THE COURSE OF THE POLYPEPTIDE CHAIN OF TRICHOSANTHIN MOLECULE

The course of the polypeptide chain of Trichosanthin has been determined from the electron density map at 4 resolution. The structure belongs to α+β type. It contains eight α-helices (39% of total residues) and four β-sheets (32% of total residues) which are made up of thirteen β-strands. The α-helices are relatively in the centre of the molecule and surrounded by β-sheets. This is the characteristic feature of Trichosanthin structure. This kind of structural arrangement has not been reported before. The transformation matrix and translation vector, which superpose two molecules in one asymmetric unit, were obtained. The root mean square error is 1.31 for this superposition.     

A DYNAMIC MECHANICAL ANALYSIS STUDY OF THE TRANSITION BEHAVIOUR OF I-PP/EPDM BLENDS

The transition behaviour of the blends of isotactic polypropylene (i-PP) with ethylene-propylene terpolymer (EPDM) containing 42 wt% propylene was investigated by dynamic mechanical analysis technique (DMA). Owing to its high propylene content, EPDM is compatible with i-PP to some degree. The interaction between the two components was strengthened. As expected, for partially compatible system the glass transition temperature of i-PP in the blends shifted to lower temperature. It was found that there existed two transitions, αEPDM and βEPDM, for the EPDM used in this work. The former was considered to be the glass transition of the random chain segments of EPDM, while the latter the local motion of the long ethylene sequences in EPDM. The unusual transition behaviour of αEPDM in the blends was explained in terms of the greater thermal expansion of EPDM and the compatibility of the two components. On the other hand, the βEPDM changed with the composition of the blends in a regular manner.     

WAXD STUDY OF POLYMORPHOUS ISOTACTICPOLYPROPYLENE BY COMPUTER PEAK-RESOLUTION METHOD

Polymorphous i-PP containing a and βphases were studied by WAXD and computer peak-resolution method (CPRM). The asymmetric Gaussian-Cauchy functions (a-GC) were adoptedto fit the profiles of both amorphous and crystalline peaks. For the crystalline peaks, the fitting resultsof a-GC are better than the symmetric-GC; for the amorphous peaks, they are better than polynomialand exponential functions, etc. Using the retarded least-square procedure (RLSP) on microcom-puter the results of peak-resolution are rather satisfatory. For the polymorphous samples containingαand βphases, the relations between phase-state, crystallinity, crystalline size, the ratio of α,βrelativecontent and the crystallization temperature T_c were studied by CPRM. The ratio of α,βrelativecontents obtained by CPRM and Turner-Jones eq. have been carefully compared. There are manyimprovements in this work. A simple estimation method of WAXD peak areas, both for amorphousand crystalline peaki, is suggested.     

THE MOLECULAR MECHANISM OF PHOTOPORPHYRIN (YHPD)''''S PHOTOSENSITIZATION——LASER RAMAN SPECTROSCOPIC STUDY OF MICROCOSMIC AND PHOTOSENSITIVE DAMAGE OF YHPD TO PROTEIN

Laser Raman spectroscopy was used to investigate the microcosmic and photosensitive damage of YHPD to lysozyme, of which the three-dimensional structure has been elucidated. The experimental results shown by various damages of the main-chain and side-chain of lysozyme are as follows: (ⅰ) Phe and Cys are also damaged by photosensitization of YHPD, except for Trp, Tyr, Met, 1/2Cys and His; (ⅱ) the order of the photosensitized sensitivity of various groups of these amino acids have been described; (ⅲ) Trp and Tyr buried in the three-dimensional structure of the protein are damaged very greatly, and (ⅳ) the main-chain conformation of the protein has changed considerably, such as a decrease in orderly structure (α-helix, β-sheet and β-turn) and a simultaneous increase in random coil.     

Structure Analysis of Streptococcal Protein G Fc Binding Domain

The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant     

IDENTIFICATION OF PARTIAL MUTATIONAL SITES IN HUMAN MITOCHONDRIAL DNA IN THE CHINESE AND ITS SIGNIFICANCE

A simple method for the identification of mutational sites in human mitochondrial DNA (mtDNA) was described. It was based on the human Cambridge sequence as a relative standard sequence and a single base pair substitution in mtDNA as a unique mutational form. The partial mutational sites can be determined using this method which was characterized by combining the restriction mapping with the analysis for the table of human mtDNA potential mutational sites with rapidity and simplicity. In the meanwhile, six mtDNA mutational sites found in Chinese population were identified by means of this method.     

