In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain.

MEKK2 and MEKK3 are mitogen-activated protein kinase kinase kinases (MAP3 kinases) of 70 and 71 kDa respectively that are markedly homologous (94%) in their kinase domains. Both MEKK2 and MEKK3 are able to activate the Jun kinase pathway in vivo. However, following routine immunoprecipitation in Triton X-100, MEKK2 but not MEKK3 is able to effectively phosphorylate both SEK-1 and MEK-1 and to undergo autophosphorylation. Unexpectedly, both MEKK2 and MEKK3 are functional in an in vitro kinase assay when cells are solubilized with the closely related detergent, NP-40. Given the high homology between these kinases, we set out to relate this differential sensitivity to Triton X-100 to differences in primary structure. A set of chimeric molecules were generated and the loss of activity in Triton X-100 mapped to kinase domain II/III and specifically to serine 390 of MEKK3 and valine 384 of MEKK2, residues immediately N-terminal to the active site lysine. Mutation of serine 390 of MEKK3 to a valine (as is found in MEKK2) conferred catalytic activity to MEKK3 in Triton X-100 whereas the reciprocal alteration of valine 384 of MEKK2 to a serine conferred lack of activity in Triton X-100 to MEKK2. Search of the protein database identified only three kinases, MEKK3, Pbs2p and Dd-PKI, with a serine or threonine at this site. The presence of a serine or threonine adjacent to the active site lysine in protein kinases is rare and, in MEKK3, results in detergent instability.

MEK kinase 1 gene disruption alters cell migration and c-Jun NH2-terminal kinase regulation but does not cause a measurable defect in NF-kappa B activation.

MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase that, in addition to regulating the c-Jun NH(2)-terminal kinase (JNK) pathway, is involved in the control of cell motility. MEKK1(-/-) mice are defective in eyelid closure, a TGFalpha-directed process involving the migration of epithelial cells. MEKK1 expression in epithelial cells stimulates lamellipodia formation, a process required for cell movement. In addition, mouse embryo fibroblasts derived from MEKK1(-/-) mice are inhibited in their migration relative to MEKK1(+/+) fibroblasts. MEKK1 is required for JNK but not NF-kappaB activation in response to virus infection, microtubule disruption, and stimulation of embryonic stem cells with lysophosphatidic acid. MEKK1 is not required for TNFalpha or IL-1 regulation of JNK or NF-kappaB activation in macrophages or fibroblasts. Thus, MEKK1 senses microtubule integrity, contributes to the regulation of fibroblast and epithelial cell migration, and is required for activation of JNK but not NF-kappaB in response to selected stress stimuli.

Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.

A 5-week-old male infant presented with severe bacterial infections and poor wound healing, suggesting a neutrophil defect. Neutrophils from this patient exhibited decreased chemotaxis, polarization, azurophilic granule secretion, and superoxide anion (O(2)(-)) production but had normal expression and up-regulation of CD11b. Rac2, which constitutes >96% of the Rac in neutrophils, is a member of the Rho family of GTPases that regulates the actin cytoskeleton and O(2)(-) production. Western blot analysis of lysates from patient neutrophils demonstrated decreased levels of Rac2 protein. Addition of recombinant Rac to extracts of the patient neutrophils reconstituted O(2)(-) production in an in vitro assay system. Molecular analysis identified a point mutation in one allele of the Rac2 gene resulting in the substitution of Asp57 by an Asn (Rac2(D57N)). Asp57 is invariant in all defined GTP-binding proteins. Rac2(D57N) binds GDP but not GTP and inhibits oxidase activation and O(2)(-) production in vitro. These data represent the description of an inhibitory mutation in a member of the Rho family of GTPases associated with a human immunodeficiency syndrome.

Increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and TRAIL.

Chemotherapeutic genotoxins induce apoptosis in epithelial-cell-derived cancer cells. The death receptor ligand TRAIL also induces apoptosis in epithelial-cell-derived cancer cells but generally fails to induce apoptosis in nontransformed cells. We show here that the treatment of four different epithelial cell lines with the topoisomerase II inhibitor etoposide in combination with TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) induces a synergistic apoptotic response. The mechanism of the synergistic effect results from the etoposide-mediated increase in the expression of the death receptors 4 (DR4) and 5 (DR5). Inhibition of NF-kappaB activation by expression of kinase-inactive MEK kinase 1(MEKK1) or dominant-negative IkappaB (DeltaIkappaB) blocked the increase in DR4 and DR5 expression following etoposide treatment. Addition of a soluble decoy DR4 fusion protein (DR4:Fc) to cell cultures reduced the amount of etoposide-induced apoptosis in a dose-dependent manner. The addition of a soluble TNF decoy receptor (TNFR:Fc) was without effect, demonstrating the specificity of DR4 binding ligands in the etoposide-induced apoptosis response. Thus, genotoxin treatment in combination with TRAIL is an effective inducer of epithelial-cell-derived tumor cell apoptosis relative to either treatment alone.

Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation.

Chemotherapeutic drugs that damage DNA kill tumor cells, in part, by inducing the expression of a death receptor such as Fas or its ligand, FasL. Here, we demonstrate that epidermal growth factor (EGF) stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial (HEK293) cells protects these cells from Fas-induced apoptosis. EGF stimulation of epithelial cells also inhibited Fas-induced caspase activation and the proteolysis of signaling proteins downstream of the EGF receptor, Cbl and Akt/protein kinase B (Akt). EGF stimulation of Akt kinase activity blocked Fas-induced apoptosis. Expression of activated Akt in MCF-7 breast adenocarcinoma cells was sufficient to block Fas-mediated apoptosis. Inhibition of EGF-stimulated extracellular signal-regulated kinase (ERK) activity did not affect EGF protection from Fas-mediated apoptosis. The findings indicate that EGF receptor stimulation of epithelial cells has a significant survival function against death receptor-induced apoptosis mediated by Akt.

Postpartum retinal detachment. A case report.

A postpartum progression of preeclampsia ultimately resulted in bilateral serous retinal detachment.

Treatment of cervical pregnancy with methotrexate.

A case of cervical pregnancy was treated successfully with methotrexate. We used serial beta-human chorionic gonadotropin levels, magnetic resonance imaging, and sonography to diagnose the pregnancy and monitor therapy. Hysterectomy was avoided, and the patient's reproductive capability was preserved. Toxicity was limited to transient elevation of liver transaminases.

Neu-Laxova syndrome: a case report.

Neu-Laxova syndrome is a rare form of congenital malformation characterized by intrauterine growth retardation, microcephaly with bizarre facial features, short neck, apparent edema, scaly skin, and perinatal death. Chromosomal analysis in reported cases has revealed a normal karyotype, and an autosomal recessive transmission has been postulated. We present a case of Neu-Laxova syndrome. The pathologic features and the prenatal radiographic appearance are described.

Noncardiogenic pulmonary edema as a rare complication of blood transfusions. A case report.

A woman was treated for leukoagglutinin-induced noncardiogenic pulmonary edema resulting from a blood transfusion.

Hyperimmunoglobulinemia E and pregnancy: a case report.

Hyperimmunoglobulinemia E is characterized by recurrent bacterial sinopulmonary and skin infections from birth or early childhood, with IgE levels at least 10 times greater than the upper limits of normal. The following case describes a young black woman with hyperimmunoglobulinemia E syndrome who had an uneventful pregnancy and delivery. The infant has been diagnosed as suffering from hyperimmunoglobulinemia E syndrome as well.

Endocervical curettage: does it contribute to the management of patients with abnormal cervical cytology?

Routine endocervical curettage has been advocated in the colposcopic evaluation of patients with abnormal cervical cytology. To assess the usefulness of this procedure, we reviewed the records of 518 patients referred to the Colposcopy Clinic with abnormal Pap smears. Data was reviewed retrospectively in 411 patients and collected prospectively in 107 patients. Dysplasia was present in 1.4% of ECC specimens obtained in patients with conclusive colposcopic examinations, and in 25.7% of specimens in patients with inconclusive examinations. Invasive cancer was not detected in any ECC specimen. Eighty patients with inconclusive colposcopic examinations underwent conization of the cervix; in this group, the final pathologic diagnosis was CIN III in 51.2%, microinvasive cancer in 2.5%, and invasive cancer in 1.2%. In patients with conclusive colposcopic examinations, the final pathologic diagnosis was CIN III in 17.2%, and no cases of microinvasive or invasive cancer were present. When the colposcopic examination is conclusive, the incidence of positive ECC is low and may not warrant performing ECC in all patients. When the examination is inconclusive, ECC rarely obviates the need for diagnostic conization as ECC rarely yields a diagnosis of invasive cancer.