[Evaluating the efficacy of fractional exhaled nitric oxide and impulse oscillometry in screening out cough variant asthma from patients with subacute cough].

Objective: To evaluate the clinical value of fractional exhaled nitric oxide (FeNO) and impulse oscillometry (IOS) in screening out cough variant asthma (CVA) from patients with subacute cough. Methods: Patients with subacute cough were included from the outpatient department of Respiratory Medicine of Zhujiang Hospital of Southern Medical University from May to October in 2016. Based on "the guidelines for the diagnosis and treatment of cough (2015 edition)" , patients were classified into CVA group, and non CVP group with other causes of subacute cough. Lung function, bronchial provocation test, FeNO and IOS were measured. The diagnostic efficiency and optimal cut-off points of FeNO and IOS indicators to diagnose CVA from subacute cough were respectively assessed by the receiver operating characteristic (ROC) curves. Results: A total of 85 patients with subacute cough were included. Among them, 35 patients were diagnosed with CVA (CVA group), the others are classified as non CVP group (n=50). In CVA group, the levels of FeNO and total respiratory impedance (Zrs) were significantly higher, while maximal mid expiratory flow (MMEF)%pred, and mid expiratory flow (MEF)75/50/25%pred, reactance at 5 Hz (X5) levels were significantly lower than those in non CVP group (all P<0.05). Furthermore, the FeNO had a positive correlation with Zrs and Fres (ρ=0.312, P=0.003 and ρ=0.318, P=0.003, respectively), had a negative correlation with X5 (ρ=-0.288, P=0.007). A ROC analysis indicated that the area under ROC curve (AUC) of FeNO in diagnosis of CVA was 0.786 (95% CI: 0.684-0.889), the best cut-off point of FeNO volume ratio was 24.5×10(-9). When FeNO volume ratio=24.5×10(-9,) the sensitivity of in diagnosing CVA was 77.8%, specificity was 70.0%. The AUC for Zrs and X5 were 0.679 and 0.687, respectively. The combination of FeNO and X5 had a greater AUC than other indicators (AUC: 0.817, 95% CI: 0.726-0.908), the sensitivity and specificity were 80.6% and 66.0%, respectively. Conclusion: Both FeNO level and IOS index can be used to screen CVA in patients with subacute cough, and the combination of both have better value in diagnosing CVA.

Effect of early enteral nutrition (EN) on endotoxin in serum and intestinal permeability in patients with severe acute pancreatitis.

OBJECTIVE: This work aimed at studying the effect of early enteral nutrition (EN) on serum endotoxin and intestinal permeability in patients with severe acute pancreatitis. PATIENTS AND METHODS: 70 cases of patients with severe acute pancreatitis were cured in our hospital from April 2015 to January 2016. Patients selected were randomly divided into two groups including a group of patients having parenteral nutrition (group PN) and that had enteral nutrition (group EN). The results were assessed by: 1) the differences of serum endotoxin level; 2) the differences of the lactulose/mannitol ratio of urine, before intervention and one and two weeks after the intervention. RESULTS: Before the intervention, both groups had similar levels of serum endotoxin and the same lactulose/mannitol excretion rate of urine (p>0.05). One and two weeks after the intervention, the serum endotoxin level and the lactulose/mannitol excretion rate of urine of the group PN were significantly higher than the group EN (p<0.05). CONCLUSIONS: Compared with PN, EN has a bigger effect on serum endotoxin and intestinal permeability in patients with severe acute pancreatitis. EN can better promote the elimination of serum endotoxin and reduce intestinal permeability. Therefore, EN deserves clinical expansion.

Uterine expression of implantation serine proteinase 2 during the implantation period and in vivo inhibitory effect of its antibody on embryo implantation in mice.

The aim of the present study was to examine the uterine expression pattern of implantation serine proteinase 2 (ISP2) protein during early pregnancy in mice and the effects of anti-ISP2 antibody on embryo implantation. Expression of ISP2 protein was found to be specifically up-regulated in mouse uterine endometrial glands following the initiation of embryo implantation. Similarly, ISP2 protein expression was observed during pseudopregnancy, indicating that its expression is not embryo dependent. In other experiments, rabbit anti-ISP2 IgG was infused into the mouse uterine lumen on Day 3 or 4 of pregnancy to examine its effects on embryo implantation, whereas vehicle (saline) or unspecific rabbit IgG served as controls. The mean number of implanted embryos from anti-ISP2-IgG-treated mice was significantly lower than that from control mice. These results suggest that ISP2 may play an important role during embryo implantation.

