Simultaneous phenotyping of pig plasma α-protease inhibitors (PI1, PO1A, PO1B, PI2) and four other proteins (PO2, TF, CP, HPX) by a simple method of 2D horizontal electrophoresis

A simple and rapid method of 2D agarose gel (pH 5.4)-horizontal polyacrylamide gel (pH 9.0) electrophoresis was developed for the simultaneous phenotyping of pig plasma alpha-protease inhibitors (protease inhibitor-1 and -2; postalbumin-1A and -1B), postalbumin-2, transferrin, ceruloplasmin and haemopexin. These eight plasma proteins were clearly visible on gels stained with Coomassie Brilliant Blue G250. The 2D patterns and mobilities of several variants of alpha-protease inhibitors were described. By using two agarose gels and 10 polyacrylamide gels, 120 samples were easily analysed in a day. Since alpha-protease inhibitors show extensive polymorphism and as the gene for postalbumin-2 is closely linked to the halothane sensitivity locus Hal, this method is a useful tool for conducting parentage control and for predicting Hal genotypes of individual pigs.     

Genetic variation at a pig serum protein locus, Po-2 and its assignment to the Phi, Hal, S, H, Pgd linkage group

Two-dimensional agarose gel (pH 5.4)-polyacrylamide gel (pH 9.0) electropho-resis of pig serum samples revealed a new serum protein (postalbumin-2, PO-2) polymorphism. Family data supported the hypothesis that the three PO-2 phenotypes observed were controlled by two codominant, autosomal alleles ( Po-2^F and Po-2^S ) at a single locus. The frequency of Po-2^F in Swedish Landrace and in Swedish Yorkshire breeds was estimated at 0.74 and 0.65, respectively. Evidence was presented for close genetic linkage between Po-2 and the red cell phospho-hexose isomerase locus ( Phi ). A recombination frequency of 3.2 % was obtained from double backcross material. Data obtained in a Danish Landrace material showing linkage between the Po-2 locus and the H blood group locus, the Pgd locus and Hal (locus for halothane sensitivity) are also given. A total of seven re-combinants were observed. They show that Po-2 is a new locus in a previously established linkage group. The likely sequence of the loci is: Phi, Hal, S, H, Po-2, Pgd .     

Localization of the Po2 locus in the S, Phi, Hal, H, Po2, Pgd linkage group in pigs

The localization of the Po2 locus controlling a polymorphic serum postalbumin was studied in 41 families of the Czech Landrace breed. The haplotypes involving six closely linked loci (S, Phi, Hal, H, Po2, Pgd) were determined for each family member. The crossovers observed between the H, Po2 and Pgd loci indicated that Po2 is located between H and Pgd. The Po2 locus appears to be closer to H [theta = 0.54% (0.06%-1.92%)] than to Pgd [theta = 4.02% (1.67%-7.96%)]. A strong Ha-Po2S association (r = 0.96, P less than 0.001) and H-PO2 linkage disequilibrium (D = 0.2218, P less than 0.01, D/DMax = 0.98) were found.     

Genetic polymorphism and close linkage of two α1-protease inhibitors in horse serum

Two-dimensional electrophoretic analysis of horse serum proteins was done by a first-dimension separation in agarose gel (pH 5.4) followed by a second-dimension separation in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many α-globulins. Two groups of aj-globulins, designated Pi1 and Pi2, were found to be protease inhibitors. Preliminary studies indicated that Pi1 and Pi2 proteins differed from each other in molecular weight and in protease inhibiting spectra. Extensive polymorphism was observed for both these proteins. Family data supported the hypothesis that Pi1 and Pi2 types were controlled by autosomal codominant alleles. For both Pi1 and Pi2 systems, most of the homozygous types showed two fractions each while the heterozygous types had 4 fractions. Six Pi1 and five Pi2 alleles were observed in two breeds of Swedish horses. Complete genetic linkage was observed for Pi1 and Pi2 loci as no recombinant type was observed in 40 informative matings studied.     

Polymorphic serum prealbumin (Pa) of pig, identified as an a!-protease inhibitor

Pig serum proteins were analysed by horizontal polyacrylamide gel electrophoresis, with a discontinuous buffer system (pH 9.0). A 12 % acrylamide concentration in the separation gel was used. Each of the two prealbumin (Pa) alleles gave rise to two closely migrating fractions. The polymorhic Pa was identified as an a,-protease inhibitor as the Pa fractions inhibited the esterolytic activity of both bovine trypsin and chymotrypsin. Therefore, it has been proposed that the locus symbol for this prealbumin be changed to Pi-1. The protease inhibitory spectra and electrophoretic mobility of the Pa (Pi-1) fractions suggested that this protein was probably the same as the pig serum a,-protease inhibitor described in some earlier studies and that it corresponds to human serum a,-protease inhibitor (Pi).     

