Differential regulation of the two mRNA species of the rodent negative acute phase protein alpha 1-inhibitor 3.

Screening of two rat liver cDNA libraries, one of which was constructed using an alpha 1-inhibitor 3 (alpha 1-13) specific primer, yielded overlapping cDNA clones which correspond to the full length cDNA for alpha 1-13 mRNA. On the basis of sequence microheterogeneity existing throughout the cDNA sequence we identified two alpha 1-13 mRNA species whose sequences are so grossly different in their bait regions that the amino acid homology therein is only 30%. Using oligonucleotide probes derived from their respective bait regions we investigated the regulation of the two alpha 1 I3 mRNA species and demonstrated that only one of them, alpha 1-I3 variant I, is regulated pretranslationally following experimentally induced inflammation.     

Immunological and chemical properties of mouse alpha 1-protease inhibitors

We previously described the isolation and purification of two similar alpha 1-protease inhibitors from mouse plasma termed alpha 1-PI(E) and alpha 1-PI(T) because of their respective affinities for elastase and trypsin. Some of the biochemical and immunological properties of these proteins are reported. Both are acidic glycoproteins with pI's of 4.1-4.2. The plasma half-life of each inhibitor, determined after administration of the 125I-protein, is approximately 4 h both in normal mice and in mice after induction of the acute phase reaction. The two proteins have almost identical amino acid compositions and similar CNBr peptide maps. Tryptic maps, however, are considerably different. Reverse-phase chromatography separated alpha 1-PI(E) into three distinct isoforms, each eluting with approximately 60% acetonitrile. Under these conditions alpha 1-PI(T) shows a single peak, clearly different from those of alpha 1-PI(E). The three alpha 1-PI(E) isoforms have the same molecular weights on sodium dodecyl sulfate-gel electrophoresis and the same tripeptide sequence at their N-terminus, and appear to be immunologically identical. Polyclonal, monospecific antibodies to each native inhibitor, prepared in rabbits, showed no cross-reactivity when tested by functional assay or crossed immunoelectrophoresis. Interestingly, each antibody recognized epitopes on the C-terminal portion of its respective antigen. These studies confirm that alpha 1-PI(E) and alpha 1-PI(T), although highly similar, are products of different genes. Like human alpha 1-PI, the two mouse inhibitors are partially inactivated by mild oxidation with chloramine-T, losing all elastase inhibitor and lesser amounts of antichymotryptic and antitryptic activity. However, unlike the human protein, neither alpha 1-PI(E) nor alpha 1-PI(T) was found to have a methionine residue at its P1 site.     

Genetic polymorphism and close linkage of two alpha 1-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 alpha-globulins. Two groups of alpha 1-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.     

Carbohydrate composition of normal and variant human alpha 1-protease inhibitors.

Carbohydrate composition of normal human alpha 1-protease inhibitor (PiM₁) and several variant inhibitors (PiM₂, PiM₃, PiA, PiS, and PiZ) was determined by methanolysis of the samples followed by quantitative analysis of both neutral and amino sugars using gas-liquid chromatography. All normal and variant inhibitors contained nine mannose, seven galactose, ten N-acetylglucosamine, and eight N-acetylneuraminic acid residues per molecule, and no significant difference was found in their carbohydrate compositions. PiA is a variant with the fastest anodal electrophoretic mobility, and PiZ is a variant with the slowest mobility thus far reported. The differences in electrophoretic mobility of these Pi variants are entirely due to their amino acid substitutions determined previously. These amino acid substitutions have no effect on the carbohydrate structure of the protease inhibitor.     

