Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1.

The role of cationic amino acids in the Na(+)/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased K(m) for succinate of 1.8 mM, compared with 0.3 mM in the wild-type NaDC-1. The R349A mutant had Na(+) and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

The emerging role of mass spectrometry in molecular biosciences: studies of protein phosphorylation in fly eyes as an example.

Modern mass spectrometry (MS) streamlined with two-dimensional gel electrophoresis, in-gel digestion and HPLC-interfaced electrospray ionization quadrupole MS or matrix-assisted laser desorption ionization time-of-flight MS enables us to analyse proteins at a minuscule scale. We present here two examples of MS applications in which (1) we identified the in vivo phosphorylation site of Drosophila arrestin, phosrestin I (PRI), and (2) we revealed the identity of an 80 kDa phosphoprotein (80K) in Drosophila eyes to be the InaD gene product, a member of the PDZ domain proteins. Available evidence suggests that PRI quenches the activation of rhodopsin and that the InaD protein adjusts photoreceptor responsiveness by assembling/disassembling components involved in photoreceptor transduction in flies. PRI undergoes a reversible phosphorylation at a single site, and 80K at multiple sites. The phosphorylation states of PRI and 80K depend on the intensity and/or duration of light stimuli. From these results we postulate that these proteins function as a molecular switch adjusting the signalling cascade through phosphorylation. The combination of two-dimensional gel electrophoresis with MS will be a powerful tool for detailed investigation of such complex switching processes. The techniques described here can be applied also to other complex signalling systems.

Predominant presence of beta-arrestin-1 in small sensory neurons of rat dorsal root ganglia.

Reverse transcription-polymerase chain reaction and western immunoblot analyses were performed to demonstrate the presence of beta-arrestin-1 in rat dorsal root ganglion. beta-Arrestin-1 existed as two alternatively spliced variants, although predominantly in its untruncated form. Several factors affected the visualization of the truncated version on a sodium dodecyl sulfate-polyacrylamide gel; however, the isoform was clearly detected on a two-dimensional gel. We further localized beta-arrestin-1 immunoreactivity in the sensory neurons of the 5th lumbar dorsal root ganglia. Beta-arrestin-1-immunoreactive neurons accounted for approximately 60% of the sensory neurons, and approximately 88% of the beta-Arrestin-1 immunoreactive neurons fell into a category of small neurons having a diameter of 10-30 microm. Members of the arrestin superfamily play crucial roles in the desensitization of G protein-coupled receptors. Our data demonstrating the presence of beta-arrestin-1 in the rat dorsal root ganglion at both messenger RNA and protein levels support the idea that beta-arrestin- participates in receptor desensitization in the sensory neurons. Furthermore, because small-size neurons of dorsal root ganglion are often implicated in nociception, the predominant presence of beta-arrestin-1 immunoreactivity in small-size sensory neurons suggests that beta-arrestin-1 may have a role modulating nociceptive signals.

Determinants of substrate and cation affinities in the Na+/dicarboxylate cotransporter.

The Na+/dicarboxylate cotransporter (NaDC-1) couples the transport of sodium and tricarboxylic acid cycle intermediates, such as succinate and citrate. The rabbit and human homologues (rbNaDC-1 and hNaDC-1, respectively) are 78% identical in amino acid sequence but exhibit several differences in their functional properties. rbNaDC-1 has a greater apparent affinity for citrate and sodium than hNaDC-1. Furthermore, unlike hNaDC-1, rbNaDC-1 is inhibited by low concentrations of lithium. In this study, chimeric transporters were constructed to identify the protein domains responsible for the functional differences between rbNaDC-1 and hNaDC-1. Individual substitutions of transmembrane domain (TMD) 7, 10 or 11 produced transporters with intermediate properties. However, substitution of TMD 7, 10, and 11 together resulted in a transporter with the citrate Km of the donor, suggesting that interactions between these domains determine the differences in apparent citrate affinities. TMDs 10 and 11 are most important in determining the differences in apparent sodium affinities, and TMD 11 determines the sensitivity to lithium inhibition. We conclude that transmembrane domains 7, 10, and 11 in NaDC-1 may contain at least one of the cation binding sites in close proximity to the substrate binding domain.

Phosrestide-1, a peptide derived from the Drosophila photoreceptor protein phosrestin I, is a potent substrate for Ca2+/calmodulin-dependent protein kinase II from rat brain.

