A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.     

Transportin Mediates Nuclear Entry of DNA in Vertebrate Systems

Delivery of DNA to the cell nucleus is an essential step in many types of viral infection, transfection, gene transfer by the plant pathogen Agrobacterium tumefaciens and in strategies for gene therapy. Thus, the mechanism by which DNA crosses the nuclear pore complex (NPC) is of great interest. Using nuclei reconstituted in vitro in Xenopus egg extracts, we previously studied DNA passage through the nuclear pores using a single-molecule approach based on optical tweezers. Fluorescently labeled DNA molecules were also seen to accumulate within nuclei. Here we find that this import of DNA relies on a soluble protein receptor of the importin family. To identify this receptor, we used different pathway-specific cargoes in competition studies as well as pathway-specific dominant negative inhibitors derived from the nucleoporin Nup153. We found that inhibition of the receptor transportin suppresses DNA import. In contrast, inhibition of importin β has little effect on the nuclear accumulation of DNA. The dependence on transportin was fully confirmed in assays using permeabilized HeLa cells and a mammalian cell extract. We conclude that the nuclear import of DNA observed in these different vertebrate systems is largely mediated by the receptor transportin. We further report that histones, a known cargo of transportin, can act as an adaptor for the binding of transportin to DNA.     

CD74 is a regulator of hematopoietic stem cell maintenance

Hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that have the capacity for self-renewal and differentiate into all blood cell lineages. These cells are the most useful cells for clinical transplantations and for regenerative medicine. So far, it has not been possible to expand adult hematopoietic stem cells (HSCs) without losing their self-renewal properties. CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), and its mRNA is known to be expressed in HSCs. Here, we demonstrate that mice lacking CD74 exhibit an accumulation of HSCs in the bone marrow (BM) due to their increased potential to repopulate and compete for BM niches. Our results suggest that CD74 regulates the maintenance of the HSCs and CD18 expression. Its absence leads to induced survival of these cells and accumulation of quiescent and proliferating cells. Furthermore, in in vitro experiments, blocking of CD74 elevated the numbers of HSPCs. Thus, we suggest that blocking CD74 could lead to improved clinical insight into BM transplant protocols, enabling improved engraftment.

Hematopoietic stem and progenitor cells (HSPCs) can self-renew and differentiate into all blood cell lineages, making them useful for clinical transplantations and regenerative medicine. This study shows that blocking the MIF receptor CD74 increases the accumulation of HSPCs and could improve the efficacy of bone marrow transplantation protocols.     

The influence of mouse sera, regenerating liver extracts and bacterial products on the abilities of different cells in vitro.

In the complexity of host tumor relations, the regeneration of the tissue in which the tumor is growing, or in some other tissue in the organism, could influence the maturation of tumor cells, i.e. tumor reversion. Clinical observations and experiments on plants, lower animals, or animal embryos, performed by several authors, and our results on the influence of regenerating mouse liver on the abilities of tumor transplanted there or elsewhere in the organism led us to study the in vitro growth of different cells or bacteria exposed to the extracts of normal or regenerating liver and/or sera from these animals. Further, sterile used bacterial media were added to bacterial or cell cultures, respectively. Depending on the model, liver extracts-particularly extracts and sera from mice with regenerating liver-were shown to inhibit radioactive thymidine incorporation in the cells. In these experiments, the number of bacteria or cells per culture was lower than in otherwise treated corresponding cultures. Further, used sterile media of bacterial cultures stimulated the growth of bacteria but inhibited thymidine incorporation into fibrosarcoma cells in vitro. Whether this means that one or several common regulators exist in nature appears as an intriguing, but still completely open question. The idea of controlling tumor growth by using such regulatory growth factors seems very provocative.     

The characterization of LeNUC1, a nuclease associated with leaf senescence of tomato

Induction of nuclease and RNase activities, together with decreases in nucleic acid content are considered to be characteristics of senescence in higher plants. However, little is known about the specific identities or functions of the enzymes involved or the mechanisms controlling their activation. Here we report the identification of a 41-kDa-tomato nuclease, LeNUC1, which is specifically induced during tomato leaf senescence but not in ripening fruits. LeNUC1 is a glycoprotein, which can degrade both RNA and DNA and has optimal activity at pH 7.5–8. EDTA inhibits the activity of LeNUC1, while the addition of Co²⁺ or Mn²⁺ can restore its activity in the presence of the chelating agent. Interestingly, the activity of LeNUC1 is also induced in young leaves upon treatment with ethylene, which is known to be a senescence-promoting hormone in tomato. Constitutive activity of a 39-kDa nuclease, LeNUC2, similar in its biochemical requirements to LeNUC1, was also detected. LeNUC2 is not induced by ethylene and does not seem to be glycosylated. Based on their characteristics, LeNUC1 and LeNUC2 can be classified as Nuclease I enzymes. LeNUC1 may be involved in nucleic acid metabolism during tomato leaf senescence.     

The effect of prostaglandin synthetase inhibitors on renal function: Studies in normal rats during sodium or water diuresis and in rats with chronic renal insufficiency

The effect of acute infusion of the prostaglandin synthetase inhibitors — meclofenamate or indomethacin — was examined in awake rats. Studies were performed in normal rats undergoing either sodium or water diuresis and in salt-replete rats with chronic renal insufficiency. Prostaglandin synthetase inhibitors had no effect on renal plasma flow, glomerular filtration rate or fractional excretion of sodium in any of the groups. Absolute urinary excretion rates for sodium and potassium decreased only in the normal, salt-replete rats. In contrast, prostaglandin synthetase inhibitors consistently decreased urinary flow and osmolar clearance under all experimental conditions studied. In the normal, salt-replete rats the fall in urine flow was preceded by an increase in urinary excretion of cyclic AMP. These results show that inhibitors of prostaglandin synthesis enhance the ability of the kidney to reabsorb water. This effect may be secondary to increased cyclic AMP generation and to increased urea recirculation resulting in higher urea accumulation in the renal medulla.