Coypu insulin. Primary structure, conformation and biological properties of a hystricomorph rodent insulin.

Insulin from a hystricomorph rodent, coypu (Myocaster coypus), was isolated and purified to near homogeneity. Like the other insulins that have been characterized in this Suborder of Rodentia, coypu insulin also exhibits a very low (3%) biological potency, relative to pig insulin, on lipogenesis in isolated rat fat-cells. The receptor-binding affinity is significantly higher (5-8%) in rat fat-cells, in rat liver plasma membranes and in pig liver cells, indicating that the efficacy of coypu insulin on receptors is about 2-fold lower than that of pig insulin. The primary structures of the oxidized A- and B-chains were determined, and our sequence analysis confirms a previous report [Smith (1972) Diabetes 21, Suppl. 2, 457-460] that the C-terminus of the A-chain is extended by a single residue (i.e. aspartate-A22), in contrast with most other insulin sequences, which terminate at residue A21. In spite of a large number of amino acid substitutions (relative to mammalian insulins), computer-graphics model-building studies suggest a similar spatial arrangement for coypu insulin to that for pig insulin. The substitution of the zinc-co-ordinating site (B10-His----Gln) along with various substitutions on the intermolecular surfaces involved in the formation of higher aggregates are consistent with the observation that this insulin is predominantly 'monomeric' in nature. The c.d. spectrum of coypu insulin is relatively similar to those of casiragua insulin and of bovine insulin at low concentration.     

The intermediary cell: Minor-vein anatomy and raffinose oligosaccharide synthesis in the Scrophulariaceae

Minor-vein anatomy, sugar content, sugar synthesis, and translocation were studied in mature leaves of nine members of the Scrophulariaceae to determine if there is a correlation between companion-cell type and class of sugar translocated. Three types of companion cell were found: intermediary cells with extensive plasmodesmatal connections to the bundle sheath; transfer cells with wall ingrowths and few plasmodesmata; and ordinary companion cells with few plasmodesmata and no wall ingrowths. Alonsoa warscewiczii Regal., Verbascum chaixi Vill., and Mimulus cardinalis Dougl. ex. Benth. have intermediary cells and ordinary companion cells in the minor veins. These plants synthesize large amounts of raffinose and stachyose as well as sucrose. Nemesia strumosa Benth., and Rhodochiton atrosanguineum Zucc. have both intermediary cells and transfer cells and make proportionately less raffinose oligosaccharide than the species above. In N. strumosa, a single sieve element may abut both an intermediary cell and a transfer cell. The minor veins of Asarina scandens (Cav.) Penn. have transfer cells and what appear to be modified intermediary cells that have fewer plasmodesmata than other species, and occasional wall ingrowths. Asarina scandens synthesizes little raffinose or stachyose. Cymbalaria muralis P. Gaertn et al. and Linaria maroccana Hook.f. have only transfer cells and Digitalis grandiflora Mill. has only ordinary companion cells; these species make a trace of galactinol and raffinose, but no stachyose. Translocation experiments indicate that there is long-distance movement of raffinose oligosaccharide in these plants, even when it is synthesized in very small quantities in the leaves. We conclude that intermediary cells are as distinct a cell type as the transfer cell. In contrast to transfer cells, which are specialized for uptake of solute from the apoplast, intermediary cells are specialized for symplastic transfer of photoassimilate from the mesophyll and for synthesis of raffinose oligosaccharide. This supports our contention that raffinose oligosaccharide synthesis and symplastic phloem loading are mechanistically linked (Turgeon and Gowan 1990, Plant Physiol. 94, 1244–1249). Minor-vein anatomy and sugar synthesis may be useful characters in determining the phylogenetic relationships of plants in this family.We thank Andrea Wolfe and Wayne Elisens for helpful discussions on the taxonomy of the Scrophulariaceae. This research was supported by National Science Foundation grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 92-37306-7819, and Hatch funds.     

HIV Viremia and T-Cell Activation Differentially Affect the Performance of Glomerular Filtration Rate Equations Based on Creatinine and Cystatin C

Background

Serum creatinine and cystatin C are used as markers of glomerular filtration rate (GFR). The performance of these GFR markers relative to exogenously measured GFR (mGFR) in HIV-positive individuals is not well established.

