Identification and transcriptional activity analysis of core regulatory region of human guanylate binding protein 5 gene promoter / 中国生物制品学杂志

@#Objective To construct luciferase reporter plasmids of truncated fragments of different lengths of human guanylate binding protein 5(GBP5)gene promoter and analyze the transcriptional activity of each fragment to determine the core regulatory region.Methods GBP5promoter sequence was amplified by PCR,truncated into five fragments of different lengths and connected to pGL3-basic plasmid.The constructed recombinant plasmids pGL3-GBP5-11/21/31/41/51were transfected into 293FT cells and detected for luciferase activity.The binding sites of transcription factors in GBP5promoter region were predicted by JASPAR software,and Yin-Yang transcription factor 1(YY1)targeting the core regulatory region was selected and verified for the transcriptional regulatory activity.The CDS sequence of YY1 was amplified by PCR to construct the overexpression plasmid pIRES2-EGFP-YY1,which was then co-transfected to 293FT cells with plasmids pGL3-GBP5-21(-1 623 ～ +47 bp)and internal reference plasmid pRL-CMV,and detected for luciferase activity to analyze the regulation of transcription factor YY1 on GBP5 promoter activity.Results Colony PCR and double enzyme digestion identification proved that the plasmid of human GBP5 promoter reporter gene was correctly constructed;JASPAR software predicted that there were multiple transcription factor binding sites such as STAT1,YY1 and Foxp3 in GBP5promoter region.Double luciferase activity assay showed that pGL3-GBP5-21(-1 623 ～ +47 bp)showed the highest promoter activity,while the promoter activity of pGL3-GBP5-41(-520 ～ +47 bp)decreased significantly,suggesting that the core region of GBP5 promoter was located at upstream-1 623 ～-520 bp of 5 'UTR;Overexpression of YY1 significantly activated the GBP5 promoter activity and regulated the expression of GBP5.Conclusion The core regulatory region of human GBP5 promoter was located in upstream-1 623 ～-520 bp of the 5 'UTR,with a binding site of transcription factor YY1 existing in this region.Meanwhile,overexpression of YY1 significantly effected the activity of GBP5 promoter.

Investigation on newly emerging and re-emerging snail habitats in Anhui, 2017-2021 / 中国热带医学

@#Abstract: Objective　To understand the changes of newly emerging and re-emerging snail areas in Anhui from 2017 to 2021 and analyze its related influencing factors, so as to provide scientific basis for formulating snail control strategies. 　　Methods　According to the historical snail survey data, the information of newly emerging and re-emerging snails from 2017 to 2021 were collected, the causes for the formation of newly emerging and re-emerging snails flourish environments were investigated and analyzed retrospectively. Results During 2017-2021, in Anhui, the area with newly emerging snails was respectively 840.41 hm2, 559.14 hm2 (66.53%) in lake and marshland areas and 281.27 hm2 (33.47%) in hilly areas; the area of re-emerging snails was respectively 1 176.87 hm2, 669.39 hm2 (56.88%) in lake and marshland areas and 507.48 hm2 (43.12%) in hilly and mountainous areas. The sum of newly emerging snail area in Chizhou, Anqing and Wuhu accounted for 89.35% of all, the sum of re-emerging snail area accounted for 88.82%. In 2021, the areas with newly emerging and re-emerging snails peaked at 611.52 hm2 and 976.84 hm2, respectively. The newly emerging and re-emerging snail habitats were mainly distributed in the transmission interruption areas, accounting for 65.54% and 84.30%, respectively. The newly emerging and re-emerging snail habitats were mainly found in fluvial marsh, accounting for 65.54% and 52.12%, respectively. In recent 5 years, the longest time interval of re-emerging snails was more than 50 years. The main causes of newly emerging snails were natural factors such as flood disaster and snail drift along river system. Natural factors, such as flood disaster and snail drift along river system, were the main reasons for the newly emerging snail habitats, accounting for 71.44% and 21.75%, respectively. Human factors, such as soil extraction from snail habitats, construction of water conservancy facilities, farmland abandonment and seedling transplanting, could also lead to the newly emerging snail habitats. Flood disaster was the main cause of re-emerging snail habitats, accounting for 72.29%. In addition, the re-emerging snail habitats were caused by historical snail residue, soil collection in snail habitats for construction projects, land abandonment, seedling transplanting, prohibition of snail control in ecological protection areas, and construction of water conservancy facilities. Conclusions　Flood disaster is an important factor for snail newly emerging and re-emerging. Human factors such as engineering construction and seedling transplanting are also easy to cause snail newly emerging and re-emerging. In order to timely detect and deal with newly emerging and re-emerging snail habitats and prevent snail diffusion, it is necessary to investigate snail distribution after flood disaster, the routine monitoring of historical snail habitats should be strengthened; in engineering construction and seedling transplanting, the disposal of soil with snails should be done well.

