Analysis of the efficacy of azelastine nasal spray combined with mussel mucin in the treatment of allergic rhinitis and the influence of peripheral blood CCL26 and CCR3 levels.

OBJECTIVE: A retrospective study was conducted to investigate the efficacy of azelastine nasal spray combined with mussel mucin in the treatment of allergic rhinitis (AR) and the effects of CCL26 and CC chemokine receptor-3 (CCR3). PATIENTS AND METHODS: A total of 80 patients with AR admitted to our hospital from March 2020 to March 2022 were included as the research objects. All subjects were divided into two groups according to the different therapeutic strategies by reviewing the patient's treatment. The control group (n = 40) was given azelastine nasal spray, while the study group (n = 40) was treated with a combination of mussel mucin and azelastine nasal spray. The clinical efficacy, clinical symptoms, and sleep quality improvement of the two groups were calculated and compared retrospectively. The serological indexes were compared, and the incidence of adverse reactions between the two groups was calculated retrospectively based on the patient's medical records. RESULTS: In the study and control groups, the effective rate was 95.00% and 72.50%. After treatment, the symptom scores of nasal congestions, nasal itching, sneezing, and runny nose and the total score of Pittsburgh sleep quality index (PSQI) in the study group were remarkably less. After treatment, the serum levels of sVCAM-1, interleukin-4 (IL-4), and immunoglobulin E (IgE) were decreased, and the levels of IL-12 were upregulated. Following treatment, Minimum nasal cross-section (NMCA) and total nasal resistance (TNR) at 75Pa in the study group were reduced more noticeably (p < 0.05). After treatment, the expression levels of CCL26 and CCR3 in peripheral blood were significantly decreased. In the control and study groups, the incidence of adverse reactions was 7.50% and 10.00%. CONCLUSIONS: Azelastine nasal spray combined with mussel mucin is effective in the treatment of allergic rhinitis, which can effectively improve patients' clinical symptoms, alleviate nasal ventilation disorders, reduce inflammatory reactions, and improve sleep quality. This strategy of combined treatment is safe and, therefore, worth advocating.

Sequence analyses of mitochondrial gene may support the existence of cryptic species within Ascaridia galli.

Ascaridia galli (Nematoda: Ascaridiidae) is the most common intestinal roundworm of chickens and other birds with a worldwide distribution. Although A. galli has been extensively studied, knowledge of the genetic variation of this parasite in detail is still insufficient. The present study examined genetic variation in the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene among A. galli isolates (n = 26) from domestic chickens in Hunan Province, China. A portion of the cox1 (pcox1) gene was amplified by polymerase chain reaction separately from adult A. galli individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of pcox1 is 441 bp. Although the intra-specific sequence variation within A. galli is 0-7.7%, the inter-specific sequence differences among other members of the infraorder Ascaridomorpha were 11.4-18.9%. Phylogenetic analyses based on the maximum likelihood method using the sequences of pcox1 confirmed that all of the Ascaridia isolates were A. galli, and also resolved three distinct clades. Taken together, the findings suggest that A. galli may represent a complex of cryptic species. Our results provide an additional genetic marker for the management of A. galli in chickens and other birds.

A study of regulatory effects of TLR4 and NF-κB on primary biliary cholangitis.

