[Effect of baseline LH/FSH ratio in PCOS on IVF-ET outcomes: a retrospective cohort study].

Objective: To exlplore the association between the baseline luteinizing hormone/follicle stimulating hormone (LH/FSH) ratio of polycystic ovary syndrome (PCOS) and in vitro fertilisation-embryo transfer outcomes. Methods: This was a retrospective cohort study. A total of 2 868 PCOS patients were enrolled, all of the participants were patients in The First Affiliated Hospital of Anhui Medical University Hospital from October 2015 to October 2021. Propensity score matching (1∶2.5) was conducted to regulate the non-random allocation of patients. Data were extracted from the hospital's medical records. Patients with baseline LH/FSH ratio>2 were deemed as study group, patients with baseline LH/FSH ratio≤2 were deemed as control group. Single factor analysis was applied to compare the differences of pregnancy outcomes between two groups. Results: After propensity score matching (1∶2.5), there were no statistically significant differences in baseline data between the two groups (all P>0.05), indicating that the data were comparable. In the study group, the total dose of gonadotropin (Gn) and duration of Gn were lower than those of the control group (t=4.989, P<0.001; t=3.267, P=0.001), the rate of in vitro maturation was higher than that of the control group (χ2=4.938, P=0.026), the number of retrieved oocytes and cleavage were higher than those of the control group (t=-2.305, P=0.021; t=-2.816, P=0.005), but there were no differences in the number and rate of high-quality embryos between the two groups (t=-1.636, P=0.102; t=-0.123, P=0.902). The incidence of moderate to severe ovarian hyperstimulation syndrome in the study group was significantly higher than that in the control group (χ2=17.277, P<0.001). Regardless of fresh embryo transfer or frozen-thawed embryo transfer cycles, the incidences of gestational diabetes mellitus in the study group were higher than those in the control group (χ2=9.174, P=0.002; χ2=4.204, P=0.040) of singleton pregnancy. In the fresh embryo transfer cycle, the clinical pregnancy rate [30.30% (20/66) vs 47.75% (53/111)] and delivery rate [30.30% (20/66) vs 46.85% (52/111)] in the study group were lower than those in the control group (χ2=5.198, P=0.023; χ2=4.695, P=0.030). In the frozen-thawed embryo transfer cycle, the delivery rate in the study group was higer than that in the control group [59.41% (423/712) vs 55.04% (1 053/1 913); χ2=7.526, P=0.023]. The clinical pregnancy rate and delivery rate of fresh embryo transfer cycle in the study group were significantly lower than those of frozen-thawed embryo transfer cycle (χ2=21.308, P<0.001; χ2=20.871, P<0.001), but there were no significant differences in the control group (all P>0.05). Conclusions: PCOS patients with a higher basal LH/FSH ratio are more likely to develop moderate to severe ovarian hyperstimulation syndrome after controlled ovarian stimulation and have a higher incidence of gestational diabetes mellitus. Better pregnancy outcome could be obtained by frozen-thawed embryo transfer.

[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.

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.

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.

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 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.

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.

Cloning and sequencing of bullfrog growth hormone complementary DNA.

Total mRNA was isolated from the pituitary glands of bullfrog (Rana catesbeiana), purified by affinity chromatography with oligo(dT)-cellulose columns. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The cDNA library was screened by hybridization with 32P-labeled duck growth hormone (GH) cDNA. A positive clone was selected and sequenced. The full-length bullfrog GH cDNA contains 950 nucleotide pairs with an open reading frame coding for the precursor GH of 215 amino-acid residues. The partial amino-acid sequence from the protein confirms that derived from the cDNA, with Phe as the first residue in the mature bullfrog GH preceded by a 25-residue hydrophobic signal peptide. The bullfrog GH shares sequence homology with those of other vertebrate species in the following order: duck (61% protein sequence homology; 67% cDNA homology), rat (56%; 61%), human (47%; 57%) and salmon (42%; 50%).

Purification of duck growth hormone and cloning of the complementary DNA.