Linkage between the porcine genes encoding immunoglobulin heavy-chain allotypes and some serum α-protease inhibitors: a conserved linkage in pig, mouse and human

Linkage between the genes coding for immunoglobulin heavy-chain allotypes and some serum alpha-protease inhibitors was demonstrated in pigs by means of segregation data in families. A recombination frequency of about 8% was estimated. This represents evolutionary conservation of an autosomal linkage group as linkage between the homologous loci has previously been reported, in humans and in mice.     

Extensive genetic polymorphism of four plasma α-protease inhibitors in pigs and evidence for tight linkage between the structural loci of these inhibitors

Summary. Two-dimensional horizontal gel electrophoresis of pig plasma samples (under non-denaturing conditions) using Immobiline pH gradient gels 4.0–6.0 for the first dimension separation, resulted in clear resolution of the variants of four different α-protease inhibitors (protease inhibitor -1 and -2, P11 and P12; postalbumin -1A and -1B, PO1A and PO1B). All these variants were readily visualized by general protein staining. About 900 families each of Swedish Landrace (SL) and Yorkshire (SY) breeds were studied. The extensive inheritance data, including the recombinants encountered, indicated that each of these four inhibitors is controlled by a separate, autosomal locus and that the four loci are tightly linked (spread over a distance of 1–1.5 cM) with the order as Pil-Po1A-Po1B-Pi2 . The alleles observed were two of Pil, 14 of Po1A , 11 of Po1B and 8 of Pi2 . About 40 haplotypes were observed in each of the two breeds. The allele frequencies at Po1A, Po1B and Pi2 loci were remarkably different in the two breeds; the alleles at these three loci showed a very strong linkage disequilibrium (0.8–1.0). The females showed much higher recombination frequencies than the males in the Po1A-Pi2 interval, suggesting that gene conversion-like events may be occurring at these loci. This linkage in pigs and similar ones comprising some plasma α-protease inhibitor genes in humans and in rodents, reported recently in the literature, indicate evolutionary conservation of a homologous linkage group in these species.     

Expression and characterization of the human 3β-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b)

The human hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST), is highly expressed in liver and adrenal cortex and displays reactivity towards a broad range of hydroxysteroids including 3β-hydroxysteroids, 3-hydroxysteroids, estrogens with a 3-phenolic moiety, and 17-hydroxyl group of androgens. In contrast, characterization of the newly described human hydroxysteroid sulfotransferase SULT2B1 isoforms shows that these enzymes are selective for the sulfation of 3β-hydroxysteroids, such as pregnenolone, epiandrosterone, DHEA, and androstenediol. There was no activity detected towards testosterone, dexamethasone, β-estradiol, androsterone, or p-nitrophenol. The SULT2B1 gene encodes two isoforms, SULT2B1a and SULT2B1b, which are generated by alternate splicing of the first exon; therefore the SULT2B1 isoforms differ at their N-terminals. Northern Blot analysis detected a SULT2B1 message in RNA isolated from the human prostate and placenta. No SULT2B1 message was observed in RNA isolated from human liver, colon, lung, kidney, brain, or testis tissue. Purified SULT2B1a was used to generate a specific rabbit polyclonal anti-SULT2B1 antibody. The anti-SULT2B1 antibody did not react with expressed human EST, P-PST-1, M-PST, DHEA-ST, or ST1B2, during immunoblot analysis. The substrate specificity of the expressed SULT2B1 isoforms suggests that these enzymes are capable of regulating the activity of adrenal androgens in human tissues via their inactivation by sulfation.     

Multiple restriction fragment length polymorphisms in the porcine calcium release channel gene (CRC): assignment to the halothane (HAL) linkage group

Two cDNA probes for the porcine calcium release channel gene (CRC) were used in restriction fragment length polymorphism (RFLP) analysis in an attempt to develop genetic markers linked to the malignant hyperthermia (stress susceptibility) gene (HAL). Three TaqI RFLPs, denoted CRC1-CRC3, each composed of two alleles, were detected. RFLPs were also detected with MspI and PvuII, but the MspI RFLP correlated completely with CRC3 in this material and the PvuII RFLP could not be scored reliably due to a minute size difference between the two allelic fragments. The autosomal codominant inheritance of these RFLP loci was confirmed by family analyses. Significant evidence for genetic linkage between the CRC1/CRC3 loci and the A1BG locus in the HAL linkage group confirmed a previous assignment of the CRC gene to chromosome 6 in the pig.     

