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From insects to mammals, metallothionein genes are induced in response to heavy metal load by the transcription factor MTF-1, which binds to short DNA sequence motifs, termed metal response elements (MREs). Here we describe a novel and seemingly paradoxical role for MTF-1 in Drosophila in that it also mediates transcriptional activation of Ctr1B, a copper importer, upon copper depletion. Activation depends on the same type of MRE motifs in the upstream region of the Ctr1B gene as are normally required for metal induction. Thus, a single transcription factor, MTF-1, plays a direct role in both copper detoxification and acquisition by inducing the expression of metallothioneins and of a copper importer, respectively.

Homologous recombination (HR) is an indispensable tool to modify the genome of yeast and mammals. More recently HR is also being used for gene targeting in Drosophila. Here we show that HR can be used efficiently to engineer chromosomal rearrangements such as pericentric and paracentric inversions and translocations in Drosophila. Two chromosomal double-strand breaks (DSBs), introduced by the rare-cutting I-SceI endonuclease on two different mobile elements sharing homologous sequences, are sufficient to promote rearrangements at a frequency of 1% to 4%. Such rearrangements, once generated by HR, can be reverted by Cre recombinase. However, Cre-mediated recombination efficiency drops with increasing distance between recombination sites, unlike HR. We therefore speculate that physical constraints on chromosomal movement are modulated during DSB repair, to facilitate the homology search throughout the genome., (Copyright 2004 Cold Spring Harbor Laboratory Press ISSN)

Bloom's syndrome (BS) is a genomic instability disorder characterized by cancer susceptibility. The protein defective in BS, BLM, belongs to the RecQ family of DNA helicases. In this study, we found that BLM interacts with hp150, the largest subunit of chromatin assembly factor 1 (CAF-1), in vitro and in vivo. Colocalization of a proportion of the cellular complement of these two proteins is found at specific nuclear foci coinciding with sites of DNA synthesis in the S phase. This colocalization increases in the presence of agents that damage DNA or inhibit DNA replication. In support of a functional interaction between BLM and CAF-1, we show that BLM inhibits CAF-1-mediated chromatin assembly during DNA repair in vitro. Although CAF-1 activity is not altered in BLM-deficient cells, the absence of BLM does impair the ability of CAF-1 to be mobilized within the nucleus in response to hydroxyurea treatment. Our results provide the first link between BLM and chromatin assembly coupled to DNA repair and suggest that BLM and CAF-1 function in a coordinated way to promote survival in response to DNA damage and/or replication blockade.

Metallothioneins (MTs) are short, cysteine-rich proteins for heavy metal homeostasis and detoxification; they bind a variety of heavy metals and also act as radical scavengers. Transcription of mammalian MT genes is activated by heavy metal load via the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein whose elimination in mice leads to embryonic lethality due to liver decay. Here we characterize the Drosophila homolog of vertebrate MTF-1 (dMTF-1), a 791-amino-acid protein which is most similar to its mammalian counterpart in the DNA-binding zinc finger region. Like mammalian MTF-1, dMTF-1 binds to conserved metal-responsive promoter elements (MREs) and requires zinc for DNA binding, yet some aspects of heavy metal regulation have also been subject to divergent evolution between Drosophila and mammals. dMTF-1, unlike mammalian MTF-1, is resistant to low pH (6 to 6.5). Furthermore, mammalian MT genes are activated best by zinc and cadmium, whereas in Drosophila cells, cadmium and copper are more potent inducers than zinc. The latter species difference is most likely due to aspects of heavy metal metabolism other than MTF-1, since in transfected mammalian cells, dMTF-1 responds to zinc like mammalian MTF-1. Heavy metal induction of both Drosophila MTs is abolished by double-stranded RNA interference: small amounts of cotransfected double-stranded RNA of dMTF-1 but not of unrelated control RNA inhibit the response to both the endogenous dMTF-1 and transfected dMTF-1. These data underline an important role for dMTF-1 in MT gene regulation and thus heavy metal homeostasis.

Objective: To report a case of hepatitis induced by two histamine (H2)-receptor blockers, famotidine and cimetidine., Case Summary: A 51-year-old obese woman with a duodenal ulcer developed symptomatic, famotidine-induced hepatitis. Other causes for hepatitis were ruled out. Famotidine was discontinued and the patient took omeprazole for a month without incident. Cimetidine therapy was then initiated and shortly thereafter, the patient developed another episode of symptomatic hepatitis that resolved upon drug discontinuation. Two abdominal ultrasound examinations six months apart and an abdominal computed tomography scan obtained later showed what appeared to be a stable, fatty liver., Discussion: This appears to be the first reported case of clinical hepatitis related to famotidine and the first reported case where clinical hepatitis followed the administration of two different H2-receptor blockers. The fatty liver probably was secondary to obesity and incidental to the hepatitis episodes., Conclusions: Famotidine can cause clinical hepatitis, and drug-induced hepatitis can occur after the administration of two different H2-receptor blockers.