OBJECTIVE: To investigate the regulatory effects of the Toll-like receptor 4 (TLR4) and the nuclear factor kappa-light-chain-enhancer of the activated B cells (NF-κB) on primary biliary cholangitis (PBC) and to analyze the possible mechanisms. MATERIALS AND METHODS: A total of 24 C57BL/6 mice were randomly divided into M group (n=12, intraperitoneally injected with polyinosinic acid-polycytidine acid (PolyI:C) for 12 consecutive weeks, 2 times/week) and C group (n=12, intraperitoneally injected with the same volume of normal saline). After 12 weeks, the mice were sacrificed to collect liver tissues. Then, an enzyme-linked immunosorbent assay (ELISA) kit was used to detect the content of interleukin-6 (IL-6), IL-10, and tumor necrosis factor-alpha (TNF-α) in liver tissues. Hematoxylin-eosin (HE) staining assay was performed to observe the pathological changes of liver tissues, and measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in peripheral blood of mice. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) staining was applied to determine cell apoptosis in liver tissues. The relative messenger ribonucleic acid (mRNA) expression levels of TLR4 and NF-κB in liver tissues were detected by quantitative Polymerase Chain Reaction (qPCR). Western blotting was adopted to measure the protein expressions of TLR4, NF-κB, myeloid differentiation factor 88 (MyD88), B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax), and Caspase-3. RESULTS: Compared with that in C group, the content of IL-6 and TNF-α in liver tissues in M group was significantly increased (p<0.01), but the level of IL-10 was statistically downregulated (p<0.01). According to HE staining, liver damage of mice in M group was evidently severer than that in C group, and the levels of ALT and AST in M group were significantly higher than those in C group (p<0.01). The amount of TUNEL-positive cells in liver tissues in M group was significantly greater than that in C group (p<0.01). The levels of TLR4 and NF-κB mRNA in liver tissues from M group were significantly elevated in comparison with the C group (p<0.01). Compared with those in C group, the expressions of TLR4, NF-κB, MyD88, and Caspase-3 proteins in M group showed statistical increases in liver tissues (p<0.01), whereas that of Bcl-2/Bax was significantly declined (p<0.01). CONCLUSIONS: PBC activates the TLR4/MyD88/NF-κB signaling pathway, induces the release of inflammatory factors and produces a large number of apoptotic proteins, which results in liver damage and cell apoptosis in mice.

Effect of nicotine withdrawal on pain sensitivity in rats to mechanical stimulation and thermal stimulation.

OBJECTIVE: To establish an improved rat model of nicotine withdrawal and dependence by subcutaneous injection of pure nicotine, and observe the effect of nicotine withdrawal on the pain sensitivity in rats. MATERIALS AND METHODS: 30 SD rats were randomly divided into 5 groups with 6 rats in each group, including the control group, normal saline group (NS group), nicotine group of 3 mg/kg/d (NT3 group), nicotine group of 9 mg/kg/d (NT9 group) and nicotine group of 18 mg/kg/d (NT18 group). The 5 groups were respectively subcutaneously injected with nothing, normal saline, 1 mg/kg nicotine, 3 mg/kg nicotine and 6 mg/kg nicotine with 3 times per day for 7 consecutive days. 60 min after last injection in the 7th d, 1 mg/kg mecamylamine was subcutaneously injected. The body weight change, survival and nicotine withdrawal score of rats were observed during injection of nicotine and after withdrawal. Mechanical withdrawal threshold (MWT) and Thermal withdrawal latency (TWL) in the right hind sole of another 18 rats selected from the control group, NS group and NT9 group (6 rats from each group) were respectively tested in 7d after injection of normal saline or nicotine. RESULTS: Compared with the NT3 group, the body weight of rats in the NT9 group and NT18 group were slowly increased in 7d after injection of nicotine (p < 0.05), but were rapidly increased in 1d and 2d after withdrawal (p < 0.01). Rats in the NT9 group and NT18 group had more withdrawal symptoms after stimulation with mecamylamine (p < 0.01), but the mortality of rats in the NT18 group reached 17%. Compared with the control group, MWT in the rats of the NT9 group were significantly decreased in 1-7d after nicotine withdrawal (p < 0.01), and were particularly significantly decreased in 1d and 2d (p < 0.01); TWL was also significantly decreased (p < 0.01), and was most significantly decreased in 4d (p < 0.01). CONCLUSIONS: An improved rat model of nicotine dependence and withdrawal can be successfully established by intermittent subcutaneous injection of nicotine at 9 mg/kg/d for 7 days, and the pain sensitivity in rats is increased after nicotine withdrawal.

[Advances in the expression of foreign genes in Hansenula polymorpha].

Hansenula polymorpha is a potential host for foreign gene expression, which has been applied widely in academic studying and industry application. It has a number of advantages of expressing genes derived from eukaryotic organisms, such as mitotically stable recombinant strains, faithful processing of the produced polypeptides, and high productivity et al. Numerous foreign proteins with high commercial value have been expressed successfully in H. polymorpha, among which some have been launched on the market. In this review, the favorable characteristics of this system for foreign gene production and new advances are described.

Synthesis and structural characterization of the novel cluster compound.

