A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture

The purpose of this study was to design a synthetic nanofibrillar matrix that more accurately models the porosity and fibrillar geometry of cell attachment surfaces in tissues. The synthetic nanofibrillar matrices are composed of nanofibers prepared by electrospinning a polymer solution of polyamide onto glass coverslips. Scanning electron and atomic force microscopy showed that the nanofibers were organized into fibrillar networks reminiscent of the architecture of basement membrane, a structurally compact form of the extracellular matrix (ECM). NIH 3T3 fibroblasts and normal rat kidney (NRK) cells, when grown on nanofibers in the presence of serum, displayed the morphology and characteristics of their counterparts in vivo. Breast epithelial cells underwent morphogenesis to form multicellular spheroids containing lumens. Hence the synthetic nanofibrillar matrix described herein provides a physically and chemically stable three-dimensional surface for ex vivo growth of cells. Nanofiber-based synthetic matrices could have considerable value for applications in tissue engineering, cell-based therapies, and studies of cell/tissue function and pathology.     

Dual effect of temperature on the human epithelial Na+ channel

The amiloride-sensitive epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption in the distal segments of the nephron, in the colon and in the airways. Its activity is regulated by intracellular and extracellular factors but the mechanisms of this regulation are not yet completely understood. Recently, we have shown that the fast regulation of ENaC by the extracellular [Na⁺], a phenomenon termed self-inhibition, is temperature dependent. In the present study we examined the effects of temperature on the single-channel properties of ENaC. Single-channel recordings from excised patches showed that the channel open probability (P _o, estimated from the number of open channels N·P _o, where N is the total number of channels) increased on average two- to threefold while the single-channel conductance decreased by about half when the temperature of the perfusion solution was lowered from ~30 to ~15 °C. The effects of temperature on the single-channel conductance and P _o explain the changes of the macroscopic current that can be observed upon temperature changes and, in particular, the paradoxical effect of temperature on the current carried by ENaC.     

Genetics of reovirus: the relationship of interference to complementation and reassortment of temperature-sensitive mutants at nonpermissive temperature.

Temperature-sensitive mutants of reovirus type 3 are capable of interfering with the replication of wild-type reovirus type 3. The interfering activity correlated with the ability of pairs of mutants to complement at 39°: Pairs of noninterfering mutants (tsD × tsE) yielded efficient complementation (indexes of 10–50); pairs of interfering mutants (including members of groups ts A, B, G) did not produce significant complementation (indexes ～ 1). The ability of pairs of mutants to reassort at 39° generally followed a similar pattern. Thus interference is an important property of ts mutants of reovirus and needs to be considered when genetic interactions are being studied at 39°.     

Thymic-dependent anti-hapten response in congenitally athymic (nude) mice immunized with DNP-thymosin.

Immunization of congenitally athymic (nu/nu) and adult thymectomized, irradiated bone marrow, reconstituted (TxBm) mice with DNP5-thymosin (dinitrophenylated-bovine thymosin fraction 5) was found to elcit IgM and IgG anti-DNP plaque-forming cells in these animals. Further studies indicated that this response was antigen specific and not due to polyclonal activation. Since the hormonal properties of the thymosin were retained following linkage with hapten and DNP-thymosin was immunogenic in CBA/N and CBA/N female X DBA/2 male)F1 male mice, animals previously shown to have an X-linked inability to respond to thymus-independent antigens, it was concluded that DNP-thymosin functions both as a hormone and as a T-dependent antigen in eliciting an immune response in nu/nu and TxBm mice. Additional support for this conclusion was provided by the demonstration that DNP-thymosin could specifically prime for and elicit an anamnestic response in nu/nu mice. These results indicate that further investigation of the immune activities of DNP-thymosin may provide valuable insight in characterizing the maturation of helper T cells and their subsequent interaction with B cells.     

Bacteremia and urinary tract infection associated with CDC group Vd biovar 2.

A case of urinary tract infection and bacteremia caused by CDC group Vd biovar 2 in a 23-year-old woman with Hodgkin's disease is described. This is the first report of CDC group Vd biovar 2 isolated from a clinical specimen and considered as a pathogen. Detailed antimicrobial susceptibility data are presented.     

Inherited prothrombotic defects in Budd-Chiari syndrome and portal vein thrombosis: a study from North India

We studied 57 patients with Budd-Chiari syndrome (BCS) and 48 with portal vein thrombosis (PVT) for underlying inherited prothrombotic defects such as protein C, protein S, and antithrombin III deficiencies. Genetic mutations for factor V Leiden, prothrombin gene 20210A, and methyltetrahydrofolate reductase (MTHFR) C677T were studied in 29 patients in each group. Inherited prothrombotic defects were detected in 16 (28%) of 57 patients with BCS and 7 (15%) of 48 patients with PVT. Factor V Leiden mutation was the most common prothrombotic defect in BCS (5/29 [17%]) followed by protein C deficiency (7/57 [12%]) and protein S deficiency (4/57 [7%]), whereas in PVT, protein C deficiency was the most common inherited prothrombotic defect (4/48 [8%]) followed by protein S deficiency (2/48 [4%]). The factor V Leiden mutation was detected in only 1 (3%) of 29 cases of PVT. The heterozygous MTHFR C677T mutation was detected in 7 (24%) of 29 patients with BCS and 6 (21%) of 29 patients with PVT. Antithrombin III deficiency, homozygous MTHFR C677T mutation, and prothrombin G20210A mutation were not detected in any patients.     

Abnormalities of cholesterol metabolism in autism spectrum disorders.

Although Smith-Lemli-Opitz Syndrome (SLOS), a genetic condition of impaired cholesterol biosynthesis, is associated with autism [Tierney et al., 2001; Am J Med Genet 98:191-200.], the incidence of SLOS and other sterol disorders among individuals with autism spectrum disorders (ASD) is unknown. This study investigated (1) the incidence of biochemically diagnosed SLOS in blood samples from a cohort of subjects with ASD from families in which more than one individual had ASD and (2) the type and incidence of other sterol disorders in the same group. Using gas chromatography/mass spectrometry, cholesterol, and its precursor sterols were quantified in 100 samples from subjects with ASD obtained from the Autism Genetic Resource Exchange (AGRE) specimen repository. Although no sample had sterol levels consistent with SLOS, 19 samples had total cholesterol levels lower than 100 mg/dl, which is below the 5th centile for children over age 2 years. These findings suggest that, in addition to SLOS, there may be other disorders of sterol metabolism or homeostasis associated with ASD.     

Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts.To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17±4 g hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8±2.3 to 45.1±3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7±89.8 to 2,181±135.4 mmHg/s in the contraction rate, and 713±60.2 to 1,364±137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.