In some soybean (Glycine max (L.) Merr.) cultivars, fruit removal does not delay the apparent loss of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activity and abundance or the decline in photosynthesis. Analysis of leaf extracts from defruited plants indicated a time-dependent increase in both Rubisco activity and abundance in a 30000 · g pellet fraction in cultivars which had been reported to lose all Rubisco protein from the supernatant fraction. Attempts to solubilize the pelleted Rubisco by increasing the buffer volume/tissue ratio or by adding alkylphenoxypolyethoxyethanol (Triton X-100), ethylenediaminetetraacetic acid (EDTA), or NaCl were unsuccessful. However, treatment of the pellets with denaturants such as 8 M urea or 5% (w/v) sodium dodecyl sulfate (SDS) did release Rubisco from the pellet. Redistribution of protein to the pellet fraction appeared to be specific for Rubisco since the amount of ribulose-5-phosphate kinase (EC 2.7.1.19) found in the pellet fraction of leaf extracts of control and defruited plants was small and constant over time. The loss of soluble Rubisco, and the concomitant increase in insoluble Rubisco, in response to fruit removal varied with genotype and was reproducible in both field and greenhouse environments. In addition, the effect was influenced by node position and light; lower and-or shaded leaves exhibited less Rubisco in the pellet fraction than leaves from the top of the plant that was fully exposed to sunlight. When isolated by sucrose-density-gradient centrifugation, the insoluble Rubisco was found to co-purify with a 30-kDa (kilodalton) polypeptide. These results indicate that alteration of the source/sink ratio by removing fruits results in the formation of an insoluble form of Rubisco in leaf extracts of soybean. Whether or not Rubisco exists as an insoluble complex with the 30-kDa polypeptide in intact leaves of defruited plants remains to be determined.

The abundances of ribulose-1,5-bisphosphate carboxylate/oxygenase (Rubisco) and ribulose-5-phosphate (Ru5P) kinase in field-grown soybean (Glycine max L. Merr.) leaves were quantified by a Western blot technique and related to changes in chlorophyll and photosynthetic capacity during senescence. Even though the leaf content of Rubisco was approximately 80-fold greater than that of Ru5P kinase, the decline in the levels of these two Calvin cycle enzymes occurred in parallel during the senescence of the leaves. Moreover, the decrease in the content of Rubisco was accompanied by parallel decreases of both the large and small subunits of this enzyme but not by an accumulation of altered large or small subunit isoforms. With increasing senescence, decreases in abundances of Rubisco, Ru5P kinase and chlorophyll were closely correlated with the decline in photosynthetic capacity; thus, the specific photosynthetic capacity when expressed per abundance of any of these parameters was rather constant despite an 8-fold decrease in photosynthetic capacity. These results suggest that during senescence of soybean leaves the chloroplast is subject to autolysis by mechanisms causing an approximately 80-fold greater rate of loss of Rubisco than Ru5P kinase.

Shoplifting is one of the most frequent crimes in the United States, yet there is no agreement about effective prevention procedures. Since most prevention strategies are aimed at either increasing public awareness of the severity of the consequences or increasing the threat of detection, procedures that contain these elements were evaluated. Posting signs around a department of a department store pointing out that shoplifting is a crime, etc., partially reduced shoplifting rates. When merchandise that was frequently taken was identified by signs and stars, shoplifting decreased to near zero. Publicity campaigns to inform the public of consequences for shoplifting may produce desirable results, but identifying likely shoplifting targets, which may increase the likelihood of detection, effectively reduces shoplifting rates.

The effects of source-sink alterations on the translocation patterns to soybean ("Fiskeby V") pods were studied using a pod leakage technique. The distribution of assimilates from a source leaf using double pulse experiments was followed at the pods at the source node and the node immediately below. Alterations were made by shading, illuminating, or excising two-thirds of the leaf area of the leaf at the node below. In control experiments both pulses exhibited identical time-course patterns at both nodes. Shading the lower leaf during the first half of the experiment and illuminating during the second reduced the distribution of (14)C-assimilate to the lower node's pods from the source leaf by approximately 30 to 50% while having no effect at the source node. Illuminating the lower leaf during the first half of the experiment followed by excision of two-thirds of that leaf's area and shading increased the import from the source leaf by 4- to 33-fold relative to the control while reducing the distribution to the source node by up to 40%. The change in distribution pattern took place in less than 30 minutes with no apparent change in the source leaf net photosynthesis or in the rate of movement to the pods. The results indicate that any alterations in the source-sink balance will quickly produce a change in the distribution patterns to the pods.