Reaction of [Mo3Y(mu-S)3(dtp)4(H2O)] (Y = O, S; dtp = S2P(OC2H5)2(-)) with HgI2 gave the novel compound [[Mo3S7(dtp)3]4 x I][(HgI3)3] x 4H2O (1), which contains a [[Mo3S7(dtp)3]4 x I] tetramer and (HgI3)-. Compound 1 has been characterized by IR, Raman, UV/Vis, and NMR spectroscopy and single-crystal X-ray diffraction analysis. It is shown that this formation process can be referred to as a new cluster reaction. The structure and spectroscopic data of the tetramer is also compared with that of the related discrete cluster [Mo3S7(dtp)3 x I]. Crystal data: space group F23, a = 26.786(3) A, V = 19218.7(4) A3, Z = 4, R = 0.059.

Cloning, sequencing, and functional analysis of H-OLE1 gene encoding delta9-fatty acid desaturase in Hansenula polymorpha.

H-OLE1, a gene encoding delta9-fatty acid desaturase (FAD) in Hansenula polymorpha strain CBS 1976, was isolated by hybridization based upon its homology with the P-OLE1 gene cloned earlier from a related species, Pichia angusta IFO 1475. The sequence of the H-OLE1 gene revealed high structural conservation with delta9-FADs from various organisms. A putative 451-amino acid polypeptide encoded by the gene, like all other delta9-FADs, contained two domains: an N-terminal catalytic domain containing three conserved histidine clusters, and a C-terminal cytochrome b5-like domain which has been suggested to be involved in electron transport in desaturation reactions. The whole H-OLE1 gene complemented a H. polymorpha fad1 mutation leading to a defect in delta9-FAD. However, the unsaturated fatty acid requirement that the Saccharomyces cerevisiae ole1 mutant displays was complemented by only the open reading frame of H-OLE1 driven by S. cerevisiae glyceroaldehyde-3-phosphate dehydrogenase promoter, but not by the intact H-OLE1, suggesting that the H. polymorpha delta9-FAD was compatible with the desaturation system of S. cerevisiae whereas the promoter of the H-OLE1 gene had no activity in heterologous cells. It was shown by Northern hybridization that transcription of the H-OLE1 gene in H. polymorpha was slightly repressed by exogenous delta9-unsaturated fatty acid. An H. polymorpha disruption mutant (deltaH-OLE1) was created by transformation of an fad1/FAD1 diploid with disrupted H-OLE1::S-LEU2 linear DNA. It was shown by genetic and molecular analyses that input DNA was integrated in several copies into the chromosomal target to replace the mutated fad1 allele. Gas chromatography analysis showed identical fatty acid compositions in cells of both fad1 and deltaHOLE1 disruption mutants.

Syntheses of (R)- and (S)-2- and 6-fluoronorepinephrine and (R)- and (S)-2- and 6-fluoroepinephrine: effect of stereochemistry on fluorine-induced adrenergic selectivities.

Several routes to the enantiomers of fluoronorepinephrines (1) and fluoroepinephrines (2) were explored. A catalytic enantioselective oxazaborolidine reduction and a chiral (salen)Ti(IV) catalyzed asymmetric synthesis of silyl cyanohydrins proved efficacious in the key stereo-defining steps of two respective routes. Binding studies of the catecholamines with alpha(1)-, alpha(2)-, beta(1)-, and beta(2)-adrenergic receptors were examined. The assays confirmed that fluorine substitution had marked effects on the affinity of (R)-norepinephrine and (R)-epinephrine for adrenergic receptors, depending on the position of substitution. Thus, a fluoro substituent at the 2-position of (R)-norepinephrine and (R)-epinephrine reduced activity at both alpha(1)- and alpha(2)-receptors and enhanced activity at beta(1)- and beta(2)-receptors, while fluorination at the 6-position reduced activity at the beta(1)- and beta(2)-receptors. The effects of fluorine substitution on the S-isomers were less predictable.

Synthesis and characterization of three tetranuclear clusters containing a [Mo3OS3Sn]6+ cubane-like core.