Incorporation of the YRHQ motif into CD3ζ chain enhances the antitumor activity of HER2-targeted CAR-T cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨在靶向HER2的CAR的CD3ζ链胞内区引入YRHQ基序对CAR-T细胞的特异性杀伤活性及免疫记忆形成的影响。方法：通过DNA合成获得包含靶向HER2的编码抗原受体H28ζ或H28ζ(YRHQ)的DNA片段，通过慢病毒载体将不同CAR的DNA片段分别转导健康人外周血T细胞，制备靶向HER2的H28ζ-CAR-T及H28ζ(YRHQ)-CAR-T细胞。扩增过程中对不同CAR-T细胞进行计数，FCM检测CAR的表达率。将CAR-T细胞分别与HER2阳性的SKOV3、MDA-MB-453或HER2阴性的MCF-7细胞共培养，LDH释放法检测其杀伤活性，ELISA法检测IL-2、IFN-γ和颗粒酶B的水平，WB法检测STAT3磷酸化水平及免疫检查点分子TIM-3和PD-1的表达，通过FCM检测CCR7、CD45RO的表达，分析CAR-T细胞的表型。结果：H28ζ-CAR-T和H28ζ(YRHQ)-CAR-T细胞扩增能力较好，体外培养7 d时扩增4~5倍。H28ζ-CAR和H28ζ(YRHQ)-CAR表达率分别为（33.3±2.85）%和（28.30±3.2）%。H28ξ(YRHQ)-CAR-T细胞的杀伤活性较H28ζ-CAR-T细胞更高（P<0.05）。经HER2抗原刺激后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞的STAT3磷酸化水平较H28ξ-CAR-T细胞明显升高（P<0.01）；而两者间PD-1和TIM-3的表达无明显差异。未经抗原刺激的CAR-T细胞CCR7和CD45RO表达与正常T细胞比较差异无统计学意义（均P>0.05），与SKOV3细胞共培养后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞中TEM细胞比例明显增加、TCM细胞比例明显减少（均P<0.05）。结论：在CD3胞内区引入YRHQ基序可在一定程度上提高CAR-T细胞的杀伤潜力。

Research progression on the first-line biological target therapy of advanced / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To develop a new type of CD7 chimeric antigen receptor modified T cell (CD7-CAR-T) for the treatment of CD7 positive acute myeloid leukemia (AML), and to observe its killing effect on CD7 positive AML cells. Methods: The CD7-CAR lentiviral vector was constructed based on the CD7 Nanobody sequence and costimulatory domain sequence of CD28 and 4-1BB. The lentiviral particles were packaged and used to co-transfect human T cells with protein expression blocker (PEBL), so as to prepare CD7- CAR-T cells. Real time cellular analysis (RTCA) was used to monitor the cytotoxicity of CD7-CAR-T cells on CD7 overexpressed 293T cells. Flow cytometry assay was used to detect the effect of CD7-CAR-T cells on proliferation and cytokine secretion of AML cells with high, medium and low CD7 expressions (KG-1, HEL and Kasumi-1 cells, respectively). Results: CD7-CAR-T cell was successfully constructed and its surface expression of CD7 was successfully blocked. Compared with T cells, CD7-CAR-T cells could significantly inhibit the proliferation of CD7-293T cells and promote the release of TNF, Granzyme B and INF-γ; in addition, CD7-CAR-T cells also significantly promoted the apoptosis (t=147.1, P<0.01; t=23.57, P<0.01) and cytokine release (P<0.05 or P<0.01) in CD7 positive KG-1 and HEL cells, but had little effect on Kasumi-1 cells that only expressed minimal CD7 antigen (t=0.7058, P>0.05). Conclusion: CD7-CAR-T cells can specifically kill CD7-positive AML cells in vitro.

