Analysis of the structural specificity of the lactose permease toward sugars

The sugar specificity properties of the lactose permease were investigated. Free galactose was shown to competitively inhibit the lactose permease yielding a Ki value of 7.4 mM. This value was severalfold higher than the observed Km for lactose (1.3 mM). A variety of other monosaccharides also showed significant inhibition of lactose transport. With regard to -OH groups along the galactose ring it appears that the relative importance is OH-3 greater than OH-4 greater than OH-6 greater than OH-2 greater than OH-1. In general, galactosides with alpha-linkages exhibited significantly higher affinities compared with their beta-linked counterparts. An optimal size for the aglycone portion of the galactoside was reached with aglycones containing hexose residues or a benzene ring. The preferred size of the aglycone appears to be hexose, benzene ring greater than methyl group greater than no aglycone much greater than disaccharide greater than trisaccharide. However, neither the specific structure of the aglycone nor its relative hydrophobicity appeared to be important factors in permease recognition. For example, the hydrophobic beta-nitrophenyl-galactosides had lower affinities compared with lactose (a beta-galactoside), whereas the alpha-nitrophenylgalactosides generally had higher affinities compared with melibiose (an alpha-galactoside). In addition, no consistent preference was seen when considering the location of the nitro group on the benzene ring. From this work, a model is presented which depicts the binding of galactosides to the lactose permease.     

Retrovirus-mediated immunosuppression. II. FeLV-UV alters in vitro murine T lymphocyte behavior by reversibly impairing lymphokine secretion

Murine splenocytes and cloned murine T cells were used to study the in vitro immunosuppressive effects of UV-inactivated feline leukemia virus (FeLV-UV) on lymphokine secretion. FeLV-UV can significantly depress the accumulation of IL 2 in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive helper T cell clone B6D/2-2m plus Con A. Inhibition of lymphokine accumulation in these cultures could not be attributed to absorption or inactivation of IL 2 by the FeLV-UV or to the FeLV-UV-induced production of substances which interfere with the IL 2 bioassay. Thus, FeLV-UV appears to block production and/or secretion of IL 2 by a direct inhibitory effect on IL 2-secreting murine T lymphocytes. Additional studies indicate that FeLV-UV impairs IL 2 production only if added very soon after lymphocyte contact with lymphokine-inducing agents and that IL 2 secretion resumes when FeLV-UV is removed from the culture. FeLV-UV also impairs accumulation of MAF (interferon-gamma?) in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive cytotoxic T lymphocyte clone B6D/2-7c plus Con A. The latter observation again suggests that FeLV-UV impairs lymphokine secretion by a direct effect on lymphokine-producing T lymphocytes. Furthermore, it suggests that FeLV-UV does not selectively impair production of IL 2 nor does it have selective inhibitory effects on helper T cells. Rather, FeLV-UV appears to have a general inhibitory effect on lymphokine production by T lymphocytes. Finally, concentrations of FeLV-UV which suppress MAF production by the CTL clone have little influence on the cytolysis mediated by the same cloned T cell population. Thus, the immunosuppressive influence of FeLV-UV is selective for phenomena associated with induction of new T lymphocyte functions, such as lymphokine secretion, and spares other immune functions already expressed by the same cells.     

Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

Why cellular stress suppresses adipogenesis in skeletal tissue,but is ineffective in adipose tissue: Control of mesenchymal cell differentiation via integrin binding sites in extracellular matrices

This Perspective addresses one of the major puzzles of adipogenesis in adipose tissue, namely its resistance to cellular stress. It introduces a concept of “density” of integrin binding sites in extracellular matrix, proposes a cellular signaling explanation for the observed effects of matrix elasticity and of cell shape on mesenchymal stem cell differentiation, and discusses how specialized integrin binding sites in collagen IV-containing matrices guard two pivotal physiological and evolutionary processes: stress-resistant adipogenesis in adipose tissues and preservation of pluripotency of mesenchymal stem-like cells in their storage niches. Finally, it proposes strategies to suppress adipogenesis in adipose tissues.     

Two-Dimensional Nanoscale Structural and Functional Imaging in Individual Collagen Type I Fibrils

The piezoelectric properties of single collagen type I fibrils in fascia were imaged with sub-20 nm spatial resolution using piezoresponse force microscopy. A detailed analysis of the piezoresponse force microscopy signal in controlled tip-fibril geometry revealed shear piezoelectricity parallel to the fibril axis. The direction of the displacement is preserved along the whole fiber length and is independent of the fiber conformation. It is shown that individual fibrils within bundles in skeletal muscle fascia can have opposite polar orientations and are organized into domains, i.e., groups of several fibers having the same polar orientation. We were also able to detect piezoelectric activity of collagen fibrils in the high-frequency range up to 200 kHz, suggesting that the mechanical response time of biomolecules to electrical stimuli can be ∼5 μs.