CTX, a Xenopus thymocyte receptor, defines a molecular family conserved throughout vertebrates

CTX, a cortical thymocyte marker in Xenopus, is an immunoglobulin superfamily (Igsf) member comprising one variable and one constant C2-type Igsf domain, a transmembrane segment and a cytoplasmic tail. Although resembling that of the TCR and immunoglobulins, the variable domain is not encoded by somatic rearrangement of the gene but by splicing of two half-domain exons. The C2 domain, also encoded by two exons, has an extra pair of cysteines. The transmembrane segment is free of charged residues, and the cytoplasmic tail (70 amino acids) contains one tyrosine and many glutamic acid residues. ChT1, a chicken homologue of CTX, has the same structural and genetic features, and both molecules are expressed on the thymocyte surface. We cloned new mouse (CTM) and human (CTH) cDNA and genes which are highly homologous to CTX/ChT1 but not lymphocyte specific. Similarity with recently described human cell surface molecules, A33 antigen and CAR (coxsackie and adenovirus 5 receptor), and a number of expressed sequence tags leads us to propose that CTX defines a novel subset of the Igsf, conserved throughout vertebrates and extending beyond the immune system. Strong homologies within vertebrate sequences suggest that the V and C2 CTX domains are scions of a very ancient lineage.     

A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes

We have isolated a novel cDNA clone encoding interferon (IFN) consensus sequence-binding protein in adult T-cell leukemia cell line or activated T cells (ICSAT); this protein is the human homolog of the recently cloned Pip/LSIRF. ICSAT is structurally most closely related to the previously cloned ICSBP, a member of the IFN regulatory factor (IRF) family of proteins that binds to interferon consensus sequences (ICSs) found in many promoters of the IFN-regulated genes. Among T-cell lines investigated, ICSAT was abundantly expressed in human T-cell leukemia virus type 1 (HTLV-1)-infected T cells. When the HTLV-1 tax gene was expressed or phorbol myristake acetate-A23187 stimulation was used, ICSAT expression was induced in Jurkat cells which otherwise do not express ICSAT. When the binding of ICSAT to four different ICSs was tested, the relative differences in binding affinities for those ICSs were determined. To study the functional role of ICSAT, we performed cotransfection experiments with the human embryonal carcinoma cell line N-Tera2. ICSAT was demonstrated to possess repressive function over the gene activation induced by IFN stimulation or by IRF-1 cotransfection. Such repressive function is similar to that seen in IRF-2 or ICSBP. However, we have found that ICSAT has a different repressive effect from that of IRF-2 or ICSBP in some IFN-responsive reporter constructs. These results suggest that a novel mechanism of gene regulation by "differential repression" is used by multiple members of repressor proteins with different repressive effects on the IFN-responsive genes.     

Testing candidate genes that may affect susceptibility to leprosy

Due to increased chloroquine resistance, the antifolate/sulpha drug combinations are becoming increasingly important in the chemotherapy of falciparum malaria. However, point mutations in the dihydrofolate reductase gene lead to resistance to the antifolate drugs. We therefore investigated the prevalence of the 6 reported point mutations in this gene among field isolates of Plasmodium falciparum from Kenya, to determine if the mutations correlated with resistance to pyrimethamine and the biguanides cycloguanil and chlorcycloguanil. We used a mutation-specific polymerase chain reaction technique to test for these reported mutations in 21 Kenyan isolates and 4 reference lines. We also amplified and directly sequenced the dihydrofolate reductase coding sequence from these parasites to confirm the results and test for other possible mutations. Of the reported mutations, we found S108N, which is the central mutation of pyrimethamine resistance, and mutations N51I and C59R, which modulate the levels of resistance and may confer decreases in response to cycloguanil that are folate and p-aminobenzoic acid dependent. No isolate possessed the paired point mutations S108T and A16V, or I164L and S108N, which have been associated with cycloguanil resistance in previous studies. These results provided supportive evidence for the combined use of a cycloguanil-class drug (e.g., chlorproguanil) and a sulpha drug (e.g., dapsone) against P.falciparum malaria in Kenya.     