Three heterometallic cubane-like clusters, [Mo3(mu 3-O)(mu 3-S)3(SnCl3)(dtp)3(py)3] (dtp = S2P(OC2H5)2-, py = C5H5N) (1), (PPN)[Mo3(mu 3-O)(mu 3-S)3(SnCl3)(dtp)3(mu-OAc)(py)] (OAc = CH3COO-, PPN = (C6H5)3PNP(C6H5)3+) (2), and (Et4N)[Mo3(mu 3-O)(mu 3-S)3(SnCl3)(dtp)2(mu-OAc)2(py)] (3) have been prepared by the reaction of [Mo3(mu 3-O)-(mu-S)3(dtp)4(H2O)] (4), [Mo3(mu 3-O)(mu-S)3(dtp)3(OAc) (py)] (5), and [Mo3(mu 3-O)(mu-S)3(dtp)2(OAc)2 (py)] (6) with SnCl2, respectively. They have been characterized by IR, UV-vis, 31P NMR, 95Mo NMR, and X-ray structure analysis. All of these heterometallic clusters have a [Mo3OS3Sn]6+ core but contain a different arrangement of peripheral ligands. As far as the neutral cluster 1 is concerned, there is no bridging OAc ligand, while only one bridging OAc ligand is observed for cluster 2 and two are for cluster 3. The Mo-Mo distances are about 0.03-0.04 A shorter than those of the starting trimolybdenum clusters. This indicates that the incorporation of SnCl3- fragment into (Mo3) clusters makes the Mo-Mo bonding enhanced. Crystal data for 1: triclinic, space group P-1, a = 10.7423(2) A, b = 14.0357(1) A, c = 16.9346(2) A, alpha = 84.054(1) degrees, beta = 87.095(1) degrees, gamma = 84.517(1) degrees, V = 2525.82(6) A3, Z = 2, R = 0.038 for 5584 reflections (I > 2.0 sigma(I)). Crystal data for 2: triclinic, space group P-1, a = 12.9529(1) A, b = 15.6324(2) A, c = 19.6355(1) A, alpha = 92.083(1) degrees, beta = 97.908(1) degrees, gamma = 110.337(1) degrees, V = 3677.41(6) A3, Z = 2, R = 0.034 for 8665 reflections (I > 2.0 sigma(I)). Crystal data for 3: monoclinic, space group P2(1)/n, a = 14.0852(5) A, b = 15.1324(5) A, c = 23.2691(7) A, beta = 97.371(1) degrees, V = 4918.7(3) A3, Z = 4, R = 0.049 for 4970 reflections (I > 2.0 sigma(I)).

Specificity of ascorbate analogs for ascorbate transport. Synthesis and detection of [(125)I]6-deoxy-6-iodo-L-ascorbic acid and characterization of its ascorbate-specific transport properties.

Cellular ascorbic acid accumulation occurs in vitro by two distinct mechanisms: transport of ascorbate itself or transport and subsequent intracellular reduction of its oxidized product, dehydroascorbic acid. It is unclear which mechanism predominates in vivo. An easily detectable compound resembling ascorbate but not dehydroascorbic acid could be a powerful tool to distinguish the two transport activities. To identify compounds, 21 ascorbate analogs were tested for inhibition of ascorbate or dehydroascorbic acid transport in human fibroblasts. The most effective analogs, competitive inhibitors of ascorbate transport with K(i) values of 3 microM, were 6-deoxy-6-bromo-, 6-deoxy-6-chloro-, and 6-deoxy-6-iodo-L-ascorbate. No analog inhibited dehydroascorbic acid transport. Using substitution chemistry, [(125)I]6-deoxy-6-iodo-L-ascorbate (1.4 x 10(4) mCi/mmol) was synthesized. HPLC detection methods were developed for radiolabeled and nonradiolabeled compounds, and transport kinetics of both compounds were characterized. Transport was sodium-dependent, inhibited by excess ascorbate, and similar to that of ascorbate. Transport of oxidized ascorbate and oxidized 6-deoxy-6-iodo-L-ascorbate was investigated using Xenopus laevis oocytes expressing glucose transporter isoform GLUT1 or GLUT3. Oxidation of ascorbate or its analog in media increased uptake of ascorbate in oocytes by 6-13-fold compared with control but not that of 6-deoxy-6-iodo-L-ascorbate. Therefore, 6-deoxy-6-iodo-L-ascorbate, although an effective inhibitor of ascorbate transport, either in its reduced or oxidized form was not a substrate for dehydroascorbic acid transport. Thus, radiolabeled and nonradiolabeled 6-deoxy-6-iodo-L-ascorbate provide a new means for discriminating dehydroascorbic acid and ascorbate transport in ascorbate recycling.

