Cloning and Characterization of δ-Guaiene Synthase Genes Encoding a Sesquiterpene Cyclase from Aquilaria microcarpa Cell Cultures

Three cDNA clones encoding δ-guaiene synthase, a sesquiterpene cyclase, were isolated from tissue cultures of Aquilaria microcarpa, and data mining analysis of the orthologous genes suggested that 10 and 9 amino acid residues of N- and C-terminal ends of the translated products of these clones remained undefined. The recombinant enzyme proteins, to which the putative missing Nand C-terminal amino acid sequences (MSSAKLGSAS and ALLRHAIEI, respectively) were ligated, exhibited the catalytic activities of sesquiterpene biosynthesis. Among these three δ-guaiene synthases, two isoforms were capable of liberating α-guaiene, δ-guaiene, β-elemene plus α-humulene as a minor product, while remaining one isoenzyme generated α-, δ-guaiene and β-elemene but not α-humulene. Although the enzyme protein solely lacking in the N-terminal 10 amino acid residues was capable of synthesizing the sesquiterpenoids, the protein without 9 amino acids at Cterminal did not exhibit the catalytic activity. These results suggest that two types of δ-guaiene synthase;α-, δ-guaiene, β-elemene-producing type, and α-, δ-guaiene, β-elemene plus α-humulene-producing type;concomitantly occur in A. microcarpa cell cultures, and several amino acid residues at C-terminal of the synthase protein are essential to exhibit the catalytic activities as the sesquiterpene cyclase.

Low Frequency of Leisure-Time Activities Correlates with Cognitive Decline and Apathy in Patients with Parkinson’s Disease

Objective: The purpose of this study is to investigate habitual leisure-time activities (physical and non-physical leisure activities) in patients with Parkinson’s disease (PD), and to determine any potential benefits of these activities on cognitive functions and emotional symptoms. Methods: Thirty-two patients with PD and 25 demographically-matched healthy controls participated in the present study. Neuropsychological tests (Wisconsin Card Sorting Test, Trail Making Test, Digit Span Test, Verbal Fluency Test, and Japanese Verbal Learning Test), assessment of emotional sym- ptoms, and interviews for confirmation of habitual leisure-time activities were conducted for all participants. Results: Patients with PD significantly showed the lower frequency of both physical and non-physical leisure activities than healthy controls. Compared to patients who engaged in physical leisure activities, patients who did not engage in such activities performed worse on the Trail Making Test (TMT-B, and TMT-B minus A). Moreover, patients who engaged in non-physical leisure activities were less apathetic than patients who did not engage in such activities. Conclusions: Our preliminary study shows that habitual leisure-time activities correlate with cognitive function and emotions in patients with PD. Reducing sedentary lifestyles and promoting habitual leisure-time activities may be helpful for preventing cognitive decline and apathy.

Pulmonary Large Cell Carcinoma Displays High Expression of EMMPRIN and VEGF

OBJECTIVE To investigate the expression of extracellular matrix metalloproteinase inducer(EMMPRIN) and vascular endothelial growth factor(VEGF) in lung carcinomas,and to clarify their roles in carcinoma progression.METHODS Expression of EMMPRIN and VEGF was examined with tissue microarrays(TMAs) of lung carcinomas(n = 181),and their suppression in adjacent normal lung samples(n = 40) were determined by immunohistochemistry.The results were compared with clinicopathological fi ndings for the same tumors.RESULTS Both EMMPRIN and VEGF were occasionally ex-pressed in pseudostratif ied columnar epithelium and frequently in lung carcinomas.Histologically,EMMPRIN and VEGF displayed higher levels in large(LCC) cell carcinomas than adenocarcinoma(AD),squamous(SQ) and small cell carcinomas(SCC)(P < 0.05).EMMPRIN was more highly expressed in SQ as compared with AD(P < 0.05),while the converse was true for VEGF(P < 0.05).Binding was generally more intense for EMMPRIN in samples from male compared to female patients(P < 0.05),whereas the lat-ter tended to exhibit more VEGF expression(P < 0.05).Positive as-sociations of VEGF expression with the TNM stage and amounts of EMMPRIN were noted in the lung carcinomas(P < 0.05).CONCLUSION EMMPRIN and VEGF possibly contribute to physiological repair of normal lung and histogenesis of lung carci-noma.Both proteins might be involved in the molecular basis for differences in the incidence of lung carcinoma between men and women.