Active polyamine metabolism occurs in Glycine max (L.) seeds during development. Most (>/=97%) of putrescine (Put), spermidine (Spd), spermine (Spm), and cadaverine (Cad) are present as free forms in the growing embryo. In the cotyledon or embryonic axis, Put decreases to a nearly undetectable level, while Spd level sharply increases as seed dry weight accumulation progresses. Spm level in the axis also increases along with the Spd level. There is little change in Spm level in the cotyledons. Maturation and dehydration results in a slight reduction of Spd level in the cotyledons. Cad is present in relatively large quantities (5.5-12 micromoles per gram dry weight) in the axes of mature soybean seeds. Only traces of Cad, as expressed on a dry weight basis, are found in the developing or mature cotyledons. The synthesis and accumulation of Cad in the axis begins at the time when the axis or the seed accumulates 30 to 50% of its maximum dry weight. The Cad accumulation (0.8 nanomole per axis per day) proceeds until the later stages of dehydration. When soybean plants are subjected to complete defoliation and shade during the midpoint of seed maturation, Cad accumulation in the axis and seed dry weight accumulation ceased almost immediately. The treatment, however, does not affect the viability of soybean seeds.

The effect of anoxia on subsequent uptake and transport of K, Rb, and Na was examined with seedlings of barley (Hordeum vulgare L.), corn (Zea mays L.), and tall fescue (Lolium x Festuca hybrid derivative) to further our understanding of xylem loading. Roots were incubated in solutions depleted of O(2) by flushing with N(2) gas. After 1 hour exposure, plants were returned to aerated solutions for 16 hours prior to measuring uptake and transport. For each species, anoxia pretreatment significantly enhanced Na transport to the shoot. The rate of Na accumulation into roots, however, was not affected. There was no enhancement of either K or Rb accumulation in shoots, indicating specificity for Na transport. A minimum exposure to anoxia of 30 minutes and a minimum of 12 hours elapsed time was necessary to achieve the maximum rate of Na transport to the shoot in barley seedlings. Accumulation of Na in the shoot of both the control and anoxia pretreated barley plants was inhibited by anoxia and by addition of the proline analog, l-azetidine-2-carboxylic acid, during the uptake period. Enhancement of Na transport was associated with a proportional increase in the rate of synthesis of a membrane bound protein with a molecular weight of 78,000 daltons.

Radioactive photosynthetic assimilates, translocated to a soybean (Glycine max [L.] Merr. ;Fiskeby V') pod can be measured directly by excising the stylar tip of the pod under 20 mm ethylenediaminetetraacetate solution (pH 7.0) and allowing the material to leak into the solution. Pods at the source node received approximately 50% of the (14)C exported from the source leaf to the pod and leaked approximately 1 to 3% of this into the solution. More than 90% of the (14)C that leaked from the pods was found in the neutral fraction and, of this, about 93% was in sucrose. Fifteen amino acids were identified in the leakage including: alanine, arginine, asparagine, gamma-aminobutyric acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tyrosine, and valine. The majority of the (14)C in the basic fraction was found in serine ( approximately 30%) and asparagine ( approximately 23%). The inorganic ions K, Ca, P, Mg, Zn, and Fe were found in the leakage component. Nitrate was not detectable in the collected leakage solution. The absence of NO(3) (-) and the large proportion of the label in sucrose suggest a possible phloem origin for most of the material. The technique provides an uncomplicated, reproducible means of analyzing the material translocated into and through the soybean pod, as well as following the time course of label arrival at the pod.

Three cultivars of soybean (Glycine max [L.] Merr. cvs Harper, McCall, and Maple Amber) were grown in the field and kept continuously deflowered throughout the normal seedfill period. For all cultivars, deflowering led to delayed leaf abscission and a slower rate of chlorophyll loss. Compared to control plants, photosynthesis and ribulose 1,5-bis-phosphate carboxylase/oxygenase (Rubisco) level declined slightly faster for deflowered Harper, but for both McCall and Maple Amber, leaves of deflowered plants maintained approximately 20% of maximum photosynthesis and Rubisco level 1 month after control plants had senesced. Deflowering led to decreased leaf N remobilization and increased starch accumulation for all cultivars, but cultivars differed in that for McCall and Maple Amber, N and starch concentrations slowly but steadily declined over time whereas for Harper, N and starch concentrations remained essentially constant over time. SDS-PAGE of leaf proteins indicated that for all cultivars, deflowering led to accumulation of four polypeptides (80, 54, 29, and 27 kilodaltons). Western analysis using antisera prepared against the 29 and 27 kilodalton polypeptides verified that these polypeptides were the glycoproteins previously reported to accumulate in vacuoles of paraveinal mesophyll cells of depodded soybean plants. The results indicated that depending on the cultivar, sink removal can lead to either slightly faster or markedly slower loss of photosynthesis and Rubisco. This difference, however, was not associated with the ability to synthesize leaf storage proteins. For any particular cultivar, declines in chlorophyll, photosynthesis, and Rubisco were initiated at approximately the same time for control and deflowered plants. Thus, even though cultivars differed in rate of decay of photosynthetic rate and Rubisco level in response to sink removal, the initiation of leaf senescence was not influenced by presence or absence of developing fruits.