Androgen receptor in mouse brain: sex differences and similarities in autoregulation.

The androgen receptor (AR) is generally considered an autoregulated protein. However, studies in brain have produced mixed results regarding sex differences, which should be present given the higher endogenous levels of androgens in males, and the effects of gonadectomy, which presumably should lead to a loss of AR. Resolving these issues is a necessary step in developing a model of AR regulation in the central nervous system and, more broadly, in determining how regulation of this receptor may mediate neural target tissue responsiveness to androgen. To further investigate these issues, the distribution, density, and regulation of neural AR were compared among male and female mice that were intact, gonadectomized, or gonadectomized and given testosterone propionate (TP) through immunocytochemical and Western blot analyses. Four brain areas that have been linked to the regulation of male-typical behavior were evaluated: bed nucleus of the stria terminalis, posterior aspect, medial preoptic area, and dorsal and ventral aspects of the lateral septum. In the immunocytochemical study, integrated particle density, which reflects the average intensity of AR staining, was assessed among the six groups 24 h after surgery using PG-21, a peptide-based AR antiserum. Major findings included regional differences in the intensity of immunostaining; a robust sexual dimorphism in each region, with males exhibiting more intense staining than females; a loss of AR in both sexes after gonadectomy, with more dramatic changes evident in males; and significant up-regulation of AR in response to TP that was equivalent in both sexes. The Western blot analyses of AR in limbic system extracts prepared from the six groups showed a pattern of differences that mirrored the immunocytochemical results, indicating that PG-21 recognized both liganded and unliganded AR. In addition, a dose-response study, in which gonadectomized males and females were administered from 25-1000 microg TP, demonstrated a significant linear trend in up-regulation of AR in both males and females, with no sexual dimorphism in the response to hormone treatment. These results demonstrate that the regulation of AR in both male and female neural tissue is comparable and that the critical determinant of AR expression is the presence or absence of androgen.

Mutation analysis of the 6-pyruvoyl-tetrahydropterin synthase gene in Chinese hyperphenylalaninemia caused by tetrahydrobiopterin synthesis deficiency.

Hyperphenylalaninemia (HPA) may be caused by deficiency of phenylalanine hydroxylase or tetrahydrobiopterin (BH4), the essential cofactor for the aromatic amino acid hydroxylases. 6-Pyruvoyl-tetrahydropterin synthase (PTPS) deficiency is a major cause of BH4 deficient HPA. In this study, seven single base mutations at nucleotides 73 (C>G), 155 (A>G), 166 (G>A), 209 (T>A), 259 (C>T), 286 (G>A), and 317 (C>T) on PTPS cDNA were detected in Chinese PTPS-deficient HPA by polymerase chain reaction and solid phase DNA sequencing. These nucleotide alterations result in R25G, N52S, V56M, V70D, P87S, D96N, and T106M amino acid substitutions, respectively. The R25G, V56M, V70D, and T106M were novel mutations found in PTPS gene. By analysis of 38 PTPS mutant alleles from 19 unrelated Chinese PTPS-deficient HPA families, the allele frequency of these mutations in Chinese PTPS-deficient HPA were determined to be approximately 5.3% (R25G), 34.2% (N52S), 7.9% (V56M), 2.6% (V70D), 36.8% (P87S), 7.9% (D96N), and 2.6% (T106M), respectively. Two common mutations, N52S and P87S, were found to account for 71% of the Chinese PTPS mutant alleles. The N52S mutation accounts for 48% of the southern Chinese PTPS mutation, but only one (9%) of the northern Chinese PTPS mutant allele was found to be N52S, which suggested that the N52S mutation might be southern Chinese. Clinically, the V56M mutation was found to associate with the mild form of PTPS deficiency. However, the R25G, N52S, P87S, and D96N were found mainly in the patients with severe clinical symptom. Using polymerase chain reaction-based mutation analysis, a fetus at risk of PTPS deficiency was diagnosed prenatally to be a carrier of N52S mutation.

Testosterone and its metabolites modulate 5HT1A and 5HT1B agonist effects on intermale aggression.

