[Clinical application of a classification based on crucial curvature of coronal imbalance in degenerative lumbar scoliosis].

Objective: To present efficacy of clinical application of a classification based on crucial curvature of coronal imbalance in degenerative lumbar scoliosis (DLS). Methods: A case series study. Clinical data of 61 cases (8 males, 53 females) who underwent posterior correction surgery for DLS from January 2019 to January 2021 were retrospectively analyzed. The mean age was (71.7±6.2) years (ranged 60-82 years). According to the direction of C7 plumb line (C7PL) deviated from central sacral vertical line (CSVL) and orientation of L4 coronal tilt, the author determined which one was the crucial curve. If C7PL deviated from CSVL in the same direction as concave side of the thoracolumbar curve and L4 coronally tilts opposite direction of C7PL deviates from CSVL, then the crucial curve was thoracolumbar curve (type 1). On the contrary, if C7PL deviated from CSVL in the same direction as concave side of the lumbosacral curve and L4 coronally tilts consist with direction of C7PL deviates from CSVL, then the crucial curve was lumbosacral curve (type 2). According to absolute value of coronal balance distance (|CBD|), each type of patients was divided into two groups, respectively, namely coronal balance (CB) (|CBD|≤3 cm) and coronal imbalance (CIB) (|CBD|>3 cm). Changes of Cobb angles of thoracolumbar curve and lumbosacral curve and CBD were recorded and analyzed. Results: The rate of preoperative CIB was 55.7% (34/61) in all the patients. Of the patients, 23 cases were classified as type 1 and 38 cases as type 2. The rate of preoperative CIB was 34.8% (8/23) in type 1 patients and 68.4% (26/38) in type 2. The rate of postoperative CIB was 27.9% (17/61) in all the patients, with 13.0% (3/23) in type 1 and 36.8% (14/38) in type 2. The |CBD| of CB group in type 1 patients decreased from (2.6±1.4) cm before the operation to (1.5±1.0) cm after (P=0.015); and the correction rate of thoracolumbar curve (68.8%±18.4%) was significantly higher than that of lumbosacral curve (34.5%±23.9%) (P=0.005). The |CBD| of CB group in type 2 patients decreased from (2.6±3.0) cm before the operation to (1.6±1.2) cm after (P=0.027); the correction rate of lumbosacral curve (71.3%±18.6%) was higher than that of thoracolumbar curve (57.3%±21.1%), but the difference was not statistically significant (P=0.546). There was no significant difference in |CBD| of CIB group in type 2 patients before and after the operation (P=0.222); the correction rate of lumbosacral curve (38.3%±14.8%) was significantly lower than that of thoracolumbar curve (53.6%±16.0%) (P=0.001). There was a correlation between the change of CBD (3.8±1.5) cm and the difference in correction rate between thoracolumbar and lumbosacral curve (32.3%±19.6%) in CB group in type 1 patients after surgery (r=0.904, P<0.001). There was a correlation between the change of CBD (1.9±2.2) cm and the difference in correction rate between lumbosacral and thoracolumbar curve (14.0%±26.2%) in CB group in type 2 patients after surgery (r=0.960, P<0.001). Conclusion: Clinical application of a classification based on crucial curvature of coronal imbalance in DLS is satisfactory, and its combination with matching correction can effectively prevent the occurrence of coronal imbalance after spinal correction surgery.

Enhancement of charge-mediated magnetoelectric coupling in Fe3O4/SrTiO3/Ba0.6Sr0.4TiO3 heterostructure.

The in-plane magnetic hysteresis loops of Fe3O4/SrTiO3(STO) and Fe3O4/STO/Ba0.6Sr0.4TiO3(BSTO) heterostructures have been investigated at 200 K under various electric fields. The bottom BSTO layer of the STO/BSTO bilayer is used to improve the dielectric properties of the top STO layer. The polarization of the STO/BSTO bilayer is ∼78% larger than that of the STO layer at room temperature due to the improvement of surface topography and the contribution of electrostatic interlayer coupling. A significant enlargement (∼70%) in the magnetoelectric response of Fe3O4/STO/BSTO heterostructure has been achieved at 200 K and 300 kV cm-1 after introducing the BSTO layer, since the STO/BSTO bilayer with larger dielectric constant supplies more polarization charges at its interface to the Fe3O4 layer than the STO layer. It indicates that the dielectric bilayer improves the polarization and thus benefits the magnetoelectric coupling in the multiferroic heterostructure.