Multifunctional Ca2+/calmodulin-dependent protein kinase type II (CaMK II) plays a crucial role in mediation of cellular responses to rising cytosolic Ca2+ levels. We find that the novel peptide substrate PGTIEKKRSNAMKKMKSIEQHR serves as a highly potent substrate for CaMK II enzymes purified from both Drosophila and rat. The peptide is derived from a photoreceptor-specific protein, phosrestin I, of the Drosophila compound eye and is designated as phosrestide-1. Using saturating substrate concentrations, the enzymes from both species transfer the gamma-phosphoryl group of ATP to phosrestide-1 at a level three to ten times greater than to the commercially available mammalian-derived CaMK II substrates, autocamtide-3 and syntide-2. This indicates a conservation of substrate preferences for CaMK II derived from distantly related species, a dipteran fly and a mammal. Although phosrestide-1 contains two potential serine residues for CaMK II phosphorylation, we find that only the C-terminal serine is phosphorylated by rat CaMK II. However, removal of the upstream sequence containing the N-terminal serine substantially reduced the potency of phosrestide-1 as a CaMK II substrate to a level comparable to that of syntide-2 or autocamtide-3. We also find that a peptide representing the N-terminal segment of phosrestide-1 does not inhibit either CaMK II. Therefore, the enhanced potency of phosrestide-1 as a CaMK II substrate is likely to be due to a preferred conformation of the peptide induced by the N-terminal segment rather than to a specific binding of the enzymes to the N-terminus of the peptide. To the best of our knowledge, phosrestide-1 is the first CaMK II substrate which is designed based on an invertebrate sequence. The high phosphorylation level of phosrestide-1 by CaMK II of mammalian origin may reflect highly conserved CaMK II signaling cascades between vertebrates and invertebrates.

Nonradioactive phosphopeptide assay by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: application to calcium/calmodulin-dependent protein kinase II.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) was used to quantify the phosphopeptide produced by calcium/calmodulin-dependent protein kinase II (CaMK II). MALDI-TOF measurements were performed in a linear and positive ion mode with delayed extraction excited at various laser powers and at different sampling positions, i.e., different loci of laser illumination. We find that the ratio of the peak area of the substrate (S) to that of its monophosphorylated form (SP) for a given mixture is constant, independent of the laser powers and/or of the sample loci illuminated by the laser. We also find that the fraction of phosphorylation determined by MALDI-TOF, or fMALDI-TOF, is proportionally smaller than that determined by HPLC, or fHPLC; the ratio fMALDI-TOF/fHPLC was 0.797 +/- 0.0229 (99% confidence limit, n = 7) for a 30-mer peptide substrate used in this study. A low mass gate, which turns off the detector temporarily, improved the ratio fMALDI-TOF/fHPLC to 0.917 +/- 0.0184 (99% confidence limit, n = 7). Our interpretation of this result is that the reduction of the phosphopeptide peak in the MALDI-TOF measurement is likely to be caused by a temporal loss of detector function rather than by a lower efficiency of ionization for the phosphopeptide compared with its parent species. In these measurements the experimental errors, up to the 50% phosphorylation state, were less than 5%. After an adjustment made based on the fMALDI-TOF/fHPLC ratio of 0.917, MALDI-TOF gave an accurate measurement for the kinetics of the CaMK II phosphorylation reaction. Since only a small volume of the reaction mixture, typically containing 3 to 50 pmol of substrate, is required for the MALDI-TOF measurement, this method can be adapted to a nonradioactive microscale assay for CaMK II and also for other protein kinases.

Maternal nutrition and the outcome of pregnancy. A renaissance in research.

Research over the past decade indicates that aspects of maternal nutrition may play a greater role in immediate and long-term health of offspring than was thought previously. The effects of nutritional exposures on outcomes likely depend primarily on the timing of insults during pregnancy and may not have to be severe to have a long-term impact on health. If the rate of change in knowledge of the effects of maternal nutritional exposures on outcomes continues at its current pace, work initiated in the past decade may well be looked back upon as representing the renaissance of maternal nutrition research.

Calcium/calmodulin-dependent kinase II phosphorylates Drosophila visual arrestin.

Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca2+ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor-specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. We are interested in identifying the protein kinase responsible for the phosphorylation of phosrestin I to link the transmembrane signaling to the light-adaptive response. Type II Ca2+/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. We show here that partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca2+/calmodulin-dependent kinase II and that the kinase activity exhibits regulatory properties that are unique to Ca2+/calmodulin-dependent kinase II such as Ca2+ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca2+/calmodulin-dependent kinase II autoinhibitory domain. We also show that Ca2+/calmodulin-dependent kinase KII activity is present in Drosophila eye preparations. These results are consistent with our hypothesis that Ca2+/calmodulin-dependent kinase II phosphorylates phosrestin I. We suggest that Ca2+/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation.

Profet, profits, and proof: do nausea and vomiting of early pregnancy protect women from "harmful" vegetables?

