Wiring diagrams of MAPK regulation by MEKK1, 2, and 3.

Mitogen-activated protein kinase (MAPK) pathways are activated by a plethora of stimuli. The literature is filled with papers describing the activation of different MAPKs by almost any stimulus or insult imaginable to cells. In this review, we use signal transduction wiring diagrams to illustrate putative upstream regulators for the MAPK kinase kinases, MEKK1, 2, and 3. Targeted gene disruption of MEKK1, 2, or 3 defined phenotypes for each MEKK associated with loss of specific MAPK regulation. Genetic analysis of MEKK function clearly defines specific components of the wiring diagram that require MEKK1, 2, or 3 for physiological responses. We propose that signal transduction network wiring diagrams are valuable tools for hypothesis building and filtering physiologically relevant phenotypic responses from less connected protein relations in the regulation of MAPK pathways.     

Parasympathetic innervation of canine tracheal smooth muscle

To investigate whether efferent parasympathetic fibers to the trachealsmooth muscle course through the pararecurrent nerve rather than therecurrent or the superior laryngeal nerve, we stimulated all threenerves in anesthetized dogs. We also recorded the pararecurrentnerve activity response to bronchoconstrictor stimuli and compared itwith pressure changes inside a saline-filled cuff of an endotrachealtube. Electrical stimulation (30 s, 100 Hz, 0.1 ms, 10 mA) increasedtracheal cuff pressure by 21.0 ± 3.2 and 1.3 ± 0.7 cmH₂O for the pararecurrent and the recurrent laryngealnerve, respectively. Stimulation of the superior laryngeal nerveincreased tracheal cuff pressure before, but not after, sectioning ofthe ramus anastomoticus, which connects it to the pararecurrent nerve.Intravenous administration of sodium cyanide increased pararecurrentnerve activity by 208 ± 51% and tracheal cuff pressure by14.4 ± 3.5 cmH₂O. Elevation of end-tidalPCO₂ to 50 Torr increased pararecurrent nerveactivity by 49 ± 19% and tracheal cuff pressure by 8.4 ± 3.6 cmH₂O. Further elevation to 60 Torr increasedpararecurrent nerve activity by 101 ± 33% and tracheal cuffpressure by 11.3 ± 2.9 cmH₂O. These results lead usto the conclusion that parasympathetic efferent fibers reach the smoothmuscle of the canine trachea via the pararecurrent nerve.

     

Ectopic Expression of the Tetratricopeptide Repeat Domain of SPINDLY Causes Defects in Gibberellin Response

The SPINDLY (SPY) protein of Arabidopsis is a negative regulator of gibberellin (GA) response. The SPY protein has 10 copies of the tetratricopeptide repeat (TPR) at the N terminus. TPR motifs function as protein-protein interaction domains. Several spy alleles are affected only in the TPR region suggesting that protein-protein interactions mediated by this domain are important for proper GA signaling. We have used a reverse genetics approach to further investigate the role of the TPR domain. The TPR domain of SPY was overexpressed in wild-type, gai, and spy plants. Expression of the TPR domain alone is not sufficient to rescue spy mutants. Expression of the TPR domain in a wild-type background produces phenotypes similar to those caused by loss-of-function spy mutants including resistance to GA biosynthesis inhibitors, short hypocotyl length, and early flowering. The dwarfing of the floral shoot internodes caused by the gai mutation was suppressed by expression of the TRP domain. Expression of the TPR domain had no effect on the abundance of endogenous SPY mRNA. The TPR domain was found to interact with SPY both in vitro and in yeast two-hybrid assays. These data indicate that the TPR domain of SPY can participate in protein-protein interactions and that these interactions are important for the proper functioning of SPY.     

Effect of elevating serum lipids on luteinizing hormone response to gonadotrophin releasing hormone challenge in energy-deficient anestrous heifers

A study was conducted to evaluate the effect of feeding a bypass fat on luteinizing hormone (LH) response to gonadotrophin releasing hormone (GnRH) in noncyclic Holstein heifers. Twelve cyclic Holstein heifers were fed a complete diet at 40% net energy for maintenance (NE(m)) until cessation of ovarian activity. Based on weights and condition scores, heifers were assigned to either a control or treatment diet containing 0.45 kg bypass fat and fed at an energy level of 85% NE(m). Diet adjustments were made following weekly weighings. GnRH challenges were conducted at four periods: prior to initial energy deprivation, at termination of 40% NE(m) feeding, and twice more at 21-d intervals after 85% NE(m) feeding began. Blood was sampled via a jugular catheter every 15 min for 5 h, and GnRH was injected after the fourth sample. None of the heifers exhibited estrous activity after the initial energy deprivation. Heifers on the bypass fat diet continued to lose weight during the treatment period, while the control heifers gained a slight amount of weight. Baseline and peak concentrations of LH were not significantly affected by time or diet. Time to GnRH-induced LH peak was longer (53 vs 130 min, P < 0.01) after 40% NE(m) and remained greater at all times thereafter. Serum lipid levels increased 82.5% among heifers being fed the bypass fat. Energy restriction had no effect on the magnitude of LH response to GnRH but did delay response time.     