Local structure, nucleation sites and crystallization behavior and their effects on magnetic properties of Fe81Si x B10P8-xCu1 (x = 0~8).

In this work, an attempt has been made to reveal critical factors dominating the crystallization and soft magnetic properties of Fe81Si x B10P8-xCu1 (x = 0, 2, 4, 6 and 8) alloys. Both melt spun and annealed alloys are characterized by differential scanning calorimetry, X-ray diffractometry, Mössbauer spectroscopy, transmission electron microscopy, positron annihilation lifetime spectroscopy and magnetometry. The changes in magnetic interaction between Fe atoms and chemical homogeneity can well explain the variation of magnetic properties of Fe81Si x B10P8-xCu1 amorphous alloys. The density of nucleation sites in the amorphous precursors decreases in the substitution of P by Si. Meanwhile, the precipitated nanograins gradually coarsen, but the inhibiting effect of P on grain growth diminishes causing the increase of the crystallinity. Moreover, various site occupancies of Si are observed in the nanocrystallites and the Si occupancy in bcc Fe decreases the average magnetic moment of nanograins. Without sacrificing amorphous forming ability, we can obtain FeSiBPCu nanocrystalline alloy with excellent soft magnetic properties by optimizing the content of Si and P in the amorphous precursors.

Fluorescence enhancement and multiple protein detection in ZnO nanostructure microfluidic devices.

In this study, different morphological ZnO nanostructures, those of sharp nanowires (NWs), rod NWs, and hexahedral-puncheon nanostructures, were grown in microfluidic channels on the same glass substrate. Characterizations of correspondent biomolecule binding properties were simulated and demonstrated. The surface was modified using 3-ammineopropyl-triethoxysilane (3-APTES) and biotin-N-hydroxysuccinimide ester (NHS-biotin). Different concentrations (4.17pM to 41.7nM) of dye-conjugated streptavidin were simultaneously infused through the second microfluidic channels, which lie 90° from the first microfluidic channels. The florescent intensity at the crossover areas showed good agreement with simulations, with sharp ZnO NWs exhibiting the largest dynamic range and the highest fluorescent intensity. We further characterize correspondent protein detection using sharp ZnO NWs. The surfaces of these ZnO NWs were modified with mouse immunoglobulin G (IgG), infused through the second microfluidic channels with dye-conjugated (Alexa 546) anti-mouse IgG in different concentrations. Concentrations ranging from 417fM to 41.7nM can be resolved using sharp ZnO NWs. Finally, multiple protein detection was demonstrated using a five-by-eight microfluidic channel array. Fluorescence images present clear multiple detections at the crossover areas when using the sharp ZnO NWs for simultaneous dye-conjugated anti-mouse IgG and dye-conjugated anti-rabbit IgG (Alexa 647) detection.

Serum levels of IL-33 and its receptor ST2 are elevated in patients with ankylosing spondylitis.

OBJECTIVES: The aims of this study were to measure the levels of interleukin (IL)-33 and ST2 and T-helper (Th)2-associated cytokines (IL-13, IL-4, IL-5) in patients with ankylosing spondylitis (AS), and examine the correlation of serum cytokine levels with disease activity and laboratory parameters. METHOD: Serum IL-33, IL-13, IL-4, and IL-5 levels were assessed by sandwich enzyme-linked immunosorbent assay (ELISA), and the mRNA levels of IL-33 and ST2 were quantified by real-time quantitative polymerase chain reaction (RT-qPCR), in 43 AS samples and compared with 27 age- and sex-matched healthy controls. RESULTS: Serum IL-33, IL-13, and IL-4 levels were increased significantly in AS patients compared with controls (p < 0.01); moreover, serum IL-33 and IL-13 levels were significantly higher in patients with active AS than in those with inactive AS (p < 0.05). The serum levels of IL-5 showed no significant difference between AS patients and controls (p > 0.05). Serum IL-33 levels were positively correlated with both IL-13 (r = 0.306, p < 0.01) and IL-4 levels (r = 0.432, p < 0.01). The mRNA levels of IL-33 and ST2 were significantly different between AS patients and controls (p < 0.01) but not between active and inactive AS patients. CONCLUSIONS: Serum levels of IL-33 could partially reflect AS disease activity and indicate that IL-33/ST2 signalling plays an important role in the pathogenesis of AS.