Expression of regulator of G-protein signalling protein 2 (RGS2) in the mouse uterus at implantation sites.

Establishment of an active dialogue between the maternal endometrium and the implanting blastocyst is essential for successful implantation. The aim of this study was to identify genes that are explicitly expressed at implantation sites of the mouse uterus by subtractive hybridization. One expressed sequence tag of the genes identified showed 92% identity to the regulator of G-protein signalling protein 2 (RGS2). The full cDNA sequence of this gene was amplified by PCR and subsequently registered in GenBank. The sequence of its open reading frame encoding 211 amino acids was the same as that of mouse RGS2, with the exception of four amino acids. Northern blot analysis showed that the expression of this gene was much higher at implantation sites than at inter-implantation sites on days 5-8 of pregnancy. In situ hybridization localized this mRNA predominantly to the stromal cells at the implantation sites. These results indicate that RGS2 has a role during implantation, possibly by regulating the intracellular Ca(2+) mobilization and T-cell proliferation at the maternal-fetal interface.

[Immunocytochemical localization of fertilin beta on human sperm].

Fertilin is a kind of sperm plasma membrane protein that mimics snake venom protein. It belongs to the ADAMs family of surface proteins that contain a disintegrin and a metalloprotease domain. Fertilin functions in the sperm-egg binding process by connecting the sperm to the egg plasma membrane via a binding site in the disintegrin domain of fertilin beta (HF93). Its localization on the sperm is in the change. In this study, the monoclonal antibody against human fertilin beta was prepared and used to analyze the localization of fertilin beta on capacitated and acrosome-reacted sperm by immunofluorescence and immunoelectron microscopy techniques. The results were as follows: (1) fertilin beta became restricted to the anterior head during the course of capacitation. (2) During the course of acrosome reaction, the expression and localization of fertilin beta changed immensely on the anterior head and restricted to the lateral of posterior head at last. The restrictions of fertilin beta to the anterior head of capacitated sperm of human beings indicated that fertilin beta may be involved in the binding the sperm to the epithelial cells of the oviduct; the restrictions of fertilin beta to the posterior head domain of acrosome-reacted sperm implied its function in sperm-egg binding and fusion.

Human mu-opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.

Human mu-opioid receptor (HmuOR) with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells. The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 +/- 0.7 and 6.52 +/- 0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by mu-selective agonists [D-Ala2, N-methyl-Phe4, glyol5]enkephalin (DAGO), Ohm, and morphine, but neither by delta nor by kappa selective agonist. Na+ (100 mM) and GTP (50 microM) could reduce HmuOR agonists etorphine and Ohm affinity binding to the overexpressed HmuOR. mu-selective agonists DAGO and Ohm effectively stimulated [35S]GTP-gammaS binding (EC50 = 2.7 nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin (PTX). These results demonstrated that HmuOR overexpressed in Sf9 insect cells functionally coupled to endogenous G(i/o) proteins.

[Biosynthesis of a single peptide chain containing human chorionic gonadotropin beta and C3D of complement].

In view of the strong immunity-enhancing function of HEL-C3d3 designed by Dr. Paul W. Dempsey, we made our efforts to produce a similar recombinant protein of hCG beta. With polymerase chain reaction, we introduced a Bam HI restriction site into the 3' terminal of hCG beta cDNA. The new cDNA and its terminal's correctness has been confirmed by sequencing. Then we have it covalently attached to the C3d3 cDNA at the pre-designed Bam HI/Bgl II site. Having the chimeric DNA correctly cloned into the protein nuclear polyhedrosis virus (AcNPV) expression vector pVL1393, we constructed the expression vector pVL1393-(hCG beta-C3d3). The insect cells were co-transfected with the expression vector and linearized nuclear polyhedrosis virus DNA, and recombinant viruses AcNPV-(hCG beta-C3d3) were screened out. Through anti-hCG beta immunoaffnity chromatography, the recombinant hCG beta-C3d3 chimera polypeptide was purified from culture supernatant of insect cells infected by the recombinant viruses. In RIA test, the expressed product competitively inhibits the binding of 125I-hCG beta to hCG beta-antibody. On SDS-PAGE and Western blot, the recombinant peptide hCG beta-C3d3 obviously appears to be with a molecular weight of 116KD. Therefore, we arrive at a conclusion that it has a normal immunogenic ability.