Transient receptor potential vanilloid 4-dependent calcium influx and ATP release in mouse and rat gastric epithelia

AIM: To explore the expression of transient receptor potential vanilloid 4(TRPV4) and its physiological meaning in mouse and rat gastric epithelia. METHODS: RT-PCR and immunochemistry were used to detect TRPV4 m RNA and protein expression in mouse stomach and a rat normal gastric epithelial cell line(RGE1-01), while Ca2+-imaging and electrophysiology were used to evaluate TRPV4 channel activity. ATP release was measured by a luciferin-luciferase assay. Gastric emptying was also compared between WT and TRPV4 knockout mice. RESULTS: TRPV4 m RNA and protein were detected in mouse tissues and RGE1-01 cells. A TRPV4-specific agonist(GSK1016790A) increased intracellular Ca2+ concentrations and/or evoked TRPV4-like current activities in WT mouse gastric epithelial cells andRGE1-01 cells, but not TRPV4 KO cells. GSK1016790 A or mechanical stimuli induced ATP release from RGE1-01 cells while TRPV4 knockout mice displayed delayed gastric emptying in vivo. CONCLUSION: TRPV4 is expressed in mouse and rat gastric epithelium and contributes to ATP release and gastric emptying.

Clinical significance of computed tomography assessment for third molar surgery

Surgical extraction of the third molar is the most commonly performed surgical procedure in the clinical practice of oral surgery.Third molar surgery is warranted when there is inadequate space for eruption,malpositioning,or risk for cyst or odontogenic tumor formation.Preoperative assessment should include a detailed morphologic analysis of the third molar and its relationship to adjacent structures and surrounding tissues.Due to developments in medical engineering technology,computed tomography(CT)now plays a critical role in providing the clear images required for adequate assessment prior to third molar surgery.Removal of the maxillary third molar is associated with a risk for maxillary sinus perforation,whereas removal of the mandibular third molar can put patients at risk for a neurosensory deficit from damage to the lingual nerve or inferior alveolar nerve.Multiple factors,including demographic,anatomic,and treatment-related factors,influence the incidence of nerve injury during or following removal of the third molar.CT assessment of the third molar prior to surgery can identify some of these risk factors,such as the absence of cortication between themandibular third molar and the inferior alveolar canal,prior to surgery to reduce the risk for nerve damage.This topic highlight presents an overview of the clinical significance of CT assessment in third molar surgery.

Edge and Core Impurity Transport Study With Spectroscopic Instruments in LHD

Impurity transport was investigated at both edge and core regions in large helical de- vice （LHD） with developed spectroscopic instruments which can measure one- and two-dimensional distributions of impurities. The edge impurity behavior was studied recently using four carbon resonant transitions in different ionization stages of CIII （977A）, CIV （1548A）, CV （40.3A） and CVI （33.7A）. When the line-averaged electron density, ne, is increased from 1 to 6 × 10^13 cm-3, the ratio of （CIII^CIV）/ne increases while the ratio of （CV＋CVI）/ne decreases. Here, CIII^CIV （CV＋CVI） expresses the sum of CIII （CV） and CIV （CVI） intensities. The CIII＋CIV indicates the carbon influx and the CV＋CVI indicates the emissions through the transport in the ergodic layer. The result thus gives experimental evidence on the impurity screening by the ergodic layer in LHD, which is also supported by a three-dimensional edge particle simulation. The core impu- rity behavior is also studied in high-density discharges （ne 〈 1 × 10^15 cm 3） with multi H2-pellets injection. It is found that the ratio of V/D （V： convection velocity, D： diffusion coefficient） decreases after pellet injection and Zeff profile shows a flat one at values of 1.1,-1.2. These results confirm no impurity accumulation occurs in high-density discharges. As a result, the iron density, rife, is analyzed to be 6 × 10^-7（=- nFe/ne） of which the amount can be negligible as radiation source even in such high-density discharges. One- and two-dimensional impurity distributions from space-resolved VUV and EUV spectrometers newly developed for further impurity transport study are also presented with their preliminary results.

