Beta A2 Research Articles

Immunolocalization of corneous beta proteins of the Epidermal Differentiation Complex in the developing claw of the alligator

Knowledge on the sharpness, mechanical and hydration resistance of the corneous material of claws requires information on its constituent proteins. The present immunohistochemical study has localized some of the main corneous beta proteins (CBPs, formerly termed beta-keratins) indicated to be present in alligator claws only by genomic data. Using specific antibodies we show the immunolocalization of representative claws CBPs of the Epidermal Differentiation Complex (Beta A1 group) during late stages of claw development in alligator. Intense but asymmetric proliferation, revealed by 5BrdU-immunolabeling, determines the formation of a curved dorsal part (unguis) and a linear ventral part (sub-unguis). The large beta-cells generated in the unguis and their packing into a solid corneous layer occur before thinner beta-cells appear in the sub-unguis. In the latter, CBPs are also immune-detected but with less intensity compared to the unguis, and corneocytes remain separated and desquamate. It is suggested that at the tip of the developing claw beta-corneocytes move downward into the initial part of the sub-unguis. This circular movement contributes to sharpen the claw as these cells fully cornify and are desquamated from the sub-unguis. Corneocytes of the unguis contain 10−16 kDa proline-serine-rich proteins that also possess high percentages of glycine, cysteine, tyrosine, valine and leucine. Cysteines likely give rise to numerous SS bonds in the constituent hard horny material, tyrosine contribute to packing proteins into a dense horny material while glycine, valine and leucine increase the hydrophobic property of claws in these water-adapted predators.

Polymorphisms in PLA2G6 and PLA2G4C genes for calcium-independent phospholipase A2 do not contribute to attenuated niacin skin flush response in schizophrenia patients

We hypothesized that attenuated niacin skin flushing in schizophrenia patients might be associated with polymorphic variants in PLA2G6 and PLA2G4C genes (rs4375 and rs1549637 variations) which encode calcium-independent phospholipase A2 beta (iPLA2β) and cytosolic phospholipase A2 gamma (cPLA2γ) enzymes. The iPLA2β and cPLA2γ may play an important role in niacin-mediated signaling; in addition to their major role – mediating phospholipids remodeling, which alters membrane receptors and signal transduction, they regulate the reservoir of arachidonic acid for prostaglandins synthesis. Skin response to topical niacin of 0.1M, 0.01M, 0.001M and 0.0001M concentrations in 75 schizophrenia patients was rated using the method of volumetric niacin response (VNR). Neither PLA2G6 nor PLA2G4C gene polymorphisms were significantly associated with VNR values. Furthermore, polymorphisms׳ synergy on niacin skin flushing was also not detected.

Differential Expression Patterns of Crystallin Genes during Ocular Development of Olive Flounder (Paralichthys olivaceus).

Olive flounder Paralichthys olivaceus is one of the most widely cultured fish species in Korea. Although olive flounder receive attention from aquaculture and fisheries and extensive research has been conducted eye morphological change in metamorphosis, but little information was known to molecular mechanism and gene expression of eye development- related genes during the early part of eye formation period. For the reason of eyesight is the most important sense in flounder larvae to search prey, the screening and identification of expressed genes in the eye will provide useful insight into the molecular regulation mechanism of eye development in olive flounder. Through the search of an olive flounder DNA database of expressed sequence tags (EST), we found a partial sequence that was similar to crystallin beta A1 and gamma S. Microscopic observation of retinal formation correspond with the time of expression of the crystallin beta A1 and gamma S gene in the developmental stage, these result suggesting that beta A1 and gamma S play a vital role in the remodeling of the retina during eye development. The expression of crystallin beta A1 and gamma S were obviously strong in eye at all tested developing stage, it is also hypothesized that crystallin acts as a molecular chaperone to prevent protein aggregation during maturation and aging in the eye.

Relationship between soluble amyloid beta and secretory phospolipase A2 to the progression of Alzheimer's disease

Relationship between soluble amyloid beta and secretory phospolipase A2 to the progression of Alzheimer's disease

Platelet receptor recognition and cross-talk in collagen-induced activation of platelets.

Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin alpha(2)beta(1) and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR gamma-chain and/or FcgammaRIIa, leading to concerted and co-operative platelet activation. Each receptor has a shear-dependent role, VWF/GpIb essential at high shear, and alpha(2)beta(1) at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation.