Skills that Iowa family physicians desire in a new physician partner

BACKGROUND AND OBJECTIVES: The importance of specific skills in primary care continues to be debated. As a result, there is not consensus on which skills need to be stressed during residency training. Our project asked community-based family physicians to rate the importance of specific skills in a new family physician partner. METHODS: Data were collected through a cross-sectional survey of all active members of the Iowa Academy of Family Physicians. Participants were surveyed by mail, using a list of 83 skills pertinent to primary care. Physicians were asked to rate the importance of a new member of their practice having the individual skills on this list. RESULTS: A total of 546 family physicians (67%) completed questionnaires. Fourteen skills (seven cognitive and seven psychomotor) were reported to be "essential" or "very important" by at least 80% of the physicians. A total of 43 skills were rated as "essential" or "very important" by at least 50% of responding family physicians. Many of the hospital-based procedural skills, particularly those used in an intensive care setting, were rated as less important. The importance ratings of many skills were associated with the physicians' ages, size of their primary hospitals, and availability of other medical specialties. CONCLUSIONS: Family physicians tended to rate office-based procedural skills, counseling skills, and management skills as "essential or very important" to their practices. These rating might be used to guide residency training in family practice.     

Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency

Millions of people worldwide suffer from nutritional imbalances of essential metals like zinc. These same metals, along with pollutants like cadmium and lead, contaminate soils at many sites around the world. In addition to posing a threat to human health, these metals can poison plants, livestock, and wildlife. Deciphering how metals are absorbed, transported, and incorporated as protein cofactors may help solve both of these problems. For example, edible plants could be engineered to serve as better dietary sources of metal nutrients, and other plant species could be tailored to remove metal ions from contaminated soils. We report here the cloning of the first zinc transporter genes from plants, the ZIP1, ZIP2, and ZIP3 genes of Arabidopsis thaliana. Expression in yeast of these closely related genes confers zinc uptake activities. In the plant, ZIP1 and ZIP3 are expressed in roots in response to zinc deficiency, suggesting that they transport zinc from the soil into the plant. Although expression of ZIP2 has not been detected, a fourth related Arabidopsis gene identified by genome sequencing, ZIP4, is induced in both shoots and roots of zinc-limited plants. Thus, ZIP4 may transport zinc intracellularly or between plant tissues. These ZIP proteins define a family of metal ion transporters that are found in plants, protozoa, fungi, invertebrates, and vertebrates, making it now possible to address questions of metal ion accumulation and homeostasis in diverse organisms.     

Comparison of the structure and expression of odd-skipped and two related genes that encode a new family of zinc finger proteins in Drosophila

The odd-skipped (odd) gene, which was identified on the basis of a pair-rule segmentation phenotype in mutant embryos, is initially expressed in the Drosophila embryo in seven pair-rule stripes, but later exhibits a segment polarity-like pattern for which no phenotypic correlate is apparent. We have molecularly characterized two embryonically expressed odd-cognate genes, sob and bowel (bowl), that encode proteins with highly conserved C2H2 zinc fingers. While the Sob and Bowl proteins each contain five tandem fingers, the Odd protein lacks a fifth (C-terminal) finger and is also less conserved among the four common fingers. Reminiscent of many segmentation gene paralogues, the closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns; in contrast, the less-tightly linked bowl gene is expressed in a distinctly different pattern at the termini of the early embryo. Although our results indicate that odd and sob are more likely than bowl to share overlapping developmental roles, some functional divergence between the Odd and Sob proteins is suggested by the absence of homology outside the zinc fingers, and also by amino acid substitutions in the Odd zinc fingers at positions that appear to be constrained in Sob and Bowl     

Isolation and characterization of a new gene, sre, which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa

Multiple GATA factors - regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence - exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2. controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBSI of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre+ and sre- strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre- mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells.     