A Case of Benign Metastasizing Leiomyoma:Pathobiological Behavior Showing A Low-grade Malignant Potential

Case Report A 38-year-old woman underwent a hysterectomy for a uterine tumor. A giant circumscribed nodule with a diameter of 20 cm at its widest point was found within the myometrium of the uterine corpus without hemorrhage or necrosis. More than 10 specimens were pathologically examined. Eleven years after hysterectomy, she developed five tumors （the largest diameter = 2 cm） in the right lung and retroperitoneal tumor （the largest diameter = 9.5 cm）. Microscopic examination indicated uterine, retroperitoneal and pulmonary foci, which were characterized by a benign appearance, including nodular smoothmuscle proliferation. The smooth muscle cells had an oval shape with relatively clear cytoplasm and a few irregular nuclei. In each specimen, spindle-shaped cells showed an interlacing bundle pattern with intermediated cellularity. The mitotic index was very low and no nuclear atypia was observed （Fig. 1）. Even though the serial pathological examination did not indicate any malignant potential, we diagnosed this case as benign metastasizing leiomyoma （BML）.

Efficient Production of δ-Guaiene, an Aroma Sesquiterpene Compound Accumulated in Agarwood, by Mevalonate Pathway-Engineered Escherichia coli Cells

Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synthase genes were coexpressed in this strain. This transformant was capable of liberating an appreciable amount of δ-guaiene, an aroma sesquiterpene compound accumulated in agarwood, and its concentration was elevated to more than 30 μg/ml culture by the incubation with mevalonolactone as an isoprene precursor in a nutrient-enriched Terrific broth. Coexpression of type 1 isopentenyl diphosphate isomerase plus acetoacetyl-CoA ligase genes also enhanced δ-guaiene production, and the concentration of the compound was approximately 38 - 42 μg/ml culture in the presence of mevalonolactone or lithium acetoacetate. These results clearly indicate that mevalonate pathway-engineered E. coli cells showed an appreciable δ-guaiene producing activity in the en- riched medium in the presence of appropriate isoprene precursors.

Penile Epidermoid Cyst Consisted of Multiple Foci: A Case Report

Penile epidermoid cysts are uncommon, and a small number of cases have been reported worldwide. We present the first documented patient with a penile epidermoid cyst which consisted of multiple focuses. A 37-year-old man presented to our department with a chief complaint of an asymptomatic, soft mass in the ventral part of the penis. The mass was nontender, freely movable and measuring 3 cm within the dermis. MRI (magnetic resonance imaging) revealed a high signal intensity on both T1- and T2-weighted images. Excision of the cyst was performed under local anesthesia. Macroscopically, the cut surface of the mass appeared to be full of a cheese-like material and the cyst consisted of multiple focuses. The cyst did not contain skin appendages. The pathological diagnosis was an epidermoid cyst of the penis. No recurrence has been noted in the year since the operation.

Hernandezine promotes cancer cell apoptosis and disrupts the lysosomal acidic environment and cathepsin D maturation

Hernandezine(Her),a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum,is recognized for its range of biological activities inherent to this herbal medicine.Despite its notable properties,the anti-cancer effects of Her have remained largely unexplored.In this study,we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms.Furthermore,Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems,leading to LC3 lipidation.Our findings revealed that Her caused damage to the mitochondrial membrane,with the damaged mitochondria undergoing mitophagy,as evidenced by the elevated expression of mitophagy markers.Conversely,Her disrupted autophagic flux,demonstrated by the upregulation of p62 and accumulation of autolysosomes,as observed in the RFP-GFP-LC3 reporter assay.Initially,we determined that Her did not prevent the fusion of autophagosomes and lysosomes.However,it inhibited the maturation of cathepsin D and increased lysosomal pH,indicating an impairment of lysosomal function.The use of the early-stage autophagy inhibitor,3-methyladenine(3-MA),did not suppress LC3II,suggesting that Her also induces noncanonical autophagy in autophagosome formation.The application of Bafilomycin A1,an inhibitor of noncanonical autophagy,diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her,thereby augmenting Her-induced cell death.These observations imply that while autophagy initially plays a protective role,the disruption of the autophagic process by Her promotes programmed cell death.This study provides the first evidence of Her’s dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death.These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death,offering potential avenues for enhancing cancer prevention and therapeutic strategies.