Determination of furans in tea by headspace-gas chromatographic mass spectrometry / 预防医学

Objective@#To establish a method for the determination of furans in tea by headspace-gas chromatographic mass spectrometry. @*Methods@#The 20% sodium chloride solution and isotope internal standards were added to the crushed and weighed tea sample. Furan, 2-methylfuran, 3-methylfuran, 2,5-dimethylfuran were separated by HP-PLOT Q capillary column and then determined by gas chromatography mass spectrometry with electron impact ionization mode.@*Results@# In the range of 5-400 ng, good linear relationships were observed in the four furan compounds, with the correlation coefficients ranging from 0.999 2 to 0.999 6. The detection limits ranged from 0.2 to 1.9 μg/kg, the quantification limit ranged from 0.4 to 3.1 μg/kg. The recovery rates of furans ranged from 95.4% to 128.2% when spiked at 5.0, 20.0 and 100.0 μg/kg, and the relative standard deviations ranged from 0.8% to 11.3%. Eighty-one tea samples were determined, the concentration of four furan compounds was highest in black tea, followed by dark tea, oolong tea, green tea and scented tea. @*Conclusion@# Headspace-gas chromatography-mass spectrometry can reduce the matrix interference of tea, and meet the requirements in the linear range, recovery and precision, which is suitable for simultaneous determination of four furan compounds in tea.

Anti-mutagenicity of Swertiamarin and Its Metabolite in Incubated System of Human Intestinal Flora / 中草药·英文版

Objective To study the biotransformation regulation and pharmacological effect of swertiamarin and its metabolite in incubated system of human intestinal flora. Methods Incubated system of human intestinal flora was utilized to research the intestinal metabolism of swertiamarin. Furthermore, mutagenic test and anti-mutagenic test were carried out to research the activity relationship of swertiamarin and its metabolite. Results Gentianine was found in the metabolites of swertiamarin. The pharmacological experiment indicated that swertiamarin and its metabolite both had good anti-mutagenic effect. Conclusion Swertiamarin is partly metabolized to gentianine after oral administration. They show similar anti-mutagenicity effects.

Effects of Different Concentrations and Exposure Time of Sodium Hypochlorite on the Structural, Compositional and Mechanical Properties of Human Dentin / 华中科技大学学报（医学）（英德文版）

This study evaluated the effects of sodium hypochlorite (NaOC1) with different concentrations and exposure time on the structural,compositional and mechanical properties of human dentin in vitro.Sixty dentin slabs were obtained from freshly extracted premolars,randomly distributed into four groups (n=15),and treated with 1％,5％,10％ NaOC1 and distilled water (control group),respectively,for a total of 60 min.Attenuated total reflection infrared (ATR-IR) spectroscopy,Raman spectroscopy and X-ray diffraction (XRD) were carried out before,10 min and 60 min after the treatment.Scanning electron microscopy (SEM) and flexural strength test were conducted as well.The results showed that dentins experienced morphological alterations in the NaOC1 groups,but not in the control group.Two-way repeated-measures analysis of variance revealed that the carbonate:mineral ratio (C:M),Raman relative intensity (RRI),a-axis,c-axis length and full width at half maximum (FWHM) with the increase of time and concentration in the NaOC1 groups were not significantly different from those in the control group (P＞0.05).Nevertheless,the mineral:matrix ratio (M:M) increased and the flexural strength declined with the increase of concentration and the extension of time in the NaOC1 groups (P＜0.05).Additionally,it was found that the M:M and the flexural strength remained unchanged after 1％ NaOCl treatment (P＞0.05),and the morphology changes were unnoticeable within 10 min in 1％ NaOC1 group.These results indicated that NaOC1 has no significant effects on the inorganic mineral of human dentin;but it undermines and eliminates the organic content concentration-and time-dependently,which in turn influences the flexural strength and toughness of dentins.In addition,an irrigation of 1％NaOCl within 10 min can minimize the effects of NaOC1 on the structural and mechanical properties of dentin during root canal treatment.