Duck growth hormone (GH) was isolated and purified from duck pituitaries by salt precipitation and HPLC on reverse-phase C18 columns. The duck GH was homogeneous as shown by SDS-polyacrylamide gel electrophoresis with a molecular weight of 22,000. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length duck GH cDNA contains 820 nucleotide pairs with an open reading frame coding for the precursor form duck GH of 216 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with Phe as the first residue in mature duck GH preceded by a 27-residue hydrophobic signal peptide. The duck GH is almost completely homologous to the chicken GH, with only three conservative substitutions (Ser for Thr, His for Tyr and Lys for Arg) and one deletion (Ala) in the duck GH sequence. Comparison of amino-acid sequence of duck GH with that of various species reveals 56%, 73% and 40% homologies with GHs of human, rat and salmon, respectively.

Purification of carp growth hormone and cloning of the complementary DNA.

The growth hormone (GH) was isolated and purified from common carp (Cyprinus carpio) pituitary glands by salt precipitation and HPLC on reverse-phase C18 columns. The carp GH cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length carp GH cDNA contains 1187 nucleotide basepairs with an open reading frame coding for the precursor form carp GH of 210 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with serine as the first residue in mature carp GH preceded by a 22-residue hydrophobic signal peptide. Comparison of the amino-acid sequence of carp GH with those of various species reveals positional identity at 32.4%, 38.8%, 42.0%, 37.2%, 66%, 55% and 49% with GHs of man, rat, duck, bullfrog, salmon, tuna and yellow tail, respectively.

Biochemical comparison of epsilon [correction of gamma]-crystallins from duck and frog eye lenses.

A biochemical comparison has been made on the crystallins isolated from duck and frog lenses. Gel-permeation chromatography of lens homogenates from both classes on Fractogel TSK HW-55(S) revealed a homogeneous trimeric protein of 120 kDa in the duck lenses and a monomeric protein of 39 kDa in the frog lenses. Both crystallin fractions consist only of an approx. 38-kDa polypeptide in their subunit structures as determined by SDS gel electrophoresis. These two crystallins were compared with respect to their native molecular masses, subunit structures, peptide mapping and amino acid compositions in order to establish the identity of each crystallin. We have found differences in the protein structures of these two crystallins despite some degree of similarity in their amino acid compositions.

Physicochemical characterization of lens crystallins from the carp and biochemical comparison with other vertebrate and invertebrate crystallins.

Lens crystallins were isolated from the homogenate of carp (Cyprinus carpio) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, immunodiffusion, amino acid analysis, circular dichroism, and protein sequence analysis. Three well-defined fractions corresponding to alpha/beta-, beta-, and gamma-crystallins were obtained in relative weight percentages of 26, 22, and 52%. The native molecular masses of the purified fractions were determined to be 410, 60, and 20 kDa, respectively. The polypeptide compositions as determined by SDS gel electrophoresis revealed the substantial presence of beta-crystallin polypeptides in the alpha-crystallin fraction; this is also evident in the fractionation of amphibian crystallins but is not common in the case of higher classes of vertebrates. The circular dichroism spectra indicate a predominant beta-sheet structure in all three fractions, albeit with some contribution of alpha-helical structure in the gamma-crystallin, the amino acid composition of which bears a resemblance to that of squid crystallin. Sequence comparison of carp gamma-crystallin with frog and calf gamma-crystallins indicates a high degree of homology in their N-terminal segments despite the dissimilarity of amino acid compositions and weak immunological cross-reactivity.

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.

Purification of growth hormones by reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (HPLC) on a column of Radial-Pak C18 cartridge was utilized for the purification of a variety of growth hormone (GH) proteins from mammalian, avian, amphibian and fish pituitary glands. Recovery of GH from pituitary glands of up to 0.43% of total protein was obtained with a high degree of homogeneity as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The HPLC-purified GHs show reactions of identity or near identity by immuno-diffusion studies on agar gel. This method offers a convenient and rapid purification of vertebrate GH on an analytical or preparative scale.

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.

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 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.