The FCGR2B rs10917661 polymorphism may confer susceptibility to ankylosing spondylitis in Han Chinese: a case-control study.

OBJECTIVE: Single-nucleotide polymorphisms (SNPs) in the Fc gamma receptor IIB (FCGR2B) gene have recently been found to be associated with several human autoimmune diseases. We undertook the current study to investigate the influence of these polymorphisms on the risk of ankylosing spondylitis (AS). METHOD: A total of 306 patients with AS from Anhui, China, fulfilling the modified New York Criteria, and 300 matched healthy controls were analysed. All subjects were genotyped for two SNPs (rs1050501, rs10917661) in the FCGR2B gene, and the SNaPshot Assay was used for genotyping. RESULTS: SNP rs10917661 was significantly associated with AS [C vs. T: odds ratio (OR) 1.723, 95% confidence interval (CI) 1.086-2.733, p = 0.020; genotype: p = 0.026] whereas no association was found for rs1050501. Furthermore, no haplotype was found to be associated with AS. CONCLUSION: These findings indicated that rs10917661 may be a novel SNP involved in AS genetic predisposition in the Han Chinese population.

A meta-analysis of the relation of polymorphism at sites -1082 and -592 of the IL-10 gene promoter with susceptibility and clearance to persistent hepatitis B virus infection in the Chinese population.

BACKGROUND: Up to now, many publications about the Chinese population have evaluated the correlation between interleukin-10 (IL-10) -1082 and -592 polymorphisms and persistent hepatitis B virus (HBV) infection. However, the results remain inconclusive. In order to resolve this conflict, a meta-analysis was performed. METHODS: Seven studies were included and dichotomous data are presented as the odds ratio (OR) with a 95% confidence interval (CI). RESULTS: The results of our study suggest that carriers of the IL-10 -592A allele were more likely to clear HBV spontaneously in the Chinese pooled population (A vs. C: OR = 0.799, 95% CI = 0.678-0.941, P = 0.007; AC vs. AA: OR = 1.343, 95% CI = 1.017-1.684, P = 0.011; AA vs. AC + CC: OR = 0.736, 95% CI = 0.594-0.912; AA + AC vs. CC: OR = 0.588, 95% CI = 0.408-0.848, P = 0.004) and the IL-10 -1082A allele was associated with significantly reduced persistent HBV infection risk in Chinese (A vs. G: OR = 0.701, 95% CI = 0.494-0.996, P = 0.047; AA vs. GG + GA: OR = 0.684, 95% CI = 0.476-0.982, P = 0.040). CONCLUSIONS: Persistent HBV infection susceptibility is associated with the gene polymorphism IL-10 -1082GA in the Chinese population and the clearance of HBV is associated with the gene polymorphism IL-10 -592CA in the Chinese population.

Association of NFKB1 -94ins/delATTG promoter polymorphism with susceptibility to autoimmune and inflammatory diseases: a meta-analysis.