Our understanding of the neurochemical and neuroendocrine systems' regulating the display of offensive intermale aggression has progressed substantially over the past twenty years. Pharmacological studies have shown that serotonin, via its action at 5HT1A and/or 5HT1B receptor sites, modulates the display of intermale aggressive behavior and that its effects serve to decrease behavioral expression. Neuroendocrine investigations, in turn, have demonstrated that male-typical aggression is testosterone-dependent and studies of genetic effects, metabolic function and steroid receptor binding have shown that facilitation of behavioral displays can occur via independent androgen-sensitive or estrogen-sensitive pathways. Remarkably, there have been virtually no studies that examined the interrelationship between these facilitative and inhibitory systems. As an initial step toward characterizing the interaction between the systems, studies were conducted that assessed hormonal modulation of serotonin function at 5HT1A and 5HT1B receptor sites. They demonstrated: (1) that the androgenic and estrogenic metabolites of testosterone differentially modulate the ability of systemically administered 8-OH-DPAT (a 5HT1A agonist) and CGS12066B (a 5HT1B agonist) to decrease offensive aggression; and (2) when microinjected into the lateral septum (LS) or medial preoptic area (MPO), the aggression-attenuating effects of 1A and 1B agonists differ regionally and vary with the steroidal milieu. In general, the results suggest that estrogens establish a restrictive environment for attenuation of T-dependent aggression by 8-OH-DPAT and CGS 12066B, while androgens either do not inhibit, or perhaps even facilitate, the ability of 5HT1A and 5HT1B agonists to reduce aggression. Potential mechanisms involved in the production of these steroidal effects are discussed and emerging issues that may impact on efforts to develop an integrative neurobiological model of offensive, intermale aggression are considered.

Serotonin agonist-induced decreases in intermale aggression are dependent on brain region and receptor subtype.

Testosterone (T) and its androgenic and estrogenic metabolites modulate the ability of serotonin (5-HT)1A and 5-HT1B agonists to inhibit intermale aggressive behavior. This study tested whether the lateral septum (LS) and medial preoptic area (MPO), which are part of the neuroanatomical substrate for aggression and contain androgen, estrogen, 5-HT1A and 5-HT1B receptors, represent sites where these modulatory effects occur. Gonadectomized CF-1 male mice were given silastic implants containing diethylstilbestrol (DES, a synthetic estrogen) or dihydrotestosterone (DHT, a nonaromatizable androgen) and implanted bilaterally with guide cannula directed at the LS or MPO. They were microinjected with either CGS12066B, a 5-HT1B agonist (400 microM LS, 200 microM MPO); 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A agonist (10 microM LS, 5 microM MPO); or combined CGS + 8-OH-DPAT treatment and tested for aggression 15 min later. When microinjections were given in the LS, androgen-treated males exhibited significantly reduced attack behavior in response to CGS or to CGS + 8-OH-D PAT. The attack behavior of DES-treated males was not reduced by any of the treatments. In contrast, all agonist treatments decreased aggression when injected into the MPO in both hormone conditions. The findings demonstrate regional variation in the ability of androgens and estrogens to modulate 5-HT1A- and 5-HT1B-agonist mediated reductions in aggression.

Development and expression of hormonal systems regulating aggression.

There are multiple pathways involved in the regulation of male typical aggression by T, and the functional pathway is determined by genotype. Target-tissue sensitivity to the aggression-promoting properties of T and its estrogenic and androgenic metabolites is determined by a complex sequence of events in which steroid receptors play a critical role. To date, it appears that the relative density of AR may be an important factor in the biobehavioral effects of androgens. Regarding sensitivity to estrogens, characterization of ER-NM interactions, and understanding of the contribution of the two activating functions within ER, appears to be necessary to comprehensively describe the cellular basis for responsiveness to the aggression-promoting effect of this T metabolite. In broader terms, these observations indicate that understanding the relationship between T and the expression of aggression in humans will require models that incorporate cellular aspects of steroid hormone action, including metabolism, receptor function, and gene regulation.

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.

