Thermostable alpha-amylase from Pyrococcus furious is an important industrial enzyme in brewing and alcohol production. Eexpression of the thermostable a-amylase in plants can reduce greatly costs in the production of alcohol using crop plants. A chloroplast expression vector, p64A, containing the thermostable alpha-amylase gene from Pyrococcus furious, was constructed with clpP-trnL-petB-chlL-rp123-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spetinomycin-resistant aadA gene as select marker. The plasmid p64A was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Nine independently transformed lines were obtained by 100 mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the transgene and cultivation in the dark all showed that the a-amylase gene had been integrated into chloroplast genome of C. reinhardtii. The activity of amylase expressed in the chloroplast of C. reinhardtii was detected by amylase activity assay and found to be as high as 77.5 u/g fresh weight of cells. These experimental results demonstrated the possibility of using transgenic chloroplasts of plant as bioreactors for production of industrial enzymes.
Chlamydomonas reinhardtii ; genetics ; Chloroplasts ; genetics ; Enzyme Stability ; Plasmids ; Polymerase Chain Reaction ; Pyrococcus furiosus ; enzymology ; alpha-Amylases ; chemistry ; genetics ; metabolism

The epidemiological and spatiotemporal distribution characteristics of human leptospirosis in Zhejiang Province from 2018 to 2022 were analyzed,to provide a scientific basis for formulating leptospirosis prevention and control strategies.Data on leptospirosis were collected from the China Information System for Disease Control and Prevention,and analyzed with descriptive epidemiological methods.ArcMap 10.8 software was used for spatial autocorrelation analysis and visual display of the results.SaTScan 10.1.2 software was used for spatiotemporal scanning,to analyze and describe the spatiotemporal aggrega-tion characteristics of leptospirosis.A total of 255 human cases of leptospirosis were reported in Zhejiang Province from 2018 to 2022,including one death;the average annual incidence was 0.084 3/100 000,and the average annual mortality was 0.000 3/100 000.The highest incidence of leptospirosis in Zhejiang occurred from August to October.Most cases were in middle-aged and older people 45-86 years of age(221 cases,86.67％).The number of cases was highest among people 65-69 years of age(57 cases).The male to female ratio was 4.67∶1.The main occupation was farming(190 cases,74.51％).The cumulative number of cases in Wenzhou and Lishui in 3 years(201 cases)accounted for 78.82％of the total cases.The global spatial auto-correlation analysis from 2018 to 2022 showed that the incidence of leptospirosis in the province presented a positive spatial au-tocorrelation.Local spatial autocorrelation analysis indicated that the"high-high"gathering areas were concentrated primarily in Wenzhou,Lishui,and Taizhou in the mountainous and hilly areas of southern Zhejiang Province.Spatiotemporal scanning anal-ysis revealed three clusters with high incidence in southern Zhejiang Province.The incidence of leptospirosis in Zhejiang Prov-ince showed clear regional clustering and seasonality,and the high incidence area was mainly in the mountainous and hilly areas of southern Zhejiang Province.The cases occurred mainly in middle-aged and older male farmers.Health education among rural resi-dents should be strengthened to prevent potential epidemic risks.

OBJECTIVETo identify the uptake and biological distribution of technetium galactosyl human serum albumin diethylenetriamine pentaacetic acid injection (99mTc-GSA) in three mouse models with different degrees of hepatic injuries.

METHODSThree mouse models including hepatic fibrosis, hepatic cholestasis, and liver cancer were established. Hepatic fibrosis model was established by intraperitoneal injection of carbon tetrachloride, 0.4 ml 10%, every 48 hours for 48 days. Hepatic cholestasis model was set up by ligature of the common bile duct for 72 hours, and liver cancer model by implantation of H22 tumor cells underneath liver capsule for 10 days. On measurement, each mouse in different models and normal controls was injected with 0.1 ml (0.37 MBq)99mTc-GSA (2 microg) into vena caudalis, and 5 minutes later sacrificed by decapitation. Important organs and tissues including liver, heart, lungs, kidney, spleen, stomach, blood, bones, muscles, and intestines were taken and their different radio countings were measured. The hepatic injuries were evaluated with serum and pathological examinations.

RESULTS99mTc-GSA was concentrated in the liver in all three models and the control mice ( >40% ID x g(-1)). Compared with the control mice (90.05 +/- 10.55)% ID x g(-1), the density of 99mTc-GSA was significantly lower in the models with hepatic injuries (P < 0.001). The liver function test indicated that the injury in hepatic fibrosis model was less serious than those in the other two models. However, the concentration of 99mTc-GSA in hepatic fibrosis model [(72.20 +/- 2.13)% ID x g(-1)] was significantly higher than those in the models with cholestasis [(56.72 +/- 5.92)% ID x g(-1)] and liver cancer [(42.80 +/- 6.05)% ID x g(-1)] (P < 0.001).

CONCLUSIONS99mTc-GSA may well concentrate in liver and its concentration degree is adversely correlated with hepatic injuries. Therefore 99mTc-GSA may be clinically used as liver imaging agent. When combined with three-dimensional scanning technique, it may facilitate constructing a new three-dimensional imaging method to demonstrate the function of designed liver segments.

Animals ; Disease Models, Animal ; Female ; Humans ; Liver ; diagnostic imaging ; drug effects ; injuries ; Liver Diseases ; diagnosis ; diagnostic imaging ; Mice ; Mice, Inbred BALB C ; Radiography ; Radionuclide Imaging ; Radiopharmaceuticals ; administration & dosage ; pharmacokinetics ; Random Allocation ; Technetium Tc 99m Aggregated Albumin ; administration & dosage ; pharmacokinetics ; Technetium Tc 99m Pentetate ; administration & dosage ; pharmacokinetics