The aim of our study is to assess the association of NFKB1 -94ins/delATTG promoter polymorphism with autoimmune and inflammatory diseases using a meta-analysis. We surveyed the studies on the association of NFKB1 -94ins/delATTG promoter polymorphism with autoimmune and inflammatory diseases. Meta-analysis was performed for genotypes DD vs WW, WD vs WW, DD vs WW + WD, WD + DD vs WW, and D allele vs W allele in a fixed/random effect model. Seventeen studies (7312 cases and 6193 controls) were identified. When all groups were pooled, we found no association between NFKB1 -94ins/delATTG promoter polymorphism and autoimmune and inflammatory diseases. In ethnic subgroup analyses, we found no association between NFKB1 -94ins/delATTG promoter polymorphism and autoimmune and inflammatory diseases in the Caucasian population. However, an association of NFKB1 -94ins/delATTG promoter polymorphism with autoimmune and inflammatory diseases was found in the Asian population [D vs W: odds ratio (OR) = 0.87, 95% confidence interval (CI) = 0.77-0.99, P = 0.03; WD + DD vs WW: OR = 0.79, 95% CI = 0.65-0.95, P = 0.01; DD vs WW + WD: OR = 0.92, 95% CI = 0.73-1.16, P = 0.11; DD vs WW: OR = 0.80, 95% CI = 0.62-1.03, P = 0.09; WD vs WW: OR = 0.78, 95% CI = 0.65-0.95, P = 0.01]. In disease subgroup analyses, we found no association between NFKB1 -94ins/delATTG promoter polymorphism and inflammatory bowel disease, ankylosing spondylitis and Graves' disease. This meta-analysis suggests a possible association between NFKB1 -94ins/delATTG promoter polymorphism and certain autoimmune and inflammatory diseases in the Asian population, but not in the Caucasian population. This finding demands further investigation.

Association of SUMO4 M55V polymorphism with susceptibility to autoimmune and inflammatory diseases: a meta-analysis.

The purpose of this study was to generate large-scale evidence on whether SUMO4 M55V polymorphism is associated with autoimmune and inflammatory diseases using a meta-analysis. We surveyed studies on the association of SUMO4 M55V polymorphism with autoimmune and inflammatory diseases in PubMed. Meta-analysis was performed for genotypes AG versus AA, GG versus AA, GG versus AA + AG, AG + GG versus AA and G allele versus A allele in a fixed/random effect model. We identified 16 studies (11, 407 cases and 10, 679 controls) using PubMed search. When all groups were pooled, we detected the association of SUMO4 M55V polymorphism with autoimmune and inflammatory diseases (G versus A: OR = 1.11, 95%CI = 1.03-1.19, P = 0.005; AG +GG versus AA: OR=1.17, 95%CI=1.06-1.28, P=0.001; GG versus AA+AG: OR=1.07, 95%CI=0.94-1.21, P=0.29; GG versus AA: OR=1.15, 95%CI=1.00-1.34, P=0.06; AG versus AA: OR=1.15, 95%CI=1.08-1.23, P<0.0001). In subgroup analyses, we detected the association of SUMO4 M55V polymorphism with autoimmune and inflammatory diseases in Asian population (G versus A: OR=1.18, 95%CI=1.08-1.28, P=0.0001; AG+GG versus AA: OR=1.30, 95%CI=1.16-1.45, P<0.00001; GG versus AA+AG: OR=1.04, 95%CI=0.78-1.37, P=0.80; GG versus AA: OR=1.20, 95%CI=0.99-1.45, P=0.07; AG versus AA: OR=1.32, 95%CI=1.18-1.49, P<0.00001). But the association was not found in Caucasian population. Meanwhile, an association of SUMO4 M55V polymorphism with autoimmune diabetes was found (G versus A: OR=1.18, 95%CI=1.08-1.30, P=0.0005; AG+GG versus AA: OR=1.22, 95%CI=1.13-1.32, P<0.00001; GG versus AA+AG: OR=1.15, 95%CI=0.96-1.38, P=0.13; GG versus AA: OR=1.32, 95%CI=1.08-1.60, P=0.006; AG versus AA: OR=1.23, 95%CI=1.13-1.33, P<0.00001). This meta-analysis demonstrates the association of SUMO4 M55V polymorphism with autoimmune and inflammatory diseases, especially in Asian population.

Optimization of a thermostable lipase from Bacillus stearothermophilus P1: overexpression, purification, and characterization.

An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids. The expressed lipase was isolated and purified to homogeneity in a single chromatographic step. The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry. The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55 degrees C. It was highly stable in the temperature range of 30-65 degrees C. The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol. Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism. In addition, it was stable for 1 h at 37 degrees C in 0.1% Chaps and Triton X-100.

Structural modeling and characterization of a thermostable lipase from Bacillus stearothermophilus P1.