Methods

We assessed the performance of the chronic kidney disease epidemiology collaboration equations based on serum concentrations of creatinine (eGFR_cr), cystatin C (eGFR_cys) and both biomarkers combined (eGFR_cr-cys) in 187 HIV-positive and 98 HIV-negative participants. Measured GFR was calculated by plasma iohexol clearance. Bias and accuracy were defined as the difference between eGFR and mGFR and the percentage of eGFR observations within 30% of mGFR, respectively. Activated CD4 and CD8 T-cells (CD38+ HLA-DR+) were measured by flow cytometry.

Results

The median mGFR was >100 ml/min/1.73 m² in both groups. All equations tended to be less accurate in HIV-positive than in HIV-negative subjects, with eGFR_cr-cys being the most accurate overall. In the HIV-positive group, eGFR_cys was significantly less accurate and more biased than eGFR_cr and eGFR_{cr_cys}. Additionally eGFR_cys bias and accuracy were strongly associated with use of antiretroviral therapy, HIV RNA suppression, and percentages of activated CD4 or CD8 T-cells. Hepatitis C seropositivity was associated with larger eGFR_cys bias in both HIV-positive and HIV-negative groups. In contrast, eGFR_cr accuracy and bias were not associated with HIV-related factors, T-cell activation, or hepatitis C.

Conclusions

The performance of eGFR_cys relative to mGFR was strongly correlated with HIV treatment factors and markers of T-cell activation, which may limit its usefulness as a GFR marker in this population.     