QUANTITATIVE RELATION BETWEEN MODIFICATION OF FUNCTIONAL GROUPS OF PROTEINS AND THEIR BIOLOGICAL ACTIVITY——A COMPUTER PROGRAM FOR ASCERTAINING THE NUMBER OF ESSENTIAL RESIDUES

Based on a statistical treatment of the quantitative relation between the extents of mod-ification of protein functional groups and the decrease of their biological activity, a meth-od was proposed (Tsou, Sci. Sin., 1962, 11:1535) and widely used for the determination ofthe number of residues essential for the biological activity ot the protein modified. How-ever, the original method depends on a trial and error approach and manual calculation tofind the best fit for the data obtained. A computer program is described in this paper forthe treatment of experimental data to produce the number of essential and non-essentialgroups modified and the ratio of rates for their modification which best fit the data ob-tained. Applications of this method show that in some cases wrong conclusions were reachedin literature and in other cases quantitative conclusions can sometimes be drawn when orig-inally either no quantitative data treatment was presented or attempts at manual curve fit-ting were unsuccessful.     

Divergent pathways of β,γ-unsaturated α-diazocarbonyl compounds catalyzed by dirhodium and Lewis acids catalysts separately or in combination

β,γ-Unsaturated a-diazocarbonyl compounds possess two reactive sites for electrophilic addition-one at the diazo carbon and the other at the vinylogous γ-position.Controlled by catalyst,divergent transformations are achieved starting from the same starting materials,either by Lewis acid-catalyzed addition or by dirhodium-catalyzed metal carbene reactions.In select cases two catalysts working in combination or in sequence provide a relay for cascade transformations.In this review,we summarize advances in catalyst-dependent divergent transformations of β,γ-unsaturated α-diazocarbonyl compounds and highlight the potential of this exciting research area and the many challenges that remain.     

EXPRESSION OF SYNTHESIZED HIRUDIN GENE IN YEAST

Hirudin is a sort of polypeptides secreted from the salivary gland of medicinal leech. It is of potential importance in medicine. We designed and synthesized the hirudin gene based on the amino acid sequence of hirudin HV2, and expressed it using the yeast alpha factor expression system. The yeast strain stably carrying the hirudin expression plasmid was deduced by mutagenesis. After its cultivation in rich nutritious medium for 36—48 h, the hirudin expression product secreted into the culture fluid was 10—20 ATU/ml. The HPLCpure hirudin product could be obtained through a simpler purification procedure, its N-terminal amino acid sequence was identical with the natural product, and it showed potent anticoagulant and antithrombin activity. About 3000 ATU of pure hirudin witha specific activity of 6600 ATU/mg could be obtained from 500 ml of culture fluid.     

MOLECULAR CLONING OF A NEW VARIANT OF HUMAN PAPILLOMAVIRUS TYPE 6 ISOLATED FROM A CHINESE WOMAN AND EXPRESSION OF ITS L1 GENE IN E. coli——MOLECULAR CLONING & EXPRESSION OF HPV6 GENE

A human papillomavirus genome DNA of 7.9 kb from a Chinese woman with genital condyloma acuminata was cloned in Bam HI site of pAT153. According to the results obtained from Southern blotting, restriction mapping as well as partial DNA sequencing, the isolated genome (HPV6BV) had obvious variance and was referred to as a new variant of HPV6 found in China the first time. HPV6BV L1 gene was successfully expressed in E. coli as a fusion protein with pUR288. The β-galactosidase/L1 fusion protein reacted with both β-galactosidase antiserum and HPV antibody using Western blot technique. The E. coli-produced fusion protein, possessing HPV antigenicity, may provide a reagent for clinical diagnosis and epidemiological survey.     