The moderate thermophilic bacterium Bacillus stearothermophilus P1 expresses a thermostable lipase that was active and stable at the high temperature. Based on secondary structure predictions and secondary structure-driven multiple sequence alignment with the homologous lipases of known three-dimensional (3-D) structure, we constructed the 3-D structure model of this enzyme and the model reveals the topological organization of the fold, corroborating our predictions. We hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and identified Ser-113, Asp-317, and His-358 as the putative members of the catalytic triad that are located close to each other at hydrogen bond distances. In addition, the strongly inhibited enzyme by 10 mM PMSF and 1-hexadecanesulfonyl chloride was indicated that it contains a serine residue which plays a key role in the catalytic mechanism. It was also confirmed by site-directed mutagenesis that mutated Ser-113, Asp-317, and His-358 to Ala and the activity of the mutant enzyme was drastically reduced.

A proteomic analysis of secreted proteins from xylan-induced Bacillus sp. strain K-1.

The expression level of extracellular proteins in an alkaliphilic bacterium, Bacillus sp. strain K-1, grown in a xylan-containing medium, is significantly increased when compared with that grown in the nonxylan culture medium. A proteomic approach has been efficiently applied to separate and characterize these differentially expressed secretory proteins. Eight prominent protein spots were identified and subjected to N-terminal amino acid sequencing. The results show that three spots share considerable similarity with the xylanolytic enzymes and that two spots share considerable similarity with the GltC regulatory protein and 3-dehydroquinate dehydratase, respectively. In addition, the three other proteins show little similarity with the known proteins in the database. In conclusion, our results demonstrate that the proteomic approach is a highly efficient method to rapidly study the differential expression of the secreted proteins by Bacillus sp. strain K-1 grown under xylan-induced condition.

Pen c 1, a novel enzymic allergen protein from Penicillium citrinum. Purification, characterization, cloning and expression.

A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum. The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.

Purification, characterization and molecular cloning of trichoanguin, a novel type I ribosome-inactivating protein from the seeds of Trichosanthes anguina.

The seeds of the plant Trichosanthes anguina contain a type I ribosome-inactivating protein (RIP), designated trichoanguin, which was purified to apparent homogeneity by the combined use of ion-exchange chromatographies, i.e. first with DE-52 cellulose and then with CM-52 cellulose. The protein was found to be a glycoprotein with a molecular mass of 35 kDa and a pI of 9.1. It strongly inhibits the protein synthesis of rabbit reticulocyte lysate, with an IC50 of 0.08 nM, but only weakly that of HeLa cells, with an IC50 of 6 microM. Trichoanguin cleaves at the A4324 site of rat 28 S rRNA by its N-glycosidase activity. The cDNA of trichoanguin consists of 1039 nt and encodes an open reading frame coding for a polypeptide of 294 amino acid residues. The first 19 residues of this polypeptide encode a signal peptide sequence and the last 30 residues comprise an extension at its C-terminus. There are four potential glycosylation sites, located at Asn-51, Asn-65, Asn-201 and Asn-226. A comparison of the amino acid sequence of trichoanguin with those of RIPs such as trichosanthin, alpha-momorcharin, ricin A-chain and abrin A-chain reveals 55%, 48%, 36% and 34% identity respectively. Molecular homology modelling of trichoanguin indicates that its tertiary structure closely resembles those of trichosanthin and alpha-momorcharin. The large structural similarities might account for their common biological effects such as an abortifacient, an anti-tumour agent and anti-HIV-1 activities. Trichoanguin contains two cysteine residues, Cys-32 and Cys-155, with the former being likely to be located on the protein surface, which is directly amenable for conjugation with antibodies to form immunoconjugates. It is therefore conceivable that trichoanguin might be a better type I RIP than any other so far examined for the preparation of immunotoxins, with a great potential for application as an effective chemotherapeutic agent for the treatment of cancer.