Fluxomics with Ratiometric Metabolite Dyes

Today''s major excitement in biology centers on signaling: How can a cell or organism measure the myriad of environmental cues, integrate it, and acclimate to the new conditions? Hormonal signals and second messengers are in the focus of most of these studies, e.g., regulation of glucose transporter GLUT4 cycling by insulin, or regulation of plant growth by auxin or brassinosteroids.^1–3 In comparison, we generally assume that we know almost everything about basic metabolism since it has been studied for many decades; for example we know since the early 80s that allosteric regulation by fructose-2,6-bisphophate plays an important role in regulating glycolysis in plants and animals.⁴ This may be the reason why studies of metabolism appear to be a bit out of fashion. But if we look to other organisms such as E. coli or yeast, we rapidly realize that metabolism is controlled by complex interconnected signaling networks, and that we understand little of these signaling networks in humans and plants.^5,6 As it turns out, the cell registers many metabolites, and flux through the pathways is regulated using complex signaling networks that involve calcium as well as hormones.Key Words: flux, fluxome, glucose, glutamate, phosphate, sucrose, fluorescence resonance energy transfer, biosensorOne of the reasons for the fable for hormones lies in the simple fact that it is easier to observe macroscopic changes, such as changes in the architecture of a plant than to determine metabolite levels, but also here new tools are urgently needed that allow quantification of these small molecules. Visualization of starch levels provided a significant advance, and in combination with mutant screens allowed to identify fundamental components of starch metabolism.^7–9 The biggest advance for the signaling field was the development of advanced chemical and genetically encoded calcium dyes.^10–12 No such dyes are available for hormones or metabolites, as soon as we try to determine levels of metabolites (or signaling molecules), we run into the issues of compartmentation and cellular differences in tissues. Today, the same enzymatic assays used decades ago are still widely used to determine metabolite levels. Although significant advances in chromatography and mass spectrometry based metabolite analysis have moved the study of metabolism to ‘omics’ era, compartmentalization of metabolism still presents a major challenge. Especially the large vacuoles of plant cells are a major obstacle, since even fractionation studies suffer from contamination. Moreover, with the current set of tools it is not possible to determine the dynamic changes in metabolite levels in different subcellular compartments in real time in vivo. Radiotracers have helped a lot to identify and quantify intermediates and to assemble pathways, originally using pulse labeling followed by paper chromatography. Today ¹³C-labeling is used together with mass spectrometry to obtain insights into metabolic flux control.¹³ This tool set for the first time enabled the comparison of mutants and study regulatory networks involved in sugar signaling. While significant, advances in radiotracer experiments do not provide cellular or subcellular information and only limited temporal resolution, they do provide efficient means for studying metabolite fluxes through complex and/or not well-defined pathways. Thus there is a clear need for metabolite specific dyes that can be targeted to subcellular compartments and that would enable flux measurements in response to environmental cues helping to push metabolic research back into the focus of signaling-related biology.In 2002, we developed the first prototype “metabolic dye” FRET sensor for maltose.^14,15 A similar glucose sensor was recently employed for measuring tracer-independent transport of glucose across the ER membrane of liver cells.¹⁶ After resolving some issues such as low signal-to-noise and gene silencing in plants, we are now able to compare glucose levels between cells in an intact root in real time.¹⁷ The parallel development of sucrose and phosphate sensors complements the set of tools, in future experiments providing a comparison of sucrose, phosphate and glucose fluxes in intact tissues with both temporal (below seconds) and spatial resolution (cellular and subcellular).^18,19The first experiments already led to a big surprise: glucose supplied to the root is rapidly taken up and is rapidly metabolized.¹⁷ Roots expressing the highest affinity sensor FLIPglu170n responded to glucose perfusion suggesting that the steady state glucose level in the root is less than 100 nM, the estimated detection limit for this sensor in these first experiments. The first experiments were limited by the mixing kinetics in the bath used for perfusion, while improvement of the chamber now allow for faster for glucose exchange. We estimate that glucose levels fall from a steady state level of approximately 5 mM in the cytosol when perfused with 5 mM glucose to below 100 nM in about three minutes. For the sensor with an affinity of 600 µM the rate of glucose accumulation, which is composed of the various rates that affect the steady state in the cytosol such as metabolism, compartmentation and transport across the plasma membrane, is in the range of 527 ± 77 µM glucose/min and that for glucose removal is 317 ± 37 (Fig. 1; Chaudhuri B, Frommer WB, unpublished). Questions that arise are: Which transport systems drive uptake? How much does the vacuole contribute to the observed flux and steady state levels? Is the capacity of hexokinase at levels below its K_m still sufficient to phosphorylate glucose efficient enough to pull glucose below 100 nM or does hexokinase have different properties in vivo compared to what we know from the purified enzyme? Are there different transporters and enzymes contributing to flux in the low (1–10 mM) and the ultrahigh affinity (low µM) phases? Are there spatial differences in the root? Why do roots take up glucose so efficiently in the first place? The combination of the sensors with information from the expression-LEDs from Birnbaum and Benfey²⁰ and specific knock-out mutants should help answering some of these questions.Open in a separate windowFigure 1Quantitative analysis of glucose flux from an Arabidopsis root expressing FLIPglu-600µΔ13, a FRET sensor for glucose with an affinity of 600 µM. The root of a 10 day-old seedling was placed into a perfusion chamber and perfused with hydroponic medium with or without 5 mM glucose. eCFP was excited and emission was recorded for eCFP and eYFP every 10 seconds (essentially as decsribed in ref. 17). The emission intensities for a region-of-interest were averaged and the emission ratio was determined at the two wavelengths for each image of a time series and plotted on the Y-axis against time on the X-axis. Addition of glucose is indicated.Another big surprise is the dramatic gradient of glucose across the plasma membrane, which has important implications for our understanding of transport processes across the plasma membrane as well as the intracellular membranes.¹⁷ Information about the gradients is relevant in the context of apo- and symplasmic unloading routes in roots²¹ and the contribution of proton-coupled transporters in cellular export.²² It will thus be interesting to follow the extracellular levels using surface-anchored sensors. Now that besides high sensitivity glucose FLIPs¹⁷ we also generated nanosensors for sucrose¹⁹ and phosphate,¹⁸ complementing the similar tool sets for calcium²³ and pH,²⁴ it is possible to compare multiple parameters and to follow flux at different levels and to calibrate against other influences.The improvements of the signal-to-noise ratio of the FRET-based metabolite sensors²⁵ makes the FLIPs a standard tool for every lab interested in measuring ion-, sugar- or amino acid flux in living cells. Since the nanosensors are genetically encoded, they can be used to characterize intracellular fluxes^16,26 in any organism for which transformation protocols have been established. The existing sets of sensors are simple to use, constructs are available through Addgene and Arabidopsis lines from the Arabidopsis Stock Center. Detailed instructions for imaging can be found at: http://carnegiedpb.stanford.edu/research/frommer/research_frommer_protocols.php. These tools will hopefully become a standard system not only for physiological analyses, but in addition provide a new way for high throughput fluxomics studies.     

Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons

Distinct classes of neurons are generated from progenitor cells distributed in characteristic dorsoventral patterns in the developing spinal neural tube. We define restricted neural progenitor populations by the discrete, nonoverlapping expression of Ngn1, Math1, and Mash1. Crossinhibition between these bHLH factors is demonstrated and provides a mechanism for the generation of discrete bHLH expression domains. This precise control of bHLH factor expression is essential for proper neural development since as demonstrated in both loss- and gain-of-function experiments, expression of Math1 or Ngn1 in dorsal progenitor cells determines whether LH2A/B- or dorsal Lim1/2-expressing interneurons will develop. Together, the data suggest that although Math1 and Ngn1 appear to be redundant with respect to neurogenesis, they have distinct functions in specifying neuronal subtype in the dorsal neural tube.     