改良的Mosher法测定高烯丙醇的绝对构型

有机化合物绝对构型的测定在有机合成和药物合成方面都有着举足轻重的地位.Mosher法就是利用核磁共振波谱技术来确定复杂化合物绝对构型时所采用的一种巧妙方法.本研究即采用改良的Mosher法对无水鞘氨醇类天然产物pachastrissamine及其异构体全合成过程中的重要中间体高烯丙醇进行了绝对构型的测定.

Systemic abnormalities in liver disease

Systemic abnormalities often occur in patients with liver disease. In particular, cardiopulmonary or renal diseases accompanied by advanced liver disease can be serious and may determine the quality of life and prognosis of patients. Therefore, both hepatologists and non-hepatologists should pay attention to such abnormalities in the management of patients with liver diseases.

SRT1720, a SIRT1 Activator, Aggravates Bleomycin-Induced Lung Injury in Mice

Diagnosis and management of interstitial lung diseases (ILDs), caused by lung epithelial injury followed by apoptosis, are often challenging. It has been controversial whether the SIRT1 protein, a principal modulator of longevity due to caloric restriction, ameliorates or aggravates ILD in animal models. Here we examined the effect of SRT1720, a syn- thetic activator of SIRT1, on bleomycin-induced lung injury in a mouse model and apoptosis in cultured epithelial cells. Oral intubation of SRT1720 over a period of 15 days caused body weight loss and a high mortality rate among bleomy- cin-treated mice. Histological examinations showed that the SRT1720 load increased fibrosis in the bleomycin-treated lung. An analysis of bronchoalveolar lavage fluid revealed remarkably increased numbers of inflammatory cells in the SRT1720-treated group. Moreover, the apoptosis of A549 lung cancer cells, caused by X-ray irradiation and an anti-Fas activating antibody, was promoted by SRT1720. These results indicate that SRT1720 not only aggravates bleomy- cin-induced ILD, but stimulates the apoptosis of physically and biologically stimulated A549 cells. While SIRT1 acti- vators are considered promising for the treatment of conditions such as diabetes mellitus, fatty liver, and chronic ob- structive pulmonary diseases, an excess of food containing SIRT1 activators may be harmful depending on the disease state, especially in the case of acute inflammation.

Hyperdry human amniotic membrane application as a wound dressing for a full-thickness skin excision after a third-degree burn injury

Background:Severe burn injuries create large skin defects that render the host susceptible to bacterial infections.Burn wound infection often causes systemic sepsis and severe septicemia,resulting in an increase in the mortality of patients with severe burn injuries.Therefore,appropriate wound care is important to prevent infection and improve patient outcomes.However,it is difficult to heal a third-degree burn injury.The aim of this studywas to investigate whether hyperdry human amniotic membrane(HD-AM)could promote early granulation tissue formation after full-thickness skin excision in third-degree burn injury sites in mice.Methods:After the development of HD-AM and creation of a third-degree burn injury model,the HD-AM was either placed or not placed on the wound area in the HD-AM group or HD-AM group,respectively.The groups were prepared for evaluation on postoperative days 1,4 and 7.Azan staining was used for granulation tissue evaluation,and estimation of CD163,transforming growth factor beta-1(TGF-β1),vascular endothelial growth factor(VEGF),CD31,alpha-smooth muscle actin(α-SMA)and Iba1 expression was performed by immunohistochemical staining.Quantitative reverse-transcription polymerase chain reaction(PCR)was used to investigate gene expression of growth factors,cell migration chemokines and angiogenic and inflammatory markers.Results:The HD-AM group showed significant early and qualitatively good growth of granulation tissue on the full-thickness skin excision site.HD-AM promoted early-phase inflammatory cell infiltration,fibroblast migration and angiogenesis in the granulation tissue.Additionally,the early infiltration of cells of the immune system was observed.Conclusions:HD-AM may be useful as a new wound dressing material for full-thickness skin excision sites after third-degree burn injuries,and may be a new therapeutic technique for improving the survival rate of patients with severe burn injuries.