Casein kinase I phosphorylates the 25-kDa mRNA cap-binding protein

The 25-kDa mRNA cap-binding protein (eIF-4E) exists in both phosphorylated and dephosphorylated forms in eukaryotic cells. Phosphorylated eIF-4E appears to be preferentially associated with 48 S initiation complexes and with the 220-kDa subunit of eIF-4F. In addition, dephosphorylation of eIF-4E has been observed during heat shock and mitosis which are accompanied by decreased protein synthesis. However, the control of eIF-4E phosphorylation and its regulatory role remain poorly understood. Using eIF-4E as a substrate we have identified and purified from rabbit reticulocytes a protein kinase that phosphorylates eIF-4E in vitro. This enzyme phosphorylated eIF-4E on both serine and threonine residues with an apparent Km of 3.7 microM. The molecular mass of the enzyme and specificity for substrates other than eIF-4E suggested that this enzyme was a species of casein kinase I. This was confirmed by comparing the phosphopeptide map of the purified reticulocyte enzyme with that of rabbit skeletal muscle casein kinase I and by comparing phosphopeptide maps of eIF-4E phosphorylated in vitro by each enzyme. We conclude that casein kinase I phosphorylates eIF-4E in vitro and suggest that eIF-4E may be phosphorylated by casein kinase I in intact cells under some physiologic conditions.     

Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography—mass spectrometry

The metabolic transformation of methandienone (I) in the horse was investigated. After administration of a commercial drug preparation to a female horse (0.5 mg/kg), urine samples were collected up to 96 h and processed without enzymic hydrolysis. Extraction was performed by a series of solid—liquid and liquid—liquid extractions, thus avoiding laborious purification techniques. For analysis by gas chromatography—mass spectrometry, the extracts were trimethylsilylated. Besides the parent compound I and its C-17 epimer II, three monohydroxylated metabolites were identified: 6β-hydroxymethandienone (III), its C-17 epimer (IV) and 16β-hydroxy-methandienone (V). In addition, three isomers of 6β,16-dihydroxymethandienone (VIa–c) were discovered. Apparently, reduction of the δ⁴ double bond of 16β-hyroxymethandienone (V) in the horse yields 16β,17β-dihydroxy-17α-methyl-5β-androst-1-en-3-one (VII). Reduction of the isomers VIa–c results in the corresponding 6β,16,17-trihydroxy-17-methyl-5β-androst-1-en-3-ones (VIIIa–c). The data presented here suggest that screening for the isomers of VI and VIII, applying the selected-ion monitoring technique, will be the most successful way of proving methandienone administration to a horse.     

Development of the jamming avoidance response and its morphological correlates in the gymnotiform electric fish,Eigenmannia

The electric fish, Eigenmannia, will smoothly shift the frequency of its electric organ discharge away from an interfering electric signal. This shift in frequency is called the jamming avoidance response (JAR). In this article, we analyze the behavioral development of the JAR and the anatomical development of structures critical for the performance of the JAR. The JAR first appears when juvenile Eigenmannia are approximately 1 month old, at a total length of 13–18 mm. We have found that the establishment of much of the sensory periphery and of central connections precedes the onset of the JAR. We describe three aspects of the behavioral development of the JAR: (a) the onset and development of the behavior is closely correlated with size, not age; (b) the magnitude (in Hz) of the JAR increases with size until the juveniles display values within the adult range (10–20 Hz) at a total length of 25–30 mm; and (3) the JAR does not require prior experience or exposure to electrical signals. Raised in total electrical isolation from the egg stage, animals tested at a total length of 25 mm performed a correct JAR when first exposed to the stimulus. We examine the development of anatomical areas important for the performance of the JAR: the peripheral electrosensory system (mechano- and electroreceptors and peripheral nerves); and central electrosensory pathways and nuclei [the electrosensory lateral line lobe (ELL), the lateral lemniscus, the torus semicircularis, and the pacemaker nucleus]. The first recognizable structures in the developing electrosensory system are the peripheral neurites of the anterior lateral line nerve. The afferent nerves are established by day 2, which is prior to the formation of receptors in the epidermis. Thus, the neurites wait for their targets. This sequence of events suggests that receptor formation may be induced by innervation of primordial cells within the epidermis. Mechanoreceptors are first formed between day 3 and 4, while electroreceptors are first formed on day 7. Electroreceptor multiplication is observed for the first time at an age of 25 days and correlates with the onset of the JAR. The somata of the anterior lateral line nerve ganglion project afferents out to peripheral electroreceptors and also send axons centrally into the ELL. The first electroreceptive axons invade the ELL by day 6, and presumably a rough somatotopic organization and segmentation within the ELL may arise as early as day 7. Axonal projections from the ELL to the torus develop after day 18. Within the torus semicircularis, giant cells are necessary for the performance of the JAR. Giant cell numbers increase exponentially during development and the onset of the JAR coincides with a minimum of at least 150 giant cells and the attainment of a total length of at least 15 mm and at least 150 giant cells. Pacemaker and relay cells comprise the adult Eigenmannia pacemaker nucleus. The growth and differentiation of these cell types also correlates with the onset of the JAR in developing animals. We describe a gradual improvement of sensory abilities, as opposed to an explosive onset of the mature JAR. We further suggest that this may be a rule common in most developing behavioral systems. © 1992 John Wiley & Sons, Inc.     

