Merck Frosst Award Lecture 1998. Molecular dissection of the human multidrug resistance P-glycoprotein.

The human multidrug resistance P-glycoprotein is an ATP-dependent drug pump that extrudes a broad range of cytotoxic agents from the cell. Its physiological role may be to protect the body from endogenous and exogenous cytotoxic agents. The protein has clinical importance because it contributes to the phenomenon of multidrug resistance during chemotherapy. In this review, we discuss some of the results obtained by using molecular biology and protein chemistry techniques for studying this important and intriguing protein.     

The effect of Gla-containing proteins on the precipitation of insoluble salts

The precipitation of insoluble salts containing divalent metal ions is inhibited by Gla-containing proteins of various origin. In this paper we demonstrate that: Gla-residues are required for the inhibitory activity; the inhibition is effected by a protein which in vivo is bound to calcified tissue (osteocalcin) as well as by proteins occurring in blood plasma (factor X) and urine (the urinary Gla-protein); The inhibitor concentration required for 50% precipitation-inhibition varied slightly from one salt to the other, but no marked differences were observed between the effects of the various Gla-containing proteins used; Precipitation-inhibition occurred in all phosphates (Be, Ca, Mn and Zn) and in all calcium salts (phosphate, oxalate and carbonate) tested.     

Streptokinase therapy of myocardial infarct dependent on the severity of the disease]

A critical analysis of 14 clinical studies published since 1963 on the fibrinolytic treatment of acute cardiac infarct with streptokinase or urokinase shows that only half of them corresponds to the demands of a controlled clinical trial of therapy. The degree of seriousness of the infarct groups described in the different papers shows considerable fluctuations. The findings on letality are not uniform, there is a distinctive trend towards a reduced letality under streptokinase. It was only in recent studies that the dependence of the results of treatment on the seriousness of the disease were checked by retrospective stratification (Australian study) or by prospective stratification (European co-operative study). According to the findings of the European co-operative study those patients who are selected for the purpose of examination and affected with an infarct of medium degree of seriousness being not older than 12 hours have a significantly reduced six months letality under streptokinase on the conditions of this study.     

Effect of feed deprivation on hepatic membrane and lipoprotein fluidity and binding of lipoproteins to hepatic membranes in the chick (Gallus domesticus)

1. Male chicks were deprived of feed for 48 hr to study the effect of metabolic stress on hepatic membrane and lipoprotein fluidity and binding of radioiodinated lipoproteins to hepatic membranes. 2. Plasma levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) were markedly and slightly elevated, respectively. 3. There was a reduction in lipoprotein and hepatic membrane fluidity. 4. Binding of [125I]LDL, but not [125I]HDL, to hepatic membranes was decreased. 5. It is suggested that a reduction in the fluidity of LDL and/or hepatic membranes impedes LDL catabolism in vivo.     

Fine structure of lymphoid cells in the nasal skin of primates.

Electron microscopy of the nasal skin of primates shows that lymphoid cells are normally present in this region. No degeneration or mitosis of these cells or their transformation into other cell types was seen. These intraepidermal lymphocytes contained cytoplasmic fibrils. There was no evidence that they were phagocytic in the epidermis. The present study suggests that these lymphoid cells migrate into the lamina propria but not into the epidermis.     

Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S–O bond cleavage) or carbon (C–O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S–O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C–O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (k_cat/K_M = 4.8 × 10³ s^? 1 M^? 1) as well as arylsulfate 4-nitrophenyl sulfate (k_cat/K_M = 12 s^? 1 M^? 1). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although > 70% of the amino acids between protomers align structurally (RMSDs 1.79–1.99 Å), the oligomeric structures show distinctly different packing and protomer–protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H₂¹⁸O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S–O cleavage in alkyl sulfate esters with extreme catalytic proficiency.     

Platelet proteomics: from discovery to diagnosis

Introduction: Platelets are the smallest cells within the circulating blood with key roles in physiological hemostasis and pathological thrombosis regulated by the onset of activating/inhibiting processes via receptor responses and signaling cascades.

Areas covered: Proteomics as well as genomic approaches have been fundamental in identifying and quantifying potential targets for future diagnostic strategies in the prevention of bleeding and thrombosis, and uncovering the complexity of platelet functions in health and disease. In this article, we provide a critical overview on current functional tests used in diagnostics and the future perspectives for platelet proteomics in clinical applications.

Expert commentary: Proteomics represents a valuable tool for the identification of patients with diverse platelet associated defects. In-depth validation of identified biomarkers, e.g. receptors, signaling proteins, post-translational modifications, in large cohorts is decisive for translation into routine clinical diagnostics.     

High levels of polyandry,but limited evidence for multiple paternity,in wild populations of the western rock lobster (Panulirus cygnus)

Polyandry, where multiple mating by females results in the temporal and spatial overlap of ejaculates from two or more males, is taxonomically widespread and occurs in varying frequencies within and among species. In decapods (crabs, lobsters, crayfish, and prawns), rates of polyandry are likely to be variable, but the extent to which patterns of multiple paternity reflect multiple mating, and thus are shaped by postmating processes that bias fertilization toward one or a subset of mated males, is unclear. Here, we use microsatellite markers to examine the frequency of multiple mating (the presence of spermatophores from two or more males) and patterns of paternity in wild populations of western rock lobster (Panulirus cygnus). Our data confirm that >45% of females had attached spermatophores arising from at least two males (i.e., confirming polyandry), but we found very limited evidence for multiple paternity; among 24 clutches sampled in this study, only two arose from fertilizations by two or more males. Single inferred paternal genotypes accounted for all remaining progeny genotypes in each clutch, including several instances when the mother had been shown to mate with two or more males. These findings highlight the need for further work to understand whether polyandry is adaptive and to uncover the mechanisms underlying postmating paternity biases in this system.     

Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes.

The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mol. Cell. Biol. 16:1896-1908, 1996), where it colocalizes with structures resembling focal complexes. We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.     

Cystic Fibrosis: Channel, Catalytic, and Folding Properties of the CFTR Protein

The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large supeifamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bi-directional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.