A mathematical model and computer simulation study of insulin receptor regulation

A homeomorphic mathematical model of cell surface insulin receptor regulation is developed. The overall structure of the model is based on molecular mechanisms suggested by in vivo and in vitro experimental evidence from many different cell types. Model parameters correspond to cellular processes which are constrained by known boundry value conditions. As an example, computer simulation results are compared with published data from BC3H-1 myocytes in culture. With appropriate parameter choice, this model is able to simulate data from other cell types. Cellular processes which are explicitly represented in the model include: bound and unbound receptor endocytosis, receptor recycling, intracellular receptor degradation, and state-dependent receptor synthesis. Most of these processes are represented as first-order events. Using more complex representations of the model structure with higher order rate constants or saturable pathways does not qualitatively improve simulation results. Simulations are able to reproduce ligand-induced down and up regulation of receptors as well as the initial spontaneous display of surface insulin receptors. To demonstrate the behavior of our model and illustrate its utility for explaining insulin receptor regulation for a variety of conditions, simulations for which experimental data is unavailable for direct comparison are also shown. We believe the structure of our model is sufficient to explain insulin receptor regulation in a wide variety of cell types. In addition our model may aid in understanding the receptor component of insulin resistance (decreased sensitivity or responsiveness to insulin) seen in pathological states such as obesity and diabetes mellitus. Finally, this model may be applicable to the study of the regulation of other polypeptide hormone receptors.     

Cloning and regulation of messenger RNA for mouse apolipoprotein E

A cDNA clone for mouse apolipoprotein E has been identified from a mouse liver cDNA library by a combination of differential colony hybridization and hybrid selection-translation. The identity of the clone was unambiguously established by partial sequencing and comparison with human apolipoprotein E nucleotide and amino acid sequences. In conjunction with an in vitro translation assay for apolipoprotein E, the clone has been used to examine the relative levels of apolipoprotein E mRNA in various tissues of the mouse and the regulation of apolipoprotein E synthesis in response to a diet rich in saturated fat and cholesterol. In the tissues examined, the clone was found to hybridize to a polyadenylated RNA species of approximately 1400 nucleotides. Of the tissues involved in lipoprotein synthesis, liver is very rich (about 1% of total) in apolipoprotein E mRNA while intestine contains only trace amounts. Appreciable levels of active apolipoprotein E mRNA (up to 10% of that in liver) are also detected in peripheral tissues not associated with lipoprotein synthesis, including lung, kidney, spleen, and heart. Thus, extrahepatic apolipoprotein E synthesis may contribute significantly to the levels present in plasma, and a possible function in "reverse cholesterol transport" is considered. When mice were placed on a high lipid diet there was no discernible change in the level of apolipoprotein E mRNA in liver or intestine, although the level of the circulating protein increased about 3-fold. We conclude that in mice the effect of diet on apolipoprotein E levels in blood does not result from induction of mRNA in these tissues.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Fibroblast growth factor induces beta-amyloid precursor mRNA in glial but not neuronal cultured cells.

Treatment of PC12 and C6 cell cultures with recombinant basic fibroblast growth factor results in approximately a five to ten-fold stimulation of beta-amyloid precursor mRNA in the C6 astrocytoma cell line but only a slight induction of precursor mRNA in the PC-12 neuronal cell line. Stimulation of expression occurred at a hormone concentration of approximately 0.5 to 1 nM and was seen after 2 days. These results suggest that basic fibroblast growth factor may contribute to amyloidosis of Alzheimer's disease.     

A gene-controlling response of bone marrow progenitor cells to granulocyte-macrophage colony stimulating factors

