IL-1 and tumor necrosis factor. Similarities and differences in stimulation of expression of alternative pathway of complement and IFN-beta 2/IL-6 genes in human fibroblasts

IL-1 and TNF induced concentration-related increases in the synthesis of factor B, C3, and IFN-beta 2/IL-6 in human skin fibroblasts. Effects of both stimuli were apparent with concentrations as low as 0.1 ng/ml and maximal responses were observed between 1 and 10 ng/ml; only for IL-1 induction of IFN-beta 2/IL-6 was there a further increase in response up to 100 ng/ml. For factor B and C3, maximal increases induced by IL-1 and TNF were similar: 119- and 109-fold for factor B and 15-fold and 11-fold for C3, respectively. Although both IL-1 and TNF increase synthesis of factor B and C3 in hepatocytes, the increases observed in fibroblasts were approximately 50- and 8-fold more for factor B and C3, respectively. Neither protein synthesis nor mRNA for IFN-beta 2/IL-6 was present in HepG2 cells either before or after stimulation with IL-1 or TNF. In contrast to the similarities between the effects of IL-1 and TNF on synthesis of factor B, C3, and IFN-beta 2/IL-6, only TNF increased synthesis of factor H. Because TNF induces membrane IL-1 in fibroblasts, it is possible to speculate that the effects of TNF on fibroblasts are due to induction of IL-1. An autocrine action of TNF through IL-1 is possible for TNF-induced synthesis of IFN-beta 2/IL-6, but the effects of TNF on synthesis of factor B, C3, and factor H indicated that TNF has effects on fibroblasts separate from IL-1. The effects of IL-1 and TNF on the synthesis of factor B and C3 in fibroblasts may be a part of an acute phase response occurring at a local level. However, the large responses in synthesis of factor B and C3 to IL-1 and TNF may suggest that factor B and C3 have a role, as yet undescribed, in tissues in addition to the role these proteins are known to play in inflammation.     

Tyrosinase gene mutations associated with type IB ("yellow") oculocutaneous albinism

We have identified three different tyrosinase gene mutant alleles in four unrelated patients with type IB ("yellow") oculocutaneous albinism (OCA) and thus have demonstrated that type IB OCA is allelic to type IA (tyrosinase negative) OCA. In an inbred Amish kindred, type IB OCA results from homozygosity for a Pro----Leu substitution at codon 406. In the second family, type IB OCA results from compound heterozygosity for a type IA OCA allele (codon 81 Pro----Leu) and a novel type IB allele (codon 275 Val----Phe). In the third patient, type IB OCA results from compound heterozygosity for the same type IB allele (codon 275 Val----Phe) and a novel type IB OCA allele. In a fourth patient, type IB OCA results from compound heterozygosity for the codon 81 type IA OCA allele and a type IB allele that contains no identifiable abnormalities; dysfunction of this type IB allele apparently results from a mutation either well within one of the large introns or at some distance from the tyrosinase gene. In vitro expression of the Amish type IB allele in nonpigmented HeLa cells demonstrates that the Pro----Leu substitution at codon 406 greatly reduces but does not abolish tyrosinase enzymatic activity, a finding consistent with the clinical phenotype.     

Homozygous tyrosinase gene mutation in an American black with tyrosinase-negative (type IA) oculocutaneous albinism

We have identified a tyrosinase gene mutation in an American black with classic, tyrosinase-negative oculocutaneous albinism. This mutation results in an amino acid substitution (Cys----Arg) at codon 89 of the tyrosinase polypeptide. The proband is homozygous for the substitution, suggesting that this mutation may be frequently associated with tyrosinase-negative oculocutaneous albinism in blacks.     

