MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termedMMK2. The predicted amino acid sequence ofMMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termedMMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness ofMMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeastMPK1 (SLT2) kinase byMMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.     

Identification of the border between fibronectin type III homologous repeats 2 and 3 of the neural cell adhesion molecule L1 as a neurite outgrowth promoting and signal transducing domain

To determine the domains of the neural cell adhesion molecule L1 involved in neurite outgrowth, we have generated monoclonal antibodies against L1 and investigated their effects on neurite outgrowth of small cerebellar neurons in culture. When the 10 antibodies were coated as substrate, only antibody 557.B6, which recognizes an epitope represented by a synthetic peptide comprising amino acids 818 to 832 at the border between the fibronectin type III homologous repeats 2 and 3, was as efficacious as L1 in promoting neurite outgrowth, increasing intracellular levels of Ca²⁺, and stimulating the turnover of inositol phosphates. These findings suggest that neurite outgrowth and changes in these second messengers are correlated. Such a correlation was confirmed by the ability of Ca²⁺ channel antagonists and pertussis toxin to inhibit neurite outgrowth on L1 and antibody 557.B6. These observations indicate for the first time a distinct site on cell surface-bound-L1 as a prominent signal-transducing domain through which the recognition events appear to be funneled to trigger neurite outgrowth, increase turnover of inositol phosphates, and elevate intracellular levels of Ca²⁺. © 1995 John Wiley & Sons, Inc.     

Pilot Preclinical and Clinical Evaluation of (4S)-4-(3-[18F]Fluoropropyl)-L-Glutamate (18F-FSPG) for PET/CT Imaging of Intracranial Malignancies

Purpose

(S)-4-(3-[¹⁸F]Fluoropropyl)-L-glutamic acid (18F-FSPG) is a novel radiopharmaceutical for Positron Emission Tomography (PET) imaging. It is a glutamate analogue that can be used to measure x_C^- transporter activity. This study was performed to assess the feasibility of 18F-FSPG for imaging orthotopic brain tumors in small animals and the translation of this approach in human subjects with intracranial malignancies.

Experimental Design

For the small animal study, GS9L glioblastoma cells were implanted into brains of Fischer rats and studied with 18F-FSPG, the 18F-labeled glucose derivative 18F-FDG and with the 18F-labeled amino acid derivative 18F-FET. For the human study, five subjects with either primary or metastatic brain cancer were recruited (mean age 50.4 years). After injection of 300 MBq of 18F-FSPG, 3 whole-body PET/Computed Tomography (CT) scans were obtained and safety parameters were measured. The three subjects with brain metastases also had an 18F-FDG PET/CT scan. Quantitative and qualitative comparison of the scans was performed to assess kinetics, biodistribution, and relative efficacy of the tracers.

Results

In the small animals, the orthotopic brain tumors were visualized well with 18F-FSPG. The high tumor uptake of 18F-FSPG in the GS9L model and the absence of background signal led to good tumor visualization with high contrast (tumor/brain ratio: 32.7). 18F-FDG and 18F-FET showed T/B ratios of 1.7 and 2.8, respectively. In the human pilot study, 18F-FSPG was well tolerated and there was similar distribution in all patients. All malignant lesions were positive with 18F-FSPG except for one low-grade primary brain tumor. In the 18F-FSPG-PET-positive tumors a similar T/B ratio was observed as in the animal model.

Conclusions

18F-FSPG is a novel PET radiopharmaceutical that demonstrates good uptake in both small animal and human studies of intracranial malignancies. Future studies on larger numbers of subjects and a wider array of brain tumors are planned.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01186601","term_id":"NCT01186601"}}NCT01186601     