Differences in expression of retinal proteins between diabetic and normal rats

To compare and identify the differences in expression of retinal proteins between normal and diabetic rats, and to analyze the molecular pathogenetic mechanisms of retinal diseases caused by diabetes. Changes in protein expression of retinal tissues from diabetic and normal rats were observed using 2-dimensional polyacrylamide gel electrophoresis (2-DE). Some protein spots exhibiting statistically significant variations (P<0.05) were selected randomly and identified by tandem mass spectrometry and analyzed by bioinformatics. 2-DE showed that the expression was up-regulated in 5 retinal proteins, down-regulated in 23 retinal proteins, and disappeared in 8 retinal proteins. Eight spots were identified from the 36 spots by tandem mass spectrometry (MS/MS) and analyzed by bioinformatics. Guanylate kinase 1, triosephosphate isomerase 1, ATP synthase subunit d, albumin and dimethylarginine dimethylaminohydrolase 2 played an important role in signal transduction. Triosephosphate isomerase 1, crystallin alpha B, ATP synthase subunit d and peroxiredoxin 6 were involved in energy metabolism of retinal tissues. Guanylate kinase 1 played an important role in photoexcitation of retinal rod photoreceptor cells. Whether crystallin beta A1 plays a role in diabetic retinas is unknown so far. There are differences in expression of retinal proteins between diabetic and normal rats. These proteins may be involved in the mechanisms and prognosis of retinal diseases caused by diabetes.

Observation ofB0Meson Decay toa1±(1260)π∓

We present a measurement of the branching fraction of the decay B(0)-->a1 (+/)(1260)pi(/+) with a1 (+/)(1260)-->pi(/+)pi(+/)pi(+/). The data sample corresponds to 218 x 10(6) BB[over ] pairs produced in e+e- annihilation through the Upsilon(4S) resonance. We measure the branching fraction Beta(B(0)-->a1(+/)(1260)pi(/+))Beta(a1(+/)(1260)-->pi(/+)pi(+/)pi(+/)) = (16.6+/1.9+/1.5) x 10(-6), where the first error quoted is statistical and the second is systematic.

Proteomic and sequence analysis of chicken lens crystallins reveals alternate splicing and translational forms of beta B2 and beta A2 crystallins.

To characterize the adult chicken lens proteome using mass spectrometry and two-dimensional gel electrophoresis (2-DE). Lens proteins from 10-week old chickens were separated by gel filtration and reversed-phase chromatography, and whole protein masses were measured with electrospray mass spectrometry. Water-soluble lens proteins were separated by 2-DE and identified by tandem mass spectrometry of in-gel digests. Whole protein masses were consistent with all major chicken lens crystallin sequences, except for beta B2 and beta B3. Subsequent cDNA sequencing revealed errors in published sequences translating into 2- and 7-amino-acid differences, respectively, for beta B2 and beta B3, which were in better agreement with the measured masses. Previously uncharacterized forms of beta A2 and beta B2 were observed. The novel form of beta A2 had four fewer amino acids, was more abundant, and resulted from translation at a second start codon. The novel form of beta B2 contained 14 additional amino acids in the interdomain linker and resulted from alternate splicing within intron 4 of the transcript. All examined crystallins, except beta A3, for which data could not be obtained, were N-terminally acetylated, and all beta-crystallins lacked an initial methionine, except for the smaller beta A2 form. In-gel digests identified 29 proteins on the 2-DE map and indicated that truncation occurs within N-terminal extensions of beta-crystallins during lens maturation. The complementary techniques 2-DE, mass spectrometry, and DNA sequencing were used to provide the most complete description of the adult chicken lens proteome to date and identified alternate forms of beta A2 and beta B2.

Identification of a new genetic variant of bovine beta-casein using reversed-phase high-performance liquid chromatography and mass spectrometric analysis.

Various components of the beta-casein fraction from bovine milk were separated by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). They included the genetic variants beta A1, beta A2, beta A3, and an unknown component previously denoted beta X [S. Visser et al., J. Chromatogr. 548 (1991) 361-370]. Tryptic digests of these components were compared by RP-HPLC and most peaks were analysed by mass spectrometry (MS). The tryptic map of beta X was closest to that of beta A1, but with a few mutually different peak components. Electrospray ionisation MS revealed that in the beta X map these components had relative molecular masses of 16 higher than the corresponding ones in the beta A1 map. The main differential peaks represented the 114-169 fragments of beta A1 and beta X, respectively, which were both purified and then cleaved with cyanogen bromide. In the resulting mixtures, each of which contained three fragments, the corresponding peptides representing the 145-156 sequence showed the 16 relative molecular mass difference. In beta X this sequence contained a Leu residue at position 152 instead of the Pro-152 in beta A1, as established by fast-atom bombardment MS-MS. The Leu could be discriminated from an Ile residue by the presence of a side-chain-specific, D-type fragment ion in the MS-MS spectrum of the beta X CNBr peptide. The sequence of the two homologous 145-156 fragments was confirmed by regular amino acid sequence analysis. In accordance with internationally accepted guidelines for the nomenclature of milk proteins, the new genetic variant has been named beta-casein F-5P.