Correlative analysis between plasma BNP,CRP levels and computed tomographic manifestations in acute moderate or severe traumatic brain injury / 中国基层医药

Objective To investigate correlative analysis between plasma BNP,CRP levels and computed tomographic manifestations in acute moderate or severe traumatic brain injury.Methods According to the severe traumatic brain injury and mild traumatic brain injury criteria that onset within 24h,patients were devided into the observation group A (73 cases with moderate or severe traumatic brain injury)and observation group B(20 cases with mild traumatic brain injury ).All patients received CT scan,CT image characteristics and Rotterdam CT grading were recorded.,Plasma BNP and CRP levels were detected at the same time,healthy persons with matched age and gender within same period were selected (control group),relationships of plasma BNP and CRP levels with CT manifestations were analyzed.Results Plasma BNP and CRP levels of the observation group were obviously higher than those of the control group (P 5mm,ventricular pressure≤0.2,Rotterdam CT score >3, the BNP and CRP levels were higher than the corresponding patnents (P 3 points.Conclusion Plasma BNP and CRP levels of patients with acute moderate or severe traumatic brain injury are closely related to various characteristics of CT,and are independent predictors of Rotterdam CT scores,they can be used as a biological marker assisting to evaluate degree of severe craniocerebral injury patients.

Study on Swertia mussotii Franch. by HPLC fingerprinting method combined with chemical pattern recognition approaches / 中国药学杂志

OBJECTIVE: To study the quality control method of Swertia mussotii Franch. METHODS: A high performance liquid chromatographic method was developed to establish the fingerprint of Swertia mussotii Franch. Nineteen samples from various batches were analyzed, furthermore, PCA were used to differentiate and evaluate the whole fingerprints. Some characteristic peaks were i-dentified preliminarily with HPLC-MS method. RESULTS: Eleven peaks were identified by MS. Swertiamarin and swertianolin were the key components for quality control. CONCLUSION: The method provides an academic reference for controlling the quality of Swertia mussotii Franch.

Expression and roles of the epithelial mesenchymal transition markers E-cadherin and vimentin in hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To determine the differential protein expressions of epithelial mesenchymal transition (EMT) markers E-cadherin and vimentin in hepatocellular carcinorma (HCC) and to investigate their correlation to the molecular mechanisms of metastasis to explore their potential utility as prognostic indicators of HCC.</p><p><b>METHODS</b>Tumor tissues and patient-matched adjacent non-tumor tissues were collected from individuals diagnosed with HCC. E-cadherin and vimentin protein expressions in the tissue specimens were quantified by western blot with densitometry of fluorescence emission and comparatively analyzed to determine the associations with molecular and clinical features. The expressions of E-cadherin and vimentin, as well as the other EMT-related protein Twist, were also detected in the tissue specimens by immunohistochemistry. Statistical analyses were carried out by paired-samples t-test, Mann-Whitney test, and Spearman rank correlation analysis.</p><p><b>RESULTS</b>E-cadherin expression was significantly lower in tumor tissues (0.082 +/- 0.063 vs. adjacent non-tumor tissues: 0.226 +/- 0.215, t = -4.050, P less than 0.01), lower in patients with portal vein tumor thrombus (vs. non-thrombic HCC patients, P = 0.001), and correlated with TNM stage (III/IV > I/II, P = 0.003). Vimentin expression was significantly higher in tumor tissues (vs. adjacent non-tumor tissues, P = 0.002), negatively correlated with E-cadherin expression (t = -0.509, P = 0.004), and closely associated with some clinical parameters, such as portal vein tumor thrombus (P less than 0.01), TNM stage (P less than 0.01), and Milan criteria (P = 0.005). Immunohistochemistry showed that E-cadherin expression was very weak in tumors but very strong in the cell membranes of non-tumor tissues, and that vimentin and Twist expressions were strong in tumors but undetectable in non-tumor tissue.</p><p><b>CONCLUSION</b>Expression levels of the EMT markers E-cadherin and vimentin in HCC are related to clinical parameters, including portal vein tumor thrombus and TNM stage, and may represent useful prognostic markers of metastasis.</p>

Studies on metabolism of total terpene ketones from Swertia mussotii with human intestinal bacteria / 中国中药杂志