OBJECTIVE: The purpose of this research was to test a widely publicized theory that nausea and vomiting of pregnancy protects women from ingesting certain vegetables and other foods that produce congenital anomalies and other adverse outcomes of pregnancy. STUDY DESIGN: The theory was tested with use of data on dietary intake, illness, and pregnancy outcome obtained from 549 women participating in a prospective, population-based study. RESULTS: No relationship between intake of proscribed vegetables and other foods and the presence of nausea and vomiting in early pregnancy was identified. Intake of proscribed foods was unrelated to adverse outcomes of pregnancy. CONCLUSION: It is suggested that claims made in the popular press about food and health relationships should be evaluated by the media as fiction unless supported by scientific research.

Changes in plasma lipids and lipoproteins associated with starting or stopping postmenopausal hormone replacement therapy. Atherosclerosis Risk in Communities Study.

The Atherosclerosis Risk in Communities Study investigators examined nearly 4000 postmenopausal women from 1987 through 1989 and 3 years later to determine changes in plasma lipids occurring with the starting or stopping of hormone replacement therapy. Women who started estrogen plus progestin therapy (n = 74) had decreases of 9.8 mg/dl in low-density lipoprotein (LDL) cholesterol and 5.8 mg/dl in apolipoprotein B and increases of 1.2 mg/dl in high-density lipoprotein (HDL) cholesterol (HDL change not significant), 13.5 mg/dl in apolipoprotein A-I, and 14.0 mg/dl in triglycerides. Women who started estrogen alone (n = 149) had similar changes, except for a much larger increase in HDL cholesterol (5.8 mg/dl), principally in HDL-2. Women who stopped hormone therapy (n = 138) had lipid changes opposite to those who started therapy, but smaller in magnitude. These results confirm those of the Postmenopausal Estrogen/Progestin Interventions Trial in a community-based longitudinal cohort: women initiating estrogen plus progestin therapy have decreases in LDL cholesterol, but the increase in HDL cholesterol is less than that for starting estrogen alone. In addition, the current study extends findings to apolipoproteins and HDL subfractions.

Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors.

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae.

Repression of transcription from the silent mating loci (HML alpha and HMRa) is essential for mating ability in Saccharomyces cerevisiae. This silencing is known to require at least five proteins (SIR1, SIR2, SIR3, SIR4, and histone H4) and is accompanied by a change in chromatin structure. We show here that four positions of histone H4 (N-terminal residues 16, 17, 18, and 19) are crucial to silencing. HML alpha and HMRa are efficiently repressed when these positions are occupied by basic amino acids but are derepressed when substituted with glycine. These results suggest that acetylation of Lys-16 would lead to derepression of the silent mating loci. Three strong extragenic suppressors of the latter H4 mutations were isolated and determined to be located in SIR3. These suppressors allow high mating efficiencies in cells expressing either wild-type H4 or H4 containing single amino acid substitutions. They did not allow efficient mating in a strain that contained an H4 N-terminal deletion. These results indicate that the SIR3 mutations do not bypass the requirement for the H4 N terminus but, rather, allow repression in the presence of a less than optimal H4 N terminus. This provides a link between one of the SIR proteins and a component of chromatin.

Differential genetic requirements for in vivo and in vitro induction of T-killer and T-suppressor cells in the mutant H-2Kb system and the cross-reactivity of the T-killer clones.

Differences in the generation of anti-H-2Kb wild-type specific cytotoxic T lymphocytes (CTL) and specific suppressor T cells (SSTC) were investigated in H-2Kbm mutant mouse strains. To this end, optimal conditions for in vivo induction of highly active CTL in this mutant system were found and the T-cell origin of CTL and SSTC was confirmed using the anti-Thy-1.2 monoclonal antibody G4. Unlike the CTL, which were generated in vivo by any of the mutant strains tested (bm1, bm3, and bm4), the SSTC were only produced by bm3, whose H-2Kbm3 antigen, in contrast to the other H-2Kbm molecules, differs from wild type by serologically defined determinants. The high activity of anti-wild type bm4 CTL induced in vivo contrasted with a low activity of such CTL induced in mixed lymphocyte culture (MLC). This appeared to be the property of bm4 only, but not of bm1 or bm3, and it was reproduced in the reciprocal system B10 anti-bm4. CTL generation could be restored in the MLC by the addition of concanavalin A supernate or a mixture of bm4 and bm12 stimulator cells. Three of the six in vivo induced and in vitro propagated bm3 anti-B6 CTL clones demonstrated selective cross-reactivity to only one of the third-party H-2K molecules used, either Kk, Kd, or Kbm4. The present results indicate that (a) the SSTC and antibody recognize similar H-2Kb epitopes; (b) the H-2Kb epitopes recognized by the CTL and SSTC are not identical; (c) the genetic control of CTL generation in vivo is distinct from that in MLC, and (d) the affinities of antigen-specific receptors on T-cell clones of the same specificity may be different, leading to their individual cross-reactivity patterns.