Human mu-opioid receptor overexpressed in baculovirus system and its pharmacological characterizations.

AIM: To overexpress human mu-opioid receptor (muOR) with characteristics similar to those of mammalian origin. METHODS: Human muOR with a tag of 6 consecutive histidines at its carboxyl terminus was expressed in recombinant baculovirus infected Sf9 insect cells. Then the pharmacological characterizations of the product were studied by receptor binding assay and cAMP assay. RESULTS: The maximal binding capacity for the [3H]diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 +/- 0.7 and 6.52 +/- 0.23 nmol/g protein, respectively. The [3H]diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by alpha-selective agonists [D-Ala2, N-methyl-Phe4, glyol5] enkephalin (DAGO), Ohm, and morphine, but neither by the delta-selective agonist [D-Pen2, D-Pen5] enkephalin (DPDPE) nor by the kappa-selective agonist ¿trans-(+/-)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]¿ benzacetamide (U50488). NaCl 100 mmol.L-1 and guanosine triphosphate (GTP) 50 mumol.L-1 could reduce mu agonists Ohm and etorphine affinity binding to the expressed muOR. DAGO and Ohm effectively inhibited forskolin-stimulated cAMP accumulation. This agonist-dependent effect was blocked by opioid antagonist naloxone. CONCLUSION: The overexpression of human muOR with a tag of six consecutive histidines at its carboxyl terminus in Sf9 insect cells retained the characteristics of wild-type human muOR.

[Biosynthesis of a single peptide chain containing human chorionic gonadotropin beta and ovine common alpha subunits tandem].

hCG beta-oLH alpha chimeric cDNA was constructed by using overlapping PCR to contact the codons of C-terminal end of hCG beta with the codons of N-terminal end of oLH alpha, then it was subcloned into nuclear polyhedrosis virus (AcNPV) expression vector pVL1393 to construct expression vector pVL1393-hCG beta-oLH alpha. The insect cells (Sf9) were cotransfected by the expression vector pVL1393-hCG beta-oLH alpha and BaculoGold AcNPV linearized genomic DNA, and recombinant viruses AcNPV-hCG beta-oLH alpha were screened out by plaque assay. Further the insect cells were infected by the recombinant viruses, the recombinant hCG beta-oLH alpha was purified by immunoaffinity chromatography column coupling anti-hCG beta monoclonal antibody from the conditioned media of infected cells. The results of SDS-PAGE silver staining and western blotting showed that hCG beta-oLH alpha single peptide chain had apparent molecular weights of 40.5 kD and 38.0 kD under non-reducing and reducing conditions respectively, indicating the occurrence of disulfide bonds and significant tertiary structure in the single peptide chain. From the results of competitive inhibition of 125I-hCG beta binding we can conclude that the anti-hCG beta antibody-binding activity of hCG beta-oLH alpha chimera is lower than that of native hCG, but higher than that of native hCG beta. Therefore, we assume that the hCG beta-oLH alpha chimera should have potential application as a target antigen of anti-hCG fertility regulatory vaccine.

[Expression of murine interleukin 12(mIL-12) in insect cells].

Since human IL-12 is species-specific in its functions and elicits little biological responses from mouse lymphocytes, it is necessary to express recombinant murine IL-12 for the usage in studying the effects of this cytokine in various rodent models. Thereby, we can investigate the role of IL-12 in immune response in vivo and evaluate its potential clinical utility. Thus, we firstly constructed two expression vectors, pVL1393-mp40 and pVL1393-mp35. They were used to co-transfect the insect cells(Sf9) separately with linearized polyhedrosis virus genomic DNA. Two kinds of recombinant viruses AcNPV-mp40 and AcNPV-mp35 were visually screened out, and mp40 and mp35 were co-expressed in the insect cells co-infected by AcNPV-mp40 and AcNPV-mp35. The results of real-time Biomolecular Interaction Analysis (BIA) and Northern blot demonstrated that the recombinant mIL-12 was expressed successfully in the insect cells. The molecular weights of recombinant mp40 and mp35 were 40 KDa and 22 KDa on SDS-PAGE under reducing conditions, respectively. The apparent molecular weight of recombinant mIL-12 is 80 KDa under non-reducing conditions of Western blot. Biological activity of the recombinant product was detected in conditional medium using antibody-capture bioassay. The expression level of recombinant mIL-12 was about 10-15 micrograms/10(6) cells, as compared with the calibration curve of mIL-12.

