Drosophila melanogaster ferritin: cDNA encoding a light chain homologue, temporal and tissue specific expression of both subunit types.

Drosophila melanogaster secreted ferritin like the cytosolic ferritins of other organisms is composed of two subunits, a heavy chain homologue (HCH) and a light chain homologue (LCH). We report the cloning of a cDNA encoding the ferritin LCH of this insect. As predicted from the gene sequence, it contains no iron responsive element (IRE). Northern blot analysis reveals two mRNAs that differ in length due to the choice of polyadenylation signals. Message levels vary through the life cycle of the fly and are markedly increased by high levels of dietary iron. The gut is the main site of increased message synthesis and iron preferentially increases the amount of shorter messages. Western blotting reveals that LCH is the predominant ferritin subunit in all life stages. The amount of LCH protein corresponds well with the message levels in control animals, while in iron-fed animals LCH does not increase proportionally with the message levels. In contrast, the amount of HCH is less than that would be predicted from message levels in control animals, but corresponds well in iron-fed animals. Ferritin is abundant in gut and hemolymph of larvae and adults and in ovaries of adult flies. At pupariation, ferritin becomes more abundant in hemolymph than in other tissues.

Iron availability dramatically alters the distribution of ferritin subunit messages in Drosophila melanogaster.

Insect ferritins have subunits homologous to the heavy and light chains of vertebrate ferritins. Cloning and sequence of the heavy chain homologue (HCH) of Drosophila melanogaster ferritin subunit have been reported earlier. When Northern blots of D. melanogaster RNA were probed with a cDNA for this HCH, three bands were observed. It was shown that these represented at least four classes of mRNA of various lengths. The polymorphism results from alternative splicing of an intron in the 5' untranslated region (UTR) that contains the iron-responsive element (IRE) and from two alternative polyadenylation sites in the 3' UTR. This has also been reported by others [Lind, M. I., Ekengren, S., Melefors, O. & Söderhäll, K. (1998) FEBS Lett. 436, 476-482]. By hybridizing Northern blots with specific probes, it has been shown that the relative proportions of the messages vary with the life stage and especially with iron supplementation of the diet. Iron significantly increases the amount of ferritin HCH messages and dramatically shifts the balance toward those messages that lack an IRE and/or have a short 3' UTR. In the larvae this change takes place in the gut, but not in the fat body. We speculate that this dramatic shift in message distribution may result from an effect of iron on the rate of transcription or message degradation, or from an effect on the splicing process itself. Synthesis of ferritin HCH subunit mRNAs that lack an IRE may be important under conditions of iron overload.

Drosophila melanogaster transferrin. Cloning, deduced protein sequence, expression during the life cycle, gene localization and up-regulation on bacterial infection.

Drosophila melanogaster transferrin cDNA was cloned from an ovarian cDNA library by using a PCR fragment amplified by two primers designed from other dipteran transferrin sequences. The clone (2035 bp) encodes a protein of 641 amino acids containing a signal peptide of 29 amino acids. Like other insect transferrins, Drosophila transferrin appears to have a functional iron-binding site only in the N-terminal lobe. The C-terminal lobe lacks iron-binding residues found in other transferrins, and has large deletions which make it much smaller than functional C-terminal lobes in other transferrins. In-situ hybridization using a digoxigenin labeled transferrin cDNA probe revealed that the gene is located at position 17B1-2 on the X chromosome. Northern blot analysis showed that transferrin mRNA was present in the larval, pupal and adult stages, but was not detectable in the embryo. Iron supplementation of the diet resulted in lower levels of transferrin mRNA. When adult flies were inoculated with bacteria (Escherichia coli), transferrin mRNA synthesis was markedly increased relative to controls.

Isolation and properties of Drosophila melanogaster ferritin--molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome.

Ferritin was purified from iron-fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed.

Induction of diaphorase-1 by dicoumarol in Drosophila virilis larvae.

Drosophila diaphorase-1 (DIA-1) is an enzyme similar to mammalian DT-diaphorase and is inhibited in vitro by dicoumarol. However, a ten-fold increase in DIA-1 activity was observed when third instar Drosophila virilis larvae were fed on a diet containing 0.1 M dicoumarol for 48 h. This induction was shown to be dose dependent and immunoprecipitation experiments with DIA-1 anti-serum demonstrated an increase in the DIA-1 protein level in dicoumarol-treated larvae. The induction of DIA1 by dicoumarol was found to be blocked by actinomycin D, which suggests a transcriptional mechanism of regulation. The opposite effect of dicoumarol on DIA-1 in vitro vs. in vivo suggests that a metabolic conversion takes place after the ingestion of this compound by D. virilis larvae.

