Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A.

Protein kinases of the DYRK ('dual-specificity tyrosine-regulated kinase') family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319-Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

Trends in domestic violence service and leadership: implications for an integrated shelter model.

Domestic violence is a dangerous and prevalent social problem affecting up to 4 million women and countless children annually. Shelters offer safety and an opportunity for change during the crisis of family violence. These individuals also have the potential for retraumatization if leadership within the program recapitulates the abuse and coercion felt at home. This article reviews three related trends through the lens of power and control--domestic violence policy and service, models of leadership, and the study of traumatic stress disorders and recovery--and describes their implications for modern shelter service delivery.

Hierarchy of sorting signals in chimeras of intestinal lactase-phlorizin hydrolase and the influenza virus hemagglutinin.

Lactase-phlorizin hydrolase (LPH) is an apical protein in intestinal cells. The location of sorting signals in LPH was investigated by preparing a series of mutants that lacked the LPH cytoplasmic domain or had the cytoplasmic domain of LPH replaced by sequences that comprised basolateral targeting signals and overlapping internalization signals of various potency. These signals are mutants of the cytoplasmic domain of the influenza hemagglutinin (HA), which have been shown to be dominant in targeting HA to the basolateral membrane. The LPH-HA chimeras were expressed in Madin-Darby canine kidney (MDCK) and colon carcinoma (Caco-2) cells, and their transport to the cell surface was analyzed. All of the LPH mutants were targeted correctly to the apical membrane. Furthermore, the LPH-HA chimeras were internalized, indicating that the HA tails were available to interact with the cytoplasmic components of clathrin-coated pits. The introduction of a strong basolateral sorting signal into LPH was not sufficient to override the strong apical signals of the LPH external domain or transmembrane domains. These results show that basolateral sorting signals are not always dominant over apical sorting signals in proteins that contain each and suggest that sorting of basolateral from apical proteins occurs within a common compartment where competition for sorting signals can occur.

Victimization of women and its impact on assessment and treatment in the psychiatric emergency setting.

An understanding of victimization is critical to the practice of emergency psychiatry. Victimization histories are disturbingly common among women presenting to the PES, particularly among frequent service users. The sequelae of victimization are both psychological and physical and often impair health and functioning across numerous domains. PTSD, BPD, and substance-use disorders are often seen among women with victimization histories, which can be particularly challenging for PES providers. Screening for trauma on PES presentation or history should not be overlooked in any person, including severely mentally ill, homeless, disabled, or elderly women. PES clinicians should remember to ask about victimization and pose questions privately in a direct and an open-ended format while conveying empathic validation. Clinical assessment of women with victimization histories in the PES should be guided by the principles of standard emergency psychiatry and be informed by an understanding of trauma. This includes a working knowledge of trauma dynamics, adherence to sound professional boundaries, and care not to retraumatize patients or re-enact perpetrator-victim dynamics. Voyeurism and regression should be avoided, particularly when eliciting trauma history. The PES should be a place for screening and acute intervention, not for conducting intensive trauma therapy. In the PES, the focus should remain on triage and treatment priorities, those of safety and stabilization, and carefully evaluating for substance use and psychosis. The PES ideally provides a "holding environment" that affords a balance of nurturing, limits, consistency, and communication. A basic knowledge of cognitive-behavioral interventions affording "crisis survival strategies," such as DBT, can be particularly useful to PES clinicians. Clinicians also need to monitor issues of countertransference and the potential to be dismissive to these women with complex, comorbid, and chronic problems and diseases. The role for the use of psychotropic medication in PES cohorts with victimization histories should target acute symptoms. Involving regular providers of these decisions is advised to coordinate care and minimize splitting and risks of polypharmacy. Although the SSRIs are effective in symptom management of disorders related to victimization, patients must be reminded of the side-effect profile, particularly sexual dysfunction and withdrawal and discontinuation syndromes.

Protein domains implicated in intracellular transport and sorting of lactase-phlorizin hydrolase.

The roles of various domains of intestinal lactase-phlorizin hydrolase (pro-LPH) on its folding, dimerization, and polarized sorting are investigated in deletion mutants of the ectodomain fused or not fused with the membrane-anchoring and cytoplasmic domains (MACT). Deletion of 236 amino acids immediately upstream of MACT has no effect on the folding, dimerization, transport competence, or polarized sorting of the mutant LPH1646MACT. By contrast, LPH1646, an anchorless counterpart of LPH1646MACT, is not transported beyond the ER and persists as a mannose-rich monomer during its entire life cycle. The further deletion of 87 amino acids generates a correctly folded but transport-incompetent monomeric LPH1559MACT mutant. The results strongly suggest that dimerization and transport of pro-LPH implicate a stretch of 87 amino acids in the ectodomain between LPH1646MACT and LPH1559MACT. In addition, dimerization of pro-LPH requires at least two further criteria: (i) a correctly folded ectodomain of pro-LPH and (ii) the presence of the transmembrane region. Neither of these requirements alone is sufficient for dimerization. Finally, the sorting of pro-LPH appears to be mediated by signals located between the cleavage site of pro-LPH and the LPH1646MACT mutant.

Drug-induced toxic epidermal necrolysis in children. Report of two cases.

Two cases of toxic epidermal necrolysis occurring in children are presented herein. In both, multiple drugs were administered before the onset of the skin eruption. Cultures for staphylococcus were negative. Histopathologic examination of the first case revealed separations at the dermal-epidermal junction. While more commonly due to staphylococcal exfoliatoxin, a drug must be ruled out as the cause of toxic epidermal necrolysis in children.