Characterization of sulfate transport in Desulfovibrio desulfuricans

Uptake of ³⁵S-labelled sulfate was studied with a new isolate of Desulfovibrio desulfuricans, strain CSN. Micromolar additions of sulfate (1–10 M or nmol/mg protein) to cell suspensions incubated in 150 mM KCl at-1°C were almost completely taken up and accumulated about 5,000-fold. Accumulation was not influenced by incubation in NaCl instead of KCl, by acidic pH (5.5) or by incubation under air for 10 min. In alkaline milieu (pH 8.5), after prolonged contact with air (2 h), or after growth with excess sulfate or thiosulfate as electron acceptor, the amount taken up was diminished approximately by half. Pasteurization inhibited sulfate uptake completely. With increasing concentrations of added sulfate (0.1 to 2.5 mM) the intracellular concentration increased only slowly up to 25 mM, and the accumulation factor decreased down to 8. Sulfate transport was reversible. Accumulated sulfate was rapidly lost from the cells after addition of excess non-labelled sulfate or after addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The ATPase inhibitor dicyclohexylcarbodiimide (DCCD) specifically inhibited sulfate reduction but had no immediate influence on sulfate accumulation. Addition of the phosphate analogue arsenate (5 mM) was without effect. These results were not in favour of an ATP-dependent transport system. The K⁺-H⁺-antiporter nigericin (in 150 mM KCl) and the Na⁺-H⁺-antiporter monensin (in 150 mM NaCl) caused partial inhibition of sulfate accumulation, whereas the K⁺-transporter valinomycin (in 150 mM KCl) and the Na⁺-H⁺ exchange inhibitor amiloride (2 mM) were without effect. The permeant thiocyanate anion (150 mM) inhibited sulfate uptake by 60% at pH 7, and completely at pH 8.5. Although the effects of the different ionophores on the chemiosmotic gradients have not been studied so far, the results indicated that probably both, pH and drive sulfate accumulation and that sulfate is taken up electrogenically in symport with more than 2 protons. The structural sulfate analogues tungstate and molybdate (0.1 mM, each) did not affect sulfate accumulation, although molybdate inhibited sulfate reduction. Chromate completely blocked both of these activities. Sulfite and selenite caused little or no decrease of sulfate accumulation, whereas with thiosulfate and selenate significant inhibition was observed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide     

Sulfate transport in Desulfobulbus propionicus and Desulfococcus multivorans

Uptake of ³⁵S-labelled sulfate was studied with two sulfate-reducing bacteria, the freshwater species Desulfobulbus propionicus and the marine species Desulfococcus multivorans. Both bacteria were able to highly accumulate micromolar additions (2.5 M) of sulfate, if the reduction of sulfate to H₂S was prevented by low temperature (0° C) or oxygen. Sulfate accumulation was highest (accumulation factors 10³ to 10⁴) after growth under sulfate-limiting conditions, while cells grown with excess sulfate revealed accumulation factors below 300. With increasing sulfate concentrations added (up to 25 mM), the accumulation factors decreased down to 1.4. Sulfate accumulation in both strains was sensitive to carbonyl cyanide m-chlorophenylhydrazone (CCCP) and thiocyanate, but not directly correlated to the ATP content of the cells. Pasteurized cells did not accumulate sulfate. Sulfate transport was reversible. Accumulated ³⁵S-labelled sulfate was quickly released after addition of non-labelled sulfate or structural sulfate analogues (thiosulfate, selenate, chromate, less effect by molybdate, tungstate, sulfite, selenite). In D. propionicus, sulfate accumulation was independent of the presence or absence of Na⁺, K⁺, Li⁺, Mg²⁺, Cl^- and Br^-. Sulfate accumulation was reversibly enhanced at pH 5 and diminished at pH 9. In the marine bacterium D. multivorans, sulfate accumulation depended on the presence of Na⁺ ions. Na⁺ could partially be replaced by Li⁺. Sulfate accumulation in D. multivorans was sensitive to the Na⁺/H⁺ antiporter monensin and the Na⁺/H⁺ antiport inhibitor amiloride. It is concluded that in D. propionicus sulfate is accumulated electrogenically in symport with at least three protons, whereas for D. multivorans electrogenic symport with sodium ions is proposed. In both species, more than one sulfate transport system must be present. High affinity transport systems appear to be derepressed under sulfate limitation only. The high affinity transport system must be regulated to avoid energy-spoiling accumulation at high sulfate concentrations.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide     

Pressure regulation in the microcirculation.

