Regulation of Raf-1-dependent signaling during early Xenopus development

The Raf-1 gene product is activated in response to cellular stimulation by a variety of growth factors and hormones. Raf-1 activity has been implicated in both cellular differentiation and proliferation. We have examined the regulation of the Raf-1/MEK/MAP kinase (MAPK) pathway during embryonic development in the frog Xenopus laevis. We report that Raf-1, MEK, and MAPK activities are turned off following fertilization and remain undetectable up until blastula stages (stage 8), some 4 h later. Tight regulation of the Raf-1/MEK/MAPK pathway following fertilization is crucial for embryonic cell cycle progression. Inappropriate reactivation of MAPK activity by microinjection of oncogenic Raf-1 RNA results in metaphase cell cycle arrest and, consequently, embryonic lethality. Our findings demonstrate an absolute requirement, in vivo, for inactivation of the MAPK signaling pathway to allow normal cell cycle progression during the period of synchronous cell divisions which occur following fertilization. Further, we show that cytostatic factor effects are mediated through MEK and MAPK.     

The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis

Brassinosteroids (BRs) are steroidal plant hormones essential for normal plant growth and development. Mutants in the biosynthesis or perception of BRs are usually dwarf. The tomato Dwarf gene (D), which was predicted to encode a cytochrome P450 enzyme (P450) with homology to other P450s involved in BR biosynthesis, was cloned previously. Here, we show that DWARF catalyses the C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate precursor of brassinolide. To do this, we first confirmed that the D cDNA complemented the mutant light- and dark-grown phenotypes of the extreme dwarf (dx) allele of tomato. To identify a substrate for the DWARF enzyme, exogenous application of BR intermediates to dx plants was carried out. C-6 oxoBR intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR, 6-deoxoCS, had little effect. Quantitative analysis of endogenous BR levels in tomato showed mainly the presence of 6-deoxoBRs. Furthermore, dx plants were found to lack CS and had a high level of 6-deoxoCS in comparison to D plants that had CS and a lower level of 6-deoxoCS. Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS was obtained by functional expression of DWARF in yeast. In these experiments, the intermediate 6alpha-hydroxycastasterone was identified, indicating that DWARF catalyzes two steps in BR biosynthesis. These data show that DWARF is involved in the C-6 oxidation in BR biosynthesis.     

Interactions between jointless and wild-type tomato tissues during development of the pedicel abscission zone and the inflorescence meristem

The jointless mutation of tomato results in the formation of flower pedicels that lack an abscission zone and inflorescence meristems that revert to vegetative growth. We have analyzed periclinal chimeras and mericlinal sectors of jointless and wild-type tissue to determine how cells in different meristem layers (L1, L2, and L3) and their derivatives interact during these two developmental processes. Cells in the inner meristem layer, L3, alone determined whether the meristem maintained the inflorescence state or reverted to vegetative growth. Moreover, L3 derivatives determined whether a functional pedicel abscission zone formed. Limited and disorganized autonomous development of wild-type L2-derived cells occurred when they overlay mutant tissue. Adjacent mutant and wild-type L3-derived tissues in pedicels developed autonomously, indicating little or no lateral communication. Only the outermost L3-derived cells within the pedicel were capable of orchestrating normal pedicel development in overlying tissues, revealing the special status of those cells as coordinators of development for L1- and L2-derived cells, whereas the innermost L3-derived cells developed autonomously but did not influence the development of other cells.     

Xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper fruits (Capsicum annuum L.)

In analysis, comparison and classification of conformations of proteins, a common computational task involves extractions of similar substructures. Structural comparisons are usually based on either of two measures of similarity: the root-mean-square (r.m.s.) deviation upon optimal superposition, or the maximal element of the difference distance matrix. The analysis presented here clarifies the relationships between different measures of structural similarity, and can provide a basis for developing algorithms and software to extract all maximal common well-fitting substructures from proteins. Given atomic coordinates of two proteins, many methods have been described for extracting some substantial (if not provably maximal) common substructure with low r.m.s. deviation. This is a relatively easy task compared with the problem addressed here, i.e., that of finding all common substructures with r.m.s. deviation less than a prespecified threshold. The combinatorial problems associated with similar subset extraction are more tractable if expressed in terms of the maximal element of the difference distance matrix than in terms of the r.m.s. deviation. However, it has been difficult to correlate these alternative measures of structural similarity. The purpose of this article is to make this connection. We first introduce a third measure of structural similarity: the maximum distance between corresponding pairs of points after superposition to minimize this value. This corresponds to fitting in the Chebyshev norm. Properties of Chebyshev superposition are derived. We describe relationships between the r.m.s. and minimax (Chebyshev) deviations upon optimal superposition, and between the Chebyshev deviation and the maximal element of the difference distance matrix. Combining these produces a relationship between the r.m.s. deviation upon optimal superposition and the maximal element of the difference distance matrix. Based on these results, we can apply algorithms and software for finding subsets of the difference distance matrix for which all elements are less than a specified bound, either to select only subsets for which the r.m.s.deviation is less than or equal to a specified threshold, or to select subsets that include all subsets for which the r.m.s. deviation is less than or equal to a threshold.     

Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of beta-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon

The possibility that extracellular matrix-cell surface interaction might lead to signalling was examined in human mononuclear cells. In mononuclear cells loaded with Indo-1 acetoxymethylester, collagen binding elicited a 2-3 fold increase in cytosolic free calcium concentration within seconds. Plasma fibronectin, serum albumin and heparin did not cause any significant change in intracellular calcium levels. Collagen I, collagen IV and glucosylated collagen I evoked an increase in intracellular Ca++ both in the presence and absence of extracellular calcium. However, in the presence of 2mM EDTA, the increase in cytosolic free calcium concentration caused by collagen I and collagen IV was partly abolished, suggesting the requirement of a cation-dependent interaction of collagen with mononuclear cells. Glucosylated collagen induced intracellular calcium mobilization even in the presence of EDTA suggesting a cation-independent interaction. These results indicate that collagen binding induces rapid mobilization of calcium in human mononuclear cells, apparently from intracellular sources.     

Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons

The phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins, may influence the development and plasticity of axons and dendrites in mammalian neuron in response to appropriate extracellular stimuli. In particular, high molecular weight microtubule-associated protein 2, has been implicated in dendrite growth and synaptic plasticity and is thought to be modulated by phosphorylation and dephosphorylation. We have previously determined that threonines 1620/1623 on the microtubule-associated protein 2 molecule become phosphorylated in vivo and are targets for proline-directed protein kinases in vitro. Using the phosphorylated site-specific antibody 305, we now report the decreased phosphorylation state of high molecular weight microtubule-associated protein 2 during the development of cultured cerebellar granule neurons. Phosphorylation of high molecular weight microtubule-associated protein 2 at this site is significantly inhibited by lithium in short-term cultured neurons, which suggests the implication of glycogen synthase kinase-3. In long-term cultured neurons, it is also partially inhibited by PD098059, an inhibitor of extracellular signal-regulated protein kinase activation, which indicates an additional contribution of this kinase to high molecular weight microtubule-associated protein 2 phosphorylation at this stage. Both in short-term and long-term cultured neurons, okadaic acid augments high molecular weight microtubule-associated protein 2 phosphorylation at this site through the inhibition of protein phosphatases 1 and/or 2A. Finally, glutamate receptor activation leads to a dephosphorylation of high molecular weight microtubule-associated protein 2 at this site which can also be effectively prevented by okadaic acid. These results are consistent with the participation of glycogen synthase kinase-3, extracellular signal-regulated protein kinases and protein phosphatases 1 and 2A, in the regulation of microtubule-associated protein 2 phosphorylation within living neurons, which may be modulated by extracellular signals like the neurotransmitter glutamate.     

Regulation of progesterone biosynthesis in human placental mitochondria by Krebs cycle metabolites

1. 2-Oxoglutarate, succinate, fumarate, malate and citrate, cis-aconitate and isocitrate stimulate conversion of cholesterol to progesterone in human placental mitochondria. 2. The stimulatory effect of dicarboxylic and tricarboxylic acids depends on the activity of malate dehydrogenase (decarboxylating) (NADP+) (EC 1.1.1.40) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42), respectively.     

The correlation between two dietary assessments of carotenoid intake and plasma carotenoid concentrations: application of a carotenoid food-composition database

A newly available carotenoid food-composition database providing specific carotenoid values for > 2300 foods was linked to dietary data on 57 male nonsmokers to examine the association between dietary carotenoid intake and plasma carotenoid concentrations over 3 wk when free-living. Carotenoid intake was estimated from a food-frequency questionnaire (FFQ) and 7 d of food diaries with concurrent analysis of plasma carotenoid concentrations. After adjustment for energy intake, percentage of energy from alcohol, and plasma lipid concentrations, significant diet-plasma correlations for the FFQ and the food diaries (FD) included alpha-carotene (r = 0.29 and 0.43), beta-carotene (r = 0.36 FFQ only), beta-cryptoxanthin (r = 0.46 and 0.44), lutein (r = 0.44 FD only), and lycopene (r = 0.53 FD only). Dietary carotenoid intakes were associated with plasma carotenoid concentrations for all the carotenoids except for beta-carotene when food diaries were used whereas the diet-plasma correlation for the provitamin A carotenoids were consistently significant when the FFQ was used.     

