Function of the conserved triad residues in the class C beta-lactamase from Citrobacter freundii GN346

The conserved KTG triad in the class C beta-lactamase from Citrobacter freundii GN346 was examined as to its function by means of site-directed mutagenesis. The following conversions were performed; Lys-315 to arginine, alanine or glutamic acid, Thr-316 to valine, and Gly-317 to alanine, proline or isoleucine. The resultant mutant enzymes revealed that a basic amino acid at position 315 and a small uncharged residue at position 317 are essential for the enzyme activity, but a hydroxyl group at residue 316 is not required for the enzymatic catalysis. The kinetic properties of the purified Arg-315 and Val-316 enzymes provided information on the function of these residues.     

Association of the interferon-γ receptor variant (Val14Met) with systemic lupus erythematosus

 Genetic factors seem to play a significant role in susceptibility to systemic lupus erythematosus (SLE). The purpose of this study was to investigate whether the amino acid polymorphism (Val14Met) found within the IFN-γ receptor gene (IFNGR1) plays a prominent role in susceptibility to SLE. We found Val14Met located at the COOH terminal of the signal peptide of the IFN-γ receptor. There was a significant difference in this polymorphism frequency between SLE patients and healthy populations. To clarify whether this amino acid substitution resulted in the alteration of the receptor function, we evaluated the induction of HLA-DR antigen expression on B cells by IFN-γ stimulation. There was also a significant difference in the induction of HLA-DR by IFN-γ stimulation between B cells. Furthermore, an intracellular cytokine assay indicated that the Th1/Th2 balance of Th cells bearing the variant receptor shifted to Th2. The genetic polymorphism found within the IFN-γ receptor gene (Val14Met) may result in a shift to Th2, and this shift may increase susceptibility to SLE. Received: 13 April 1998 / Revised: 30 July 1998     

Mast cell chymase regulates dermal mast cell number in mice

Chymase inhibitor reduced the increase in the number of dermal mast cells in 2,4-dinitrofluorobenzene-induced dermatitis in a dose-dependent manner. Intradermal injection of human chymase to mouse ear significantly increased histamine content, the marker for mast cell number in the skin. These results suggest that chymase released by mast cells may participate in local mast cell accumulation in a positive feedback fashion. Immunohistochemical analysis revealed that the intradermal injection of chymase reduces expression of stem cell factor (SCF) on surface of the skin keratinocytes. In addition, incubation of human keratinocytes with chymase in vitro resulted in release of SCF into the culture medium. Since soluble SCF is thought to regulate mast cell number, the chymase-induced mast cell accumulation may occur via the ability of chymase to process membrane-bound SCF on the epidermal keratinocytes.     

Electrophysiologic characteristics of the Ca-permeable channels, ECaC and CaT, in the kidney

To investigate the molecular mechanism of Ca transport in the kidney, we have isolated Ca-permeable channels, rECaC (rat ECaC) and mCaT (mouse CaT1), from rodent kidney, which are recently reported as Ca-transporting proteins. RT-PCR suggested the presence of CaT1 in medullary tubules. It showed 67% homology with rECaC constructing a family. Whole cellular currents in Chinese hamster ovary (CHO) cells were measured by patch clamp. Expression of both proteins exhibited a similar large cation current, a high permeability to Ca, a time-dependent rapid inactivation, and a "run-down." When the pipet contained EGTA, the inactivation and the run-down did not occur. Addition of db-cAMP activated and following rp-cAMPS recovered the mCaT-induced current significantly, whereas no influence was observed in the rECaC-induced one. We conclude that ECaC and CaT are a molecular family of ion channel with similar characteristics, contributing Ca transport in the kidney.     

Molecular cloning of a novel brain-type Na(+)-dependent inorganic phosphate cotransporter

We have isolated a human cDNA encoding a protein, designated DNPI, that shows 82% amino acid identity and 92% similarity to the human brain-specific Na(+)-dependent inorganic phosphate (Na(+)/P(i)) cotransporter (BNPI), which is localized exclusively to neuron-rich regions. Expression of DNPI mRNA in Xenopus oocytes resulted in a significant increase in Na(+)-dependent P(i) transport, indicating that DNPI is a novel Na(+)/P(i) cotransporter. Northern blot analysis shows that DNPI mRNA is expressed predominantly in brain, where the highest levels are observed in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia-rich region of corpus callosum. In other regions examined, both mRNAs are moderately or highly expressed. These results indicate that BNPI and DNPI, which coordinate Na(+)-dependent P(i) transport in the neuron-rich regions of the brain, may form a new class within the Na(+)/P(i) cotransporter family.     

Aph-1 contributes to the stabilization and trafficking of the gamma-secretase complex through mechanisms involving intermolecular and intramolecular interactions

Gamma-secretase cleaves type I transmembrane proteins, including beta-amyloid precursor protein and Notch, and requires the formation of a protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2 for its activity. Aph-1 is implicated in the stabilization of this complex, although its precise mechanistic role remains unknown. Substitution of the first glycine within the transmembrane GXXXG motif of Aph-1 causes a loss-of-function phenotype in Caenorhabditis elegans. Here, using an untranslated region-targeted RNA interference/rescue strategy in Drosophila Schneider 2 cells, we show that Aph-1 contributes to the assembly of the gamma-secretase complex by multiple mechanisms involving intermolecular and intramolecular interactions depending on or independent of the conserved glycines. Aph-1 binds to nicastrin forming an early subcomplex independent of the conserved glycines within the endoplasmic reticulum. Certain mutations in the conserved GXXXG motif affect the interaction of the Aph-1.nicastrin subcomplex with presenilin that mediates trafficking of the presenilin.Aph-1.nicastrin tripartite complex to the Golgi. The same mutations decrease the stability of Aph-1 polypeptides themselves, possibly by affecting intramolecular associations through the transmembrane domains. Our data suggest that the proper assembly of the Aph-1.nicastrin subcomplex with presenilin is the prerequisite for the trafficking as well as the enzymatic activity of the gamma-secretase complex and that Aph-1 functions as a stabilizing scaffold in the assembly of this complex.     