Endogenous inhibitors (GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2: derivatives of adrenally produced corticosterone and cortisol

Two isoforms of 11β-HSD exist; 11β-HSD1 is bi-directional (the reductase usually being predominant) and 11β-HSD2 functions as a dehydrogenase, conferring kidney mineralocorticoid specificity. We have previously described endogenous substances in human urine, “glycyrrhetinic acid-like factors (GALFs)”, which like licorice, inhibit the bi-directional 11β-HSD1 enzyme as well as the dehydrogenase reaction of 11β-HSD2.

Many of the more potent GALFs are derived from two major families of adrenal steroids, corticosterone and cortisol. For example, 35-tetrahydro-corticosterone, its derivative, 35-tetrahydro-11β-hydroxy-progesterone (produced by 21-deoxygenation of corticosterone in intestinal flora); 35-tetrahydro-11β-hydroxy-testosterone (produced by side chain cleavage of cortisol); are potent inhibitors of 11β-HSD1 and 11β-HSD2-dehydrogenase, with IC₅₀'s in range 0.26–3.0 μM, whereas their 11-keto-35-tetrahydro-derivatives inhibit 11β-HSD1 reductase, with IC₅₀'s in range 0.7–0.8 μM (their 35β-derivatives being completely inactive).

Inhibitors of 11β-HSD2 increase local cortisol levels, permitting it to act as a mineralocorticoid in kidney. Inhibitors of 11β-HSD1 dehydrogenase/11β-HSD1 reductase serve to adjust the set point of local deactivation/reactivation of cortisol in vascular and other glucocorticoid target tissues, including adipose, vascular, adrenal tissue, and the eye. These adrenally derived 11-oxygenated C₂₁- and C₁₉-steroidal substances may serve as 11β-HSD1- or 11β-HSD2-GALFs. We conclude that adrenally derived products are likely regulators of local cortisol bioactivity in humans.     

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products

Epidemiological studies testing the effect of β-carotene in humans have found a relative risk for lung cancer in smokers supplemented with β-carotene. We investigated the reactions of retinal and β-apo-8′-carotenal, two β-carotene oxidation products, with 2′-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N²-etheno-2′-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with β-carotene or β-carotene oxidation products, significantly increased levels of 1,N²-etheno-2′-deoxyguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of β-carotene oxidation products.     

Biochemical genetics of Es-14 (formerly Es-Si) and a new esterase variation,Es-15, of the laboratory rat (Rattus norvegicus): Biochemistry,tissue expression,and linkage to Es-1 in linkage group V

The segregation of rat esterases controlled by loci residing in linkage group V (LGV) has been studied in two backcross series, (LEW/Han × BN/Han)F₁ × LEW/Han and (LEW/Han × LE/Han)F₁ × LEW/Han. Es-14 (formerly Es-Si) was shown to be closely linked to Es-1. A new esterase locus, Es-15, was described which codes for a liver isozyme. The distribution pattern of three alleles at the Es-15 locus is presented for 52 independent inbred strains. Close linkage of Es-15 to Es-14 and to Es-1 was established, proposing the following gene order: [Es-2, Es-10]—[ES-1, ES-14, ES-15]. The esterase loci on LGV are thus separated into two gene clusters, cluster 1 and cluster 2. These conclusions are supported by the strain distribution patterns of the two RI strain series, LXB and DXE.Otto von Deimling was supported by the Deutsche Forschungsgemeinschaft (De 315/2-1, communication No. 56).     

Polymorphic plasma postalbumins of some domestic animals (pig PO2, horse Xk and dog Pa proteins) identified as homologous to human plasma α1B-glycoprotein

Pig, horse and dog plasma proteins, separated by horizontal polyacrylamide gel electrophoresis (pH 9.0) and electrophoretically transferred to nitrocellulose membranes, were tested for cross-reaction with antiserum to human plasma alpha 1B-glycoprotein (alpha 1B). The results showed that one previously reported polymorphic plasma postalbumin in each of these species (pig PO2, horse Xk and dog Pa protein) was homologous to human plasma alpha 1B. In the light of the previously known genetic linkages in these species, this implied: (1) alpha 1B gene is close linked to Phi, Pgd and Hal (halothane sensitivity locus) loci in pigs; and (2) alpha 1B gene is linked to ME1 and Phi loci in horses. This suggested that the alpha 1B gene may also be found to be closely linked to gene(s) controlling susceptibility to malignant hyperthermia in humans and other mammals.     