The results of direct pressure measurements are described which demonstrate that pressures in a certain fraction of mesenteric capillaries remain remarkably constant during large changes in systemic pressure. The results of isogravimetric studies, reported in the literature, are also described which indicate that this phenomenon may also occur in the intestine. The question is raised whether capillary pressures may therefore be regulated. Pressures recorded from mesenteric arterioles and capillaries are shown which indicate that maintenance of a constant capillary pressure is primarily the consequence of the vascular architecture peculiar to this tissue, and is merely a secondary reflection of mechanisms associated with flow regulation. The results of direct pressure measurements recorded in the microcirculation of intestinal muscle are also shown. These data indicate that capillary pressures in innervated, denervated, and xylocaine-treated intestinal muscle change in direct proportion to variations in arterial pressure. It is concluded that capillary pressures in the intestinal muscle layers are therefore not regulated, so that the observation that capillary pressures may be maintained is probably a phenomenon unique to the mesentery. Pressures recorded from capillaries in the mucosal villi are also shown and compared to capillary pressures measured in the microvasculature of mesentery and intestinal muscle. When systemic pressure was normal (107 +/- 10 mm Hg), capillary pressure in the mesentery averaged 30 to 33 mm Hg; capillary pressures in the intestinal muscle averaged 22 to 24 mm Hg; and capillary pressures in the mucosal villi averaged 13 to 15 mm Hg. These data suggest that mesenteric capillaries are primarily a filtering network; intestinal muscle capillaries are normally in fluid balance; whereas at rest mucosal capillaries are primarily absorptive. These pressures, recorded from the three major regions of the rat intestine, were used to calculate a weighted average for the whole organ. The calculated value, based on assumed values for relative capillary densities, was 17 mm Hg. This result compares favorably with data from whole organ, isogravimetric studies, and may clarify some of the apparent discrepancies between previous isogravimetric and servopressure studies.     

Detection of the messenger RNA coding for preproenkephalin A in bovine adrenal by in situ hybridization

The messenger RNA (mRNA) coding for the adrenal precursor of enkephalins (preproenkephalin-A) has been detected in bovine adrenal medulla cells using in situ hybridization with 32P-labelled preproenkephalin A (PPA) complementary DNA. In formaldehyde- and Carnoy-fixed tissue sections, an intense elective labelling restricted to the cells located at the periphery of the adrenal medulla can be detected after hybridization procedure, using X-ray film and classical autoradiographic procedure. Adequate controls show that this labelling is obtained only using PPA complementary DNA, inserted or not in its vector. Distribution of PPA mRNA appears identical to that of its immunoreactive end products, namely Met-enkephalin and BAM22 peptide, detected by immunohistochemistry. Norepinephrine, detectable using monoamine histofluorescence, appears restricted to the cells of the center of the gland unlabelled for PPA mRNA and its end-products. Cultured bovine adrenomedullary cells that exhibited enkephalin immunoreactivity also contain PPA mRNA located in their cytoplasm.     

Isolation and characterization of the bovine hypothalamic corticotropin-releasing factor

A 41 amino acid peptide with high intrinsic corticotropin-releasing activity was isolated from 1000 bovine hypothalami by means of immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The primary structure of the amino terminal 39 amino acids was characterized by gas phase sequence analysis. The sequence of the amidated carboxyl terminal dipeptide was established by digestion of the intact natural product with Staphylococcus aureus V8 protease, dansylation of the digest and comparative reverse phase liquid chromatography studies with the synthetic dansylated dipeptides Ile-Ala-NH2, Ile-Ala-OH, Ala-Ile-NH2 and Ala-Ile-OH. The complete structure of the bovine corticotropin-releasing factor was established as: Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val- Leu- Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Asn-Asn-Arg-Lys-Leu- Leu- Asp-Ile-Ala-NH2 using approximately 650 pmol of material.