Tyrosine 394 is phosphorylated in Alzheimer's paired helical filament tau and in fetal tau with c-Abl as the candidate tyrosine kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimer's disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl) pyrazolo[3,4-d] pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimer's disease.

Subcellular localization of the steroid receptor coactivators (SRCs) and MEF2 in muscle and rhabdomyosarcoma cells

Skeletal muscle differentiation and the activation of muscle-specific gene expression are dependent on the concerted action of the MyoD family and the MADS protein, MEF2, which function in a cooperative manner. The steroid receptor coactivator SRC-2/GRIP-1/TIF-2, is necessary for skeletal muscle differentiation, and functions as a cofactor for the transcription factor, MEF2. SRC-P belongs to the SRC family of transcriptional coactivators/cofactors that also includes SRC-1 and SRC-3/RAC-3/ACTR/ AIB-1. In this study we demonstrate that SRC-P is essentially localized in the nucleus of proliferating myoblasts; however, weak (but notable) expression is observed in the cytoplasm. Differentiation induces a predominant localization of SRC-P to the nucleus; furthermore, the nuclear staining is progressively more localized to dot-like structures or nuclear bodies. MEF2 is primarily expressed in the nucleus, although we observed a mosaic or variegated expression pattern in myoblasts; however, in myotubes all nuclei express MEF2. GRIP-1 and MEF2 are coexpressed in the nucleus during skeletal muscle differentiation, consistent with the direct interaction of these proteins. Rhabdomyosarcoma (RMS) cells derived from malignant skeletal muscle tumors have been proposed to be deficient in cofactors. Alveolar RMS cells very weakly express the steroid receptor coactivator, SRC-P, in a diffuse nucleocytoplasmic staining pattern. MEF2 and the cofactors, SRC-1 and SRC-3 are abundantly expressed in alveolar and embryonal RMS cells; however, the staining is not localized to the nucleus. Furthermore, the subcellular localization and transcriptional activity of MEF2C and a MEF2-dependent reporter are compromised in alveolar RMS cells. In contrast, embryonal RMS cells express SRC-2 in the nucleus, and MEF2 shuttles from the cytoplasm to the nucleus after serum withdrawal. In conclusion, this study suggests that the steroid receptor coactivator SRC-P and MEF2 are localized to the nucleus during the differentiation process. In contrast, RMS cells display aberrant transcription factor SRC localization and expression, which may underlie certain features of the RMS phenotype.

The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochleae

Messenger RNAs coding for growth factors and receptor tyrosine kinases were measured by quantitative competitive and by semi-quantitative reverse-transcription polymerase chain reaction in whole and dissected chick inner ears. The fibroblast growth factor (FGF) receptor 1 chick embryonic kinase (CEK) 1 was expressed in all structures examined (otocyst, hatchling whole cochlea, cochlear nerve ganglion, and cochlear and vestibular sensory epithelia), although slightly more heavily in the otocyst. The related fibroblast growth factor receptors CEK 2 and 3 were preferentially expressed in the nerve ganglion and in the vestibular sensory epithelium, respectively. FGF 1 mRNA was low in early development, increasing to mature levels at around embryonic age 11 days, while FGF2, mRNA was expressed at constant levels at all ages. In response to ototoxic damage, FGF1 mRNA levels were increased in the early damaged cochlear sensory epithelium. Immunohistochemistry for CEK1 showed that normal hair cells expressed the receptor heavily on the hair cell stereocilia, while with early damage, CEK1 came to be expressed heavily on the apical surfaces of the supporting cells. In normal chicks, the CEK4 and CEK8 eph-class receptor tyrosine kinases were expressed relatively heavily by the cochlear nerve ganglion, and CEK10 was expressed relatively heavily by the cochlear hair cell sensory epithelium. The results suggest that the FGF system may be involved in the response of the cochlear epithelium to ototoxic damage. The eph-class receptor tyrosine kinase CEK10 may be involved in cell interactions in the cochlear sensory epithelium, while CEK4 and CEK8 may play a role in the cochlear innervation.

FHA domain boundaries of the Dun1p and Rad53p cell cycle checkpoint kinases

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues), In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks, This indicates that the non-catalytic FI-IA domain plays an important role in the transcriptional function of the Dun1p protein kinase. (C) 2000 Federation of European Biochemical Societies.

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3 Delta 977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3 Delta 977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

A family of cytokine-inducible inhibitors of signalling

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation(1). Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members af the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters(1-4). Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two-relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS (ref. 5) form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases

The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/ threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK There are now more than 50 proteins shown to be substrates for JNK These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.

Family orientated delivery of routine nursing care in hospital

Hospital nursing may be better deployed to acute clinical patient care. The recruitment of family assistance will facilitate this process in patients in hospital awaiting placement and without acute care issues.

Prevention of child behavior problems through universal implementation of a group behavioral family intervention.

The aim of this mental health promotion initiative was to evaluate the effectiveness of a universally delivered group behavioral family intervention (BFI) in preventing behavior problems in children. This study investigates the transferability of an efficacious clinical program to a universal prevention intervention delivered through child and community health services targeting parents of preschoolers within a metropolitan health region. A quasiexperimental two-group (BFI, n=804 vs. Comparison group, n=806) longitudinal design followed preschool aged children and their parents over a 2-year period. BFI was associated with significant reductions in parent-reported levels of dysfunctional parenting and parent-reported levels of child behavior problems. Effect sizes on child behavior problems ranged from large (.83) to moderate (.47). Positive and significant effects were also observed in parent mental health, marital adjustment, and levels of child rearing conflict. Findings are discussed with respect to their implication for significant population reductions in child behavior problems as well as the pragmatic challenges for prevention science in encouraging both the evaluation and uptake of preventive initiatives in real world settings.