Regulation of skeletal muscle perfusion during exercise

OBJECTIVE: To compare the composition and mechanical properties of the newly developed bladder acellular matrix graft (BAMG) with the normal urinary bladder in rat, pig and human. MATERIALS AND METHODS: Rat, pig and human urinary bladders were harvested and divided into control and experimental groups. For the latter, BAMGs were prepared, and light and transmission electron microscopic studies performed. Strips from the normal bladders and the BAMGs (10 in each group) were tested under tension, and the ultimate tensile strength, maximum strain, and elastic modulus were determined from stress/strain curves. RESULTS: Both types I and III collagen, as well as elastic fibres, were observed as major components of the matrix scaffold. There were more collagen type I fibres in the rat than in the pig and human BAMGs, whereas the pig, and particularly the human, both showed higher levels of type III collagen and elastic fibres. These different matrix scaffold patterns were confirmed by electron microscopy. Results from biomechanical testing showed no significant differences for strength, strain or elastic modulus between BAMG and control bladder strips, except in the rat where the maximum strain values were significantly lower. CONCLUSION: There are variations in the acellular matrix structure with similar biomechanical properties between the BAMG and the normal urinary bladder in three different species. These results may underscore the potential of the BAMG. Furthermore, this in vitro model provides a suitable method to study the mechanical properties of the urinary bladder and may serve as a diagnostic tool for various investigations.     

Regulation of hsp expression during rodent spermatogenesis

As part of an effort to develop peptides with selective kappa-opioid antagonist activity, a series of N-alkylated [D-Pro10]dynorphin A-(1-11) derivatives were made through solid-phase peptide synthesis: R-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-LysOH, where R = N-benzyl, N-cyclopropylmethyl, N,N-dicyclopropylmethyl, or N,N-diallyl. These derivatives and dynorphin A-(1-13)NH2 were evaluated for kappa-opioid receptor binding affinity and potency as inhibitors of adenylyl cyclase. Equilibrium competition binding experiments using [3H]diprenorphine (approximately 600 pM) were performed on membranes prepared from cultured Chinese hamster ovary (CHO) cells stably expressing the rat kappa-opioid receptor. Tissue prepared from this cell line was used to evaluate opioid peptide inhibition of forskolin-stimulated (50 microM) adenylyl cyclase activity. Displacement of [3H]diprenorphine specific binding by these peptides was observed with a rank order of affinity (Ki, nM) = [D-Pro10]dynorphin A-(1-11) (0.13) > dynorphin A-(1-13)NH2 (0.34) > N-cyclopropylmethyl- (1.4) > N,N-dicyclopropylmethyl- (12.6) approximately N-benzyl- (18.3) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11) (26.0). A similar rank order was observed for potency of adenylyl cyclase inhibition (IC50, nM): [D-Pro10]dynorphin A-(1-11) (0.12) approximately dynorphin A-(1-13)NH2 (0.19) > N-cyclopropylmethyl- (2.7) > N,N-dicyclopropylmethyl- (13.2) approximately N,N-diallyl- (18.0) approximately N-benzyl-[D-Pro10]dynorphin A-(1-11) (36.4). The peptides differed in their percent maximal inhibition of adenylyl cyclase activity: dynorphin A-(1-13)NH2 (100%) approximately N-cyclopropylmethyl- (94.3%) approximately [D-Pro10]dynorphin A-(1-11) (87.9%) > N-benzyl- (71.4%) > N,N-dicyclopropylmethyl- (23.6%) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11)(18.9%). As the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) derivatives were found to have only weak partial agonist activity with respect to adenylyl cyclase inhibition, they were evaluated for their ability to reverse dynorphin A-(1-13)NH2 (10 nM) inhibition of adenylyl cyclase activity. N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) reversed dynorphin A-(1-13)NH2 inhibition to levels equal to the maximal inhibition produced by N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) alone. This weak partial agonism combined with nanomolar potency render the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) compounds promising leads for further attempts to synthesize peptide kappa-opioid receptor antagonists.     

Regulation of DNA-replication origins during cell-cycle progression

We have shown previously that chromosome VI of Saccharomyces cerevisiae contains nine origins of DNA replication that differ in initiation frequency and replicate sequentially during the S phase of the cell cycle. Here we show that there are links between activation of these multiple origins and regulation of S-phase progression. We study the effects of a DNA-damaging agent, methyl methane sulphonate (MMS), and of mutations in checkpoint genes such as rad53 on the activity of origins, measured by two-dimensional gel analysis, and on cell-cycle progression, measured by fluorescence-activated cell sorting. We find that when MMS slows down S-phase progression it also selectively blocks initiation from late origins. A rad53 mutation enhances late and/or inefficient origins and releases the initiation block by MMS. Mutation of rad53 also results in a late origin becoming early replicating. We conclude that rad53 regulates the timing of initiation of replication from late origins during normal cell growth and blocks initiation from late origins in MMS-treated cells. rad53 is, therefore, involved in the cell's surveillance of S-phase progression. We also find that orc2, which encodes subunit 2 of the origin-recognition complex, is involved in suppression of late origins.     

