The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895–1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis.

SLC13 family of Na⁺-coupled di- and tri-carboxylate/sulfate transporters

The SLC13 family comprises five genes (SLC13A1, SLC13A2, SLC13A3, SLC13A4, and SLC13A5) encoding structurally related multi-spanning transporters (8-13 transmembrane domains) with orthologues found in prokaryotes and eukaryotes. Mammalian SLC13 members mediate the electrogenic Na(+)-coupled anion cotransport at the plasma membrane of epithelial cells (mainly kidney, small intestine, placenta and liver) or cells of the central nervous system. While the two SLC13 cotransporters NaS1 (SLC13A1) and NaS2 (SLC13A4) transport anions such sulfate, selenate and thiosulfate, the three other SLC13 members, NaDC1 (SLC13A2), NaCT (SLC13A5) and NaDC3 (SLC13A3), transport di- and tri-carboxylate Krebs cycle intermediates such as succinate, citrate and α-ketoglutarate. All these transporters play a variety of physiological and pathophysiological roles in the different organs. Thus, the purpose of this review is to summarize the roles of SLC13 members in human physiology and pathophysiology and what the therapeutic perspectives are. We have also described the most recent advances on the structure, expression, function and regulation of SLC13 transporters.

The SLC1 high-affinity glutamate and neutral amino acid transporter family

Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells throughout the body with glutamate for metabolic purposes. The high-affinity glutamate transporters EAAC1 (SLC1A1), GLT1 (SLC1A2), GLAST (SLC1A3), EAAT4 (SLC1A6), and EAAT5 (SLC1A7) mediate the cellular uptake of glutamate by the co-transport of three sodium ions (Na(+)) and one proton (H(+)), with the counter-transport of one potassium ion (K(+)). Thereby, they protect the CNS from glutamate-induced neurotoxicity. Loss of function of glutamate transporters has been implicated in the pathogenesis of several diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. In addition, glutamate transporters play a role in glutamate excitotoxicity following an ischemic stroke, due to reversed glutamate transport. Besides glutamate transporters, the SLC1 family encompasses two transporters of neutral amino acids, ASCT1 (SLC1A4) and ASCT2 (SLC1A5). Both transporters facilitate electroneutral exchange of amino acids in neurons and/or cells of the peripheral tissues. Some years ago, a high resolution structure of an archaeal homologue of the SLC1 family was determined, followed by the elucidation of its structure in the presence of the substrate aspartate and the inhibitor d,l-threo-benzyloxy aspartate (d,l-TBOA). Historically, the first few known inhibitors of SLC1 transporters were based on constrained glutamate analogs which were active in the high micromolar range but often also showed off-target activity at glutamate receptors. Further development led to the discovery of l-threo-β-hydroxyaspartate derivatives, some of which effectively inhibited SLC1 transporters at nanomolar concentrations. More recently, small molecule inhibitors have been identified whose structures are not based on amino acids. Activators of SLC1 family members have also been discovered but there are only a few examples known.

The changing values of children and preferences regarding family size in south Africa

This study extends the ongoing studies on values of children and intergenerational relations into an African context with the following key questions: To what extent can the structure of values of children (VOC) identified in previous studies be found in South Africa and what are women's preferences regarding the size of small, large, and ideal families? The sample consisted of three cohorts of Black South African women from the Limpopo Province. Factor analyses showed that the dimensions of VOC found in South Africa are a conglomeration of different values. Identified dimensions were Social/Emotional and Traditional/Utilitarian with respect to reasons for wanting children. Regarding reasons for not wanting children, Child as Source of Constraints and Constraints to Have a Child were identified. Intergenerational comparisons showed variations in the importance of value of children dimensions and in preferences regarding family size. Values of children of younger mothers were related to the number of children these women had and to their preferences regarding family size. The findings are discussed within the changing socio-political context of South Africa.

Improving Family Functioning Through Family Preservation Services: Results of the Los Angeles Experiment

This article describes a study of the outcomes of home-based family preservation services for abusive and neglectful families in Los Angeles County. It focuses on changes in family functioning during the 3 month service period and one year after case closing. Families known to the public child welfare agency were referred to the project based on caseworker judgement of the need for services rather than on the criteria of imminent risk of placement. Two hundred forty families were randomly assigned to either the service group receiving family preservation services from two non-profit agencies or to the comparison group receiving regular public agency services. Both caseworkers and families reported small but significant improvements in family functioning for the service group families, but not for the comparison group families. Study findings also suggest the aspects of family functioning most changed by services, the characteristics of families most affected by services, and variables which predicted service success.