Analysis of two constitutive forms of microsomal heme oxygenase in different rat tissues.

AIM: To isolate and purify the heme oxygenase (HO) isoform in microsomal fractions of Sprague-Dawley rat liver and brain in order to understand the characteristics of the two constitutive forms and the mechanism of the occurrence of hyperbilirubinemia. METHODS: After induction by hematin and phenylhydrazine, the rat liver and brain microsomal fractions were isolated and purified by DEAE-Sephacel and hydroxyapatite. Activity and the apparent molecular weight of the two isoforms [heme oxygenase 1 (HO-1) and heme oxygenase-2 (HO-2)] were measured. Kunming mice were used to prepare antiserum against purified liver HO-2. Rat liver HO-1 and brain HO-2 preparations were analyzed by the western immunoblotting technique. RESULTS: Two isoforms were purified and identified in the treated rat liver, and HO-1 was the predominant form with a ratio of 2:1. In the native state, HO-2 activity was detectable but HO-1 activity was increased in response to hematin and phenylhydrazine, while HO-2 activity was fully refractory to these agents. The apparent molecular weights of HO-1 and HO-2 were about Mr 30000 and Mr 36000 under reducing conditions, respectively. In the untreated liver and treated brain, only one peak of HO activity exhibiting an elution profile similar to that of HO-2 of the treated liver was detected. The presence of an activity peak was not found in the elution profile at the region where the inducible isoform of HO (HO-1) was expected. The apparent molecular weight in treated brain preparation was identical to that of the purified liver HO-2. Cross-reactivity of HO-2 in the brain microsomal preparation was established, but a reactivity of HO-1 in the liver was not observed by western immunoblotting analysis when antiserum to liver HO-2 was applied. CONCLUSION: Two constitutive forms of HO, designated as HO-1 and HO-2, exist in the treated rat liver. HO-1 is an inducible enzyme. In the treated rat brain only HO-2 exists and is a molecular entity similar to that found in liver. The two constitutive forms were different in molecular weight and in inducibility and immunochemical properties.

[Expression of cDNA of human chorionic gonadotropin beta-subunit (beta-hCG) cDNA in insect cells and effect of expressed product on mouse lymphocytes in vitro].

Expression vector pVL 1393-hCG beta containing beta-hCG cDNA has been constructed using an unfused protein nuclear polyhedrosis virus (AcNPV) expression vector. The insect cells (Sf 9) were cotransfected by the expression vector and nuclear polyhedrosis virus genomic DNA, and recombinant virus AcNPV-hCG beta was screened out, beta-hCG cDNA was expressed in insect cells infected by recombinant virus and recombinant beta-hCG (r beta-hCG) was secreted into medium. The purity of r beta-hCG, purified by immuno-affinity chromatography, was about 90% and the molecular weight of r beta-hCG was 22,500 Da. Like hCG, r beta-hCG suppressed significantly proliferation of induced lymphocytes, as well as production of IL-2 to some extent, on a parallel with suppression of lymphoproliferation.

[Expression of the extracellular domain (1-341) of rat ovarian lutropin receptor in insect cells and preliminary characterization of the expressed product].

Extracellular domain residues 1-341 (designed R 341) of luteinizing hormone/human chorionic gonadotropin receptor (LH/hCG receptor) has high binding affinity for ligand. This paper describes expression of cDNA coding for R 341 in insect cells and preliminary identification of the expressed protein. SDS-PAGE silver staining and immunoblotting analysis show expressed product appears in two bands, major band has molecular weight 38.5 Kd, and weak band, 40.0 Kd. Ligand binding immunoblotting and 125I-hCG-binding blotting analysis indicate that expressed product R 341 has specific binding affinity for ligand. Ligand binding assay and Scatchard analysis indicate that recombinant receptor R 341 has high binding affinity for hCG and Kd is 5.68 x 10(-10) mol/L.

[Cloning and overexpression of rat ovary LH/hCG receptor cDNA in insect cells].