Lys-17 is the amine-donor substrate site for transglutaminase in beta A3-crystallin.

The bovine lens protein beta A3-crystallin has recently been shown to be an amine-donor (Lys) substrate for tissue-type transglutaminase, using a newly developed amine-acceptor hexapeptide as a probe (Groenen, P.J.T.A., Seccia, M., Smulders, R.H.P.H., Gravela, E., Cheeseman, K.H., Bloemendal, H., and de Jong, W.W. (1993) Biochem. J. 295, 399-404). In the present study, the reactive amine-donor site has been identified by site-directed mutagenesis of the putative substrate lysine. The mutation Lys-17-->Arg abolishes the substrate capacity. This residue, located in the N-terminal extension of the polypeptide, thus acts as the sole amine-donor substrate in beta A3-crystallin. Our finding reinforces the notion that, in the crystallins, all amine-donor as well as amine-acceptor substrate sites reside in the N- or C-terminal arms. Transglutaminase-mediated cross-linking of beta A3-crystallin also gives rise to a beta A3 dimer, presumably due to the fact that Lys-17 can be cross-linked to the previously established Gln-7 or Gln-8 amine-acceptor site.

Exposure of β H-crystallin to hydroxyl radicals enhances the transglutaminase-susceptibility of its existing amine-donor and amine-acceptor sites

beta H-crystallin was exposed to radiolytically generated hydroxyl radicals at defined radical concentrations, and its capacity to act as an amine-acceptor substrate and as an amine-donor substrate for transglutaminase were investigated. [14C]Methylamine was used as a probe for labelling amine-acceptor sites; a novel biotinylated hexapeptide was used to label amine-donor sites. The results demonstrate that both primary amine incorporation and hexapeptide incorporation by transglutaminase are considerably increased after oxidative attack on the crystallin. The identity of the labelled subunits was established, and it is shown that, in both cases, this increased incorporation is not due to the production of new substrates, but that the existing incorporation sites become more susceptible. Moreover, using the newly developed probe, we could identify, for the first time, the major crystallin subunits active as amine-donor substrates (both before and after treatment) to be beta B1-, beta A3- and beta A4-crystallin. These data support the proposal that oxidative stress and transglutaminase activity may be jointly involved in the changes found in lens crystallins with age and in the development of cataract.

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

The eye lens beta-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized beta-crystallin sequences (beta A2, beta A3/A1, beta A4, beta B1, beta B2, beta B3), isolated from a bovine lens cDNA library, to Southern blots on which EcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic beta B-crystallins gave hybridization signals in more species than the acidic beta A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for beta A2 was found in trout and lamprey, for beta A4 in trout, and for beta A3/A1 only in toad. The results demonstrate that the duplications leading to the beta-crystallin gene family occurred before or during the earliest stages of vertebrate evolution.

Biochemical identification of I-J as a novel dimeric surface molecule on mouse helper and suppressor T cell clones

A monoclonal anti-I-Jk antibody JK10-23 was capable of precipitating the putative I-Jk molecule from NP-40 lysates of 125I-surface labelled mouse T cell clones with either helper or suppressor functions. The I-J molecule detected by specific immunoprecipitation and subsequent one- or two-dimensional gel analysis was a Mr 84-90 K dimer composed of 42-46 K glycopeptide subunits having isoelectric point pH 5.3 to 6.4. A monomeric form of I-J also existed in some of the T cell clones. The I-J subunit was a glycosylated polypeptide with a 41 K backbone having at least two glycosylation sites. I-J was distinguishable from other known dimeric T cell surface molecules with comparable molecular size, that is, T cell receptor alpha beta heterodimer, A1 and YE molecules expressed on a T cell leukemia EL4, and mouse CD28. The I-Jk molecule was precipitable from T cell clones with I-Ak and I-Ek restriction specificities including a clone derived from an H-2b----H-2bxkF1 radiation bone marrow chimera. None of the H-2b-restricted T cell clones from H-2b and its F1 showed the I-Jk immunoreactivity. T cell clones having either I-Ab or I-Ek restriction specificities derived from intra-H-2 recombinant mouse B10.A(5R) were positive for the I-Jk, while an I-Ab-restricted T cell clone from B10.A(3R) was negative in the I-Jk immunoprecipitation. The results indicate that I-J is a novel dimeric surface molecule, most likely to be a homodimer, expressed on T cells according to the major histocompatibility complex.