What is Family Preservation and Why Does it Matter?

This paper describes competing ideas about family preservation, defined both as a defined program of social services and a philosophical approach to helping troubled families. A straightforward definition has become almost impossible because the phrase has taken on so many different meanings, provoking controversy about its "real" meaning and value. Indeed, "family preservation" has become the proverbial elephant whose splendors and horrors are described with great certainty by those impressed by only one of its aspects. While skirmishes between "child savers" and "family preservers" have been part of the child welfare field since its beginning at the turn of the last century, recent debates over family preservation have been especially heated, generating more confusion and animosity than might be expected from the ranks of the small and usually mild-mannered social work profession. The debate is so heated that the director of one of the nation's largest child welfare agencies said recently that he is afraid to "even use the two words on the same page." <1> While the debate about the value of family preservation is unresolved, experimentation with different approaches to service delivery over the last two decades has helped to lay the groundwork for a resurgence of interest in family and community-centered reforms. Better understanding of the family preservation "debates" may be helpful if these reforms are to be successful over the long term. The paper discusses the competing ideas, values, and perceptions that have led observers to their different understandings of family preservation. It briefly chronicles the history of child welfare and examines key theories that have helped lay the groundwork for the resurgence of interest in family-centered services. It concludes with observations about how the competing values at stake in family preservation may affect the next generation of reforms.

Positional cloning and characterization of the human aniridia and murine small eye genes

Aniridia (AN) is a congenital, panocular disorder of the eye characterized by the complete or partial absence of the iris. The disease can occur in both the sporadic and familial forms which, in the latter case, is inherited as an autosomal dominant trait with high penetrance. The objective of this study was to isolate and characterize the genes involved in AN and Sey, and thereby to gain a better understanding of the molecular basis of the two disorders.^ Using a positional cloning strategy, I have approached and cloned from the AN locus in human chromosomal band 11p13 a cDNA that is deleted in two patients with AN. The deletions in these patients overlap by about 70 kb and encompass the 3$\sp\prime$ end of the cDNA. This cDNA detects a 2.7 kb mRNA encoded by a transcription unit estimated to span approximately 50 kb of genomic DNA. The message is specifically expressed in all tissues affected in all forms of AN, namely within the presumptive iris, lens, neuroretina, the superficial layers of the cornea, the olfactory bulbs, and the cerebellum. Sequence analysis of the AN cDNA revealed a number of motifs characteristic of certain transcription factors. Chief among these are the presence of the paired domain, the homeodomain, and a carboxy-terminal domain rich in serine, threonine and proline residues. The overall structure shows high homology to the Drosophila segmentation gene paired and members of the murine Pax family of developmental control genes.^ Utilizing a conserved human genomic DNA sequence as probe, I was able to isolate an embryonic murine cDNA which is over 92% homologous in nucleotide sequence and virtually identical at the amino acid level to the human AN cDNA. The expression pattern of the murine gene is the same as that in man, supporting the conclusion that it probably corresponds to the Sey gene. Its specific expression in the neuroectodermal component of the eye, in glioblastomas, but not in the neural crest-derived PC12 pheochromocytoma cell line, suggests that a defect in neuroectodermal rather mesodermal development might be the common etiological factor underlying AN and Sey. ^

Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type.

With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the "mycoides cluster'.

The Family Pasteurellaceae

This chapter describes the systematics and evolution of Pasteurellaceae with emphasis on new information generated since the 3rd edition of The Prokaryotes which only included chapters dealing with Haemophilus, Actinobacillus, and Pasteurella. A major source of new information for the current chapter has been provided by whole genome sequences now available for many taxa of the family. Some 100 species and species-like taxa have been documented and 18 genera of Pasteurellaceae reported so far. Members of the family include specialized commensals, potential pathogens, or pathogens of vertebrates and mainly survive poorly in other habitats including the external environment. The pathogenic members are of major importance to animal production and human health. Members of Pasteurellaceae have relatively small genomes, probably as a result of adaption to a special habitat. The most important species in veterinary microbiology include Pasteurella multocida, Actinobacillus pleuropneumoniae, [Haemophilus] parasuis, Mannheimia haemolytica, Bibersteinia trehalosi, and Avibacterium paragallinarum, while Haemophilus influenzae and Aggregatibacter actinomycetemcomitans represent the most important species as to human disease. Traditional isolation techniques are still used in both human and veterinary clinical diagnostic laboratories although genetically based diagnostic methods have replaced traditional biochemical/physiological methods for characterization and identification. For all species, MALDI-TOF can now be used as a diagnostic tool. As control and if MALDI-TOF equipment is not at hand, PCR-based specific detection is possible for Pasteurella multocida, Actinobacillus pleuropneumoniae, [Haemophilus] parasuis, Mannheimia haemolytica, Avibacterium paragallinarum, Gallibacterium anatis, Haemophilus influenzae, and Aggregatibacter actinomycetemcomitans. A lot of work has been directed towards identification of virulence factors and understanding host microbe interactions involved in disease.

