Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells

Background

Plant phloem consists of an interdependent cell pair, the sieve element / companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants.

Results

In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs.

Conclusion

A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

     

Molecular monitoring of the Leu-164 mutation of dihydrofolate reductase in a highly sulfadoxine/pyrimethamine-resistant area in Africa

Background

Procalcitonin (PCT) is closely correlated with parasite burden and clinical outcome in falciparum malaria. The role of PCT in tertian malaria has not previously been investigated.

Patients and methods

PCT serum levels in 37 patients with tertian malaria were analysed. Clinical and laboratory parameters were assessed and statistically correlated both to the initial PCT levels and during the course of the disease.

Results

PCT levels rose for one day after commencing treatment and declined thereafter. However, there was no significant correlation with parasite burden, clinical parameters, laboratory values, or the presence of semi-immunity. Before treatment, the majority of patients showed normal or slightly elevated PCT levels (< 2.5 ng/ml), but PCT was markedly elevated (4.8 – 47 ng/ml) in one third of the population. The two groups did not differ by any other of the assessed parameters. Thus, while the post-treatment course of PCT resembles falciparum malaria, the lack of correlation between disease severity and even high PCT levels in a large proportion of patients is intriguing.

Conclusions

There is a fundamental difference in the relationship of PCT with tertian malaria not seen in other infectious diseases in which elevated PCT levels have been observed. This suggests distinct pathophysiological pathways in malaria.     

Intestinal zinc uptake in two marine teleosts,squirrelfish (Holocentrus adscensionis) and gulf toadfish (Opsanus beta)

Zinc is a vital micronutrient, yet as an environmental toxicant it can be deleterious to aquatic organisms such as fish. Consequently, the study of zinc uptake mechanisms is essential for understanding nutrition, toxicity, and metabolism of this metal. Intestinal zinc uptake was studied in two marine teleosts, using both in vitro (in vitro perfusion and intestinal sacs) and in vivo techniques (in situ bolus). Female squirrelfish (Holocentrus adscensionis) exhibited significantly increased epithelial zinc uptake associated with enhanced hepatic zinc accumulation. This confirms this zinc-hyperaccumulating teleost as a potential model of zinc absorption. Intestinal zinc uptake in the gulf toadfish (Opsanus beta) was biphasic with respect to zinc concentration (0.3-500 microM), exhibiting both saturable and passive uptake components. In both species, the passage of zinc into the postintestinal compartment was highly dependent on technique. Decreased proportions of postintestinal zinc in vivo, coupled with concentration-dependent distribution of zinc accumulation, suggested mechanisms may act to control the movement of zinc into the circulation. In addition, the results of this study were used to reinterpret previous findings of zinc uptake in freshwater fish and allowed a critique of techniques used to study intestinal metal uptake.     

The medical therapy of prostatic symptoms study: what will we learn?

Until recently, benign prostatic hyperplasia (BPH) was thought of as a relatively uncomplicated disease process resulting from age-related enlargement of the prostate gland, which was thought to cause all of the symptoms associated with the disease. Prostatectomy was considered the state-of-the art treatment of BPH. Advances in our understanding of the pathogenesis of BPH and the relationship among clinical signs and symptoms has allowed for the development of pharmacologic therapies for this disorder, namely, selective alpha-blockers and 5-alpha-reductase inhibitors. These 2 classes of drugs have been demonstrated to be safe and effective for the treatment of BPH. Evidence supports different mechanisms of action, suggesting that, when used in combination, these therapies may act synergistically. The Medical Therapy of Prostatic Symptoms study was designed, in part, to test this hypothesis. Initial results of the trial are presented in this article.     

Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum

A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.     

Estimating domain orientation of two human antibody IgG4 chimeras by crystallohydrodynamics

A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(4): cB72.3(4A), devoid of inter-chain disulfide bridging, and cB72.3(4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.Presented at the conference for Advances in Analytical Ultracentrifugation and Hydrodynamics, 8–11 June 2002, Grenoble, France     

IGF-I and IGFBP-3 transport in the rat heart

Specific binding of IGF-binding protein (IGFBP)-3 was shown to be present in the isolated, beating rat heart. The uptake of perfused (125)I-labeled IGF-I in the beating heart was decreased to 9% by blocking IGF-I binding sites with the IGF-I analog Long R(3) (LR(3)) IGF-I. When LR(3) was perfused with complexes of (125)I-IGF-I. IGFBP-3, uptake of (125)I-IGF-I was decreased to 41%, which was significantly greater than LR(3) and (125)I-IGF-I (41 vs. 9%). These data suggest that both microvessel IGF-I and IGFBP-3 binding sites contribute to the transport of IGF-I in the perfused rat heart. This also suggests a novel and plausible mechanism whereby circulating IGFs reach sites of IGF bioactivity.