Equus przewalskii plasma protease inhibitor (Pi) system

A detailed biochemical characterization of four of the five previously described alleles of the plasma protease inhibitor (Pi) system of Equus przewalskii was performed using both one- and two-dimensional electrophoretic techniques. The proteins have been characterized in terms of isoelectric point, relative molecular mass, inhibitory activity to bovine trypsin and chymotrypsin, immunochemical cross-reactivity, terminal sialic acid content and enzyme:inhibitor complex formation and the oxidation sensitivity of this interaction. Using these functional criteria, only three loci (Spi 1, 2 and 3) were found to control the plasma Pi proteins of the E. przewalskii haplotypes. In contrast a fourth locus, Spi 4, was found in some E. caballus haplotypes. The significance of these results with respect to the complexity of the protein pattern exhibited by the equine Pi multigene family is discussed.     

The plasma protease inhibitor system (Pi) of Standardbred horses

The plasma protease inhibitor system (Pi) of Standardbred horses was studied by thin-layer, high-voltage, acid polyacrylamide gel electrophoresis (pH 4.6) followed by protein staining and staining for trypsin and chymotrypsin inhibition. In addition to the eight Thoroughbred alleles ( Pi^{F, g, i, L, N, S1, S2, u)} , another 10 alleles, designated PiH, j, k, ^o, ^p, ^q , ^r, ^v, ^{x, z}, were postulated to account for the 98 Pi types which were observed in Standardbreds. Detailed inhibitory spectra of the 'new' alleles were determined and further exceptions to the Pil, Pi2 classification of Juneja et al. (1979) were found. Limited family data demonstrated the genetic nature of the 'new' variants and confirmed the allelic inheritance of the 'new' Pi variants.     

ISO-DALT characterization of 12 'new' equine plasma protease inhibitor (Pi) alleles

Twelve equine protease inhibitory alleles, PiE, H, J, K, L2, O, P, Q, R, V, X, Z, have been characterized in terms of isoelectric point, molecular mass and inhibitory activity to bovine trypsin and chymotrypsin by ISO-DALT electrophoresis. Protein maps for 20 Pi alleles including those of the eight 'Thoroughbred' alleles (PiF, G, I, L, N, S1, S2, U) have now been determined. Five pairs of alleles, S1/S2, G/K, L/L2, P/R and U/Z, possessed varying numbers of common proteins ranging from one protein in the case of G/K and L/L2 to six in the case of U/Z. Based on these results and studies of the abnormal expressions of PiF, PiL and PiS1, a theory of at least three closely linked loci has been postulated to account for the marked heterogeneity of the equine protease inhibitory system.     

Polymorphism of pig serum alpha-protease inhibitor-3 (PI3) and assignment of the locus to the Pi1, Po1A, Po1B, Pi2, Igh linkage group

Polymorphism of an alpha-protease inhibitor, PI3, in pig serum samples was detected using 2D agarose gel (pH 5.4)--polyacrylamide gel (pH 9.0) electrophoresis. Evidence was obtained that the five variants observed (A, B1, B2, C and D) are under genetic control by codominant alleles (Pi3A, Pi3B1, Pi3B2, Pi3C and Pi3D) at one autosomal locus. Variants A, B1, B2 and C inhibited chymotrypsin; there was no appreciable inhibition of trypsin and papain. Variant D did not inhibit chymotrypsin, and therefore its classification as a PI3 variant was put in question. PI3 typing was not possible in about 50% of the studied pigs since in those cases the PI3 variants were either too weak or absent. On the basis of backcross matings and haplotyping in complete families for protease inhibitor loci Pi1, Po1A, Pi2 and Pi3 it was proved that the Pi3 locus belongs to the protease inhibitor gene cluster, and the position of the locus in the linkage group was proposed as being Pi1-Po1A-(Po1B)-Pi3-Pi2-(Igh1, Igh2, Igh3, Igh4).     

Anencephaly with incomplete twinning (diprosopus)

A case of diprosopus with anencephaly is presented. It is suggested that such concurrence of neural tube defects and incomplete twinning corroborates the notion that a single pathogenetic mechanism may be common to both neural tube defects and monozygotic twinning.     

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.Yiwen Deng and Xudong Zhu contributed equally to this work.     

Genetic linkage between loci for a red cell alloantigen (U) and serum protease inhibitor (Pi) in the horse

Preliminary evidence for the fifth autosomal linkage group in the horse, comprised of the loci for a red cell alloantigen (U) and serum protease inhibitor (Pi), was demonstrated by means of paternal half-sib groups in thoroughbred, standardbred and Arabian breeds. Recombination frequency in males was estimated to be 0.125 +/- 0.019.     

An additional locus (PI4) in the protease inhibitor (PI) gene cluster of pigs

A further α-protease inhibitor system, PI4, was detected in porcine sera using either 2D agarose gel, pH 5.0-PAGE, pH 9.0, or ID PAGE followed by immunoblotting with rabbit anti-porcine PI2 or PI3 antisera. PI4 inhibited chymotrypsin, but not trypsin. Seven allelic variants of PI4 were described. By haplotyping of a-protease inhibitor systems in 52 complete families it was shown that PI4 locus belongs to the PI gene cluster. The probable order of the PI loci was: PI1, PO1A, PI2, PI4, PI3.     

Genetics and molecular mapping of a male fertility restoration locus (Rfg1) in rye (Secale cereale L.)