MICA polymorphism in South American Indians

We have studied the MICA alleles of 196 unrelated subjects from three South American Indian tribes (Toba, Wichi and Terena). They are members of isolated tribes located in the Gran Chaco area in northeastern Argentina and in Mato Grosso do Sul in South Central Brazil. Of 55 previously known alleles, nine were observed in South American Indians, compared with 16 that were found in North American Caucasians, suggesting a more restricted allelic distribution of MICA in these tribes. In South American Indians, MICA*00201 was the most frequent allele, with a gene frequency of 33% in Toba, 47% in Wichi and 44% in Terena. MICA*00201, MICA*027 (external domain sequence like MICA*008/TM allele A5) and MICA*010 accounted for more than 90% of all the MICA genes in South American Indians. In North American Caucasians, MICA*00801 (*008/A5.1) accounted for 42% of the genes and was the most common allele. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of HLA-B in the South American Indian populations. Phylogenetic trees constructed using gene frequencies of the transmembrane short tandem repeats in the populations reported here, and in other populations taken from published reports, suggest that South American Indians are more closely related to Asians than to Europeans.     

Immunogenicity and protective efficacy of three DNA vaccines encoding pre-erythrocytic- and erythrocytic-stage antigens of Plasmodium cynomolgi in rhesus monkeys

Although several malaria vaccine candidate antigens have been identified, the most suitable methods for their delivery are still being investigated. In this regard, direct immunization with DNA encoding these vaccine target antigens is an attractive alternative. Here, we have investigated the immune responses to DNA immunization with three major vaccine target antigens: the apical membrane antigen-1 and the 19-kDa C-terminal fragment of merozoite surface protein-1 from the erythrocytic stage, and the thrombospondin-related adhesive protein from the pre-erythrocytic stage of Plasmodium cynomolgi in rhesus monkeys. Antigen-specific antibodies were developed in all the immunized monkeys and peripheral blood mononuclear cells from all immunized monkeys proliferated to different extents upon in vitro stimulation with the corresponding recombinant proteins. The immunized monkeys were challenged with P. cynomolgi sporozoites. All of the immunized animals developed infection but although there was no significant difference between the control and vaccinated animals in terms of pre-patent period, total duration of patency and primary peak parasitemia, the vaccinated animals had significantly lower secondary peak parasitemia than the control animals.     

Deimination of myelin basic protein. 1. Effect of deimination of arginyl residues of myelin basic protein on its structure and susceptibility to digestion by cathepsin D

The effect of deimination of arginyl residues in bovine myelin basic protein (MBP) on its susceptibility to digestion by cathepsin D has been studied. Using bovine component 1 (C-1) of MBP, the most unmodified of the components with all 18 arginyl residues intact, we have generated a number of citrullinated forms by treatment of the protein with purified peptidylarginine deiminase (PAD) in vitro. We obtained species containing 0-9.9 mol of citrulline/mol of MBP. These various species were digested with cathepsin D, a metalloproteinase which cleaves proteins at Phe-Phe linkages. The rate of digestion compared to component 1 was only slightly affected when 2.7 or 3.8 mol of citrulline/mol of MBP was present. With 7.0 mol of citrulline/mol of MBP, a large increase in the rate of digestion occurred. No further increase was observed with 9.9 mol of citrulline/mol of MBP. The immunodominant peptide 43-88 (bovine sequence) was released slowly when 2.7 and 3.8 mol of citrulline/mol of MBP was present, but it was released rapidly when 7.0 mol of citrulline/mol of MBP was present. The dramatic change in digestion with 7.0 mol of citrulline/mol of MBP or more could be explained by a change in three-dimensional structure. Molecular dynamics simulation showed that increasing the number of citrullinyl residues above 7 mol/mol of MBP generated a more open structure, consistent with experimental observations in the literature. We conclude that PAD, which deiminates arginyl residues in proteins, decreases both the charge and compact structure of MBP. This structural change allows better access of the Phe-Phe linkages to cathepsin D. This scheme represents an effective way of generating the immunodominant peptide which sensitizes T-cells for the autoimmune response in demyelinating disease.