Characterization of gamma-crystallin from the eye lens of bullfrog: complexity of gamma-crystallin multigene family as revealed by sequence comparison among different amphibian species.

gamma-Crystallin is the major and most abundant lens protein present in the eye lens of lower vertebrates such as amphibian and piscine species. To facilitate structural characterization of gamma-crystallins isolated from the lens of the bullfrog (Rana catesbeiana), a cDNA mixture was synthesized from the poly(A)+mRNA isolated from fresh eye lenses. cDNA encoding gamma-crystallin was then amplified using polymerase chain reaction (PCR) based on two primers designed according to the relatively conserved N- and C-terminal sequences of known gamma-crystallins from teleostean fishes. PCR-amplified product corresponding to gamma-crystallin isoforms was obtained, which was then subcloned in pUC18 vector and transformed into Escherichia coli strain JM109. Plasmids containing amplified gamma-crystallin cDNAs were purified and prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing several clones containing DNA inserts of about 0.54 kb revealed the presence of two isoforms with an open reading frame of 534 base pairs, covering two gamma-crystallins each with a deduced protein sequence of 177 amino acids including the translation-initiating methionine. These gamma-crystallins of pI 6.364 and 6.366 contain a low-methionine content of 2.81%, in contrast to 11-16% obtained for those gamma-crystallins with high-methionine content from most teleostean lenses. Pairwise sequence comparison of bullfrog gamma-crystallins with those published sequences of gamma-crystallins from carp, shark, Xenopus and another Rana frog, bovine, and human lenses indicates that there is only 46-63% sequence similarity among these species, revealing that amphibians possess a very complex and heterogeneous group of gamma-crystallins even from closely related species of Rana frogs. The sequence analysis and comparison of various isoforms of the frog gamma-crystallin family provide a firm basis for identifying these lens proteins as members of a multigene family more complex than that reported for mammalian gamma-crystallins.

Sequence analysis of one major basic beta-crystallin (beta Bp) of amphibian lenses: evolutionary comparison and phylogenetic relatedness between beta- and gamma-crystallins.

beta Bp-Crystallin, a major basic beta-crystallin of vertebrate eye lens, is developmentally regulated during the process of amphibian metamorphosis. In order to facilitate the determination of the primary sequence of this ubiquitous crystallin present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+ mRNA isolated from bullfrog eye lenses. A protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding beta Bp-crystallin by polymerase chain reaction (PCR). PCR-amplified product corresponding to beta Bp-crystallin was then ligated into pGEM-T vector and then transformed into E. coli strain JM109. One complete full-length reading frame of 615 base pairs, which covers a deduced protein sequence of 205 amino acids, including the universal initiating methionine, was revealed by automatic nucleotide sequencing with a fluorescence-based dideoxynucleotide chain-termination method. It shows 83, 74, 78 and 80 percent sequence similarity to the homologous beta 2 crystallins of chicken, rat, bovine, and human species, respectively, revealing the close structural relationship among beta Bp-crystallins even from remotely related species. In this study phylogenetic trees based on nucleotide and protein sequences of various beta- and gamma-crystallins from different vertebrate classes are constructed using a combination of distance matrix and approximate parsimony methods, which corroborate the previous supposition that beta- and gamma-crystallins form a superfamily with a common ancestry.

Sequence analysis of frog alpha B-crystallin cDNA: sequence homology and evolutionary comparison of alpha A, alpha B and heat shock proteins.

alpha-Crystallin is a major lens protein present in the lenses of all vertebrate species. Recent studies have revealed that bovine alpha-crystallins possess genuine chaperone activity similar to small heat-shock proteins. In order to facilitate the determination of the primary sequence of amphibian alpha B-crystallin, cDNA encoding alpha B subunit chain was amplified using a new "Rapid Amplification of cDNA Ends" (RACE) protocol of Polymerase Chain Reaction (PCR). PCR-amplified product corresponding to alpha B subunit was then subcloned into pUC18 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the automatic fluorescence-based dideoxynucleotide chain-termination method. Sequencing more than five clones containing DNA inserts coding for alpha B-crystallin subunit constructed only one complete full-length reading frame of 522 base pairs similar to that of alpha A subunit, covering a deduced protein sequence of 173 amino acids including the universal translation-initiating methionine. The frog alpha B crystallin shows 69, 66 and 56% whereas alpha A crystallin shows 83, 81 and 69% sequence similarity to the homologous chains of bovine, chicken and dogfish, respectively, revealing a more divergent structural relationship among these alpha B subunits as compared to alpha A subunits. Structural analysis and comparison of alpha A- and alpha B-crystallin subunits from eye lenses of different classes of vertebrates also shed some light on the evolutionary relatedness between alpha B/alpha A crystallins and the small heat-shock proteins.