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.     

Immunogenicity, macrophage sensitivity, and therapeutic response to C. parvum of fibrosarcomas induced in C. parvum-treated and untreated mice

Summary No correlation has been observed between the therapeutic response of isogeneic transplants of methylcholanthrene-induced murine fibrosarcomas to Corynebacterium parvum (CP) and the sensitivity of the tumour cells to the cytotoxic action of CP-activated macrophages (Mø) in vitro. Neither of these parameters appeared to be influenced by repeated administration of CP during induction of the primary tumour or by whether the carcinogen was administered in solution or as crystals on a Millipore disc. On the other hand, tumours which developed in CP-treated mice after injection of a small dose (0.1 mg) of methylcholanthrene (MC) were more immunogenic than those which developed after the same dose of MC in untreated mice, as judged by the capacity of an injection of an irradiated tumour cell suspension to protect isogeneic mice against subsequent challenge with viable cells. Further work is needed to explain these findings.     

Effects of testosterone on messenger ribonucleic acid and protein synthesis in rat seminal vesicle

In a previous report [Higgins et al. (1976) Biochem. J. 158, 271–282] we described the effects of alterations in androgen status on the synthesis of two basic secretory proteins of the rat seminal vesicle. In the present paper we examine the effects of testosterone on the activity of mRNA in the seminal vesicle. Total cellular poly(A)-rich RNA was isolated and translated in a cell-free system prepared from wheat germ. Translation products were separated on denaturing polyacrylamide gels and the protein bands corresponding to the two basic secretory proteins were identified immunologically. Incorporation of radioactive methionine into these bands was taken as a measure of the individual mRNA activities. Total mRNA activity was estimated by radioactivity in total acid-precipitable material. The results show that 1 to 2 weeks after castration the activities of mRNA molecules for the basic secretory proteins were decreased 10–20-fold on a tissue basis. Testosterone given in vivo rapidly and substantially restores mRNA activity to normal. Since these changes correlate closely with variations in the rates of synthesis of the secretory proteins in whole cells it suggests that androgenic steroids control protein synthesis chiefly via mRNA availability. In this respect their action resembles those of other steroid hormones acting in other systems. However, these effects of testosterone on the mRNA molecules for the major secretory proteins could not be distinguished from those on total mRNA. Thus the proportion of the total mRNA population accounted for by the two specific mRNA molecules showed less than a 2-fold variation with androgen status. Similarly the two secretory proteins always accounted for 25–33% of general protein synthesis. This is in sharp contrast with the markedly differential effects of other steroid hormones controlling synthesis of major proteins in other well-studied systems. We interpret our results as indicating that testosterone regulates the mRNA population of the seminal vesicle as a whole.     

The Ecology of Seasonal Stress and the Origins of Agriculture in the Near East

The time, place, and reasons for the first domestication of cereals and legumes in the Near East can now be securely identified using combined evidence from paleoenvironmental studies, models of ecosystem dynamics, and regional archeology. The heartland of domestication was the Jordan Valley and surrounding region in the Southern Levant. Approximately 10,000 years ago, people began planting crops where the wild ancestral species had proliferated over two millenia. Impetus for domestication came from the synergistic effects of climatic change, anthropogenic environmental change, technological change, and social innovation. At the end of the Pleistocene, after a long period of climatic instability, a mediterranean climate more strongly seasonal than any today emerged with hyper-arid summers that selected for annual species of cereals and legumes. This occurred long after people had invented tools suitable for grinding hard seeds, but the new, lengthy dry season and consequent need to use stored foods encouraged sedentism among human groups who subsequently depleted their immediate environments of wild resources. These preconditions facilitated the development of agriculture. The scenario developed here is specific to the Near East, for such case studies of specific factors in independent regions of domestication are essential before we attempt to explain cases all over the world with reference to global causes.     

