Identification of a UPTG inhibitor protein from maize endosperm: high homology with sucrose synthase protein

A thermolabile UPTG inhibitor protein (IP) was isolated and purified from a developing maize endosperm preparation. High homology of two internal peptides of IP with known plant sucrose synthase (SS) sequences suggested that IP might be related somehow with SS. IP and SS activities were found in the same preparation and showed thermolability between 60-65 degrees C. IP and SS activities presented the same ionic charge and molecular mass in native conditions (Mono Q and Superose-12 columns chromatographies). Western blot experiments with an anti-SS antibody as well as with an anti-IP antibody showed a single 80 kDa polypeptide band where IP and SS activities were present. Anti-SS antibody can neutralize SS as well as IP activities in a neutralization assay. It was found that in the maize mutant shrunken-1, lacking SS1 protein, the UPTG activity was not inhibited. Furthermore, the solubilized preparation of the sh1 endosperm is unable of inhibiting UPTG activity from potato tuber. The high correlation between IP and SS properties suggests that IP might be in fact a form of SS. Moreover, the relation between IP and the SS1 isoform is discussed. So, a new biological activity of SS is suggested.     

Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42 degreesC. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.     

Molecular cloning of starch synthase I from maize (W64) endosperm and expression in Escherichia coli

Vertebrate myelin is enriched in the lipid galactocerebroside (GalC) and its sulfated derivated sulfatide. To understand the in vivo function of these lipids, we analyzed myelination in mice that contain a null mutation in the gene encoding UDP-galactose:ceramide galactosyltransferase, the enzyme responsible for catalyzing the final step in GalC synthesis. Galactolipid-deficient myelin is regionally unstable and progressively degenerates. At postnatal day 30, demyelination is restricted to the midbrain and hindbrain, but by postnatal day 90, it spreads throughout the central nervous system. Activated microglial cells and reactive astrocytes appear with the loss of myelin in older animals. Nonetheless, major myelin protein gene mRNA levels are normal throughout the life of these animals, suggesting that widespread oligodendrocyte death is not the primary cause of demyelination. The developmental switch in myelin-associated glycoprotein isoform expression, however, does not occur normally in these mice, suggesting an alteration in oligodendrocyte maturation. Taken together, these findings indicate that GalC and sulfatide are required for the long-term maintenance of myelin and that their absence may have subtle effects on the development of oligodendrocytes.     

Sec-independent protein translocation by the maize Hcf106 protein

The bacterial Sec and signal recognition particle (ffh-dependent) protein translocation mechanisms are conserved between prokaryotes and higher plant chloroplasts. A third translocation mechanism in chloroplasts [the proton concentration difference (DeltapH) pathway] was previously thought to be unique. The hcf106 mutation of maize disrupts the localization of proteins transported through this DeltapH pathway in isolated chloroplasts. The Hcf106 gene encodes a receptor-like thylakoid membrane protein, which shows homology to open reading frames from all completely sequenced bacterial genomes, which suggests that the DeltapH pathway has been conserved since the endosymbiotic origin of chloroplasts. Thus, the third protein translocation pathway, of which HCF106 is a component, is found in both bacteria and plants.     

Genetic regulation of entry into meiosis in Caenorhabditis elegans

The Caenorhabditis elegans germline is composed of mitotically dividing cells at the distal end that give rise to meiotic cells more proximally. Specification of the distal region as mitotic relies on induction by the somatic distal tip cell and the glp-1 signal transduction pathway. However, the genetic control over the transition from mitosis to meiosis is not understood. In this paper, we report the identification of a gene, gld-2, that has at least two functions in germline development. First, gld-2 is required for normal progression through meiotic prophase. Second, gld-2 promotes entry into meiosis from the mitotic cell cycle. With respect to this second function, gld-2 appears to be functionally redundant with a previously described gene, gld-1 (Francis, R., Barton, M. K., Kimble, J. and Schedl, T. (1995) Genetics 139, 579-606). Germ cells in gld-1(o) and gld-2 single mutants enter meiosis at the normal time, but germ cells in gld-2 gld-1(o) double mutants do not enter meiosis. Instead, the double mutant germline is mitotic throughout and forms a large tumor. We suggest that gld-1 and gld-2 define two independent regulatory pathways, each of which can be sufficient for entry into meiosis. Epistasis analyses show that gld-1 and gld-2 work downstream of the glp-1 signal transduction pathway. Therefore, we hypothesize that glp-1 promotes proliferation by inhibiting the meiosis-promoting functions of gld-1 and gld-2.     