Kinetics of the Hb A to Hb C switch and erythropoietin plasma levels in sheep.

The induction of Hb A (alpha 2 beta A2) and Hb C (alpha 2 beta C2) synthesis in three adult sheep has been sequentially analysed, in relation to the reduction of the haematocrit (Ht) and to the changes of erythropoietin (Epo) concentration in plasma. Hb A production is detected in peripheral reticulocytes when the Ht approaches 70% of its initial value in correspondence with the first rise of EPO plasma level, whereas HB C synthesis appears when the Ht is further reduced to about 50%, at an Epo concentration two to three times higher. The assumption that the cell committed to HB C synthesis is close to the erythroid colony-forming unit (CFU-e) progenitor is also discussed.

The cat hemoglobin polymorphism: Southern blot analysis of the beta-globin gene region from cats of various Hb A/Hb B phenotypes.

The molecular basis for the genetic control of variable proportions of the two cat hemoglobins Hb A(alpha 2 beta A2) and Hb B (alpha 2 beta B2) was investigated. Ratios of Hb A/Hb B vary between 50/50 and 90/10 among members of the mongrel cat population, with clusters around 50, 35, and 10% Hb B. Genomic DNA from cats of 50/50, 70/30, and 90/10 phenotypes were cut by restriction endonucleases HindIII, EcoRI, BamHI, BglII, and Pstl and hybridized to a fragment of the human beta-globin gene. The results of the Southern blots suggested a pattern of homozygote, heterozygote, homozygote for the respective cat phenotypes, 50/50, 70/30, and 90/10. Therefore, the cat hemoglobin polymorphism seems to result from the possible combinations of an allelic gene pair.

Evolutionary and functional relationships between the basic and acidic β-crystallins

beta-Crystallins are complex oligomers composed of many related subunits. In order to understand their interactions we have built molecular models of several bovine beta-crystallins, based on their sequence similarity to the well-defined gamma-II crystallin structure, using interactive computer graphics techniques. Their common origin with gamma-crystallin is displayed in both the retention of four-fold sequence repeats of critical residues involved with stabilizing a folded beta-hairpin and the conservation of core-filling hydrophobic side-chains. The beta-crystallins have been built as bilobal molecules with each domain composed of two 'Greek key' motifs which associate about an approximate two-fold axis to form beta-sheets. The beta-crystallin sequences have previously been shown to comprise two families, the basic and acidic subunits, which have extensions of sequence. The three-dimensional models show how the two families appear to stabilize the folded beta-hairpin in the N- and C-terminal domains in ways which suggest that they have diverged from a common ancestor in different ways. Acidic beta-crystallins, like gamma-crystallins, have a regular array of charges on their N-terminal domain which has been interrupted in basic beta-crystallins by hydrophobic residues which may be related to the presence of a C-terminal extension. beta-Crystallins are more highly charged than gamma-crystallins although their charge density is higher in certain regions of the N-terminal domain, particularly in beta B1-crystallin. beta-crystallins also differ from gamma-crystallins in the virtual absence of core-filling sulphydryl groups whereas they have numerous sulphur-containing side-chains together with tryptophan and histidine rings protruding from the globular domains, particularly in the acidic subunits. The burial of these residues in subunit contacts is consistent with their spectroscopic and electrostatic properties. Protein subunit aggregation commonly occurs through hydrophobic interaction or beta-sheet extension. Analysis of the subunit surfaces has identified an N-terminal hydrophobic region common to beta B1 and beta B2 whereas a C-terminal hydrophobic loop region is common to beta B1 and beta A1 and may be correlated with their association properties. It is suggested that the polar C-terminal domain of beta B2 contributes towards the solubility of higher aggregates by interactions involving beta-sheet structure.

Molecular basis for ATP/2,3-bisphosphoglycerate control switch-over (poikilotherm/homeotherm) an intermediate amino-acid sequence in the hemoglobin of the great Indian rhinoceros (Rhinoceros unicornis, Perissodactyla).