Characterization of gammaS-crystallin isoforms from a catfish: evolutionary comparison of various gamma-, gammaS-, and beta-crystallins.

gammaS-Crystallin from catfish eye lenses, formerly designated betas-crystallin in mammalian lenses, is structurally characterized in this study by cDNA cloning and sequencing. To facilitate sequence characterization of gammaS-crystallin with structural properties lying between beta- and gamma-crystallins, a cDNA mixture was constructed from the poly(A)+ mRNA isolated from catfish eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain nucleotide segments encoding multiple gammaS-crystallin isoforms. Sequencing several positive clones revealed that at least two distinct isoforms exist in the gammaS-crystallin class of this teleostean fish, similar to the authentic gamma-crystallin family characterized previously in species of the piscine class. Comparison of protein sequences encoded by two representative catfish gammaS1 and gammaS2 cDNAs with the published sequences of beta-, gamma-, and gammaS-crystallins from shark, carp, bullfrog, bovine, and human lenses indicates that there is about 20-50% sequence homology between catfish gammaS-crystallins and various members of the related beta/gamma-crystallin superfamily from different evolutionary classes, with a higher sequence similarity being found between catfish gammaS- and mammalian gamma-crystallins than between catfish gammaS- and bovine or carp gammaS-crystallins. Phylogenetic trees constructed on the basis of the nucleotide and protein sequence divergence among various beta-, gamma-, and gammaS-crystallins corroborate the closer relatedness of catfish gammaS- to authentic gamma-crystallin than to bovine and carp gammaS-crystallins. The results suggest that evolution of catfish gammaS-crystallins follows a different path from that of bovine and carp gammaS-crystallins and may represent a more ancient offshoot from the ancestral gamma/gammaS coding gene than carp and bovine gammaS-crystallins.

Characterization of phospholipase A2 (PLA2) from Taiwan Cobra: isoenzymes and their site-directed mutants.

Extracellular and secretory phospholipase A2 (PLA2), a class of phospholipid digesting enzyme, is widely distributed in animal venoms of reptiles and insects. Two cDNAs encoding PLA2 isoenzymes from Taiwan Cobra (Naja naja atra) were cloned into pQE-30 plasmid vector and expressed in Escherichia coli. The recombinant products were subjected to refolding using sulfonation under reduction/oxidation conditions with glutathione and enterokinase removal of His-tag, resulting in the active recombinant PLA2 with the same molecular masses of native enzymes as determined by mass spectrometry. The recombinant PLA2 was also shown by circular dichroism to possess a secondary structure similar to native PLA2. The enzymatic activity of the major isoenzyme (PLA2-1) is higher than the other minor isoenzyme (PLA2-2), which shows two amino acid difference from PLA2-1. Site-directed mutagenesis was used to probe the structure/function relationship of two highly conserved residues among all reported PLA2, i.e., His-47 and Asp-93. Replacement of His-47 residue by either Ala or Arg resulted in the complete loss of activity. Similarly, the mutant Asp-93 --> Asn (D93N) also retained little activity. These results suggest that both His-47 and Asp-93 are essential for the catalytic activity of PLA2. Computer graphic study, based on homology modelling, highlights the differences between native PLA2 isoenzymes and their site-directed mutants, which may account for the differences in the observed biological activity.

Characterization of gamma S-crystallin isoforms from lip shark (Chiloscyllium colax): evolutionary comparison between gamma S and beta/gamma crystallins.

gamma S-Crystallin from shark eye lenses, formerly termed beta s crystallin in mammalian lenses, is structurally characterized in this study by cDNA cloning and sequencing. To facilitate sequence characterization of gamma S-crystallin possessing intermediate structural properties between beta- and gamma-crystallins, cDNA mixture was constructed from the poly(A)+ mRNA isolated from shark eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain nucleotide segments encoding multiple shark gamma S-crystallins. Sequencing several positive clones revealed that a multiplicity of isoforms exists in the gamma S-crystallin class of this cartilaginous fish, similar to authentic gamma-crystallin family characterized from the same shark species. Comparison of protein sequences encoded by two representative shark gamma S1 and gamma S2 cDNAs with those published sequences of beta-, gamma-, and gamma S crystallins from bovine, human, bullfrog and carp lenses indicated that there is about 35-64% sequence homology between shark gamma S crystallins and structurally related crystallins from different evolutionary classes, with a higher sequence similarity between shark gamma S and mammalian gamma-crystallins than that of shark gamma S and carp gamma S or bovine gamma S crystallins. A phylogenetic tree constructed on the basis of the sequence divergence among various beta-, gamma-, and gamma S crystallins corroborates the closer relatedness of shark gamma S to authentic gamma-crystallin than to mammalian and teleostean gamma S crystallins. It further strengthens the supposition that ancestral precursors of gamma S-crystallins were present in the shark lens long before the appearance of present-day teleostean and mammalian gamma S-crystallins.