Organization and nucleotide sequences of the human tyrosinase gene and a truncated tyrosinase-related segment

We have isolated and sequenced the gene encoding human tyrosinase, the key enzyme in pigment biosynthesis. The human tyrosinase gene contains five exons and spans more than 50 kb of DNA on chromosome segment 11q14----q21. We have also isolated a second segment in the human genome that is closely related to tyrosinase. The tyrosinase-related segment, located on 11p11.2----cen, contains only exons 4 and 5 plus adjacent noncoding regions. This segment is present in all human ethnic groups analyzed, and the noncoding nucleotide sequences shared by the 11q tyrosinase gene and the 11p tyrosinase-related segment differ by only 2.6%. This suggests that this segment of the tyrosinase gene was duplicated approximately 24 million years ago.     

Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

Role of Herpes Simplex Virus VP11/12 Tyrosine-Based Motifs in Binding and Activation of the Src Family Kinase Lck and Recruitment of p85, Grb2, and Shc

Previous studies have shown that the abundant herpes simplex virus 1 (HSV-1) tegument protein VP11/12, encoded by gene UL46, stimulates phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling: it binds the Src family kinase (SFK) Lck, is tyrosine phosphorylated, recruits the p85 subunit of PI3-kinase, and is essential for the activation of Akt during HSV-1 infection. The C-terminal region of VP11/12 contains tyrosine-based motifs predicted to bind the SH2 domains of SFKs (YETV and YEEI), p85 (YTHM), and Grb2 (YENV) and the phosphotyrosine-binding (PTB) domain of Shc (NPLY). We inactivated each of these motifs in the context of the intact viral genome and examined effects on binding and activation of Lck and recruitment of p85, Grb2, and Shc. Inactivating the p85, Grb2, or Shc motif reduced (p85) or eliminated (Grb2 and Shc) the interaction with the cognate signaling molecule without greatly affecting the other interactions or activation of Lck. Inactivating either SFK motif had only a minor effect on Lck binding and little or no effect on recruitment of p85, Grb2, or Shc. In contrast, inactivation of both SFK motifs severely reduced Lck binding and activation and tyrosine phosphorylation of VP11/12 and reduced (p85) or eliminated (Grb2 and Shc) binding of other signaling proteins. Overall, these data demonstrate the key redundant roles of the VP11/12 SFK-binding motifs in the recruitment and activation of SFKs and indicate that activated SFKs then lead (directly or indirectly) to phosphorylation of the additional motifs involved in recruiting p85, Grb2, and Shc. Thus, VP11/12 appears to mimic an activated growth factor receptor.     

Sra-1 interacts with Kette and Wasp and is required for neuronal and bristle development in Drosophila

Regulation of growth cone and cell motility involves the coordinated control of F-actin dynamics. An important regulator of F-actin formation is the Arp2/3 complex, which in turn is activated by Wasp and Wave. A complex comprising Kette/Nap1, Sra-1/Pir121/CYFIP, Abi and HSPC300 modulates the activity of Wave and Wasp. We present the characterization of Drosophila Sra-1 (specifically Rac1-associated protein 1). sra-1 and kette are spatially and temporally co-expressed, and both encoded proteins interact in vivo. During late embryonic and larval development, the Sra-1 protein is found in the neuropile. Outgrowing photoreceptor neurons express high levels of Sra-1 also in growth cones. Expression of double stranded sra-1 RNA in photoreceptor neurons leads to a stalling of axonal growth. Following knockdown of sra-1 function in motoneurons, we noted abnormal neuromuscular junctions similar to what we determined for hypomorphic kette mutations. Similar mutant phenotypes were induced after expression of membrane-bound Sra-1 that lacks the Kette-binding domain, suggesting that sra-1 function is mediated through kette. Furthermore, we could show that both proteins stabilize each other and directly control the regulation of the F-actin cytoskeleton in a Wasp-dependent manner.     

Monolayer formation of human osteoblastic cells on vertically aligned multiwalled carbon nanotube scaffolds

Monolayer formation of SaOS‐2 (human osteoblast‐like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non‐toxicity and the flat spreading with monolayer formation of the SaOs‐2 on VACNT scaffolds surface indicate that they can be used for biomedical applications.