Ionic strength effect on the thermal unfolding of alpha-spectrin peptides

In previous work, we have shown that the ionic strength-mediated differences found for the hydrodynamic dimensions of the human erythrocyte spectrin are not caused by secondary structural changes, but are caused more probably by subtle changes in tertiary interactions (LaBrake, C. C., Wang, L., Keiderling, T. A., and Fung, L. W.-M. (1993) Biochemistry 32, 10296-10302.). The substructure of spectrin has been suggested to be composed largely of triple alpha-helical bundle structural domains in tandem. In the present study, we used fluorescence and circular dichroism methods to study ionic strength effects on intact spectrin dimers and on recombinant peptides of spectrin domains of different lengths. We observed little ionic strength effect on the thermal unfolding temperature, Tm, values in these systems. However, we found that ionic strength-induced cooperativity in the unfolding processes was similar for the spectrin dimer and for peptides with two or three domains, as measured by entropy changes (DeltaSm). Although single-domain peptides exhibited rather variable DeltaSm values, depending on the specific domain, they showed little salt effects on the DeltaSm values themselves. This suggests that spectrin undergoes subtle ionic strength-induced conformational changes, probably near the interdomain regions of the molecule. These conformational changes may be responsible for the observed hydrodynamic and unfolding properties in intact spectrin under different ionic strength conditions. We suggest that recombinant peptides of various lengths may serve as models for studying the structural flexibility in spectrin.     

Parental antecedents of adult codependency

Two aspects of codependency were investigated among 442 undergraduates. First, parental antecedents were examined by subjects completing measures of codependency, perceived parental dysfunctions (compulsivity, chemical dependency, and codependency), and parental styles (coercion, control, and non-nurturance). As expected, correlations between adult codependency and parental coercion, control, non-nurturance, and maternal compulsivity were significant. However, correlations between codependency and parental chemical dependency were not significant. A multiple regression analysis identified parental codependency and maternal coercion as significant predictors of subject codependency. To examine the second aspect of codependency, which assumes that codependency was identified over 40 years ago by Karen Horney, subjects completed a loss of self measure which correlated highly with codependency.     

Over one-half billion years of head conservation? Expression of an ems class gene in Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa)

We report the isolation of an empty spiracles class homeodomain-containing gene, Cn-ems, from the hydrozoan Hydractinia symbiolongicarpus, the first gene of this class characterized in a lower metazoan. Cn-ems was found to be expressed in the head of gastrozooids, specifically in endodermal epithelial cells of the taeniolae of the hypostome. Cn-ems is not expressed in gonozooids, which lack taeniolae. Experimental conversion of the posterior region of the planula larva into head structures up-regulates expression of the gene. These findings establish that the association of ems-class genes with head structures preceded the evolution of bilateral symmetry.     

Genetic dissection of a mammalian replicator in the human beta-globin locus

The timing and localization of DNA replication initiation in mammalian cells are heritable traits, but it is not known whether initiation requires specific DNA sequences. A site-specific recombination strategy was used to show that DNA sequences previously identified as replication initiation sites could initiate replication when transferred to new chromosomal locations. An 8-kilobase DNA sequence encompassing the origin of DNA replication in the human beta-globin locus initiated replication in the simian genome. Specific deletions within the globin origin did not initiate replication in these chromosomal sites. These data suggest that initiation of DNA replication in mammalian cells requires specific sequence information and extend the replicon hypothesis to higher eukaryotes.     

Reactive arthritis in patients attending an urban sexually transmitted diseases clinic

OBJECTIVE: To assess the prevalence, clinical manifestations, associated genital infections, and HLA associations of reactive arthritis (ReA) among patients attending an urban sexually transmitted diseases (STD) clinic. METHODS: Using a standardized questionnaire, 271 consecutive adults, primarily black, with possible or proven Chlamydia trachomatis genital infection were screened for symptoms of ReA. A followup questionnaire was administered 6 weeks later by mail. Patients who reported at least 1 symptom were evaluated by a rheumatologist. HLA-B typing was performed on patients with objective ReA features. RESULTS: Nine of 217 patients (4.1%) with genital infection/inflammation had objective ReA features. Chlamydial or nongonococcal STD syndromes were diagnosed in 8 of these 9 patients (88%). Genital infection/inflammation was asymptomatic in 78% of patients with ReA features. HLA-B27 or other B7-cross-reactive group antigens were not associated with the occurrence of ReA. CONCLUSION: Nongonococcal genital infections, often asymptomatic, can trigger a relatively mild ReA in a larger number of exposed patients than previously thought, irrespective of the individual's HLA status.     