A rapid protein sample preparation method based on organic-aqueous microwave irradiation technique

Fast and efficient sample preparation methods are a prerequisite for protein identification in bottom-up proteomics. Here, an innovative microwave irradiation sample preparation method was developed based on an optimized organic-aqueous solvent system for protein identification. Specifically, protein solutions containing high-concentration acetonitrile were subjected to 5 min microwave irradiation. After cooling down, trypsin was added and the digestion was performed with 30 s microwave irradiation, and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS). A shortened processing time of only 5.5 min is needed with this method(more than 12 h is necessary in the traditional overnight protein sample preparation). Moreover, due to the absence of urea and other chaotropic reagents, the digests can be readily identified by MALDI-TOF MS. When an assessment of this method was performed by digesting a model protein BSA, 69% ± 3% sequence coverage corresponding to 47 ± 3 peptides was obtained, which shows better protein identification than that from the standard overnight protein sample preparation method(51% ± 2% sequence coverage and 23 ± 1 peptides). Another model protein α-casein was used for the analysis of protein phosphorylation with the newly developed method that yielded 4 phosphopeptides with 8 phosphorylation sites, whereas 3 phosphopeptides with 2 phosphorylation sites were obtained from the traditional overnight approach. Moreover, the organic-aqueous microwave irradiation method provides effective digestion for proteins down to fmol.     

RETENTION RULE IN THE LIQUID CHROMATOGRAPHY——SELECTION OF THE OPTIMUM ELUTION COMPOSITION OF SOME PTH AMINO ACIDS ON SILICA GEL

The minimal relative retention α' and the required relative retention α'_(min) of the pair of substances being most difficult to separate in a multicomponent mixture have been discussed on the basis of the fundametal retention equation ln k'=b_0 b_1C_B b_2ln C_B and resolution criterion. The selection principle to optimize elution composition means that selected a value must be greater than α'_(min) and that the capacity factor of last peak should be held to a minimum. A rapid method to select optimum elution composition has been established in which it is necessary to do a few experiments. An example for the 5 pairs of phenylthiohydantion amino acids (PTH-AA) using the ternary mobile phase of methanol, ether and hexane is also demonstrated in this paper. The results show that theoretical prediction is good in agreement with the experiments.     

MOLECULAR CHARACTERIZATION OF RICE Wx GENE

The complete nucleotide (nt) sequence of the rice waxy(Wx) gene, which is responsible for the synthesis of amylose in endosperm and pollen, has been determined by a combination of restriction mapping and nt sequence analysis of two overlapping genomic DNA clones. The entire gene is about 5.5 kb in length. The alignment of the nt sequence of the Wx gene from rice with those of maize (Klsgen, R. B. et al.) and barley (Rohde, W. et al.) revealed the presence of thirteen introns and fourteen exons. The full-length of Wx protein in cluding transit peptide is 609 amino acid (aa) residues. The calculated molecular weight of rice Wx preprotein is about 72 kD. There is no significant difference between the similarity scores of the aa sequence deduced from the rice Wx gene compared with those of maize and barley. However, the nt sequences of the 5'-end upstream, 3'-end downstream and introns of the rice Wx gene, as well as the aa sequence of the transit peptide region of the Wx preprotein have low similarity scor     

STATISTICAL PROPERTIES OF LONG-RANGE CONTACTS IN GLOBULAR PROTEINS

The analysis of residue-residue contacts in protein structures can shed some light on our understanding of the folding and stability of proteins. In this paper, we study the statistical properties of long-range and short-range residue-residue contacts of 91 globular proteins using CSU software and analyze the importance of long-range contacts in globular protein structure. There are many short-range and long-range contacts in globular proteins, and it is found that the average number of long-range contacts per residue is 5.63 and the percentage of residue-residue contacts which are involved in long-range ones is 59.4%. In more detail, the distribution of long-range contacts in different residue intervals is investigated and it is found that the residues occurring in the interval range of 4-10 residues apart in the sequence contribute more long-range contacts to the stability of globular protein. The number of long-range contacts per residue, which is a measure of ability toform residue-residue contacts, is also calculated for 20 different amino acid residues. It is shown that hydrophobic residues (including Leu, Val, Ile, Met, Phe, Tyr, Cys and Trp) having a large number of long-range contacts easily form long-range contacts, while the hydrophilic amino acids (including Ala, Gly, Thr, His, Glu, Gln, Asp, Asn, Lys, Ser, Arg, and Pro) form long-range contacts with more difficulty. The relationship between the Fauchere-Pliska hydrophobicity scale (FPH) and the number of short-range and long-range contacts per residue for 20 amino acid residues is also studied. An approximately linear relationship between the Fauchere-Pliska hydrophobicity scale (FPH) and the number of long-range contacts per residue CL is found and can be expressed as     

