Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit

Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin is a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present in larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3′ end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5′-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail. © 1995 Wiley-Liss, Inc.     

Identification of a PstI polymorphism in the p21Cip1/Waf1 cyclin-dependent kinase inhibitor gene

A PstI polymorphism in the 3 flanking region of the p21^CiP1/Waf1 cyclin-dependent kinase inhibitor gene is described. DNA sequencing analysis identified a CT base substitution in the 3 flanking region of the gene. This substitution leads to the destruction of a PstI site and results in a biallelic DNA polymorphism. This restriction fragment length polymorphism (RFLP) provides the first known genetic marker for this cell cycle regulatory gene.     

Normal levels of zinc and sulfhydryls in morphologically abnormal populations of spermatozoa from moderately zinc-deficient rats

Zinc is required for spermatogenesis in mammals and is concentrated in the dense outer fibers of the sperm tail, where it is associated with cysteine-rich protein. To investigate the effects of marginal zinc deficiency upon dense fiber formation and upon sperm quality in general, weanling Sprague-Dawley rats were administered a commercial low-zinc diet, supplemented with phytate, for approximately 60 days, and were compared with controls fed the same diet plus 50 ppm zinc in their drinking water. The following characteristics of the zinc-deficient rats were significantly lower than in the controls: body weight, testis weight, epididymis weight, seminal vesicle weight, sperm content of the cauda epididy-midis, sperm motility, testis zinc, and hair zinc. By contrast, the levels of sperm zinc and sperm sulfhydryls were the same in the zinc-deficient and control rats. The zinc-deficient rats displayed a highly variable spectrum of sperm defects, which included decapitation, disorganized and redundant tail elements, and superfluous cytoplasm. However, abortive dense fiber development was only rarely observed. Apparently, even when availability of zinc is limited and reduced sperm production ensues, elaboration of dense fibers rich in zinc and sulfhydryls continues to be obligatory for the completion of spermiogenesis.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

Flow field-flow fractionation as a methodology for protein separation and characterization

Flow field-flow fractionation is introduced as a new tool applicable to protein studies. Specific advantages of this method are discussed, including the capability for measuring diffusivities and Stokes radii directly, even for trace components. The theoretical equations of flow FFF are summarized and expanded to include an explicit dependence on the Stokes radius. Several native proteins are retained. The retention is shown to be systematically controllable by changes in cross flow and the results are in quantitative agreement with theory. Fractograms of different rat plasmas are then shown to produce coincident peaks, while human plasma exhibits several systematic peak shifts with respect to the fractogram of the rat plasma. Finally, changes in the Stokes radii of ferritin peaks are shown after various forms of treatment with SDS. Flow FFF in this study demonstrates a capability of working with a mass range of ∼ 10⁵ in a single run.     

Action of the C3b-inactivator on the cell-bound C3b.

The action of C3bINA and beta 1H on cell-bound C3b is described in this paper. The alpha-polypeptide of C3b that binds covalently to cell surfaces is cleaved by the C3bINA and beta 1H into two fragments: one of 60,000 (C3b alpha-60) and another of 40,000 (C3b alpha-40) daltons. The beta-chain of C3b is unaffected by the C3bINA and beta 1H. The three polypeptides, C3b alpha-60, C3b alpha-40, and C3 beta, are held together as a single unit by disulfide bonds. This unit, referred to as C3b' is covalently bound to cell surfaces via the C3b alpha-60 polypeptide. The conversion of C3b to C3b' by C3bINA and beta 1H abolishes the ability of the C3b-bearing cells to adhere to human erythrocytes as well as the ability to form, on the cell surface, the B, D, and properdin-dependent amplification C3-convertase. However, the agglutinability of the cells with either anti-C3c or anti-C3d is not affected. Treatment of the C3b'-bearing cells with trypsin releases fragments of C3b' into solution, leaving a polypeptide of 32,000 daltons covalently linked to the membrane. Since the trypsinized cells are agglutinable by anti-C3d but not by anti-C3c, the 32,000 dalton polypeptide appears to correspond antigenically to C3d.     

Antibody-mediated immunosuppression of a cytotoxic cell response not involving a simple antigen-masking mechanism.

An in vitro cytotoxic cell response against alloantigens was inhibited by antibody directed against the alloantigens and also by anti-trinitrophenyl antibody provided that the allogeneic stimulator cells were modified with trinitrophenyl. Inhibition of an anti-allogeneic cytotoxic cell response against trinitrophenyl-coupled allogeneic stimulator cells by anti-trinitrophenyl antibody is dependent upon the intact Fc portion of inhibitory antibody. The magnitude of the difference between intact and F(ab')2 anti-trinitrophenyl antibody in immunosuppressive activity suggests that the Fc portion emits negative signals rather than simply adding to a steric hindrance effect.     

Identification of the primate papovavirus HD as the stump-tailed macaque virus.

The recently isolated primate papovavirus HD is shown to be indistinguishable from the stump-tailed macaque virus by immunofluorescent reactivity, by restriction endonuclease analysis, and by nucleic acid hybridization assay.     

Regulation of RNA synthesis in yeast III

Summary Centrifugal elutriation was used to separate cells in different stages of the cell cycle from a culture of Saccharomyces cerevisiae in balanced exponential growth. The rate of DNA and RNA synthesis was determined using a pulse-long-term label technique that is capable of distinguishing between exponential, linear, and periodic variations in the rate of synthesis through the cell cycle. It was found that while the rate of DNA synthesis varies periodically through the cell cycle, the rate of synthesis of mRNA, rRNA, and tRNA increases exponentially through the cell cycle. The implications of these findings for the control of RNA synthesis are discussed.