Heparin enhances fibrinolysis in B16 mouse melanoma cells

The fibrinolytic activity of two tumorigenic B16 mouse melanoma lines was stimulated by exogenous hog mucosal or beef lung heparin. In contrast, the activity of two normal fibroblast lines was unaffected. The degradation of ¹²⁵l-fibrin was increased up to 3.6-fold by the addition of heparin. Chondroitin-4-sulfate or dextran sulfate did not change the fibrinolytic activity of three of the cell lines, but, at concentrations where enhancement by heparin was much reduced, the activity of one of the B16 melanoma lines was somewhat elevated. Antithrombin III did not alter the plasminogen activator activity of the B16 cell lines, but, in the presence of exogenous heparin, the enhancement of fibrinolysis was greatly reduced. The polymers were not cytotoxic during the assay period, and, had little affect on the plating efficiencies of the lines.     

Correlation between cell killing and massive second-round superinfection by members of some subgroups of avian leukosis virus.

Avian leukosis viruses of subgroups B, D, and F are cytopathic for chicken cells, whereas viruses of subgroups A, C, and E are not. The amounts of unintegrated linear viral DNA in cells at different times after infection with cytopathic or noncytopathic viruses were determined by hybridization and transfection assays. Shortly after infection, there is a transient accumulation of unintegrated linear viral DNA in cells infected with cytopathic avian leukosis viruses. By 10 days after infection, the majority of this unintegrated viral DNA is not present in the infected cells. The transient cytopathic effect seen in these infected cells also disappears by this time. Low amounts of unintegrated linear viral DNA persist in these cells. Cells infected with noncytopathic viruses do not show this transient accumulation of unintegrated viral DNA. Cells infected with cytopathic viruses and subsequently grown in the presence of neutralizing antibody do not show the transient accumulation of unintegrated viral DNA or cytopathic effects. These results demonstrate a correlation between envelope subgroup, transient accumulation of unintegrated linear viral DNA, and transient cell killing by avian leukosis viruses. The cell killing appears to be the result of massive second-round superinfection by the cytopathic avian leukosis viruses.     

An enzymecytochemical study on the adrenal of the freshwater teleost, Clarias batrachus (L.).

The adrenal homologue of C. batrachus is distributed around the postcardinal vein in the pronephric head kidney. The cortical cells are round or oval in shape. They showed positive reaction for total lipid, glycogen and ascorbic acid. Their intense delta5-3beta HSDH activity indicates their capacity for steroid biosynthesis. In addition, the cortical cells of C. batrachus exhibited strong G-6-PD, NADPH diaphorase, NADH diaphorase, MAO and weak SDH and LDH activity. The presence of MAO suggests the aminergic control of the adrenal in this species and the silver positive fibres seen the cortical cells were hypertrophied, degranulated and the lipid content was also decreased. The chromaffin or medullary cells were distributed in groups among the cortical cells. They are largely oval or angular in shape. They react positively to ferric ferricyanide, chromaffin and argentaffin reactions and ascorbic acid test.     

Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys₂His₂) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.     

Estrogen contributes to seasonal plasticity of the adult avian song control system

Songbirds show dramatic neural plasticity as adults, including large-scale anatomical changes in discrete brain regions ("song control nuclei") controlling the production of singing behavior. The volumes of several song control nuclei are much larger in the breeding season than in the nonbreeding season, and these seasonal neural changes are regulated by plasma testosterone (T) levels. In many cases, the effects of T on the central nervous system are mediated by neural conversion to estradiol (E(2)) by the enzyme aromatase. The forebrain of male songbirds expresses very high levels of aromatase, in some cases adjacent to song control nuclei. We examined the effects of aromatase inhibition and estrogen treatment on song nuclei size using wild male songbirds in both the breeding and nonbreeding seasons. In breeding males, aromatase inhibition caused the volume of a telencephalic song control nucleus (HVC) to decrease, and this effect was partially rescued by concurrent estrogen replacement. In nonbreeding males, estradiol treatment caused HVC to grow to maximal spring size within 2 weeks. Overall, these data suggest that aromatization of T is an important mediator of song control system plasticity, and that estradiol has neurotrophic effects in adult male songbirds. This study demonstrates that estrogen can affect adult neural plasticity on a gross anatomical scale and is the first examination of estrogen effects on the brain of a wild animal.