<p><b>OBJECTIVE</b>To study the metabolism of total terpene ketones from Swertia mussotii with human intestinal bacteria.</p><p><b>METHOD</b>Total terpene ketones were incubated with human intestinal bacteria under an anaerobic environment and at 37 degrees C. The metabolites were extracted by ethyl acetate processing, detected by HPLC-DAD method. A qualitative analysis was made for its metabolites by HPLC-MS.</p><p><b>RESULT</b>Eight metabolites were detected from total terpene ketones from S. mussotii with human intestinal bacteria, and two of them were preliminarily identified as gentianine and mangiferin aglycon.</p><p><b>CONCLUSION</b>Total terpene ketones can be metabolized with human intestinal bacteria, which provides basis for experiments on the metabolism process total terpene ketones from S. mussotii with human intestinal bacteria.</p>

Life cycle assessment of energy consumption and greenhouse gas emissions of cellulosic ethanol from corn stover / 生物工程学报

Life Cycle Assessment (LCA) is the only standardized tool currently used to assess environmental loads of products and processes. The life cycle analysis, as a part of LCA, is a useful and powerful methodology for studying life cycle energy efficiency and life cycle GHG emission. To quantitatively explain the potential of energy saving and greenhouse gas (GHG) emissions reduction of corn stover-based ethanol, we analyzed life cycle energy consumption and GHG emissions of corn stover-based ethanol by the method of life cycle analysis. The processes are dilute acid prehydrolysis and enzymatic hydrolysis. The functional unit was defined as 1 km distance driven by the vehicle. Results indicated: compared with gasoline, the corn stover-based E100 (100% ethanol) and E10 (a blend of 10% ethanol and 90% gasoline by volume) could reduce life cycle fossil energy consumption by 79.63% and 6.25% respectively, as well as GHG emissions by 53.98% and 6.69%; the fossil energy consumed by biomass stage was 68.3% of total fossil energy input, N-fertilizer and diesel were the main factors which contributed 45.78% and 33.26% to biomass stage; electricity production process contributed 42.06% to the net GHG emissions, the improvement of technology might reduce emissions markedly.

Endothelial nitric oxide synthase gene polymorphisms associated with susceptibility to high altitude pulmonary edema in Chinese railway construction workers at Qinghai-Tibet over 4 500 meters above sea level / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To examine whether the polymorphisms of endothelial nitric oxide synthase (eNOS) gene are associated with the susceptibility to high altitude pulmonary edema (HAPE) in Chinese railway construction workers at Qinghai-Tibet where the altitude is over 4 500 m above sea level.</p><p><b>METHODS</b>A case-control study was conducted including 149 HAPE patients in the construction workers and 160 healthy controls randomly recruited from their co-workers, matching the patients in ethnicity, age, sex, lifestyle, and working conditions. Three polymorphisms of eNOS gene, T-786C in promoter, 894G/T in exon 7, and 27bp variable number tandem repeat (VNTR) in intron 4, were genotyped using polymerase chain reaction (PCR) and confirmed with DNA sequencing.</p><p><b>RESULTS</b>The frequencies of 894T allele and heterozygous G/T of the 894G/T variant were significantly higher in HAPE patients group than in the control group (P=0.0028 and P=0.0047, respectively). However, the frequencies of the T-786C in promoter and the 27bp VNTR in intron 4 were not significantly different between the two groups. Haplotypic analysis revealed that the frequencies of two haplotypes (H3,T-T-b, b indicates 5 repeats of 27 bp VNTR; H6, C-G-a, a indicates 4 repeats of 27 bp VNTR) were significantly higher in HAPE patients (both Pü0.0001). On the contrary, the frequencies of H1 (T-G-b) and H2 (T-G-a) were lower in HAPE patients than in healthy controls (both Pü0.001).</p><p><b>CONCLUSIONS</b>Two haplotypes (T-T-b and C-G-a) may be strongly associated with susceptibility to HAPE. Compared with the individual alleles of eNOS gene, the interaction of multiple genetic markers within a haplotype may be a major determinant for the susceptibility to HAPE.</p>

Viral pathogens of acute lower respiratory tract infection in China.