Soluble minerals in chemical evolution

The adsorption of 5-AMP and 5-CMP was studied in saturated solutions of several soluble mineral salts (NaCl, Na₂SO₄, MgCl₂·6H₂O, MgSO₄·7H₂O, CaCl₂·2H₂O, CaSO₄·2H₂O, SrCl₂·6H₂O, SrSO₄, and ZnSO₄·7H₂O) as a function of pH, ionic strength, and surface area of the solid salt. The adsorption shows a pH dependence; this can be correlated with the charge on the nucleotide molecule which is determined by the state of protonation of the N-1 nitrogen of 5-AMP or N-3 nitrogen of 5-CMP and the phosphate oxygens. The adsorption which results from the binding between the nucleotide molecule and the salt surface is proposed as being due to electrostatic forces. It was concluded that the adsorption was reversible in nature. The adsorption shows a strong dependence upon ionic strength and decreases with increasing ionic strength. Surface area is shown to be an important factor in evaluating and comparing the magnitude of adsorption of nucleotides onto various mineral salts. The implications of the results of the study are discussed in terms of the importance of soluble mineral salts as adsorption sites in the characterization of the adsorption reactions of an adsorbed template in biogeochemical cycles.     

Chloroplast photooxidation inhibits the expression of a set of nuclear genes

Summary Mutations or herbicides which inhibit the accumulation of carotenoid pigments in higher plants also result in the arrest of chloroplast development at a very early stage. The cause is extensive photooxidative damage within the chloroplast in the absence of protective carotenoids. Because the extent of photooxidation is dependent upon light intensity, normal chloroplast development can occur when carotenoid-deficient seedlings are grown in very dim light. Normal accumulation of chloroplastic and cytosolic mRNAs encoding chloroplast proteins proceeds only under permissive dim light conditions. Illumination with higher intensity light causes rapid chlorophyll photooxidation and the loss of two cytosolic mRNAs coding for proteins destined for the chloroplast, but does not affect another light-regulated cytosolic mRNA encoding a cytosolic protein. This experimental system may have uncovered a mechanism which coordinates the expression of genes in different cellular compartments.Abbreviations LHCP light-harvesting chlorophyll a/b protein - SSu small subunit - RuBP fibulose 1,5-bisphoshate - PEP phosphoenolpyruvate     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol-3yl-acetic acid

Phenylacetic acid (PAA), a naturally-occurring acidic plant growth substance, was readily taken up by pea (Pisum sativum L. cv. Alderman) stem segments from buffered external solutions by a pH-dependent, non-mediated diffusion. Net uptake from a 0.2 M solution at pH 4.5 proceeded at a constant rate for at least 60 min and, up to approx. 100 M, the rate of uptake was directly proportional to the external concentration of the compound. The net rate of uptake of PAA was not affected by the inclusion of indol-3yl-acetic acid (IAA) in the uptake medium (up to approx. 30 M) and, unlike the net uptake of IAA, was not stimulated by N-1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid. At an external concentration of 0.2 M and pH 4.5, the net rate of uptake of PAA was about twice that of IAA. It was concluded that the uptake of PAA did not involve the participation of carriers and that PAA was not a transported substrate for the carriers involved in the uptake and polar transport of IAA. Nevertheless, the inclusion of 3–100 M unlabelled PAA in the external medium greatly stimulated the uptake by pea stem segments of [1-¹⁴C]IAA (external concentration 0.2 M). It was concluded that whilst PAA was not a transported substrate for the NPA-sensitive IAA efflux carrier, it interacted with this carrier to inhibit IAA efflux from cells. Over the concentration range 3–100 M, PAA progressively reduced the stimulatory effect of NPA on IAA uptake, indicating that PAA also inhibited carrier-mediated uptake of IAA. The consequences of these observations for the regulation of polar auxin transport are discussed.Abbreviations IAA indol-3yl-acetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid     

Primary structure of an analog of crustacean pigment-dispersing hormone from the lubber grasshopper Romalea microptera

An octadecapeptide capable of inducing pigment dispersion in the chromatophores of the fiddler crab Uca pugilator has been isolated from lyophilized heads of the lubber grasshopper Romalea microptera. This pigment-dispersing factor (PDF) was purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase high performance liquid chromatography. Automated gas-phase sequencing, followed by the identification of the carboxyl-terminal amide, established the primary structure of this PDF as Asn-Ser-Glu-Ile-Ile-Asn-Ser-Leu-Leu-Gly-Leu-Pro-Lys-Leu-Leu-Asn-Asp-Ala- NH2. This structure was confirmed by chemical synthesis and by demonstrating that the synthetic and native PDF displayed identical chromatographic behavior and biological activity. The Romalea PDF is structurally related to the crustacean pigment-dispersing hormones (PDHs), which are also octadecapeptides. The sequence of grasshopper PDF shows 78% homology with beta-PDH (from the crabs U. pugilator and Cancer magister) and 50% homology with alpha-PDH (from the prawn Pandalus borealis). This study provides the first direct chemical evidence for the structural relatedness of insect PDF to the crustacean PDHs, thus identifying them as an authentic family of arthropod peptides.     

Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-60 and/or Asn-97. Incubation of the A form with methyltransferase and S-adenosyl-L-methionine converts about half of the A form to an electrophoretic band indistinguishable from the B form. The activity of this partly converted calmodulin rises to 30-50% that of native calmodulin. These observations imply that the methyltransferase may have a biological role in restoring activity to proteins which contain abnormal isoaspartyl peptide bonds resulting from asparagine deamidation.