Catalytic domains of tyrosine kinases determine the phosphorylation sites within c-Cbl

Catalytic (SH1) domains of protein tyrosine kinases (PTKs) demonstrate specificity for peptide substrates. Whether SH1 domains differentiate between tyrosines in a physiological substrate has not been confirmed. Using purified proteins, we studied the ability of Syk, Fyn, and Abl to differentiate between tyrosines in a common PTK substrate, c-Cbl. We found that each kinase produced a distinct pattern of c-Cbl phosphorylation, which altered the phosphotyrosine-dependent interactions between c-Cbl and CrkL or phosphatidylinositol 3'-kinase (PI3-K). Our data support the concept that SH1 domains determine the final sites of phosphorylation once PTKs reach their target proteins.     

Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis,and consequently enhances Ras/ERK signalling

Drosophila Sprouty (dSpry) was genetically identified as a novel antagonist of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signalling, ostensibly by eliciting its response on the Ras/MAPK pathway. Four mammalian sprouty genes have been cloned, which appear to play an inhibitory role mainly in FGF- mediated lung and limb morphogenesis. Evidence is presented herein that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl, and its impact on the cellular localization and signalling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signalling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signalling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpry2DeltaN11 and mSpry4, both deficient in c-Cbl binding, to instigate these effects. hSpry2 interacts specifically with the c-Cbl RING finger domain and displaces UbcH7 from its binding site on the E3 ligase. We conclude that hSpry2 potentiates EGFR signalling by specifically intercepting c-Cbl-mediated effects on receptor down-regulation.     

Abundance, sizes and developmental stages of larval krill, Euphausia superba, during winter in ice-covered seas west of the Antarctic Peninsula

Larval krill were sampled west of the Antarctic Peninsula duringthree winter cruises: September 1991, June 1993 and September1993. Larval abundances were estimated from net catches andcompared directly to visual counts (made by a SCUBA diver) oflarvae occupying the ice habitat at the same sampling stations.The number of larvae per square meter sampled with nets wasmore often greater than that observed by the diver, irrespectiveof the sampling period. However, comparisons of larval abundancewithin sampling periods were not statistically significant.Larval krill collected by divers were significantly larger thanthose collected with nets for each of the three cruises. Thestage composition of larval krill also depended on the collectionmethod: net-collected samples contained a disproportionatelyhigh number of early furcilia larvae in June 1993 (early winter),and a disproportionately low number of early juveniles duringSeptember 1991 and 1993 (late winter). These results lead usto suggest that larval/juvenile krill occupy both the watercolumn and sea ice habitat during the austral winter, and thatthere are often differences in the sizes and developmental stagesof the two groups. For larval krill that occupied the sea icehabitat, aggregations were larger and more numerous during latewinter than in early winter. In addition, larvae within aggregationsoccupied structurally complex microhabitats, provided by over-raftedice floes, more often than they occupied smooth, downward-facingice surfaces where ice was not over-rafted.     

Observations on new Myxobolus species and Kudoa species infecting the nervous system of Australian fishes

Myxobolus galaxii sp. nov. from the spinal cord of Galaxias olidus in northeast Victoria induced little pathologicaf change even when present in lare numbers. Myxobolus gadopsii sp. nov. was found in the meninx and other connective tissues of Gadopsis marmoratus popufations within all three mainland Victorian enotypes as well as in G. bispinosus. Myxobolus gadopsii also appeared to be harmless to its hosts. Kudoa sp. from the brain of Lates calcarifer in north Queensland was associated with abnormal swimming behaviour in juveniles but not in adults. A further Kudoa species, probably K. nova or K. clupeidae, was examined in Thunnus maccoyii from Western Australia, where it was found that most, if not all, of the infections developed in peripheral nerves.     

Specific glycanforms of type IX collagen accumulate in embryonic chick sterna after 17 days of development

Type IX collagen is a key component of the extracellular matrix of cartilage where it occurs at the surfaces of type II collagen fibrils as a glycanated molecule. The function of the glycosaminoglycan (GAG) side chain of the molecule is, however, unknown. We have shown that type IX collagen in chicken sternal cartilage is synthesized with a unimodal distribution of GAG chain size, but at post 17 days of development three predominant glycanforms of type IX collagen accumulate. Such accumulation did not occur in sterna from day 15 embryos. In day 17 embryos predominant glycanforms were found in the caudal region of the sternum. By day 19 of development the three predominant glycanforms are widespread throughout the caudal and cephalic regions. The results indicate that developmental and anatomical changes occur to type IX collagen that depend on the size of the GAG chain attached to the alpha2(IX) chain of the molecule.      

