Enhanced Expression of Human Platelet-Derived Growth factor in \u3cem\u3ePichia Pastoris\u3c/em\u3e

Enhanced yields of the platelet-derived growth factor (PDGF) B-chain are obtained using the Pichia pastoris yeast system. Yields of both wild-type and mutant human proteins are enhanced when the yeast is transformed with a vector containing the yeast mating factor promoter fused to a sequence encoding the mature protein. The secreted wild-type protein is indistinguishable from mature 29-32 kDa protein isolated from human and Chinese hamster ovary (CHO) cells. An RKK→EEE mutant exhibited reduced association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK→EEE mutants of either the A- or B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor binding activities, each mutant retained full mitogenic activity when applied to cultured Swiss 3T3 cells. Circular dichroism spectrophotometric analysis of the RKK→EEE mutant revealed a secondary structure indistinguishable from the wild type. Yields of PDGF protein are increased many fold and in less time over previous methods

Regulation of rat mammary gene expression by extracellular matrix components

In the mammary gland the induction and maintenance of differentiation are dependent on both lactogenic hormones and the extracellular matrix (ECM). Since mammary epithelial cells differentiate on a basement membrane in vivo we have examined the effects of basement membrane components on the expression of milk protein genes in primary rat mammary cultures. We examined the effects of a basement membrane gel derived from the Englebreth-Holm-Swarm tumor as well as its major component, laminin, on the expression of a group of milk protein genes. We demonstrate that the basement membrane gel induces [alpha]-casein and [alpha]-lactalbumin ([alpha]-LA) accumulation up to 160- and 70-fold, respectively, of that on tissue culture plastic. Laminin, a major component of the basement membrane, also caused significant induction of these same proteins. In order to determine whether these ECM effects occurred at a translational or post-translational level, pulse-chase experiments were performed. These experiments demonstrated that a laminin substratum selectively effects milk protein turnover and secretion. In order to demonstrate whether ECM effects occurred at the level of steady state accumulation of mRNA we performed dot blot and Northern analyses using cloned cDNA probes for [alpha]-, [beta]-, and [gamma]-caseins and [alpha]-LA. These studies demonstrated that ECM components induced [alpha]- and [beta]-caseins up to 10-fold and [alpha]-LA up to 3-fold, with no significant effect on [gamma]-casein. These results demonstrate that milk protein genes are not coordinately regulated by ECM components. Furthermore, since the amount of induction of milk proteins exceeds the amount of induction of mRNAs for these proteins, we conclude that in our system a major effect of ECM components is at the translational and/or post-translational levels. Based on these findings we propose a model in which basement membrane components effect mammary gene expression at multiple levels.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26485/1/0000021.pd

Nm23-H1 Regulates Glucose-Stimulated Insulin Secretion in Pancreatic β-Cells via Arf6-Rac1 Signaling Axis

Background: A growing body of evidence implicates novel roles for nm23-like proteins in the regulation of cellular functions. However, roles of these proteins in islet function and glucose-stimulated insulin secretion (GSIS) remain largely unknown. Methods: siRNA-nm23-H1 and nucleoside diphosphate kinase and histidine kinase-deficient mutants of nm23-H1 (K12Q and H118F) were used to assess roles of nm23-H1 in GSIS. Results: siRNA-mediated knockdown of the expression of nm23-H1 markedly inhibited GSIS in INS-1 832/13 cells. Nm23-H1 knockdown also resulted in significant inhibition of glucose-mediated activation of Arf6, a small G-protein, which has been implicated in GSIS. Expression of K12Q and H118F mutants of nm23-H1 in INS-1 832/13 cells led to inhibition of glucose-induced translocation and membrane association of Rac1, another small G-protein, which is downstream to Arf6 in the signaling events leading to GSIS. A significant inhibition of GSIS was also seen in these cells expressing K12Q and H118F. Conclusions: We conclude that the nm23-H1 activation step is upstream of Arf6 activation in signaling events leading to GSIS. NDP kinase and histidine kinase functions of nm23-H1 are necessary for glucose-induced membrane association of Rac1 and ensuing insulin secretion. We present the first evidence for regulation of GSIS by nm23-H1 in pancreatic β-cells

A study of some aspects of a mild wear process

http://www.archive.org/details/studyofsomeaspec00boydU.S. Navy (U.S.N.) authors.U.S. Coast Guard (U.S.C.G.) author

Pigmented Epithelioid Melanocytoma (PEM)/Animal Type Melanoma (ATM): Quest for an Origin. Report of One Unusual Case Indicating Follicular Origin and Another Arising in an Intradermal Nevus

Pigmented epithelioid melanocytoma (PEM) is a tumor encompassing epithelioid blue nevus of Carney complex (EBN of CNC) and was previously termed animal-type melanoma. Histologically PEMs are heavily pigmented spindled and epithelioid dermal melanocytic tumors with infiltrative borders, however, their origin remains unclear. Stem cells for the epidermis and hair follicle are located in the bulge area of the hair follicle with the potential to differentiate into multiple lineages. Multiple cutaneous carcinomas, including follicular cutaneous squamous cell carcinoma (FSCC), are thought to arise from stem cells in the follicular bulge. We present two cases of PEM/ATM in a 63 year-old male on the scalp with follicular origin and a 72 year-old female on the upper back arising in an intradermal nevus. Biopsy of both cases revealed a proliferation of heavily pigmented dermal nests of melanocytes with atypia. The Case 1 tumor was in continuation with the outer root sheath of the hair follicle in the bulge region. Case 2 arose in an intradermal melanocytic nevus. Rare mitotic figures, including atypical mitotic figures, were identified in both cases. We present two cases of PEM, with histologic evidence suggesting two origins: one from the follicular bulb and one from an intradermal nevus

The HGF/SF Mouse Model of UV-Induced Melanoma as an In Vivo Sensor for Metastasis-Regulating Gene

Cutaneous malignant melanoma is an aggressive and potentially lethal form of skin cancer, particularly in its advanced and therapy-resistant stages, and the need for novel therapeutics and prognostic tools is acute. Incidence of melanoma has steadily increased over the past few decades, with exposure to the genome-damaging effects of ultraviolet radiation (UVR) well-recognized as a primary cause. A number of genetically-engineered mouse models (GEMMs) have been created that exhibit high incidence of spontaneous and induced forms of melanoma, and a select subset recapitulates its progression to aggressive and metastatic forms. These GEMMs hold considerable promise for providing insights into advanced stages of melanoma, such as potential therapeutic targets and prognostic markers, and as in vivo systems for testing of novel therapies. In this review, we summarize how the HGF/SF transgenic mouse has been used to reveal metastasis-regulating activity of four different genes (CDK4R24C, survivin and NME1/NME2) in the context of UV-induced melanoma. We also discuss how these models can potentially yield new strategies for clinical management of melanoma in its most aggressive forms