Development and characterization of polyclonal antibodies against a conserved sequence in the catalytic domain of protein kinases.

Using a synthetic oligopeptide (CGGGTPEYLAPEGGK) crosslinked to keyhole limpet hemocyanin we have raised polyclonal rabbit antibodies against a 9 residue homologous region found in the catalytic domain of most protein kinases. These antibodies reacted during Western immunoblotting with cAMP dependent protein kinase catalytic subunit, phosphorylase kinase gamma subunit and calcium calmodulin dependent protein kinase II which have homologous sequences of GTPEYLAPE, GTPSYLAPE and GTPGYLSPE, respectively. Five other protein kinases did not react with anti-GTPEYLAPE antibodies during Western immunoblotting. Affinity-purified antibodies were able to detect as little as 50 ng of cAMP dependent protein kinase and 200 ng of Ca2+/calmodulin dependent protein kinase II. Immunoblotting of A431 cell plasma membrane vesicles indicated the presence of an approximately 55 kDa protein that contains the conserved sequence and is likely to be a protein kinase. Antibodies directed against conserved sequences present in protein kinases, or possibly other enzymes, may be useful in identifying previously uncharacterized enzymes at the protein level.     

Transfer of 1-pyrroline-5-carboxylate as oxidizing potential from hepatocytes to erythrocytes.

The interconversions of proline and 1-pyrroline-5-carboxylate form an intercellular cycle that is the basis of a metabolic interaction between hepatocytes and erythrocytes. The cycle transfers oxidizing potential from hepatocytes to erythrocytes, which stimulates pentose phosphate pathway in erythrocytes. This interaction depends on the differential metabolism of proline and 1-pyrroline-5-carboxylate in erythrocytes and hepatocytes and consists of the following: in hepatocytes proline oxidase converts proline into 1-pyrroline-5-carboxylate, which is released into the medium and taken up by erythrocytes; erythrocyte 1-pyrroline-5-carboxylate reductase converts 1-pyrroline-5-carboxylate into proline and concomitantly generates NADP+; the generated oxidizing potential drives glucose metabolism through the pentose phosphate pathway in erythrocytes; finally, erythrocytes release proline into the medium, enabling it to re-enter hepatocytes and repeat the cycle. The increased activity of the pentose phosphate pathway in erythrocytes may enhance the production of 5-phosphoribosyl pyrophosphate, a necessary moiety for the processing of purines.     

Bacterial ecology of an old-growth douglas fir canopy

Microbial populations associated with the major substrates of the canopy of a single 70 m old-growth Douglas fir were studied to determine potential activities. Seasonal samples from bark, foliage, epiphytic moss, lichens, and litter accumulations were collected to: (a) obtain population data, (b) isolate the major groups of microorganisms present, (c) measure enzymatic activities associated with cellulose and xylan degradation, and (d) examine the potential for nitrogen fixation. We tested 562 bacterial isolates for utilization of 25 compounds associated with the canopy substrates, and for activities in nitrogen and sulfur cycle transformations. Total bacterial populations, reflecting seasonal temperature and moisture conditions, were lowest on bark and foliage [21–266×10³ colony-forming units (CFU/g)] and highest on moss and lodged litter (19–610×10⁵ CFU/g). Lichens contained intermediate numbers of bacteria (3.3–270×10⁵ CFU/g). The majority of the bacteria were classified as species ofArthrobacter, Bacillus, Flavobacterium, andXanthomonas. Isolates ofAlcaligenes (Achromobacter), Aeromonas, Chromobacterium, Micrococcus, andPseudomonas were less common. No measurable rates of nitrogen fixation attributable to free-living bacteria were detected by acetylene reduction. Eleven species in six genera of lichens containing a blue-green algal phycobiont showed positive acetylene reduction. One species,Lobaria oregana, accounted for 51% of the total lichen biomass of the canopy. Cellulase and xylanase activity was routinely detected in moss and litter samples, and less frequently in lichens. There was a strong correlation between the two activities for moss (r=0.94) and litter (r=0.81).     

Ecology of soil arthrobacters in clarion-webster toposequences of iowa

Toposequence variations in soil properties were characterized and related to variations in populations of total isolatable bacteria and arthrobacters. Increases in soil NO₃-N, available phosphorous, NO₃-N-producing power, Arthrobacter counts, and the percentage of the total counts represented by arthrobacters were correlated with decreases in soil acidity. The total bacterial counts were not correlated with soil acidity but were associated with percentage of soil organic matter and percentage of clay. The percentage of the total counts represented by arthrobacters was lowest at the summit position and increased downslope to the highest value in the toeslope position. Factor analysis of the data revealed that 67 to 81% of the total variance exhibited by all variables per site-sampling period could be accounted for by soil acidity, soil structure, soil fertility, soil moisture, and bacterial factors. A selective medium was developed for soil arthrobacters and tested on a wide variety of central Iowa soils to determine its potential as a medium for enumeration as well as isolation. The medium developed in this study was found to be superior to the other available direct-isolation media for soil arthrobacters.     

Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.