Acute Graft-Versus-Host Disease of the Kidney in Allogeneic Rat Bone Marrow Transplantation

Allogeneic hematopoietic cell or bone marrow transplantation (BMT) causes graft-versus-host-disease (GVHD). However, the involvement of the kidney in acute GVHD is not well-understood. Acute GVHD was induced in Lewis rats (RT1^l) by transplantation of Dark Agouti (DA) rat (RT1^a) bone marrow cells (6.0×10⁷ cells) without immunosuppression after lethal irradiation (10 Gy). We examined the impact of acute GVHD on the kidney in allogeneic BMT rats and compared them with those in Lewis-to-Lewis syngeneic BMT control and non-BMT control rats. In syngeneic BMT and non-BMT control rats, acute GVHD did not develop by day 28. In allogeneic BMT rats, severe acute GVHD developed at 21–28 days after BMT in the skin, intestine, and liver with decreased body weight (>20%), skin rush, diarrhea, and liver dysfunction. In the kidney, infiltration of donor-type leukocytes was by day 28. Mild inflammation characterized by infiltration of CD3+ T-cells, including CD8+ T-cells and CD4+ T-cells, and CD68+ macrophages to the interstitium around the small arteries was noted. During moderate to severe inflammation, these infiltrating cells expanded into the peritubular interstitium with peritubular capillaritis, tubulitis, acute glomerulitis, and endarteritis. Renal dysfunction also developed, and the serum blood urea nitrogen (33.9±4.7 mg/dL) and urinary N-acetyl-β-D-glucosaminidase (NAG: 31.5±15.5 U/L) levels increased. No immunoglobulin and complement deposition was detected in the kidney. In conclusion, the kidney was a primary target organ of acute GVHD after BMT. Acute GVHD of the kidney was characterized by increased levels of urinary NAG and cell-mediated injury to the renal microvasculature and renal tubules.     

Scavenger receptor class B type I is expressed in cultured keratinocytes and epidermis. Regulation in response to changes in cholesterol homeostasis and barrier requirements.

Cholesterol is a key lipid in the stratum corneum, where it is critical for permeability barrier homeostasis. The epidermis is an active site of cholesterol synthesis, but inhibition of epidermal cholesterol synthesis with topically applied statins only modestly affects epidermal permeability barrier function, suggesting a possible compensatory role for extraepidermal cholesterol. Scavenger receptor class B type I (SR-BI) is a recently described cell surface receptor for high density lipoproteins (HDL) that mediates the selective uptake of cholesterol esters from circulating HDL. In the present study, we demonstrate that SR-BI is present in cultured human keratinocytes and that calcium-induced differentiation markedly decreases SR-BI levels. Additionally, the cell association of [(3)H]cholesterol-labeled HDL decreased in differentiated versus undifferentiated keratinocytes. Furthermore, the inhibition of cholesterol synthesis with simvastatin resulted in a 3-4-fold increase in both SR-BI mRNA and protein levels, whereas conversely, addition of 25-hydroxycholesterol suppressed SR-BI levels by approximately 50%. SR-BI mRNA is also expressed in murine epidermis, increasing by 50% in parallel with cholesterol requirements following acute barrier disruption. Because the increase is completely blocked by occlusion with a vapor-impermeable membrane, changes in epidermal SR-BI expression are regulated specifically by barrier requirements. Lastly, using immunofluorescence we demonstrated that SR-BI is present in human epidermis predominantly in the basal layer and increases following barrier disruption. In summary, the present study demonstrates first that SR-BI is expressed in keratinocytes and regulated by cellular cholesterol requirements, suggesting that it plays a role in keratinocyte cholesterol homeostasis. Second, the increase in SR-BI following barrier disruption suggests that SR-BI expression increases to facilitate cholesterol uptake leading to barrier restoration.     

The channel-lining 6′ amino acid in the second membrane-spanning region of ionotropic GABA receptors has more profound effects on 4′-ethynyl-4-<Emphasis Type="Italic">n</Emphasis>-propylbicycloorthobenzoate binding than the 2′ amino acid

The noncompetitive antagonist of ionotropic γ-aminobutyric acid (GABA) receptors 4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) is a useful tool to probe the antagonist-binding site. In the present study, four mutants of the human GABA_A receptor β3 subunit were stably expressed in S2 cells and examined for their abilities to bind [³H]EBOB to identify the binding site of EBOB. The homo-oligomeric β3 GABA receptor was used as a housefly GABA receptor model, as the β3 subunit has a high sequence similarity with the housefly Rdl subunit in the second membrane-spanning (M2) region. The A274S mutation at the -1′ position in the M2 region had no effect on [³H]EBOB binding. The A277S mutation at the 2′ position led to a decrease in the affinity of EBOB for the GABA receptor. The T281V mutant at the 6′ position and the A277S/T281V double mutant completely abolished the binding ability. A β3 GABA receptor homology model predicts these interactions between the receptor and EBOB. These results suggest that EBOB interacts with threonine 281 and alanine 277, and that threonine 281 plays a more critical role in interacting with EBOB than alanine 277.