Purification and characterization of diaphorases from some Drosophila species.

Diaphorase-1 and diaphorase-2 were isolated from two Drosophila species, D. virilis and D. melanogaster, and purified by gel filtration, affinity chromatography, immunoaffinity chromatography, and ion-exchange chromatography. The molecular weights of both enzymes were the same in each species. The molecular weight of diaphorase-1 was the same under both denaturating and nondenaturating conditions, close to 60,000, indicating a monomeric structure. Sodium dodecyl sulfate (SDS) electrophoresis of the purified diaphorase-2 revealed the presence of a single protein band of 55,000 Da, while the molecular weight of the native enzyme was found to be 67,000. The two diaphorases were further characterized by their pH optima, isoelectric points, and kinetic parameters, and antibodies were raised in rabbits against the purified enzymes from D. virilis. The antibodies showed no cross-reactions but recognized the corresponding diaphorases in D. melanogaster and D. novamexicana as well as D. virilis. The data obtained confirmed the hypothesis of an independent genetic control of diaphorase-1 and diaphorase-2 in Drosophila.

[A combined method for treating metastases of colorectal carcinoma to the liver]. / Kombiniran metod za lechenie na metastazi ot kolorektalen kartsinom v cherniia drob.

The method is a combination of operative treatment of metastases in the liver and regional intra-arterial chemotherapy. The indications for operative treatment of the underlying tumor, the metastases in the liver and the regional chemotherapy are pointed out. Two groups of patients with colorectal cancer and remote metastases in the liver are presented: one received surgical treatment alone, the other combined therapy. It is assumed that the combined treatment of the basic tumor, the metastases in the liver plus regional intra-arterial chemotherapy is a new trend in the treatment of advanced colorectal cancer metastasized in the liver.

[The tumor markers CEA and CA-19.9 in patients with colorectal carcinoma]. / Tumorni markeri CEA i CA-19.9 pri bolni s kolorektalen kartsinom.

The levels of two tumor markers--CEA and CA-19.9 were repeatedly determined in 146 patients with histologically verified colo-rectal cancer and in 58 healthy controls. The normal CA-19.9 values were up to 30 U/ml. The sensitivity of the two markers proved to be low: 48.8 per cent for CEA and 37 per cent for CA-19.9. In 26 of 35 patients with local recurrence or metastases marker increase preceded the appearance of clinical symptoms of progression of the pathologic process. The two markers appeared more informative than each of them when separately determined.

[The use of a new plastic method for consolidating a preternatural anus as prevention in paracolostomy hernias]. / Prilozhenie na nov plastichen metod za ukrepvane na protivoestestveniia anus praeter kato profilaktika na parakolostomichnite khernii.

Once the stage of extraperitoneal evasion of the sigmoid has been achieved for definitive preternatural anus, transsection is made of the aponeurosis of m. obliqu. ext. abdominis along its tendon fibers at a length of 5-6 cm. Then an aperture is made along the muscle fibers with the same length on m. obliqu. int. abdominis and m. transversus abdominis as well and finally f. transversalis is transsected. The peritoneum is intra-abdominally detached from the fascia beneath and alongside the aperture. An "Ampoxen" layer is placed around the internal aperture of the abdominal wall. The explant is sutured to the fascia and the evaded sigmoid. The latter is sutured to the aponeurosis of m. obliqu. ext. abdominis. Another Ampoxen layer is placed over this aponeurosis and sutured to the evaded intestine and in a chess-board way to the fascia. The method was applied in 22 patients with very good postoperative result.

[The results of the formation of a new anal sphincter by using the mm. graciles in acquired anal incontinence]. / Rezultati ot oformianeto na nov analen sfinkter chrez izpolzvane na mm. graciles pri pridobita analna inkontinentsiia.

The operative method of Pickrell for shaping a new anal sphincter from mm.graciles is described, emphasizing the refinements which the authors have introduced in it. In mobilizing the muscle, fascial fibers are left on both sides of the vascular-nervous bundle. They prevent the latter from overdistension when the muscle turns around the anal canal. Two arcuate lateral sections are made in the perianal area; this is followed by driving a subcutaneous tunnel, but under the perineal raphe and the anococcygeal ligaments. The two muscles are perianally wound each on 360 degrees, whereby m.gracilis dex. is fixed to tub. ossis ischii sin. and m.gracilis sin. is fixed to tub. ossis ischii dex. Before being fixed, the tendon end is inserted in the subperiosteal bone tunnel. When the tendon of the muscle is shorter, it may successfully be elongated by the explantoplast Ampoxen (applied in 3 patients). A total of 16 patients were treated. The result was good in 15 (93.8 per cent) and unsatisfactory in 1 (6.2 per cent).