Protein crystal growth and the International Space Station.

Protein structural information plays a key role in understanding biological structure-function relationships and in the development of new pharmaceuticals for both chronic and infectious diseases. The Center for Macromolecular Crystallography (CMC) has devoted considerable effort studying the fundamental processes involved in macromolecular crystal growth both in a 1-g and microgravity environment. Results from experiments performed on more than 35 U.

ATP release mechanisms, ATP receptors and purinergic signalling along the nephron.

1. Autocrine and paracrine signalling along the nephron of the kidney has been a widely held hypothesis for several decades. The lumen of the nephron is an ideal autocrine and paracrine signalling microenvironment.

Review of recent developments in the molecular characterization of recombinant alfa interferons on the 40th anniversary of the discovery of interferon.

Recombinant alfa interferons (IFN-alpha s) are approved worldwide for the treatment of a variety of cancers and diseases of virologic origin. A series of recent advances in the molecular characterization of recombinant IFN-alpha s have allowed the determination of the three-dimensional IFN-alpha 2b structure by high-resolution x-ray crystallography. We review here recent developments in our understanding of the molecular and physicochemical properties of recombinant IFN-alpha, including our current state of knowledge of the IFN-alpha gene family and the multiple species of human leukocyte IFN.

Yeast Sec14p deficient in phosphatidylinositol transfer activity is functional in vivo.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity.

Transcriptional regulation of intercellular adhesion molecule-1 in astrocytes involves NF-kappaB and C/EBP isoforms.

ICAM-1 is an inducible cell surface protein that is involved in cell extravasation into inflamed tissues as well as immune responses. ICAM-1 expression is upregulated by proinflammatory cytokines such as TNF-alpha and IL-1beta in numerous cell types including the astrocyte, which functions as an immune effector cell in the central nervous system (CNS). We investigated the mechanism by which the ICAM-1 gene is transcriptionally regulated in astrocytes in response to TNF-alpha and IL-1beta.

Inhibition of growth and proliferation of EcRG293 cell line expressing high-affinity gonadotropin-releasing hormone (GnRH) receptor under the control of an inducible promoter by GnRH agonist (D-Lys6)GnRH and antagonist (Antide).

The mechanism by which gonadotropin-releasing hormone (GnRH) agonists and antagonists inhibit tumor cell growth and proliferation is controversial. Direct mediation of the antitumor effects through the high-affinity GnRH receptors has been questioned because of the low level of expression of receptors on the tumor cells. We have developed a human kidney embryonic cell line (EcRG293) that expresses high-affinity GnRH receptor under the control of an inducible promoter activated by muristerone A.

Phosphatidylinositol transfer proteins: the long and winding road to physiological function.

Phosphatidylinositol transfer proteins (PITPs) have historically been thought to help execute lipid-sorting events by transporting phospholipid monomers between membrane bilayers. Recent data, however, indicate unanticipated roles for PITPs in the coordination and/or coupling of phospholipid metabolism with vesicle trafficking and the downregulation of signal-transduction reactions. We are only now beginning to appreciate both the identities of PITP-dependent cellular reactions and the intriguing mechanisms by which PITPs execute their functions in eukaryotic cells.

Expression and purification of Streptococcus pneumoniae hyaluronate lyase from Escherichia coli.

Pneumococcal hyaluronate lyase enzyme breaks down hyaluronan of the extracellular matrix of tissues and possibly contributes to the invasion of host tissue and to the penetration of host defenses by this bacterial pathogen. In light of the emergence of increasing numbers of antibiotic-resistant strains, the understanding of the mechanism of action of hyaluronate lyase enzyme may lead to a better understanding of interactions between a host and bacterial pathogens and may contribute to more efficient treatment of bacterial infections. The native Streptococcus pneumoniae hyaluronate lyase enzyme has a molecular mass of 107 kDa but undergoes conversion to smaller enzymatically active forms.

Large-scale preparation of the delta10 form of staphylokinase by in vitro processing of recombinant staphylokinase with purified human plasminogen.

The authors have developed a rapid and convenient method for purification of a low molecular weight form (delta 10) of the bacterial plasminogen activator, staphylokinase. Recombinant staphylokinase is expressed in Escherichia coli, with an amino terminal extension that facilitated purification by immobilized metal-affinity chromatography. Purified staphylokinase is treated with human plasminogen, and the resulting truncated form is purified using a combination of immobilized metal affinity chromatography and hydrophobic interaction chromatography.

Complications of cirrhosis. Why they occur and what to do about them.

Cirrhosis is a chronic disease of the liver in which dense bands of fibrosis enclose regenerative hepatocellular nodules. Clinical and radiologic features of advanced liver disease provide presumptive evidence for the presence of cirrhosis. Major complications are related to the increased hepatic resistance, increased sodium and water retention, and hyperdynamic changes of the circulatory system.