Plasma levels of immunoreactive ceruloplasmin and other acute phase proteins during lactation

Lactation elevates plasma copper as well as oxidase activity levels of the copper-containing, acute phase protein ceruloplasmin (Cp). The present study provides an initial inquiry into the mechanisms behind these changes. Plasma obtained from 12 lactating women, 1 month postpartum, displayed a greater percentage increase in immunoreactive Cp levels (mean increase = 89%) than in plasma copper (mean = 66%) or Cp oxidase activity (mean = 42%). Lactation did not increase plasma content of C-reactive protein or alpha 1-antitrypsin but significantly elevated haptoglobin concentrations. Plasma alpha 2-macroglobulin contents correlated with immunoreactive Cp levels in lactating women but not in controls. These results strengthen the hypothesis that plasma content of individual acute phase proteins is regulated by both overlapping and individualized processes. In addition, the present findings raise the possibility that lactation increases both Cp synthesis and plasma turnover time of Cp-bound copper.     

Differentiation in the seed protein profiles of two closely related species of Narcissus

In the present paper we have analysed the relationship between two closely related species of Narcissus L. [N. cantabricus D.C. and N. hedraeanthus (Webb & Heldr.) Colmeiro] by means of seed protein electrophoresis. We have calculated two different coefficients of similarity between the seed protein profiles of both species. With the aim of knowing the meaning of such coefficients we have compared them with the ones obtained in several other different groups.     

Evidence for a novel circulating alpha 1 protein in tumour-bearing rats. Differentiation from acute phase alpha 1 proteins and from alpha 1 fetoprotein

It has been found in this study that the serum from rats bearing a transplanted dibenzanthracene-induced tumour ($\text{RD}\text{3}$), has a high concentration of alpha₁ proteins compared with normal rat serum. These alpha₁ proteins have been isolated by an immunoabsorption method and have been compared by immunological methods with the acute phase alpha₁ proteins isolated by the same method from the serum of rats presenting an inflammatory reaction. It has been found that the isolated $\text{RD}\text{3}$ alpha₁ proteins were composed of two major proteins: one of these corresponded to an inflammatory protein, the alpha₁-AP-globulin. The other may be a new protein, as it is absent from the serum of rats with an acute phase inflammatory reaction and nor does it correspond to alpha₁ feto-protein, a carcino-embryonic protein presenting the same electrophoretic mobility.     

Partial preparative separation and properties of the isoinhibitors of human alpha 1-antitrypsin (alpha 1-protease inhibitor).

Human lapha 1-antitrypsin (alpha 1-protease inhibitor) was chromatographed on a DEAE-cellulose column at pH 6.4. After elution with a linearly increasing concentration of NaCl, five pools (pools I-V) were formed from the eluate, pool I corresponding to the lowest and pool V to the highest concentration of salt. As demonstrated by analytical isoelectric focusing, with increasing concentrations of NaCl the concentration of the cathodal isoinhibitors gradually decreased and the concentration of the anodal ones increased in the pools. Pool I contained only three cathodal and pool V only three anodal isoinhibitors with a limited overlap between the pools. In contrast with the isoinhibitor composition, the sialic acid contents of the pools did not vary with the elution conditions. In line with the chemical evidence, desialylation of the fractions did not affect their electrofocusing positions relative to one another and did not abolish the microheterogeneity of the protein.     

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.     

Differential regulation of Na,K-ATPase alpha 1, alpha 2, and beta subunit mRNA and protein levels by thyroid hormone

The purpose of this study was to determine the effect of thyroid status on the Na,K-ATPase alpha isoforms and beta in rat heart, skeletal muscle, kidney, and brain at the levels of mRNA, protein abundance, and enzymatic activity. Northern and dot-blot analysis of RNA (euthyroid, hypothyroid, and triiodothyronine-injected hypothyroids = hyperthyroids) and immunoblot analysis of protein (euthyroid and hypothyroid) revealed isoform-specific regulation of Na,K-ATPase by thyroid status in kidney, heart, and skeletal muscle and no regulation of sodium pump subunit levels in the brain. In general, in the transition from euthyroid to hypothyroid alpha 1 mRNA and protein levels are unchanged in kidney and skeletal muscle and slightly decreased in heart, while alpha 2 mRNA and protein are decreased significantly in heart and skeletal muscle. In hypothyroid heart and skeletal muscle, the decrease in alpha 2 protein levels was much greater than the decrease in alpha 2 mRNA levels relative to euthyroid indicating translational or post-translational regulation of alpha 2 protein abundance by triiodothyronine status in these tissues. The regulation of beta subunit by thyroid status is tissue-dependent. In hypothyroid kidney beta mRNA levels do not change, but immunodetectable beta protein levels decrease relative to euthyroid, and the decrease parallels the decrease in Na,K-ATPase activity. In hypothyroid heart and skeletal muscle beta mRNA levels decrease; beta protein decreases in heart and was not detected in the skeletal muscle. These findings demonstrate that the euthyroid levels of expression of alpha 1 in heart, alpha 2 in heart and skeletal muscle, and beta in kidney, heart, and skeletal muscle are dependent on the presence of thyroid hormone.     