Multinomial goodness-of-fit: large sample tests with survey design correction and exact tests for small samples

I introduce the new mgof command to compute distributional tests for discrete (categorical, multinomial) variables. The command supports largesample tests for complex survey designs and exact tests for small samples as well as classic large-sample x2-approximation tests based on Pearson’s X2, the likelihood ratio, or any other statistic from the power-divergence family (Cressie and Read, 1984, Journal of the Royal Statistical Society, Series B (Methodological) 46: 440–464). The complex survey correction is based on the approach by Rao and Scott (1981, Journal of the American Statistical Association 76: 221–230) and parallels the survey design correction used for independence tests in svy: tabulate. mgof computes the exact tests by using Monte Carlo methods or exhaustive enumeration. mgof also provides an exact one-sample Kolmogorov–Smirnov test for discrete data.

The relationship between top management team innovation orientation and firm growth: The mediating role of firm innovativeness

Upper echelon theory and research on innovation have considered top management teams and their behaviour and characteristics as important factors that positively influence innovativeness and organizational outcomes. Yet, innovation research has mostly focused on individual new product projects, and their performance and impact on firm performance. Recent research has started to apply a more holistic view in terms of innovation, by considering firm-wide innovation instead of single new products. Upper echelon research has concentrated on direct relationships between top management team characteristics and organizational outcomes. But recent research calls for mediating effects of the relationship between top management team characteristics and organizational outcomes. Hence, this study introduces firm innovativeness as a mediator between top management team innovation orientation and firm growth. Focusing on small and medium-sized firms, which often represent highly innovative firms, results show that firm innovativeness fully mediates the relationship between top management team innovation orientation and firm growth. Implications and future research are discussed.

Development of small molecular tools for the cellular study of adenosine receptors

The adenosine receptors are members of the G-protein coupled receptor (GPCR) family which represents the largest class of cell-surface proteins mediating cellular communication. As a result, GPCRs are formidable drug targets and it is estimated that approximately 30% of the marketed drugs act through members of this receptor class. There are four known subtypes of adenosine receptors: A1, A2A, A2B and A3. The adenosine A1 receptor, which is the subject of this presentation, mediates the physiological effects of adenosine in various tissues including the brain, heart, kidney and adipocytes. In the brain for instance, its role in epilepsy and ischemia has been the focus of many studies. Previous attempts to study the biosynthesis, trafficking and agonist-induced internalisation of the adenosine A1 receptor in neurons using fluorescent protein-receptor fusion constructs have been hampered by the sheer size of the fluorescent protein (GFP) that ultimately affected the function of the receptor. We have therefore initiated a research programme to develop small molecule fluorescent agonists that selectively activate the adenosine A1 receptor. Our probe design is based on the endogenous ligand adenosine and the known unselective adenosine receptor agonist NECA. We have synthesised a small library of non-fluorescent adenosine derivatives that have different cyclic and bicyclic moieties at the 6 position of the purine ring and have evaluated the pharmacology of these compounds using a yeast-based assay. This analysis revealed compounds with interesting behaviour, i.e. exhibiting subtype-selectivity and biased signalling, that can be potentially used as tool compounds in their own right for cellular studies of the adenosine A1 receptor. Furthermore, we have also linked fluorescent dyes to the purine ring and discovered fluorescent compounds that can activate the adenosine A1 receptor.

Phenotype and genotype in 103 patients with tricho-rhino-phalangeal syndrome.