Sequence characterization of gamma-crystallins from lip shark (Chiloscyllium colax): existence of two cDNAs encoding gamma-crystallins of mammalian and teleostean classes.

gamma-Crystallin is a common lens protein of most vertebrate eye lenses and the major protein component in lenses of fishes and in many mammalian species during embryonic and neonatal stages. To facilitate the structural characterization of gamma-crystallin possessing extensive charge heterogeneity, a cDNA mixture was constructed from the poly(A)+ mRNA isolated from shark eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain cDNAs encoding multiple shark gamma-crystallins. Sequencing analysis of multiple positive clones containing PCR-amplified inserts revealed the presence of a multiplicity of isoforms in the gamma-crystallin class of this cartilaginous fish. It was of interest to find that two shark cDNA sequences coexist, one encoding gamma-crystallin (gamma M1) of high methionine content (15.5%) and the other encoding one (gamma M2) of low methionine content (5.1%), each corresponding to the major teleostean and mammalian gamma-crystallins, respectively. Comparison of protein sequences encoded by these two shark cDNAs with published sequences of gamma-crystallins from mouse, bovine, human, frog, and carp lenses indicated that there is about 61-80% sequence homology between different species of the piscine class, whereas only 47-66% is found between mammals and shark. A phylogenetic tree constructed on the basis of sequence divergence among various gamma-crystallin cDNAs revealed the close relatedness between shark gamma M2-crystallin and mammalian gamma-crystallins and that between shark gamma M1 and teleostean gamma-crystallins. The results pointed to the fact that ancestral precursors of gamma-crystallins were present in the sharp lens long before the appearance of modern-day mammalian and teleostean gamma-crystallins.

Sequence analysis of four acidic beta-crystallin subunits of amphibian lenses: phylogenetic comparison between beta- and gamma-crystallins.

beta-Crystallins composed of the most heterogeneous group of subunit chains among the three major crystallin families of vertebrates, i.e. alpha-, beta- and gamma-crystallins, are less well understood at the structural and functional levels than the other two. They comprise a multigene family with at least three basic (betaB1-3) and four acidic (betaA1-4) subunit polypeptides. In order to facilitate the determination of the primary sequences of all these ubiquitous crystallin subunits present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+ mRNA isolated from bullfrog eye lenses. We report here a protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding beta-crystallin acidic subunit polypeptides by polymerase chain reaction (PCR). Four complete full-length reading frames with two each of 597 and 648 base pairs, which cover four deduced protein sequences of 198 (betaA1-1 and betaA1-2) and 215 (betaA3-1 and betaA3-2) amino acids including the universal initiating methionine, were revealed by nucleotide sequencing. They show about 96-98% sequence similarity among themselves and 76-80%, 80-83% to the homologous betaA1/A3 crystallins of bovine and human species respectively, revealing the close structural relationship among acidic subunits of all beta-crystallins even from remotely related species. In this study a phylogenetic comparison based on amino-acid sequences of various betaA1/A3 crystallins plus the major basic beta-crystallin (betaBp) and gamma-crystallin from different vertebrate species is made using a combination of distance matrix and approximate parsimony methods, which correctly groups these betaA crystallin chains together as one family distinct from basic beta-crystallins and gamma-crystallin and further corroborates the supposition that beta- and gamma-crystallins form a superfamily with a common ancestry.

LYS-49 phospholipase A2 homologs from venoms of Deinagkistrodon acutus and Trimeresurus mucrosquamatus have identical protein sequence.

The complete amino acid sequences of the Lys-49 PLA2s from the venom of Deinagkistrodon acutus (from Taiwan and China) and Trimeresurus mucrosquamatus (Taiwan habu) were solved by a facile cDNA cloning and sequencing method. The deduced amino acid sequences of the Lys-49 PLA2s of both venoms are identical, suggesting close phylogenic relationship between this two snake species of different genera. In addition, by cloning and cDNA sequencing, the mRNA coding for a Arg-49 PLA2 homolog of low expression level was also found in the venom gland of T. mucrosquamatus.