Viable maternal-effect mutations that affect the development of the nematode Caenorhabditis elegans

We carried out a genetic screen for viable maternal-effect mutants to identify genes with a critical function relatively early in development. This type of mutation would not have been identified readily in previous screens for viable mutants and therefore could define previously unidentified genes. We screened 30,000 genomes and identified 41 mutations falling into 24 complementation groups. We genetically mapped these 24 loci; only two of them appear to correspond to previously identified genes. We present a partial phenotypic characterization of the mutants and a quantitative analysis of the degree to which they can be maternally or zygotically rescued.     

Analysis of Hoxd-13 and Hoxd-11 misexpression in chick limb buds reveals that Hox genes affect both bone condensation and growth

Hox genes are important regulators of limb pattern in vertebrate development. Misexpression of Hox genes in chicks using retroviral vectors provides an opportunity to analyze gain-of-function phenotypes and to assess their modes of action. Here we report the misexpression phenotype for Hoxd-13 and compare it to the misexpression phenotype of Hoxd-11. Hoxd-13 misexpression in the hindlimb results in a shortening of the long bones, including the femur, the tibia, the fibula and the tarsometatarsals. Mutations in an alanine repeat region in the N-terminus of Hoxd-13 have recently been implicated in human synpolydactyly (Muragaki, Y., Mundlos, S., Upton, J. and Olsen, B. R. (1996) Science 272, 548-551). N-terminal truncations of Hoxd-13 which lack this repeat were constructed and were found to produce a similar, although slightly milder, misexpression phenotype than the full-length Hoxd-13. The stage of bone development regulated by Hox genes has not previously been examined. The changes in bone lengths caused by Hoxd-13 misexpression are late phenotypes that first become apparent during the growth phase of the bones. Analysis of tritiated thymidine uptake by the tibia and fibula demonstrates that Hox genes can pattern the limb skeleton by regulating the rates of cell division in the proliferative zone of growing cartilage. Hoxd-11, in contrast to Hoxd-13, acts both at the initial cartilage condensation phase in the foot and during the later growth phase in the lower leg. Ectopic Hoxd-13 appears to act in a dominant negative manner in regions where it is not normally expressed. We propose a model in which all Hox genes are growth promoters, regulating the expression of the same target genes, with some Hox genes being more effective promoters of growth than other Hox genes. According to this model, the overall rate of growth in a given region is the result of the combined action of all of the Hox genes expressed in that region competing for the same target genes.     

Expression of cartilage extracellular matrix and potential regulatory genes in a new human chondrosarcoma cell line

The ability of differentiating cells to migrate within the developing central nervous system (CNS) depends on extrinsic guidance signals, some of which are growth factors. In this study we have investigated the chemotactic response of cultured stem cells from the embryonic rat cortex to platelet-derived growth factor (PDGF). Nestin-positive stem cells from the developing CNS can be maintained and expanded in vitro under serum-free conditions in the presence of basic fibroblast growth factor (bFGF). Northern blot analysis of PDGF receptor expression revealed both alpha- and beta-receptors on bFGF-treated neural stem cells. Both PDGF-AA and PDGF-BB readily induced directed migration of cultured neuroepithelial cells as measured in a microchemotaxis assay. Blocking of the migratory response was achieved by incubation with PDGF isoform-specific antibodies. More than 90% of the migrating cells were nestin-positive and incorporation of BrdU was also seen suggesting the cells to be immature and not yet committed to a specific cell lineage. These findings suggest a role for PDGF in cell migration in the developing cortex.     

Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid

OBJECTIVE: Despite improved systemic control of metastatic breast cancer, the incidence of brain metastases from breast carcinoma continues to rise, in part because most systemically administered agents have poor central nervous system penetration. Therefore, as a method of optimizing drug delivery into the central nervous system, we studied the safety and efficacy of chemotherapy delivered locally via biodegradable polymers in a mouse model of breast carcinoma metastases to the brain. METHODS: The chemotherapeutic agents carmustine (BCNU), carboplatin, and camptothecin were incorporated into controlled release polymers and tested individually against intracranial challenges of EMT-6 breast tumor in BALB/c female mice. For each drug, four groups were tested: Group 1, empty polymer (no drug); Group 2, external beam radiotherapy (XRT) alone; Group 3, local chemotherapy from biodegradable polymer alone; and Group 4, local chemotherapy and XRT together. Polymers were implanted 5 days after intracranial tumor inoculation; XRT was administered on Days 7 through 9 (300 cGy/d). RESULTS: BCNU polymer alone (n = 10; median survival time, >200 d; P < 0.0001) and BCNU and XRT together (n = 10; median survival time, 41 d; P = 0.02) significantly improved survival in mice with intracranial EMT-6 breast cancer in comparison with control animals (n = 20; median survival time, 17 d). Carboplatin and camptothecin, either with or without XRT, and XRT alone did not have any significant effect on survival. CONCLUSION: Local delivery of BCNU with biodegradable polymers can significantly prolong survival in a murine model of intracranial metastatic breast cancer. Surgical resection and placement of BCNU polymers into the resection cavity may decrease the incidence of local recurrence of breast cancer metastases with minimal morbidity.     

DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum

To study K+ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 microg/ml) in the symmetrical high K+ (145 mm) Ringer solution. During measurement of the macroscopic K+ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K+ currents: one is an inwardly rectifying K+ current and the other is a slowly activating outwardly rectifying K+ current. The inwardly rectifying K+ current is inhibited by Ba2+. The slowly activating K+ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristate 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of ISK channel. RT-PCR analysis revealed the presence of ISK cDNA in the rat trachea epithelia. Although 0.1 mM Ba2+ only had minimal affect on short-circuit current (Isc) induced by cAMP in intact epithelia, 0.1 mM clofilium strongly inhibited it. These results indicate that ISK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia.     

A regulatory role for recombinase activating genes, RAG-1 and RAG-2, in T cell development

Effects of etoposide (VP-16) and cytosine arabinoside (Ara-C) on the cell cycle of HL-60 and THP-1 cells were studied by flow cytometry using the bromodeoxyuridine (BrdU)/DNA assay technique to investigate the efficacy of VP-16 for monocytic leukemia cells. VP-16 inhibited the proliferation of THP-1 cells more strongly than that of HL-60 cells at any concentrations used at 24 and 48 hr. VP-16 arrested HL-60 and THP-1 cells in the G2/M phase and reduced them in the G0/G1 and early S phase at higher concentrations. There was no significant difference in the percentage of G2/M phase cells at the same concentration between both cells. However, reduction in the G0/G1 and early S phase cells was more marked in THP-1 than HL-60 cells significantly. On the other hand, Ara-C perturbed the cell cycle of HL-60 cells more than that of THP-1 cells at 24 and 48 hr. These results suggest that the effects of VP-16 on the cell cycle may be more intense in THP-1 than HL-60 cells, and support the efficacy of VP-16 for treating monocytic leukemia in vivo.     

Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. Quorum-sensing bacteria produce, release, and respond to hormone-like molecules (autoinducers) that accumulate in the external environment as the cell population grows. In the marine bacterium Vibrio harveyi two parallel quorum-sensing systems exist, and each is composed of a sensor-autoinducer pair. V. harveyi reporter strains capable of detecting only autoinducer 1 (AI-1) or autoinducer 2 (AI-2) have been constructed and used to show that many species of bacteria, including Escherichia coli MG1655, E. coli O157:H7, Salmonella typhimurium 14028, and S. typhimurium LT2 produce autoinducers similar or identical to the V. harveyi system 2 autoinducer AI-2. However, the domesticated laboratory strain E. coli DH5alpha does not produce this signal molecule. Here we report the identification and analysis of the gene responsible for AI-2 production in V. harveyi, S. typhimurium, and E. coli. The genes, which we have named luxSV.h., luxSS.t., and luxSE.c. respectively, are highly homologous to one another but not to any other identified gene. E. coli DH5alpha can be complemented to AI-2 production by the introduction of the luxS gene from V. harveyi or E. coli O157:H7. Analysis of the E. coli DH5alpha luxSE.c. gene shows that it contains a frameshift mutation resulting in premature truncation of the LuxSE.c. protein. Our results indicate that the luxS genes define a new family of autoinducer-production genes.     