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities, and subdivided in TRPS I, caused by mutations in TRPS1, and TRPS II, caused by a contiguous gene deletion affecting (amongst others) TRPS1 and EXT1. We performed a collaborative international study to delineate phenotype, natural history, variability, and genotype-phenotype correlations in more detail. We gathered information on 103 cytogenetically or molecularly confirmed affected individuals. TRPS I was present in 85 individuals (22 missense mutations, 62 other mutations), TRPS II in 14, and in 5 it remained uncertain whether TRPS1 was partially or completely deleted. Main features defining the facial phenotype include fine and sparse hair, thick and broad eyebrows, especially the medial portion, a broad nasal ridge and tip, underdeveloped nasal alae, and a broad columella. The facial manifestations in patients with TRPS I and TRPS II do not show a significant difference. In the limbs the main findings are short hands and feet, hypermobility, and a tendency for isolated metacarpals and metatarsals to be shortened. Nails of fingers and toes are typically thin and dystrophic. The radiological hallmark are the cone-shaped epiphyses and in TRPS II multiple exostoses. Osteopenia is common in both, as is reduced linear growth, both prenatally and postnatally. Variability for all findings, also within a single family, can be marked. Morbidity mostly concerns joint problems, manifesting in increased or decreased mobility, pain and in a minority an increased fracture rate. The hips can be markedly affected at a (very) young age. Intellectual disability is uncommon in TRPS I and, if present, usually mild. In TRPS II intellectual disability is present in most but not all, and again typically mild to moderate in severity. Missense mutations are located exclusively in exon 6 and 7 of TRPS1. Other mutations are located anywhere in exons 4-7. Whole gene deletions are common but have variable breakpoints. Most of the phenotype in patients with TRPS II is explained by the deletion of TRPS1 and EXT1, but haploinsufficiency of RAD21 is also likely to contribute. Genotype-phenotype studies showed that mutations located in exon 6 may have somewhat more pronounced facial characteristics and more marked shortening of hands and feet compared to mutations located elsewhere in TRPS1, but numbers are too small to allow firm conclusions.

A New Family of High-Affinity Transporters for Adenine, Cytosine, and Purine Derivatives in Arabidopsis

In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant Ki = 20 to 35 μM), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.

The role of SRC family kinase activation in prostate cancer growth and lymph node metastasis

Aberrant expression and/or activation of Src Family of non-receptor protein tyrosine kinases (SFKs) occur frequently during progressive stages of multiple types of human malignancies, including prostate cancer. Two SFKs, Src and Lyn, are expressed and implicated in prostate cancer progression. Work in this dissertation investigated the specific roles of Src and Lyn in the prostate tumor progression, and the effects of SFK inhibition on prostate tumor growth and lymph node metastasis in pre-clinical mouse models. ^ Firstly, using a pharmacological inhibitor of SFKs in clinical trials, dasatinib, I demonstrated that SFK inhibition affects both cellular migration and proliferation in vitro. Systemic administration of dasatinib reduced primary tumor growth, as well as development of lymph node metastases, in both androgen-sensitive and -resistant orthotopic prostate cancer mouse models. Immunohistochemical analysis of the primary tumors revealed that dasatinib treatment decreased SFK phosphorylation but not expression, resulting in decreased cellular proliferation and increased apoptosis. For this analysis of immunohistochemical stained tissues, I developed a novel method of quantifying immunohistochemical stain intensity that greatly reduced the inherent bias in analyzing staining intensity. ^ To determine if Src and Lyn played overlapping or distinct roles in prostate cancer tumor growth and progression, Src expression alone was inhibited by small-interfering RNA. The resulting stable cell lines were decreased in migration, but not substantially affected in proliferation rates. In contrast, an analogous strategy targeting Lyn led to stable cell lines in which proliferation rates were significantly reduced. ^ Lastly, I tested the efficacy of a novel SFK inhibitor (KX2-391) targeting peptide substrate-binding domain, on prostate cancer growth and lymph node metastasis in vivo. I demonstrated that KX2-391 has similar effects as dasatinib, an ATP-competitive small molecular inhibitor, on both the primary tumor growth and development of lymph node metastasis in vivo, work that contributed to the first-in-man Phase I clinical trial of KX2-391. ^ In summary, studies in this dissertation provide the first demonstration that Src and Lyn activities affect different cellular functions required for prostate tumor growth and metastasis, and SFK inhibitors effectively reduce primary tumor growth and lymph node metastasis. Therefore, I conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer. ^