Luteinizing hormone/human chorionic gonadotropin receptor (LH/hCG receptor) is a glycosylated protein coupled with G-protein. This paper reports screening of LH/hCG receptor cDNA from rat ovary cDNA library and the overexpression of the cDNA in insect cells. Full length of LH/hCG receptor cDNA is 2403 bp encoding signal peptide and matured protein of LH/hCG receptor. The cDNA is overexpressed in insect cells using baculovirus expression vector pVL 1393. The apparent molecular weight of purified receptor using immunoaffinity chromatography is 120 Kd and 92 Kd under non-reducing and reducing conditions, respectively. Ligand binding assay and Scatchard Plot analysis indicates that Kd is 8.4 x 10(-9) mol/L which is similar to that of the purified receptor from CHO cells.

[Cell-specific expression of AFP gene is dependent on some nuclear proteins].

AFP is an oncodevelopmental protein. Its level decreases abruptly after birth and reaches almost undetectable level during normal adult life. However, reexpression of the gene can be observed during hepatocarcinogenesis. To further understand mechanism of regulating AFP expression, we checked several restriction enzyme map of 5' terminal and flanking sequences of AFP gene. There are no differences among adult rat liver, fetal liver and hepatoma cells. Using +2(-)-255 bp sequence probe of AFP gene to do southwestern blotting assay, the result showed that the gene-active cells, such as hepatoma cells, contained binding-proteins which were apparently lacking in adult rat liver, lung, spleen, heart and kidney cells. While the fractions of nuclear proteins from adult rat liver cells were devoid of any stimulatory effect on transcription, those of binding-proteins from hepatoma stimulated the transcription of AFP gene in vitro. The hepatoma binding-proteins can rescue transcription activity of fraction of nuclear proteins from adult rat liver cells. These results indicate that cell-specific expression of AFP gene is regulated by protein-factors.

Carbohydrate variant of the recombinant beta-subunit of human choriogonadotropin expressed in baculovirus expression system.

The beta-subunit of human choriogonadotropin (hCG) has two complex type N-linked and four O-linked carbohydrate chains. To further evaluate the specificity of the carbohydrate moiety on the hCG function, we have expressed hCG beta subunit in the baculovirus insect cell system to modify its carbohydrate structures. The recombinant hCG beta (rhCG beta) was efficiently secreted in the medium and was purified to homogeneity by immunoaffinity chromatography using a highly specific monoclonal antibody against hCG beta. The homogeneity of the recombinant subunit was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under reducing and nonreducing conditions and reverse phase high performance liquid chromatography. rhCG beta had molecular weights of 22,500 and 33,000 under reducing and nonreducing conditions, respectively. Digestion with N-glycanase cleaved the Mr = 22,500 protein to 18,000, while digestion with Endo H or Endo F yielded an additional protein band of 20,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carbohydrate analysis by pulse amperometry yielded the relative number of 2.5, 2.4, 3.7, and 11.3 residues of fucose, N-acetylgalactosamine, galactose, and mannose, respectively, based on a value of 4 residues for N-acetylglucosamine. Lectin binding studies showed rhCG beta to bind with concanavalin A with a high affinity and not with wheat germ agglutinin. In the studies with endoglycosidases together with the carbohydrate analysis and lectin binding properties, rhCG beta appears to have two high mannose-type N-linked and three to four O-linked carbohydrate simple disaccharide chains. The carbohydrate modification of the beta-subunit did not alter its immunopotency and its ability to combine with hCG alpha. The reconstituted hormone made up of rhCG beta and hCG alpha was found to be similar to hCG in biological properties such as receptor binding and in its ability to stimulate cAMP and steroidogenesis.

cDNA-derived amino acid sequences of choriocarcinoma alpha- and beta-subunits of human choriogonadotropin.