The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family

The complete nucleotide sequence of the genomic RNA from the insect picorna-like virus Drosophila C virus (DCV) was determined. The DCV sequence predicts a genome organization different to that of other RNA virus families whose sequences are known. The single-stranded positive-sense genomic RNA is 9264 nucleotides in length and contains two large open reading frames (ORFs) which are separated by 191 nucleotides. The 5' ORF contains regions of similarities with the RNA-dependent RNA polymerase, helicase and protease domains of viruses from the picornavirus, comovirus and sequivirus families. The 3' ORF encodes the capsid proteins as confirmed by N-terminal sequence analysis of these proteins. The capsid protein coding region is unusual in two ways: firstly the cistron appears to lack an initiating methionine and secondly no subgenomic RNA is produced, suggesting that the proteins may be translated through internal initiation of translation from the genomic length RNA. The finding of this novel genome organization for DCV shows that this virus is not a member of the Picornaviridae as previously thought, but belongs to a distinct and hitherto unrecognized virus family.

Carrageenans from Australian representatives of the family Cystocloniaceae (Gigartinales, Rhodophyta), with description of Calliblepharis celatospora sp. nov., and transfer of Austroclonium to the family Areschougiaceae

Ten Australian representatives from seven of the 10 genera presently constituting the family Cystolcloniaceae have been analyzed for their cell-wall galactans. Included in our survey are the monotypic Australian-endemic genera Austroclonium, Gloiophyllis, Erythronaema, and Stictosporum, one species of Craspedocarpus, three species of Rhodophyllis, and two species of Calliblepharis. As one of the species of the latter genus is endemic to Western Australia and presently undescribed, we illustrate its habit and anatomical features in formally proposing to name it Calliblepharis celatospora Kraft, sp. nov. All the species surveyed essentially produce typical iota (iota)-carrageenans, with the exception of Austroclonium. The sulfated galactans from Austroclonium predominantly contain the repeating units of iota-, alpha (alpha)-, and 6'-O-methylated iota- and alpha-carrageenans; whether these exist as discrete polysaccharides or a complex hybrid structure was not resolved. Thus, Austroclonium carrageenans resemble the polysaccharides from Rhabdonia, Areschougia, and Erythroclonium. Although these latter three genera are currently included in the large gigartinalean family Solieriaceae, all produce significantly different carrageenans from Solieria itself and related genera such as Eucheuma, Kappaphycus, Betaphycus, Sarcodiotheca, Agardhiella, Sarconema, and Callophycus. In consideration of these findings, as well as of significant anatomical similarities, we provisionally recommend reestablishment of the family Rhabdoniaceae Kylin (as the family Areschougiaceae J. Agardh) for Rhabdonia, Areschougia, Erythroclonium, and Austroclonium.

Advice on exercise from a family physician can help sedentary patients to become active

Objectives: To test the effectiveness, in the setting of primary health care, of verbal advice on exercise from a family physician (FP) combined with supporting written information. Design: A controlled trial with subjects allocated to a control group or one of two intervention groups using a balanced design based on day of the week. Setting: Ten general practices in Perth, Western Australia. Subjects: All sedentary patients consulting an FP. Intervention: Verbal advice on exercise from the FP and a pamphlet on exercise mailed to the patient's home address within 2 days of his/her visit to the doctor. Main outcome measure: Level of physical activity at followup. Results: 6,351 adult patients attending an FP practice completed a screening questionnaire, and 763 sedentary adults were recruited to the project. The response to follow-up, via a postal survey at 1, 6, and 12 months after the index consultation was 70%, 60%, and 57%, respectively. At 1 month a subsample of the control and intervention subjects were contacted for a telephone interview to verify self-reported levels of activity (n = 136). Treating all nonresponders as sedentary, at 1 month significantly more subjects in the combined intervention groups reported doing some physical activity (40%) compared with the control group (31%). Similarly, at 6 months, 30% of the control group and 38% of the combined intervention groups were now active. There was very little change at followup at 12 months (31% control and 36% intervention groups, respectively). Conclusion: A simple intervention aimed at the promotion of physical activity to sedentary patients in general practice can help reduce inactivity.

The case for involving the family in the treatment of childhood anxiety

Anxiety disorders are among the most common forms of psychopathology reported by children and adolescents. Anxiety disorders in children have debilitating consequences, both for the child and his/her family. In addition, research indicates that, in many cases, these disorders follow a chronic and persistent course. The efficacy of child-focused cognitive-behavioural interventions in the treatment of childhood anxiety disorders has been well demonstrated by recent, well-controlled research. This treatment outcome literature is briefly reviewed. Most recently, however, interventions used in the treatment of childhood anxiety disorders have taken the form of combined interventions aimed both at the anxious child and his or her family. This paper reviews two related bodies of research in presenting a case for the involvement of families in the treatment of childhood anxiety. First, the etiological evidence implicating the families of anxious children in the development and/or maintenance of childhood anxiety is presented. Second, the treatment outcome studies that have empirically evaluated this type of combined intervention are reviewed.