 A gene determining the restoration of cytoplasmic genic male sterility (CMS) caused by the Gülzow (G)-type cytoplasm was mapped by analyzing an F₂ and F₃ population comprising 140 and 133 individual plants, respectively. The target gene, designated Rfg1, was mapped on chromosome 4RL distally to three RFLP (Xpsr119, Xpsr167, Xpsr899) and four RAPD (XP01, XAP05, XR11, XS10) loci. Xpsr167 and Xpsr899 are known to be located on the segment of chromosome 4RL which was ancestrally translocated and is homoeologous to the distal end of other Triticeae 6S chromosomes. It is suggested that Rfg1 may be allelic to the gene determining the restoration of rye CMS caused by the Pampa (P) cytoplasm (chromosome 4RL) and to Rfc4 that on rye addition lines of chromosome 4RL restores male fertility of hexaploid wheat with T. timopheevi cytoplasm. Homoeoallelism to two loci for cytoplasmic-male-sterility restoration on chromosomes 6AS and 6BS in hexaploid wheat is also suggested. Received: 1 December 1997 / Accepted: 10 February 1998     

Protease inhibitor localization in control and streptozotocin-diabetic skeletal muscles

Summary ₁-Antitrypsin and ₁-inhibitor-3 were localized for the first time inside skeletal muscle cells. Their content, especially that of ₁-inhibitor-3, was greatly reduced following streptozotocin-induced diabetes. ₁-Antitrypsin and ₁-inhibitor-3 were also observed in the vascular components and interstitial space surrounding both control and diabetic soleus muscles as revealed by immunofluorescence. In diabetic muscles, the non-myofibre locale of ₁-inhibitor-3 was reduced, and to a lesser extent, ₁-antitrypsin. Both myofibre and extracellular patterns were reversed to control levels by insulin replacement.     

Renal Endosomal Phosphate (Pi) Transport in Normal and Diabetic Rats and Response to Chronic Pi Deprivation

Chronic renal adaptation to dietary deprivation of Pi is accompanied by increased Na⁺/Pi co-transport across the brush border membrane of the renal proximal tubule. The increased activity of this co-transport system depends on de novo protein synthesis and insulin. The present study used normal and diabetic rats to determine if the endosomal pool of Na⁺/Pi co-transporters was altered by Pi deprivation and the possible role of insulin. In response to 5 days of dietary Pi deprivation there was a significant increase in endosomal Na⁺/Pi co-transport in control rats but there was no change in diabetic rats. The increase in endosomal Pi uptake was restored in diabetic rats treated with exogenous insulin. Na⁺/Pi-independent Pi uptake and proline uptake remained unchanged in all groups. The changes in endosomal Na⁺/Pi co-transport correlated with the abundance of the specific Na⁺/Pi co-transporter protein, as determined by Western blots. The pattern of endosomal changes paralleled that observed in brush border membranes. One possibility consistent with these findings is that the endosomal fraction contains newly synthesized Na⁺/Pi co-transporters targeted for delivery to the apical brush border membrane. Increased synthesis and delivery is required to maintain the adaptation to chronic Pi deprivation. © 1997 John Wiley & Sons, Ltd.     

Regulation of the phosphate (Pi) concentration in UMR 106 osteoblast-like cells: Effect of Pi,Na+ and K+

Osteoblast-like cells possess Na-dependent transporters which accumulate orthophosphate (Pi) from the extracellular medium. This may be important in bone formation. Here we describe parallel measurements of Pi uptake and cellular [Pi] in such cells from the rat (UMR 106–01 and UMR 106–06) and human (OB), and in non-osteoblastic human fibroblasts (Detroit 532 (DET)). In UMR 106–01, cellular [Pi] was weakly dependent on extracellular [Pi] and higher than expected from passive transport alone. [³²Pi]-uptake was inhibited by Na deprivation, but paradoxically increased on K deprivation. With Na, 87 per cent of cellular ³²P was found in organic phosphorus pools after only 5 min. Na deprivation also decreased cellular [Pi], in both UMR 106–01 and DET, but the decrease was smaller than that in [³²Pi]-uptake. Ouabain decreased [³²Pi]-uptake and cellular [Pi] in DET, but not in UMR 106–01. Regulation of cellular [Pi] is therefore at least partly dependent on Na/Pi co-transport, but this does not seem to be an exclusive property of osteoblasts.     

Three twinning types in mink (Mustela vison) embryogeny

The frequency of emergence of monochorial twins in the postimplantational embryogeny of the American mink and their karyotypes were studied. Monochorial pairs were found in which embryos had different karyotypes: 2n, XX and 2n, XY or 2n and 3n. This fact contradicts the notion that monochorial twins should be monozygotic and genetically identical but confirms our earlier hypothesis that a third twinning type exists in mink: monozygotic but genetically different. The mechanism of the emergence of this twinning type in mammals is discussed. It is suggested that the high (up to 4.5%) frequency of its emergence in the American mink is related to obligate embryonic diapause, causing abnormal fertilization.     

Tammar wallaby plasma protease inhibitory (Pi) proteins

Electrophoretic examination (isoelectric focusing and polyacrylamide gel electrophoresis) of 157 plasmas from a Kangaroo Island population of tammar wallabies (Macropus eugenii) resulted in the identification of five putative condominant protease inhibitor alleles, F, I, M, P and S, which exhibited microheterogeneity due to variable terminal sialic acid content. The frequencies of the five alleles in this population were 0.041(F), 0.682(I), 0.194(M), 0.073(P) and 0.010(S). The proteins had isoelectric points in the pH range 3.94-4.38, Mr of 60,500 to 66,000 and were identified as protease inhibitors by their abilities to inhibit both trypsin and chymotrypsin. Protein blotting of the denatured proteins demonstrated cross reaction with antiserum to human alpha 1-protease inhibitor.