The production of granulocytes and macrophages from progenitor cells in the bone marrow is controlled, in part, by a family of humoral regulators, termed colony stimulating factors (CSF). We have examined genetic factors controlling this process using in vitro cloning techniques. The inbred mouse strain LP/J showed elevated colony formation (CFU-C) in response to one subtype of CSF (G,M-CSF) compared to other strains of mice examined including the strain C57BL/6J. This variation resulted in a shift to the left of the CFU-C dose-response curve for LP/J. No difference between LP/J and C57BL/6J was seen with another subtype of CSF (CSF-1). Maximal CFU-C response was similar in the two mouse strains with both types of CSF, and mixing experiments with both types of CSF gave the same maximal level of colony formation as the individual CSF. (C57BL/6J X LP/J)F1 progeny exhibited a CFU-C dose-response curve to CSF-2 that was intermediate between the parental types, indicating additive inheritance. Genetic analysis of backcross progeny suggested that the variation in CFU-C response is probably determined by a single primary gene, although the variability of the colony formation assay has complicated interpretation of genetic studies. These results suggest that CSF-1 and G,M-CSF act independently on a single bone marrow progenitor cell population. The properties of the genetic variation for G,M-CSF response are consistent with an alteration in cellular receptors for G,M-CSF.     

The role of snail aestivation in transmission of schistosomiasis in changing climatic conditions

Schistosomiasis vector snails are subjected to extreme seasonal changes, particularly in ephemeral rivers and lentic waterbodies. In the tropics, aestivation is one of the adaptive strategies for survival and is used by snails in times of extremely high temperatures and desiccation. Aestivation therefore plays an important role in maintaining the transmission of schistosomiasis. This review assesses the possible impacts of climate change on the temporal and spatial distribution of schistosomiasis-transmitting snails with special emphasis on aestivation, and discusses the effect of schistosome infection on aestivation ability. The impacts of parasite development on snails, as well as physiological changes, are discussed with reference to schistosomiasis transmission. This review shows that schistosome-infected snails have lower survival rates during aestivation, and that those that survive manage to get rid of the infection. In general, snail aestivation ability is poor and survival chances diminish with time. Longer dry periods result in fewer, as well as uninfected, snails. However, the ability of the surviving snails to repopulate the habitats is high.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Facilitation and herbivory during restoration of California coastal sage scrub

Using nurse plants to facilitate native plant recruitment in degraded habitats is a common restoration practice across various arid and semiarid environments. Living trees or shrubs are typically considered nurse plants, whereas dead shrubs left in the landscape from prolonged drought are understudied prospective facilitators for native plant recruitment. The interaction between nurse plants and biotic pressures, such as herbivory, on native recruitment is also not well understood in semiarid plant communities. We investigated the effects of facilitation and herbivory on native seedling germination, growth, and survival in the restoration of degraded coastal sage scrub (CSS) habitat. Native shrub seedlings (Artemisia californica and Salvia mellifera) were planted, and native annual species (Amsinckia intermedia, Deinandra fasciculata, Phacelia distans, and Pseudognaphalium californicum) were sown in three Shrub Type treatments (live shrub, dead shrub, and exposed areas), with a nested Cage treatment (no cage and cage) in each Shrub Type treatment. Annual species grew equally well in all Shrub Type treatments; shrub seedlings grew largest in exposed areas. While there was little evidence of facilitation for all species tested, there were strong positive effects of caging on growth and establishment of all species. Caging palatable native species or planting species with anti‐herbivory traits around target plants may be more strategic approaches compared to using nurse plants in restoring degraded CSS after extended drought.     

Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor

PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B contains this motif (RYRDVS(50)), and wild-type PTP1B (but not mutants with substitutions for Ser(50)) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3(IR) cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with [(32)P]-orthophosphate. Insulin stimulation caused a significant increase in phosphorylation of wild-type PTP1B that could be blocked by pretreatment of cells with wortmannin or cotransfection of a dominant inhibitory Akt mutant. Similar results were observed with endogenous PTP1B in untransfected HepG2 cells. Cotransfection of constitutively active Akt caused robust phosphorylation of wild-type PTP1B both in the absence and presence of insulin. By contrast, PTP1B-S50A did not undergo phosphorylation in response to insulin. We tested the functional significance of phosphorylation at Ser(50) by evaluating insulin receptor autophosphorylation in transfected Cos-7 cells. Insulin treatment caused robust receptor autophosphorylation that could be substantially reduced by coexpression of wild-type PTP1B. Similar results were obtained with coexpression of PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an impaired ability to dephosphorylate the insulin receptor. Moreover, cotransfection of constitutively active Akt significantly inhibited the ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the insulin receptor. We conclude that PTP1B is a novel substrate for Akt and that phosphorylation of PTP1B by Akt at Ser(50) may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.