The truncation that generated the v-cbl oncogene reveals an ability for nuclear transport, DNA binding and acute transformation.

The v-cbl oncogene is the transforming gene of the murine Cas NS-1 retrovirus which induces pre-B cell lymphomas and myeloid leukaemias. Sequencing of c-cbl has revealed that v-cbl was generated by a large truncation that removed 60% of the C-terminus of the corresponding protein. In this study we prepared antibodies to cbl and found that c-cbl encodes a 120 kDa protein which is localized in the cytoplasm with a cytosolic and cytoskeletal distribution. Immunofluorescence studies show a striking pattern of brightly staining vesicles in mitotic cells similar to that observed with cytokeratin antibodies. In contrast to p120c-cbl, which is exclusively cytoplasmic, the p100gag-v-cbl encoded by Cas NS-1 is localized in both the cytoplasm and the nucleus. This redistribution to the nucleus correlates with the ability of cbl to induce acute transformation. Furthermore the truncated protein encoded by v-cbl can bind DNA, unlike the full-length protein. These results suggest that the C-terminus of cbl is involved in the retention of p120c-cbl in the cytoplasm and the inhibition of DNA binding. The findings also suggest that a truncated protein encoded by c-cbl exists in the nucleus of normal cells.     

Phospholipid-protein interactions in human low density lipoprotein detected by 31P nuclear magnetic resonance.

31P nuclear magnetic resonance (NMR) spectra of human low density lipoprotein (LDL) has been obtained and the major phospholipid components identified. Analysis of the spectra revealed two phospholipid environments: one occupied by 4/5 of the phospholipid with high resolution resonances possessing properties similar to phospholipids in vesicles, and a second occupied by 1/5 of the phospholipid with broad lines indicative of immobilization. Limited trypsin treatment of the particle cleaved all of the B peptide into smaller molecular weight peptides which remained with the particle. Trypsin-treated LDL eluted from a Sepharose CL-6B column similarly to native LDL so that the modified particle remained intact. 31P NMR spectra of trypsin-treated LDL showed little or no immobilized phospholipid. The immobilization in the native LDL particle is attributed to lipid-protein interactions between 1/5 of the phospholipid and the B peptide.     

The morphology of the vertical and transverse intrinsic musculature of the tongue in the 15-week human fetus

The attachments, courses and interrelationships of the transverse and vertical intrinsic muscle masses of the tongue were examined in 28 fifteen-week fetal specimens. Observations were made from 30-micron sections cut through the tongue in one of the three standard planes of section. Both sets of muscululature are qualitatively well-developed by this time period in fetal life. The transverse fibers were found to occupy the entire length of the tongue. They attach to the lamina propria of the lateral aspect of the body of the tongue and, in the root, to perimysial and adventitial connective tissue. In addition, some fibers were observed to be confluent with the mm. palatoglossus, tonsilloglossus and pharyngis superior. Medially, transverse fibers were found for the most part to terminate in the dense ventral aspect of the median septum. Vertical fibers are present from a point slightly posterior to the tip of the tongue to the level of the foramen cecum, beyond which they become sparse. All vertical fibers attach superiorly to the dorsal lamina propria. In the free part of the body, their ventral attachment, likewise, is to lamina propria. In the middle part of the tongue and, to a greater extent, in the root (as the inferior and lateral free surface decreases) these fibers attach in either the fascial plane underlying the transverse component or to the perimysium of longitudinally-running muscle bundles.     

Impact of synthetic pyrethroid-sheep dip on the indigenous microflora of animal slurries

The chemical constituents of sheep dip in the UK are currently changing from organophosphate-based to synthetic pyrethroid-based insecticides. As a result, changes are also being made to the methods of disposal of these chemicals in the environment, such that pyrethroid sheep dips must now be diluted in animal slurry or water. To date, there is a lack of quantitative information on the impact of the insecticide on the indigenous microflora of animal slurries. This paper investigated the impact of Bayticol (synthetic pyrethroid sheep dip) over a range of concentrations on selected populations of bacteria within animal slurry. It was found that, with increasing pesticide concentration, there was up to a four orders of magnitude increase in the numbers of faecal coliforms and pathogens, such as putative Salmonella spp. These findings have implications for the disposal of sheep dip-amended animal slurries to land from several aspects: (i) the longevity of putative pathogens in the field may require re-evaluation of the time required before the return of grazing livestock to a slurry-amended field; (ii) the potential for the transfer of pathogenic bacteria and faecal coliforms into human and animal foodchains, and (iii) the increased potential for faecal coliforms being washed into streams, rivers and coastal bathing waters.