A unique transcription factor for the A alpha fibrinogen gene is related to the mitochondrial single-stranded DNA binding protein P16.

Although stimulation of hepatic cells with interleukin-6 induces the expression of fibrinogen, the molecular basis for this regulation remains largely uncharacterized. A recent examination of the A alpha fibrinogen gene promoter identified a protein, termed the A alpha-core protein, that bound constitutively to the IL-6 response element [Liu, Z. & Fuller, G.

Cation permeability of a cloned rat epithelial amiloride-sensitive Na+ channel.

1. Conductance of heterotrimeric rat epithelial Na+ channels (alpha, beta, gamma-rENaCs) for Li+ and Na+ in planar lipid bilayers was a non-linear function of ion concentration, with a maximum of 30.4 +/- 2.

Molecular structure of the human gonadotropin-releasing hormone receptor gene.

GnRH receptors belong to the family of G protein-coupled receptor proteins and have been localized to the anterior pituitary, brain and reproductive organs as well as many steroid-dependent tumor tissues. Recently, cDNAs for the GnRH receptors of several species including the human have been cloned. To determine the structure of the gene encoding the human GnRH receptor, we isolated the receptor gene clones from the human genomic libraries.

Mutational analyses of differentiation-dependent human papillomavirus type 18 enhancer elements in epithelial raft cultures of neonatal foreskin keratinocytes.

Human papillomaviruses (HPVs) reproduce only in differentiated squamous epithelia. Viral transcription is rather restricted in basal strata but increases dramatically in the spinous cells. Inopportune viral oncoprotein expression in the basal reserve cells can lead to dysplasias and carcinomas.

Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis.

One of the characteristic features of microglia is their rapid activation in response to injury, inflammation, neurodegeneration, infection, and brain tumors. This review focuses on the role of the microglia in multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the central nervous system (CNS), and in the animal model of MS, experimental allergic encephalomyelitis (EAE). Microglial activation in MS and EAE is thought to contribute directly to CNS damage through several mechanisms, including production of proinflammatory cytokines, matrix metalloproteinases, and free radicals.

Coupling chemical energy by the hsp70/tim44 complex to drive protein translocation into mitochondria.

A dynamic complex between the mitochondrial cognate of hsp70 (mthsp70) and the inner membrane protein tim44 couples energy derived from ATP hydrolysis to drive multiple steps in the mitochondrial protein import pathway: (1) The delta psi dependent import step and the mthsp70/tim44 complex cooperate to facilitate the unidirectional transfer of the mitochondrial targeting signal across the inner membrane. (2) The mthsp70/tim44 complex helps to unfold domains on precursors proteins that arrive at the import apparatus in a folded conformation on the cis side of the outer membrane. (3) Completion of import is then driven by the mthsp70/ tim44 complex in a manner that is independent of delta psi.

Insertional and deletional RNA editing in trypanosome mitochondria.

The mitochondrial mRNAs of trypanosomes are often post-transcriptionally modified by an RNA processing event, termed RNA editing, which results in the insertion or deletion of uridylate (U) residues in mRNAs. RNA editing is necessary for the formation of complete coding sequences for several essential mitochondrial proteins. The number and site of U addition and deletion is directed by small guide RNAs (gRNAs).

Regulation of intracellular calcium is closely linked to glucose metabolism in J774 macrophages.

The effects of 2-deoxy-D-glucose (2dGlc) and glucose deprivation were investigated in the J774 murine macrophage-like cell line. 2dGlc addition or glucose deprivation for 4 min led to an inhibition in the transient increase in cytoplasmic free Ca2+ ([Ca2+]i) that otherwise occurs in response to three different agonists: IgG, ATP and platelet activating factor. This inhibition was preceded by a partial release of Ca2+ from intracellular, thapsigargin-sensitive stores.

Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin.

alphaB-Crystallin, originally described as a structural lens protein, is now known to be a member of the small heat shock protein family and is expressed in a number of nonlens tissues. This highly conserved 20 kDa protein aggregates with homologous proteins, including alphaA-crystallin and the small heat shock protein HSP28, to form large heteromeric complexes. Recently, Roquemore et al.

Roles for hsp70 in protein translocation across membranes of organelles.

The family of hsp70 molecular chaperones plays an essential and diverse role in cellular physiology. Hsp70 proteins appear to elicit their effects by interaction with polypeptides that present domains which exhibit non-native conformations at distinct stages during their life in the cell. Work pertaining to the functions of hsp70 proteins in driving protein translocation across membranes is reviewed herein.