Alpha 1-antitrypsin and serum albumin mRNA accumulation in normal, acute phase and ZZ human liver

Alpha 1-Antitrypsin and albumin mRNA levels of 4 human livers were assessed using a newly sequenced cDNA clone of the carboxyterminal third of alpha 1-antitrypsin and a previously cloned albumin cDNA sequence. The relative concentration of alpha 1-antitrypsin mRNA was the same in poly(A)-containing RNA isolated from acute phase (MM) and alpha1-antitrypsin deficient (ZZ) individuals. In the acute phase liver relative to the normal (MM) liver, total RNA extracts showed a marked decrease in albumin mRNA concentration but no increase in alpha 1-antitrypsin mRNA. The ZZ liver showed decreased total and poly(A)-containing RNA content but the same proportion of alpha 1-antitrypsin to albumin mRNA as in the normal (MM) liver. This supports other evidence that ZZ alpha 1-antitrypsin deficiency is due to a defect in polypeptide processing (secretion) rather than a deficiency in mRNA accumulation.     

Phenobarbital-induced rat liver cytochrome P-450. Purification and characterization of two closely related isozymic forms

The "major" phenobarbital (PB)-induced cytochrome P-450 species present in livers of male Sprague-Dawley rats was resolved into two catalytically active heme-protein fractions on diethylaminoethyl cellulose. The two species, P-450 PB-4 (Mr = 49,000) and P-450 PB-5 (Mr = 51,000), were purified to homogeneity, and their chromatographic, spectral, catalytic, and structural properties were compared. P-450 BP-5 eluted earlier on hydroxylapatite and exhibited a more significant cholate-induced Type I spectral shift than P-450 BP-4. Very similar substrate specificity profiles were evident when the two isozymes were reconstituted with lipid, cytochrome P-450 reductase, and cytochrome b5 for oxidative metabolism of several xenobiotics, although P-450 PB-4 exhibited a higher specific catalytic activity (greater than or equal to 5-fold) with all substrates tested. Marked differences were also observed in the sensitivities of both isozymes to several P-450 inhibitors. In addition, P-450 PB-4 was greater than or equal to 10-fold more susceptible than P-450 PB-5 to suicide inactivation by two allyl-containing compounds, allylisopropylacetamide and secobarbital, providing a possible explanation of the previously observed partial inactivation by such compounds of phenobarbital-induced P-450 activity in liver microsomes. One-dimensional peptide maps of the two isoenzymes were highly similar. Antibody raised against purified Long Evans rat liver P-450b (Thomas, P. E., Korzeniowski, D., Ryan, D., and Levin, W. (1979) Arch. Biochem. Biophys. 192, 524-532) cross-reacted with P-450 PB-4 and P-450 PB-5. NH2-terminal sequence analysis demonstrated that the first 31 residues of both PB-4 and PB-5 were identical. These sequences indicated that a highly hydrophobic terminal segment, observed previously for other P-450s as well, is followed by a cluster of basic residues, suggesting that the NH2-terminal portion of these P-450s might be involved in membrane anchoring. Although it is unclear whether P-450 PB-4 and P-450 PB-5 are separate gene products or are related by post-translational modifications, this present demonstration of closely related isozymic forms suggests the possible added complexity of microheterogeneity for this family of microsomal monooxygenases.