Genetic regulation of preimplantation mouse embryo survival

The preimplantation period of mammalian development is characterized by cleavage of a one-cell embryo to a blastocyst stage embryo. During preimplantation development, 15%-50% of the embryos die as a result of factors that are largely unknown. Two parameters of preimplantation development, a fast rate of development and a low degree of fragmentation, are indicative of good embryo quality. There is mounting evidence that genes control both rate of development and degree of fragmentation. We have discovered a gene, Ped (preimplantation embryo development), which controls the rate of preimplantation embryonic cleavage. The Ped gene is encoded by two similar genes, Q7 and Q9, in the Q region of the mouse major histocompatibility complex (MHC). The Ped gene product is an MHC class Ib protein, the Qa-2 antigen. The mechanisms by which the Ped gene controls rate of embryonic cleavage division are being explored. In order to understand genetic mechanisms underlying the second criterion of embryo quality, degree of fragmentation, we have begun to assess expression of the genes that could potentially regulate apoptosis in preimplantation embryos. We have shown that staurosporine can induce apoptosis in mouse blastocysts. By using RT-PCR, we have shown that genes encoding protein in the two major gene families that regulate apoptosis, the Bcl-2 and caspase gene families, are present in preimplantation embryos. We hypothesize that there is a homeostatic mechanism by which genes that regulate cell survival and those that regulate cell death determine the overall viability of preimplantation embryos.     

Biochemical evidence for a calmodulin-stimulated calcium-dependent protein kinase in maize

We provide biochemical evidence for the presence of a Ca2+-dependent calmodulin (CaM)-stimulated protein kinase (CCaMK) from etiolated maize coleoptiles. The kinase, with a molecular mass of 72.3 kDa, was purified to homogeneity by means of ammonium sulphate precipitation, DEAE-Sephacel chromatography. CaM-Sepharose chromatography and gel purification. The purified kinase required 5 mM Mg2+ for activity and had an optimum pH of 7.5. The kinase is a Ca2+-binding protein, as was evident by 45Ca2+-binding and Ca2+ mobility-gel-shift assays. 1 microM Ca2+ stimulated the kinase activity about 12-fold and was further stimulated by the addition of exogenous CaM (approximately 100 nM). Addition of Ca2+ and CaM antagonists decreased the kinase activity. Under in vitro assay conditions the kinase phosphorylated preferentially syntide-2, histone IIIS and casein. Syntide-2 and histone IIIS were phosphorylated at serine residues, showing that the kinase belongs to the serine/threonine family of protein kinases. Autophosphorylation of CCaMK occurred on threonine residue(s) and was Ca2+ dependent. Addition of exogenous CaM had no effect on autophosphorylation. The properties of the maize kinase suggests that it is a CCaMK that shows dual stimulation with Ca2+ and CaM for substrate phosphorylation and only Ca2+ requirement for autophosphorylation. Antibodies raised against the kinase cross-reacted with maize total proteins to give a single band of 72 kDa and precipitated substrate (syntide-2 and histone IIIS)-phosphorylation and autophosphorylation activities in a specific manner. Localisation studies with antibodies showed that the kinase is ubiquitous.     