OBJECTIVES: To document the viral etiology of acute lower respiratory tract infection (ALRIs) in Chinese children. SETTING: Children Hospital, Zhejiang University, China. STUDY DESIGN: Cross-sectional. PARTICIPANTS: 34885 children with ALRI between January 2001 to December 2006. METHODS: Nasopharyngeal aspirates were collected from all subjects. Respiratory syncytial virus (RSV), adenovirus (ADV), type 1 to 3 parainfluenza viruses (PIV), and type A and B influenza virus (Flu) were detected by direct immunofluorescence. RESULTS: Viruses were identified in 32.3% cases, including RSV (23.6%), PIV 3 (4.3%), Flu A (2.0%), ADV (1.7%), PIV I (0.6%), Flu B (0.2%) and PIV 2 (0.1%). RSV and PIV 3 predominated in younger children while Flu A and Flu B predominated in older children (P<0.001, respectively). PIV 1 was more prevalent in children aged 1 to 3 years. The peak frequency of RSV, PIV 3 and Flu A were in early spring, June to August, and August and September, respectively. Flu B had a peak in the winter and spring. Adenovirus infections occurred in all seasons with a relatively constant frequency. CONCLUSIONS: Viruses are an important cause of ALRIs in Chinese children constituting 1/3 of total cases. RSV is the most common pathogen.

Construction and screening of phage display single chain antibody library against Bursaphelenchus xylophilus cellulase / 生物工程学报

A phage display single-chain variable fragment (scFv) library against Bursaphelenchus xylophilus cellulase (BXC) was constructed and used to screen the specific antibodies binding to BXC. The total RNA was extracted from fresh spleens of BALB/C mice immunized with BXC. Gene fragments encoding VH and VL were amplified by RT-PCR and assembled into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The recombinant fragments were cloned into the phagemids (pCANTABSE) and electroporated into E. coli TG1. The recombinant phagemids were rescued by reinfection of helper phage M13K07. The repertoire of the phage display antibody was about 5 x 10(4). The specific antibodies against BXC were obtained after five rounds of affinity selection. The positive phage clone was used to infect E. coli HB2151. SDS-PAGE and western blot analysis showed that the soluble scFv antibodies expressed bound specifically to BXC. The studies laid foundation for quarantine and pathological study of Bursaphelenchus xylophilu.

Clinical significance of transrectal ultrasound in detecting of rectal cancer margin and deciding of distal surgical incisal edge / 中华外科杂志

<p><b>OBJECTIVE</b>To study the role of transrectal ultrasound in detecting and deciding rectal cancer margin and surgical incisal edge.</p><p><b>METHODS</b>33 surgical specimens of rectal carcinoma were examined with transrectal ultrasound. Cancerous margin and surgical incisal edge were determined. The results were compared with pathological examination. p53 and K-ras gene mutation as tumor molecular markers of residue cancer cells were detected in incisal edge tissue with PCR-SSCP method.</p><p><b>RESULTS</b>General accuracy for cancer infiltration depth with transrectal ultrasound was 86.6%. For mucosa and submucosa infiltration lesions, the accuracy was 72.7%. For lamina muscularis, the accuracy was 90.9%. And for adventitia and peripheral tissue infiltration of rectum, the accuracy was 88.5% and 100% respectively. No remains of cancer cells and tumor molecular markers were detected at distal incisal edges of 1.0 cm, 2.0 cm and 3.0 cm determined with transrectal ultrasound.</p><p><b>CONCLUSIONS</b>Rectal cancer margine and surgical incisal edge determined with transrectal ultrasound are close to examined by pathology. Transrectal ultrasound is helpful and reliable to define incisal edge in rectal cancer surgery.</p>

MICROBIAL ENZYME CONVERSION OF L-CYSTEBSE AND L-CYSTINE / 微生物学通报

Pseudomonas sp. TS1138 isolated from soil samples was able to form L-cysteine from DL-2-Amino-△2-Thia-zoline-4-Carboxylic Acid (DL-ATC) after cultured 16 hours . The optimum carbon and nitrogen soruces of strain growth and enzyme formation are glucose and urea. This enzyme was induced by DL-ATC. The product was identified to be L-Cysteine based on thin layer chromatography, optical rotation and HPLC studies.