Cyclic (1 → 2)-β-d-glucans excreted by the glucuronan-producing strain Rhizobium meffloti M5N1CS CS (NCIM B 40472) and by the succinoglycan-producing strain Rhizobium meliloti M5N1

During fermentation, the mutant strain Rhizobium mefliloti M5N1 CS, which induces nodule formation on alfalfa roots, produces a partially acetylated (1 → 4)-β-d-glucuronan. In addition to this exopolysaccharide of high molecular weight, the mutant strain produces oligoglucoronates and cyclic (1 → 2)-β-d-glucans with degrees of polymerization from 17 to 30. Under the conditions applied, magnesium has no effect on cyclic glucan production by the mutant strain, but the succinoglycan production by the wild-type strain Rhizobium meliloti M5N1 increases.     

Mitogenic activity of particulate yeast β-(1 → 3)-d-glucan and its water-soluble derivatives

Particulate β-d-glucan was isolated from baker's yeast using autolysis and delipidization of the cells, followed by alkaline and acid treatment. The residual water-insoluble glucan termed cerevan has a β-(1→ 3)-linked backbone with β-(1 → 6)-linked short side chains. In order to achieve water solubility of the glucan, various derivatives were prepared (car☐ymethyl-, car☐yethyl-, hydroxyethyl-, sulfoethyl-), and the β-glucan was oxidized to glucuronoglucan. Their solubility, degree of substitution (DS), and molecular weight distribution (M_w) were compared. The immunomodulatory activity of these preparations was investigated in mitogenic and co-mitogenic tests on rat thymocytes. Cerevan showed higher stimulation indices compared with the known immunomodulator zymosan. Of the water-soluble derivatives, sulfoethylglucan was found to be the most active. Of the car☐ymethyl derivatives of various DS, the preparation with DS=0.75 exhibited the highest activity. Water-soluble car☐ymethyl preparations with DS > 1.0 and low-molecular-weight glucuronoglucan were inactive.     

A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein, WISH, induces Arp2/3 complex activation independent of Cdc42

We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.     

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.     

IgA1 is the premier serum glycoprotein recognized by human galectin-1 since T antigen (Galbeta1-->3GalNAc-) is far superior to non-repeating N-acetyl lactosamine as ligand

Human heart galectin-1 (HHL) was separated by high pressure liquid chromatography from endogenous glycoproteins co-purified with it during affinity chromatography. These glycoproteins offered excellent ligands for HHL binding and were rich in T antigen (Galβ1 → 3 GalNAc-) of O-linked oligosaccharides. In enzyme linked lectin assay and hemagglutination inhibition assay, human IgA1, bovine fetuin and other O-glycosylated T antigen-bearing glycoproteins bound to the lectin efficiently in contrast to single N-acetyl lactosamine (LacNAc)-bearing N-linked oligosaccharides released from them and to IgG which is not O-glycosylated. HHL binding to IgA1 and fetuin was unaffected by removal of their N-linked oligosaccharides by -mannosidase. When immobilized, O-glycosylated serum proteins but not IgG could capture HHL from its solutions. Desialylated or polymeric IgA1 was better inhibitor than monomeric IgA1. The findings suggest a possible role for galectin-1 in anchoring of microbial and cancer cells known to be rich in T antigen, in high serum IgA1 turn over and in tissue sequestering of IgA1 immune complexes especially after their microbial desialylation in IgA nephropathy and other immune complex-mediated disorders.     

Genetic variation at a pig serum protein locus, Po-2 and its assignment to the Phi, Hal, S, H, Pgd linkage group

Two-dimensional agarose gel (pH 5.4)-polyacrylamide gel (pH 9.0) electrophoresis of pig serum samples revealed a new serum protein (postalbumin-2, PO-2) polymorphism. Family data supported the hypothesis that the three PO-2 phenotypes observed were controlled by two codominant, autosomal alleles (Po-2F and Po-2s) at a single locus. The frequency of Po-2F in Swedish Landrace and in Swedish Yorkshire breeds was estimated at 0.74 and 0.65, respectively. Evidence was presented for close genetic linkage between Po-2 and the red cell phosphohexose isomerase locus (Phi). A recombination frequency of 3.2% was obtained from double backcross material. Data obtained in a Danish Landrace material showing linkage between the Po-2 locus and the H blood group locus, the Pgd locus and Hal (locus for halothane sensitivity) are also given. A total of seven recombinants were observed. They show that Po-2 is a new locus in a previously established linkage group. The likely sequence of the loci is: Phi, Hal, S, H, Po-2, Pgd.