Genetic salivary protein polymorphism in Mexican population

Genetic polymorphism is the major contributor that affects human salivary composition. In order to determine the molecular phenotypes in saliva, it is important to know the distribution of proteins with specific functions which allows the clinical diagnosis of specific diseases. Unstimulated human whole saliva samples from 120 subjects were subjected to sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE). The phenotype distribution of several molecules including MG1, MG2, alpha-Amylase, PRP-I and cystatins were similar. Qualitative and quantitative characteristics were specific in each subject.     

Genetic and environmental variance in content dimensions of the MMPI.

To evaluate genetic and environmental variance in the Minnesota Multiphasic Personality Inventory (MMPI), I studied 9 factor scales identified in the 1st item factor analysis of normal adult MMPIs in 820 adolescent and young adult co-twins. Conventional twin comparisons documented heritable variance in 6 of the 9 MMPI factors (Neuroticism, Psychoticism, Extraversion, Somatic Complaints, Inadequacy, Cynicism); significant influence from shared environmental experience was found for 4 factors (Masculinity vs Femininity, Extraversion, Religious Orthodoxy, Intellectual Interests). Genetic variance was more evident in results from sisters than those of brothers, and a developmental-genetic analysis, using hierarchical multiple regressions of double-entry matrixes of raw data, revealed that in 4 MMPI factor scales, genetic effects were significantly modulated by age and/or gender during the developmental period from early adolescence to early adulthood. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Genetic and ontogenetic variability in the noradrenergic regulation of adrenocortical function

Corticosterone levels in the blood under restriction positively correlated with the hormone response to i.c.v. norepinephrine injection in 7 genetic groups of rats. Reactions to stress and norepinephrine simultaneously decreased in adult rats after prenatal glucocorticoid treatment. Prenatal stress or corticosterone treatment have a lasting effect on typosine hydroxylase (TH) activity and adrenoligands binding in the mice brain. Prenatal treatment increased TH activity in the noradrenergic system of the brain and decreased binding of radiolabelled prazosin, clonidine and dihydroalprenolol in some brain regions as well as stress reaction in mice. Thus, individual variations in adrenocortical stress response may be related to the differences in the function of the brain noradrenergic system.     

Activity-dependent regulation of dopamine content in the olfactory bulbs of naris-occluded rats

Several lines of evidence strongly suggest that reduced olfactory nerve activity results in decreased bulb dopamine content. In the present study, high performance liquid chromatography with electrochemical detection was used to assess catecholamine levels in bulbs from postnatal day 60 rats that had undergone either unilateral naris cautery or a sham surgery on day 30. Thirty days of odor deprivation dramatically reduced dopamine and dihydroxyphenylacetic acid levels in functionally-deprived bulbs (ipsilateral to occluded nares) as compared to contralateral controls, while norepinephrine and dihydroxyphenylglycol levels were unchanged. The loss of dopamine was more severe in medial as compared to lateral aspects of experimental bulbs, while the loss of dihydroxyphenylacetic acid was similar on the two sides. To test directly the hypothesis that afferent activity regulates dopamine and dihydroxyphenylacetic acid content, 1 h of high frequency tetanic nerve stimulation was provided to the rostral-medial olfactory nerve layer in deprived olfactory bulbs, and catecholamine levels were assessed from 6 to 192 h later. Partial and temporary recovery of dopamine was observed in medial aspects of the bulb when rats were examined 96 h later, while consistent recovery of dihydroxyphenylacetic acid content was not apparent. These data corroborate evidence that olfactory nerve activity is a potent regulator of bulb dopamine and indicate that continued afferent input is necessary to maintain dopamine levels.     