Although amino acid sequences of the alpha- and beta-subunits of human choriogonadotropin (hCG) are known, only limited information is available on the disease state hCG. We have examined the amino acid sequences of the alpha- and beta-subunits of hCG from choriocarcinoma BeWo cells. The amino acid sequences were derived from the nucleotide sequences of BeWo cDNA clones of hCG alpha- and beta-subunits and were found to be identical with those of the normal subunits. It appears that the differences between the normal and the choriocarcinoma alpha- and beta-subunits of hCG reside primarily in the carbohydrates rather than the amino acid sequences. It may be pointed out that although coding and non-coding regions of BeWo cDNA clones of CG alpha and CG beta had several base changes from the hCG alpha and hCG beta cDNAs, these changes did not result in the alteration of their amino acid sequences. The longest BeWo alpha and beta cDNAs were 719 and 878 base pairs (bp) in length and lacked only 16 and 7 bp from the transcription start sites respectively. BeWo CG alpha cDNA had two base changes in the non-coding regions, one insertion of C at position 39 and another substitution of T for A at position 651, the latter change deleted one HindIII polymorphous site. The BeWo CG beta cDNA also had two base substitutions, A for G at 131 in the non-coding region and T for C at 807 position in the coding region.

A developmentally regulated hydroxyproline-rich glycoprotein in maize pericarp cell walls.

We have studied the accumulation of peptidyl hydroxyproline in the pericarp of developing maize (Zea mays L., Golden cross Bantam sweet corn) kernels. Although this hydroxyproline accumulates throughout development, it is most soluble and its content per milligram dry weight greatest at midmaturation stages of development. Salt-soluble proteins containing this hydroxyproline from isolated cell walls of developing kernels were fractionated on a CsCl density gradient and on a Chromatofocusing column, resulting in the purification of an hydroxyproline-rich glycoprotein, PC-1. PC-1 is a basic protein of approximately 65 to 70 kilodaltons in molecular weight with an isoelectric point of at least 10.2 and a density of 1.38 to 1.39 in CsCl. Amino acid composition data indicate that it is rich in hydroxyproline, threonine, proline, lysine, and glycine. Its relation to dicot extensin is discussed.

Total synthesis of yeast alanine transfer ribonucleic acid.

By a combination of chemical and enzymatic methods, small oligonucleotides with lengths varying from 2 to 8 nucleotides were synthesized from mononucleotides. The small oligonucleotides were then ligated with T4 RNA ligase into six large oligonucleotides (9 to 19 nucleotides long) which were further ligated to form two half molecules with 35 and 41 nucleotides respectively. Finally, the two synthetic half molecules were annealed and ligated to obtain the whole molecule of yeast alanine tRNA (tRNAAlay). Prior to this, two semi-syntheses were performed, i.e. ligation of the synthetic 5'-half molecule with the natural 3'-half molecule and that of the natural 5'-half molecule with the synthetic 3'-half molecule. Both the semi-synthetic tRNAAlay and the synthetic tRNAAlay occupy the same position as the natural tRNAAlay after electrophoresis on a 20% polyacrylamide gel. They have the same chemical composition (containing 9 modified nucleotides of 7 different species) and structure as the natural tRNAAlay and are biologically active, i.e. accepting and transferring alanine into proteins in a cell-free protein synthesizing system, the accepting activity of the synthetic product is 52-66% of that of the natural tRNAAlay and 91-106% of that of the reconstituted product of the two natural half molecules. The incorporation activity of alanine into proteins of the synthetic 3H-alanine tRNAAlay is 63%, corresponding to 91% of that of the natural tRNAAlay and 115% of that of the reconstituted product of the two natural half molecules. To the best of our knowledge, this is the first time that a natural RNA with biological activity is synthesized.

Assay of biological activity of synthetic yeast alanine transfer RNA (tRNAAlay).

The biological activity of the synthetic tRNAAlay was studied with an extremely sensitive method. tRNAAlay accepted alanine in the presence of rat liver aminoacyl-tRNAAlay-synthetase (this was called the accepting activity). The aminoacylated tRNAAlay was conveniently precipitated by ethanol with good recovery. The efficiency of transferring alanine from the aminoacylated tRNAAlay into the protein was determined in in vitro rabbit reticulocyte lysate cell-free protein-synthesizing system (this was called the incorporation activity). Both accepting and incorporation activities could be determined in one assay with only 5-7 pmoles of tRNAAlay either in ligation mixture or in purified form. Our results show that the accepting activities of the synthetic products were 51.6-65.6% and 91.3-106.0% of that of natural and reconstituted natural tRNAAlay respectively. The efficiency of the incorporation of alanine in the aminoacylated tRNAAlay into the protein was 61.6-63.1%, corresponding to 90.6-91.7% and 97.2-115.8% of that of the natural and the reconstituted natural tRNAAlay respectively.