The complete primary structure of the two hemoglobin components of the Great Indian Rhinoceros (Rhinoceros unicornis) is presented. The ratio for the two components B(alpha 2 beta I2): A(alpha 2 beta II2) is 6:4. Polypeptide subunits were separated by chromatography on CM-cellulose in a buffer containing 8M urea. The sequence was studied by degradation of the tryptic and hydrolytic cleavage products in a liquid phase sequencer. At position beta NA2 component B has Asp, whereas component A has Glu, an ATP-binding site in fish and reptilian hemoglobins. The other phosphate binding sites i.e. beta NA1 Val, beta EF6 Lys and beta H21 His are identical with 2,3-bisphosphoglycerate-(DPG)binding sites in mammalian hemoglobins, whereby rhinoceros hemoglobin resembles both ATP-sensitive poikilotherm hemoglobin and DPG-sensitive mammalian hemoglobin. The two components (beta I/beta II) additionally differ by exchange of Glu----Gly at position beta A3 and Gln----Lys at position beta GH3. The significance of these changes is discussed. Oxygenation properties of the two hemoglobins components and their dependence on ATP and DPG are given. The structure and function of Rhinoceros hemoglobin may give an insight into the evolution of the organic phosphate binding in vertebrate hemoglobins.

The primary structure of sperm whale hemoglobin (Physeter catodon, cetacea).

The complete primary structure of the two major hemoglobin components of sperm whale (Physeter catodon) is presented. The major components A and B account for 55% and 40% respectively whereas the minor component constitutes for 5% of the total hemoglobin. The globin chains were separated on CM-Cellulose in 8M urea buffer. The sequence was determined by automatic Edman degradation of tryptic and hydrolytic peptides in a liquid phase sequencer. Alignment of the sequence with human hemoglobin shows 22 exchanges each for the alpha I and alpha II and 21 exchanges for the beta I and beta II chains. Within the two beta-chains three differences have been located, beta NA2 His/Gln, beta A2 Gly/Ala and beta A8 Leu/Val. The two alpha-chains are characterized by heterogeneities at position alpha A8 Val/Ile or Ala/Ile (ratio of the phenylthiohydantoin derivatives of the amino acids 1:1) and alpha AB1 Asn/Ser (ratio of the phenylthiohydantoin derivatives of the amino acids 6:4). The role of these exchanges in modulating oxygen affinity is discussed.

The primary structure of hemoglobins from the domestic cat (Felis catus, Felidae).

The complete primary structure of the two hemoglobin components of the domestic cat (Felis catus) is presented. The major component (A) accounts for 60-70% whereas the minor component (B) constitutes 30-40% of the total hemoglobin. Separation of the polypeptides was carried out in buffers containing 8M urea on CM-Cellulose. The sequence was studied by Edman degradation of tryptic and cyanogen bromide cleavage products in a liquid phase sequencer. The sequence is compared for homology with human hemoglobin. The beta-chain of the minor components (beta B) has a blocked N-terminal residue identified as acetylserine whereas that of the major component (beta A) is free glycine. The two hemoglobins have identical alpha-chains and differ with respect to their beta-chains at the following positions (beta B/beta A): beta NA1 Ac-Ser/Gly, beta A1 Ser/Thr, beta H17 Ser/Asn and beta HC1 Arg/Lys. The structural and functional aspects of these exchanges are discussed.

Lens transglutaminase selects specific beta-crystallin sequences as substrate.

A Ca2+-dependent transglutaminase (EC 2.3.2.13) has been demonstrated in the eye lenses of several mammalian species [Lorand, L., Hsu, L. K. M., Siefring, G. E., Jr., & Rafferty, N. S. (1981) Proc. Natl. Acad. Sci. USA 78, 1356-1360]. Using [3H]methylamine as a convenient probe for transglutaminase activity, we have explored the action of this enzyme in the bovine eye lens. We could characterize the glutamine residues acting as acyl-donor sites in three beta-crystallin chains, which are the only substrates for lens transglutaminase among the various lens-specific structural proteins, the crystallins. A single glutamine was found to bind [3H]methylamine in each of these three chains: glutamine -9 in beta Bp (beta B2), glutamine -21 in beta B3, and glutamine -23 or -24 in beta A3. The four glutamines are all located in the NH2-terminal regions, which presumably extend from the compact two-domain structure of the beta-crystallin chains. It was, moreover, established that several components of the lens cytoskeleton are substrates for transglutaminase.