A microtubule-associated protein in maize is expressed during phytochrome-induced cell elongation

Plants can adapt their shape to environmental stimuli. This response is mediated by the reorganization of cortical microtubules, a unique element of the cytoskeleton. However, the molecular base of this response has remained obscure so far. In an attempt to solve this problem, signal-dependent changes in the pattern of microtubule-binding proteins were analysed during coleoptile elongation in maize, that is, under the control of the plant photoreceptor phytochrome. Two putative MAPs of 100 kDa (P100) and 50 kDa apparent molecular weights were identified in cytosolic extracts from non-elongating and elongating cells. Both proteins co-assembled with endogenous tubulin, bound to neurotubules and were immunologically related to the neural MAP tau: the P100 protein, depending on the physiological situation, was manifest as a double band and was always found to be heat-stable. In contrast, the 50 kDa MAP was heat-stable only for particular tissues and physiological treatments. The P100 protein was present in all tissues, however in a reduced amount in elongating coleoptiles. The 50 kDa MAP was expressed exclusively upon induction of phytochrome-dependent cell elongation. As shown by immunofluorescence double-staining, an epitope shared by both proteins colocalized with cortical microtubules in situ, but exclusively in elongating cells. In non-elongating cells, only the nuclei were stained. Partially purified nuclei from elongating cells were enriched in P100, whereas the 50 kDa MAP became enriched in a partially purified plasma membrane fraction.     

Autocrine regulation of protein secretion in mouse mammary epithelial cells

Milk secretion is under autocrine control by an inhibitory milk protein, named FIL (feedback inhibitor of lactation). Lactating mammary acini and epithelial cells cultured on reconstituted basement membrane (EHS matrix) with lactogenic hormones were used to study the characteristics of autocrine inhibition. FIL inhibited milk protein secretion in lactating acini, but not in epithelial cells on EHS matrix. The latter's insensitivity to FIL was due to formation of multicellular structures termed mammospheres, in which cell surrounded a central luminal space. Cell polarization, and the formation of tight intercellular junctions prevented FIL access to the apical cell surface, which faced the mammosphere lumina. When apical access was permitted either by incomplete mammosphere formation or EGTA treatment, FIL inhibited mammosphere protein secretion to the same extent as in lactating acini. The study shows that autocrine inhibition by FIL occurs specifically through interaction with the apical surface of the mammary epithelial cell, and suggests the presence of a FIL receptor on this, but not the basolateral cell membrane.     

Long-term regulation of renal urea transporter protein expression in rat

To test the hypothesis that the abundance of the apical urea transporter of the inner medullary collecting duct (IMCD) is regulated in vivo by factors associated with altered water balance, immunoblots of rat inner medullary membrane fractions were probed with rabbit polyclonal antibodies against the renal urea transporter (RUT) gene product. In inner medullas of Brattleboro rats, which manifest severe chronic water diuresis, a 117-kD band was seen, in addition to the previously described 97-kD band. These two bands were detectable by antibodies directed against two different regions of the RUT sequence. When Brattleboro rats were treated with a 5-d infusion of arginine vasopressin (AVP) by osmotic minipump, the 117-kD band was markedly diminished, whereas the 97-kD band was unchanged. Simultaneous infusion of the diuretic agent furosemide prevented the AVP-induced decrease in the 117-kD band. In AVP-infused Sprague Dawley rats, the 117-kD band was barely perceptible. However, when AVP-treated rats were infused with furosemide for 5 d, the 117-kD band was markedly accentuated, whereas the 97-kD band was unchanged. The abundance of the 117-kD RUT protein in the renal papilla was inversely correlated with dietary protein intake. Further immunoblotting studies revealed that the 117-kD protein is heavily expressed in IMCD cells and not in non-collecting duct components of the inner medulla, and is present in low-density microsome fractions from inner medulla. From this study, the following conclusions can be made: (1) The collecting duct urea transporter is present in at least two forms (97 and 117 kD) in the IMCD. (2) The expression level of the 117-kD urea transporter protein is regulated and is inversely correlated with medullary osmolality and urea concentration, but does not correlate with circulating AVP level. (3) Although AVP regulates RUT function on a short-term basis, long-term changes in AVP levels do not increase RUT abundance.     

Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutation chloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutant fer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that the fer gene is epistatic over the chln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. The chln gene is located on chromosome 1 and the fer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate the fer gene by map-based cloning. The isolation of the fer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.     

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.