the SRP pathway GTPases evolved to ensure that SRP and the SR engage external ligands before interacting with each other.

Preparation of levoglucosan by pyrolysis of cellulose and its citric acid fermentation.

Levoglucosan (LG), 1,6-anhydro-beta-D-glucopyranose, was produced by pyrolysis of cellulose. A response surface method was used to optimize the reaction parameters: X1, temperature; X2, time required for heating cellulose from room temperature to the designed pyrolysis temperature; and X3, vacuum, and a Box-Behnken design was employed for this purpose. The optimal temperature and time were found to be 388 degrees C and 26.2 min by fixing the vacuum at 1 mm Hg. The levoglucosan prepared was fermented to citric acid by Aspergillus niger CBX-209, which was a mutant derived by gamma-ray mutagenesis of the parent strain CBX-2. The mutant could produce increasing citric acid with increasing LG purity and had a citric acid yield of 87.5% when using purified levoglucosan as the sole carbon source in a 5 day fermentation period.

FtsY binds to the Escherichia coli inner membrane via interactions with phosphatidylethanolamine and membrane proteins.

Targeting of many polytopic proteins to the inner membrane of prokaryotes occurs via an essential signal recognition particle-like pathway. FtsY, the Escherichia coli homolog of the eukaryotic signal recognition particle receptor alpha-subunit, binds to membranes via its amino-terminal AN domain. We demonstrate that FtsY assembles on membranes via interactions with phosphatidylethanolamine and with a trypsin-sensitive component. Both interactions are mediated by the AN domain of FtsY. In the absence of phosphatidylethanolamine, the trypsin-sensitive component is sufficient for binding and function of FtsY in the targeting of membrane proteins. We propose a two-step mechanism for the assembly of FtsY on the membrane similar to that of SecA on the E. coli inner membrane.

Ancestral origins and worldwide distribution of the PRNP 200K mutation causing familial Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) belongs to a group of prion diseases that may be infectious, sporadic, or hereditary. The 200K point mutation in the PRNP gene is the most frequent cause of hereditary CJD, accounting for >70% of families with CJD worldwide. Prevalence of the 200K variant of familial CJD is especially high in Slovakia, Chile, and Italy, and among populations of Libyan and Tunisian Jews. To study ancestral origins of the 200K mutation-associated chromosomes, we selected microsatellite markers flanking the PRNP gene on chromosome 20p12-pter and an intragenic single-nucleotide polymorphism at the PRNP codon 129. Haplotypes were constructed for 62 CJD families originating from 11 world populations. The results show that Libyan, Tunisian, Italian, Chilean, and Spanish families share a major haplotype, suggesting that the 200K mutation may have originated from a single mutational event, perhaps in Spain, and spread to all these populations with Sephardic migrants expelled from Spain in the Middle Ages. Slovakian families and a family of Polish origin show another unique haplotype. The haplotypes in families from Germany, Sicily, Austria, and Japan are different from the Mediterranean or eastern European haplotypes. On the basis of this study, we conclude that founder effect and independent mutational events are responsible for the current geographic distribution of hereditary CJD associated with the 200K mutation.

SecA is required for the insertion of inner membrane proteins targeted by the Escherichia coli signal recognition particle.

Recent work has demonstrated that the signal recognition particle (SRP) is required for the efficient insertion of many proteins into the Escherichia coli inner membrane (IM). Based on an analogy to eukaryotic SRP, it is likely that bacterial SRP binds to inner membrane proteins (IMPs) co-translationally and then targets them to protein transport channels ("translocons"). Here we present evidence that SecA, which has previously been shown to facilitate the export of proteins targeted in a post-translational fashion, is also required for the membrane insertion of proteins targeted by SRP. The introduction of SecA mutations into strains that have modest SRP deficiencies produced a synthetic lethal effect, suggesting that SecA and SRP might function in the same biochemical pathway. Consistent with this explanation, depletion of SecA by inactivating a temperature-sensitive amber suppressor in a secAam strain completely blocked the membrane insertion of AcrB, a protein that is targeted by SRP. In the absence of substantial SecA, pulse-labeled AcrB was retained in the cytoplasm even after a prolonged chase period and was eventually degraded. Although protein export was also severely impaired by SecA depletion, the observation that more than 20% of the OmpA molecules were translocated properly showed that translocons were still active. Taken together, these results imply that SecA plays a much broader role in the transport of proteins across the E. coli IM than has been previously recognized.

Roles of histidine-103 and tyrosine-235 in the function of the prolipoprotein diacylglyceryl transferase of Escherichia coli.

Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is the first enzyme in the posttranslational sequence of reactions resulting in the lipid modification of lipoproteins in bacteria. A previous comparison of the primary sequences of the Lgt enzymes from phylogenetically distant bacterial species revealed several highly conserved amino acid sequences throughout the molecule; the most extensive of these was the region 103HGGLIG108 in the Escherichia coli Lgt (H.-Y. Qi, K. Sankaran, K. Gan, and H. C. Wu, J. Bacteriol. 177:6820-6824, 1995). These studies also revealed that the kinetics of inactivation of E. coli Lgt with diethylpyrocarbonate were consistent with the modification of a single essential histidine or tyrosine residue. The current study was conducted in an attempt to identify this essential amino acid residue in order to further define structure-function relationships in Lgt. Accordingly, all of the histidine residues and seven of the tyrosine residues of E. coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abilities of the respective mutant lgt alleles to complement the temperature-sensitive phenotype of E. coli SK634 defective in Lgt activity, were determined. The data obtained from these studies, in conjunction with additional chemical inactivation studies, support the conclusion that His-103 is essential for Lgt activity. These studies also indicated that Tyr-235 plays an important role in the function of this enzyme. Although other histidine and tyrosine residues were not found to be essential for Lgt activity, alterations of His-196 resulted in a significant reduction of in vitro activity.

Structure-function relationship of bacterial prolipoprotein diacylglyceryl transferase: functionally significant conserved regions.

The structure-function relationship of bacterial prolipoprotein diacylgyceryl transferase (LGT) Has been investigated by a comparison of the primary structures of this enzyme in phylogenetically distant bacterial species, analysis of the sequences of mutant enzymes, and specific chemical modification of the Escherichia coli enzyme. A clone containing the gene for LGT, lgt, of the gram-positive species Staphylococcus aureus was isolated by complementation of the temperature-sensitive lgt mutant of E. coli (strain SK634) defective in LGT activity. In vivo and in vitro assays for prolipoprotein diacylglyceryl modification activity indicated that the complementing clone restored the prolipoprotein modification activity in the mutant strain. Sequence determination of the insert DNA revealed an open reading frame of 837 bp encoding a protein of 279 amino acids with a calculated molecular mass of 31.6 kDa. S. aureus LGT showed 24% identity and 47% similarity with E. coli, Salmonella typhimurium, and Haemophilus influenzae LGT.S. aureus LGT, while 12 amino acids shorter than the E. coli enzyme, had a hydropathic profile and a predicted pI (10.4) similar to those of the E. coli enzyme. Multiple sequence alignment among E. coli, S. typhimurium, H. influenzae, and S. aureus LGT proteins revealed regions of highly conserved amino acid sequences throughout the molecule. Three independent lgt mutant alleles from E. coli SK634, SK635, and SK636 and one lgt allele from S. typhimurium SE5221, all defective in LGT activity at the nonpermissive temperature, were cloned by PCR and sequenced. The mutant alleles were found to contain a single base alteration resulting in the substitution of a conserved amino acid. The longest set of identical amino acids without any gap was H-103-GGLIG-108 in LGT from these four microorganisms. In E. coli lgt mutant SK634, Gly-104 in this region was mutated to Ser, and the mutant organism was temperature sensitive in growth and exhibited low LGT activity in vitro. Diethylpyrocarbonate inactivated the E. coli LGT with a second-order rate constant of 18.6 M-1S-1, and the inactivation of LGT activity was reversed by hydroxylamine at pH 7. The inactivation kinetics were consistent with the modification of a single residue, His or Tyr, essential for LGT activity.

Mutation in Cys662 of Escherichia coli DNA topoisomerase I confers temperature sensitivity and change in DNA cleavage selectivity.

Cys662 is one of the 12 cysteine residues proposed to be co-ordination sites for binding of three Zn(II) in Escherichia coli DNA topoisomerase I. Oligonucleotide-directed mutagenesis was used to convert Cys662 to either serine or histidine. The mutant enzymes were overexpressed and purified to homogeneity. Analysis of the purified enzymes demonstrated that the mutations resulted in loss of one tightly bound Zn(II). In vivo complementation tests and in vitro relaxation activity assays at different temperatures showed that the partial relaxation activities retained in the two mutant enzymes were temperature sensitive. Fluorescence measurements indicated that the wild-type and mutant enzymes have structural differences. When DNA cleavage specificity was examined, the mutant enzymes were found to have altered cleavage site preferences. The preferred cleavage sites of the wild-type enzyme all had a cytosine residue four nucleotides to the minus side of the break. The cleavage sites produced by the mutant enzymes did not show a preference for cytosine at that position.

Protein, cDNA, and genomic DNA sequences of the towel gourd trypsin inhibitor. A squash family inhibitor.

Two trypsin inhibitor components of the squash family were isolated and purified from the juice of the towel gourd (Luffa cylindrica) using anhydrotrypsin affinity chromatography followed by high pressure liquid chromatography. The inhibitors were sequenced and found to consist of 28 and 29 amino acid residues. The determined sequences show high similarity to other inhibitors of the squash family, especially in the location of disulfide bonds and the reactive site and also in the COOH-terminal region. A cDNA library of towel gourd was constructed and used as a template for polymerase chain reaction amplification of two cDNA fragments of the inhibitor with an overlapping sequence. A full-length cDNA sequence coding for the inhibitor was then completed. The open reading frame codes for a prepro-inhibitor protein with the pre- and pro-peptides consisting of 21 and 13 residues, respectively. The deduced amino acid sequence of 29 residues for the inhibitor is consistent with that determined by primary structure analysis. The genomic sequence of the mature inhibitor was also ascertained using the total DNA of the towel gourd as a polymerase chain reaction template. The genomic sequence is completely identical with that of the cDNA, showing no intervening sequence.