Oleavirus, a new genus in the family Bromoviridae

We investigated the efficacy of trovafloxacin, a new quinolone, in comparison with that of clindamycin in the treatment of intra-abdominal abscesses caused by Bacteroides fragilis in young and senescent mice. The development of abscess formation, the number of viable organisms, and antibiotic concentrations were measured, and the values for young and old mice were compared. Trovafloxacin was well distributed to the tissues in both young and old animals. Although the pharmacokinetics and concentrations of trovafloxacin in serum were similar between young and old mice, the levels in tissue were higher in senescent mice than in young mice. Trovafloxacin therapy sterilized abscesses in 94% of young mice and in 73% of old mice, but this difference was not significant. This therapeutic response to trovafloxacin was similar to that seen with clindamycin. These results suggest that aging may not have any adverse effect on the therapeutic outcome for intra-abdominal abscesses caused by B. fragilis.     

Growth factor independence and growth regulatory pathways in human melanoma development

This review concentrates on growth autonomy of tumor cells in relation to tumor progression. Human malignant melanoma serves as an example for progressive growth factor independence at subsequent stages of tumor progression. Mechanisms by which malignant cells acquire growth factor independence are discussed. In melanoma, deregulation of growth regulatory pathways has been described on four levels: 1) aberrant production of autocrine growth factors that substitute for exogenous growth factors (basic fibroblast growth factor [bFGF]); 2) alterations in the response to negative autocrine growth factors (interleukin [IL]-6 and transforming growth factor [TGF]-beta); 3) overexpression of epidermal growth factor receptors (EGF-R); and 4) alterations of cellular protooncogenes involved in signal transduction (RAS, MYB) and growth suppression (p53). In addition to bFGF and IL-6, multiple other growth factor genes are activated in malignant melanoma cells but not normal melanocytes. These include both chains of platelet-derived growth factor (PDGF), TGF-alpha, IL-1, IL-8, and tumor necrosis factor (TNF)-alpha. Of these, PDGF-B has been investigated in more detail. Melanoma-derived PDGF clearly does not act in a direct autocrine mode, but has important paracrine effects on normal tissue constituents, notably fibroblasts and endothelial cells, that are essential for tumor development in vivo. It is speculated that other melanoma-derived growth factors with as yet undefined functions similarly exert such paracrine or 'indirect' autocrine effects that cannot be sufficiently addressed in studies on cultured cells.     

Cloning of NKG2-F, a new member of the NKG2 family of human natural killer cell receptor genes

The NKG2 family of genes encodes at least four different type II transmembrane molecules (NKG2-A, NKG2-B, NKG2-C and NKG2-E) which contain a C-lectin domain. These proteins have been shown to be covalently associated with CD94, another C-type lectin member. The heterodimers are involved in natural killer cell-mediated recognition of different HLA-allotypes. Here we describe the cloning of a new NKG2-related gene, termed NKG2-F, localized 25 kb from NKG2-A as well as its relationship with the previously described NKG2-D cDNA. Despite the similarities with the other NKG2 genes, NKG2-F encodes a putative protein which does not contain any lectin domain. However, a conserved 24-amino acid sequence, present in all members of the NKG2 family, suggests that NKG2-F is also able to form heterodimers with CD94.