A CIRCULAR DICHROIC STUDY OF THE INTERACTION OF INSULIN A AND B CHAINS

It has been shown previously that the S-thiomethyl forms of the insulin A and B chains interact in solution leading to a partial transfer of Tyr side chains from hydrophilic to hydrophobic environments. In the present work, a circular dichroic study has indicated that the α-helix contents of the chains show a gradual increase upon mixing of the chains reaching completion in about 2h. The separated S-thiomethyl protected chains contain some ordered structure (A: α=15%, β=27%: B: α=22%, β=23%). Contrary to reports in the literature, the reduced chains also show some ordered structure and upon mixing of the reduced chains, the α-helix content also shows some increase. The ordered structure of the reduced chains decrease with increasing concentrations of dithiothreitel and in presence of a large excess of DTT both the reduced chains have very little, if any, α-helix structure. These latter results are in accord with those of Wu and Yang. In agreement with the results obtained previously with ultravio     

A Stable Vector for High-Level Expression and Secretion of Human Interferon αA in Yeast

Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10~(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.     

Effect of arginine on stability of GST-ZNF191（243-368）

For better understanding the chemical or biological information of ZNF191(243-368), we expressed the fusion protein of GST and ZNF191(243-368), and used it to obtain the binding DNA sequence of this zinc finger protein. But in the process of expression and purification, we found this fusion protein slowly degradated. For resolving this problem, we simultaneously added charged amino acids L-Arg and L-Glu to the solution of fusion protein, and demonstrated that this method can dramatically increase the stability of this fusion protein. This method can make the fusion protein suitable for the continuous works, especially for situations where high protein concentration and long-term stability without precipitate and degradation of protein are required.     

CLONING AND SEQUENCE ANALYSIS OF 3''''-END REGION OF POTATO VIRUS Y GENOME

The entire coat protein (CP) gene and part of the 3'-noncoding sequence of the potatovirus Y (PVY, the Chinese isolate) genome were synthesized with polymerase chain reaction(PCR) using cDNA of its genomic RNA as a template. A restriction endonuclease site Ncoland the initiation codon AUG were included in primer Y5 while the SalI site was includedin primer Y3. After being double digested with Ncol and SalI enzymes, the PCR product wascloned into a pGEM derivative plasmid, and the CP gene in one of the clones, pPCY6, wassequenced. Several clones were selected from the cDNA library by using the CP gene frag-ment of pPCY6 as a probe and the sequences of these clones were determined. These se-quences included part of the NIb gene, entire CP gene and 3'-noncoding region, 1317 bp alltogether.Sequence analysis indicated that the nucleotide sequence homology of the CP geneof this strain with that of the 0 strain (94.2%) was a little higher than with that of the Nstrain (89.6%), but the homology of amino acid se     

Computer simulation on kinetics of primary process in photosynthesis of algae (V) --Excitation energy transfer in phycoerythrocyanins

Excitation energy transfer in phycoerythrocyanins (PEC) was studied by use of computer simulation. The results observed from the simulation are as follows: (i) The α84 is a more efficient sensitizing chromophore than β155 and donates the excitation energy into β84 and β155 while it scarcely emits fluorescence itself, (ii) Only the 1α84 →2β84 is the sub-picosecond process in a PEC trimer, therefore it is readily to obtain the time constant from fs-level time-resolved spectral measurement. (iii) The β84 and β155 chromophores in PEC behave quite differently from those in C-PC because of the changes in α84. It is observed that 1β156→6β155 is the dominant pathway linking two trimers and both of the chromophores possess much higher fluorescence fractions, and about 80% of the total fluorescence is emitted from the β84 chromophore. (iv) A far less mean number of transfer times is observed through the fast-transfer pairs in PEC compared with that in C-PC because of slow transfer rate for the path