Regulation of progesterone biosynthesis in the human placenta by estradiol 17 beta and progesterone

The information available concerning the effects of chemotherapy administered during pregnancy is limited and consists of case reports and small series. A registry has been established at the National Cancer Institute, but there are currently only several hundred cases of neonates exposed to chemotherapy registered. All clinicians who care for women receiving chemotherapy during pregnancy should report those experiences to the National Cancer Institute to increase the data base. When chemotherapy is used during the embryogenesis period in the first trimester there is an increased rate of spontaneous abortion and major birth defects. The most toxic chemotherapeutic agents administered during pregnancy are methotrexate and aminopterin and should be avoided when possible, particularly during the first trimester. Pregnancy-related physiologic changes should be kept in mind when dosing and administering cytotoxic chemotherapy. The risk of fetal malformation when chemotherapy is administered during the second and third trimesters is probably not greater than background rate, but there may be a greater risk of stillbirth, fetal growth restriction, premature birth, and maternal and fetal myelosuppression. Breastfeeding should be avoided in women receiving chemotherapy.     

Regulation of lipid synthesis in Bradyrhizobium japonicum: low oxygen concentrations trigger phosphatidylinositol biosynthesis

Lowering oxygen tension in free-living Bradyrhizobium japonicum resulted in a dramatic switch of membrane chemistry in which phosphatidylcholine, the predominant lipid in aerated cultures, was no longer synthesized and phosphatidylethanolamine became the major lipid. Besides this change, phosphatidylinositol, a typical plant lipid rarely found in bacteria, was also synthesized.     

Regulation of ferulate-5-hydroxylase expression in Arabidopsis in the context of sinapate ester biosynthesis

OBJECTIVE: A set of guidelines to assist men with traumatic brain injury (TBI) to alleviate gender role strain was assessed to determine its effectiveness and acceptability to participants. METHOD: Four adult male participants with TBI received the intervention (the set of guidelines) for 4 months. The intervention consisted of rebuilding self-identified gendered social roles and activities. Focused interviews and participant observation were used to determine whether gender role strain changed after intervention. RESULTS: The participants reported that the intervention enabled them to (a) enhance their gender role satisfaction through newly rebuilt roles and activities, (b) attain certain long-held personal goals, (c) feel more like members of society, (d) perceive a greater congruency between their internal self-images and external postinjury roles, (e) learn more about personal skills and values as men, (f) feel more comfortable using help-seeking behaviors, (g) feel a sense of shared experience and affinity, (h) feel more understood and accepted, and (i) contribute to others through community member roles. DISCUSSION: The set of guidelines for alleviating gender role strain was effective in assisting these participants to enhance their gender role satisfaction through rebuilding desired male-gendered social roles and activities. Dating, courtship, extended family member, community member, friend, and mentor-protege roles, lost as a result of TBI, were rebuilt through gender-neutral activities that facilitated a sense of volitional control, competency, and normalcy. Nonetheless, the men continued to lack desired rites of passage leading from male adolescence to adulthood.     

Regulation of ventilatory capacity during exercise in asthmatics

In asthmatic and control subjects, we examined the changes in ventilatory capacity (VECap), end-expiratory lung volume (EELV), and degree of flow limitation during three types of exercise: 1) incremental, 2) constant load (50% of maximal exercise capacity; 36 min), and 3) interval (alternating between 60 and 40% of maximal exercise capacity; 6-min workloads for 36 min). The VECap and degree of flow limitation at rest and during the various stages of exercise were estimated by aligning the tidal breathing flow-volume (F-V) loops within the maximal expiratory F-V (MEFV) envelope using the measured EELV. In contrast to more usual estimates of VECap (i.e., maximal voluntary ventilation and forced expiratory volume in 1 s x 40), the calculated VECap depended on the existing bronchomotor tone, the lung volume at which the subjects breathed (i.e., EELV), and the tidal volume. During interval and constant-load exercise, asthmatic subjects experienced reduced ventilatory reserve, higher degrees of flow limitation, and had higher EELVs compared with nonasthmatic subjects. During interval exercise, the VECap of the asthmatic subjects increased and decreased with variations in minute ventilation, due in part to alterations in their MEFV curve as exercise intensity varied between 60 and 49% of maximal capacity. In conclusion, asthmatic subjects have a more variable VECap and reduced ventilatory reserve during exercise compared with nonasthmatic subjects. The variations in VECap are due in part to a more labile MEFV curve secondary to changes in bronchomotor tone. Asthmatics defend VECap and minimize flow limitation by increasing EELV.