Measuring the progression of foreign-body reaction to silicone implants using in vivo MR microscopy

We used in vivo magnetic resonance (MR) microscopy to follow the growth of fibrous capsule as a foreign body reaction to silicone implants in rats. Anesthetized rats were imaged 1, 7, 14, and 28 days after silicone-coated MR imaging coils were sutured to their neck muscles. On the twenty-eighth day, rats were sacrificed and coils and adjacent tissues were removed en bloc and fixed in formalin, reimaged with MR, and sectioned for conventional histology. Three-dimensional (3-D) spin-echo [3DFT] acquisition gave in-plane resolution of 32 x 32 microns in vivo and 16 x 16 microns ex vivo. All MR images showed a diffuse band of elevated signal intensity between the silicone of the coil and adjacent tissue. The border of the hyperintense band was thin and not well defined at seven days post-implantation. From 7-28 days, the band showed relatively homogeneous signal intensity and its thickness increased 44% on the rectus muscle side and 78% on the subcutaneous side. The capsule thickness determined either by MR in vivo and ex vivo microscopy or conventional histology was not significantly different, and there was a significant correlation between thickness measurements among those methods. MR in vivo microscopy provides sufficient resolution and spatial information to serially evaluate the growth of the foreign body fibrous capsule over time, thus achieving greater accuracy and consistency in measurements.     

Selective inhibition of cyclic AMP-dependent protein kinase by isoquinoline derivatives

A large series of isoquinoline derivatives was synthesised including derivatives of isoquinoline, isoquinolino[3,4-c]furazan, 1,2-dihydro-1-oxoisoquinoline, 6-oxopyrimido[1,2-d]isoquinoline, benzo[c][1,8]-naphthyridine, pyrazino[2,3-c]isoquinoline and benzimidazo[2,1-a]isoquinoline as well as further structurally related isoquinoline derivatives and pyrido-2,3-furazans. Representatives of all of these classes of isoquinolines are potent and selective inhibitors of the cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK) from rat liver. The most effective cAK inhibitors are a series of 1,3-di-substituted and 1,3,4-tri-substituted isoquinolines (IC50 values 30-50 nM) (compounds A1, A2, A3, A4 and A5) and 2-ethylcarboxy-3-amino-5,6-dihydro-6-oxobenzo[c] [1,8]naphthyridine (E1) (IC50 0.08 microM). Compounds A1-A5 inhibit cAK in a fashion that is competitive with respect to ATP as substrate. The isoquinoline inhibitors A1-A5 are ineffective or very poor inhibitors of wheat embryo Ca(2+)-dependent protein kinase (CDPK) and rat brain Ca(2+)-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). E1 is a moderately effective inhibitor of CDPK and PKC (IC50 values 30 and 61 microM, respectively). The bisisoquinoline-1(2H)-one compound B7 inhibits cAK, CDPK, PKC and MLCK (IC50 values 8, 95, 24 and 7 microM, respectively) as does J1 [2-(p-bromophenyl)pyrrolo-[2,3-c]isoquinoline-5(4H)-one] (IC50 values 2, 50, 44 and 7 microM, respectively). The very potent isoquinoline-derived cAK inhibitors found here involve substitution of the N-containing isoquinoline ring system and these inhibitors show high specificity for cAK.     

Toxicity of fotemustine in rat hepatocytes and mechanism-based protection against it

Fotemustine is a relatively novel DNA-alkylating 2-chloroethyl-substituted N-nitrosourea (CENU) drug, clinically used for the treatment of disseminated malignant melanoma in different visceral and non-visceral tissues. Thrombocytopenia has been observed in patients treated with fotemustine and liver and renal toxicities as well. In this study, firstly the metabolism of fotemustine was investigated in vitro and secondly the undesired cytotoxicity of fotemustine as well as different ways of protection against it. In rat hepatocytes, chosen as a model system, fotemustine was shown to cause lactate dehydrogenase (LDH) leakage, glutathione (GSH) depletion, GSSG-formation and lipid peroxidation (LPO). A reactive metabolite, DEP-isocyanate, is most likely responsible for these undesired cytotoxic effects. Based on the observed cytotoxicity mechanisms, chemoprotection with several sulfhydryl-containing nucleophiles and antioxidants was investigated. The sulfhydryl nucleophiles; GSH, N-acetyl-L-cysteine (NAC) and glutathione isopropylester (GSH-IP) protected almost completely against fotemustine-induced LDH-leakage and LPO. NAC and GSH protected partly against fotemustine-induced GSH-depletion. The antioxidant, vitamin E protected completely against fotemustine-induced LPO, but only partly against fotemustine-induced LDH-leakage and not against GSH-depletion. Ebselen, a peroxidase-mimetic organoselenium compound, did not show protective effects against the cytotoxicity of fotemustine, possibly because GSH is required for the bioactivation of ebselen. It is concluded that co-administration of sulfhydryl nucleophiles, in particular NAC and GSH-IP, possibly in combination with antioxidants, such as vitamin E, are effective against the toxicity of fotemustine in vitro. It might, therefore, be worthwhile to investigate the cytoprotective potency of these agents against undesired toxicities of fotemustine in vivo as well.     

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.