Differential expression and function of ABCG1 and ABCG4 during development and aging

ABCG1 and ABCG4 are highly homologous members of the ATP binding cassette (ABC) transporter family that regulate cellular cholesterol homeostasis. In adult mice, ABCG1 is known to be expressed in numerous cell types and tissues, whereas ABCG4 expression is limited to the central nervous system (CNS). Here, we show significant differences in expression of these two transporters during development. Examination of β-galactosidase-stained tissue sections from Abcg1^−/−LacZ and Abcg4^−/−LacZ knockin mice shows that ABCG4 is highly but transiently expressed both in hematopoietic cells and in enterocytes during development. In contrast, ABCG1 is expressed in macrophages and in endothelial cells of both embryonic and adult liver. We also show that ABCG1 and ABCG4 are both expressed as early as E12.5 in the embryonic eye and developing CNS. Loss of both ABCG1 and ABCG4 results in accumulation in the retina and/or brain of oxysterols, in altered expression of liver X receptor and sterol-regulatory element binding protein-2 target genes, and in a stress response gene. Finally, behavioral tests show that Abcg4^−/− mice have a general deficit in associative fear memory. Together, these data indicate that loss of ABCG1 and/or ABCG4 from the CNS results in changes in metabolic pathways and in behavior.     

Genomic Content of Neisseria Species

The physical properties of most bacterial genomes are largely unexplored. We have previously demonstrated that the strict human pathogen Neisseria gonorrhoeae is polyploid, carrying an average of three chromosome copies per cell and only maintaining one pair of replication forks per chromosome (D. M. Tobiason and H. S. Seifert, PLos Biol. 4:1069-1078, 2006). We are following up this initial report to test several predictions of the polyploidy model of gonococcal chromosome organization. We demonstrate that the N. gonorrhoeae chromosomes exist solely as monomers and not covalently linked dimers, and in agreement with the monomer status, we show that distinct nucleoid regions can be detected by electron microscopy. Two different approaches to isolate heterozygous N. gonorrhoeae resulted in the formation of merodiploids, showing that even with more than one chromosome copy, these bacteria are genetically haploid. We show that the closely related bacterium Neisseria meningitidis is also polyploid, while the commensal organism Neisseria lactamica maintains chromosomes in single copy. We conclude that the pathogenic Neisseria strains are homozygous diploids.Bacteria are unicellular organisms that exhibit a multitude of shapes and sizes and exist in a wide range of environments. Despite the extreme diversity of capabilities and physiology evidenced by different bacterial species, most bacteria are assumed to conform to the enteric model of genomic organization, chromosomal replication, and genomic segregation during cell division exemplified by Escherichia coli. In contradiction to this limited view of bacterial genome biology, some bacterial species have their genome divided between multiple DNA elements (10), and some possess linear chromosomes (2, 19). A few bacterial species have been reported to carry multiple genome copies per cell (members of the genera Azotobacter, Borrelia, Buchnera, Deinococcus, Neisseria, and Epulopiscium), with copy number estimates ranging from two copies to thousands of copies per cell (1, 7, 17, 23, 25, 26, 34, 35, 39, 46). The exact number of genomes per cell has not been determined for most of these organisms, and the mechanisms for organizing polyploid genomes and segregating them during cell division remain to be determined. An exception is Deinococcus radiodurans, which has been shown to possess four complete chromosomes during exponential growth and up to 16 genomes within the stationary phase. The polyploid genomes of D. radiodurans have been proposed to assemble into a toroidal mass in the cell (29), but the validity of this finding has been questioned (11, 13, 49). There are few obvious commonalities between these polyploid organisms, except that some Neisseria, Deinococcus, and Borrelia species utilize homologous recombination to mediate specialized processes essential for the survival of these species. In addition, members of the Azotobacter, Buchnera, and Epulopiscium genera are obligate symbionts that do not possess a free-living stage, but the reasons why obligate symbionts would possess polyploid chromosomes are unknown.Neisseria gonorrhoeae and Neisseria meningitidis are the two pathogenic members of the Neisseria genus. N. gonorrhoeae is the sole causative agent of the disease gonorrhea, and N. meningitidis is the most common cause of bacterial meningitis in adolescents and young adults. One attribute that these human-specific pathogens use to coexist and evolve within humans lies in their capacity to antigenically vary and phase vary several outer membrane structures, including pili, Opa proteins, and the lipooligosaccharide (LOS) (12, 21). Variation of the Opa and LOS antigens is mediated by polynucleotide repeat variation that modulates expression of biosynthetic genes (40, 48). These changes in polynucleotide repeat sequences are mediated through slipped-strand mispairing that occurs during normal DNA replication and therefore would not obviously benefit from polyploidy. In contrast, pilin antigenic variation is a RecA-mediated gene conversion event (27), which could be aided by having two copies of all the recombining pilin loci within a single cell to facilitate the nonreciprocal transfer of pilin sequences. Therefore, we postulated that the presence of multiple genome copies per gonococcal cell may be required to facilitate these high-frequency gene conversion events. Analysis of gonococcal genome content by flow cytometry and fluorescent microscopy indicated that there existed greater than one genome equivalent of gonococcal DNA content per cell (46). Additionally, quantitative real-time PCR and genome microarray analysis measured a marker frequency pinpointing a single DNA replication event per round of cell division. On average, each coccal unit had three genome copies per cell, and a population of cells with a single genome equivalent per cell was never observed, even under conditions of slower growth. These observations predicted a model for gonococcal replication (Fig. ​(Fig.1)1) in which each coccal unit has a minimum of two chromosomes that replicate in unison to produce four chromosomes prior to cell division and the conclusion that this species is diploid.Open in a separate windowFIG. 1.Model of gonococcal DNA replication and chromosome segregation in a monococcus. The gonococcal chromosome is indicated by a dotted line. An antibiotic resistance (Ab^R) marker recombined into a gonococcal chromosome (solid line). At time zero minutes, DNA replication begins, and after 35 min, DNA replication is complete. Gonococcal cell division occurs after 60 min. In scenario I, homozygous chromosomes are segregated together. In scenario II, heterozygous bacteria are produced.Though the analysis of the genomic content has only been reported for the gonococcus, the genus Neisseria encompasses a number of pathogenic and commensal bacteria. N. meningitidis is the leading cause of bacterial meningitis worldwide and is asymptomatically carried in the human nasopharynx (47). Most of the Neisseria species are commensal organisms that inhabit the nasopharynx and rarely cause disease (24). The most extensively studied commensal Neisseria species, N. lactamica, shares extensive homology with the pathogenic Neisseriae species and also predominately resides in the human nasopharynx (31). Only the pathogenic neisseriae, N. gonorrhoeae and N. meningitidis, have been shown to undergo pilin antigenic variation (43). Since the polyploid nature of N. gonorrhoeae has been proposed to be required for pilin antigenic variation, N. meningitidis may also have multiple genome copies per cell. The genomic copy number of other Neisseria species and the putative relationship between genomic content and pathogenesis remain to be determined.In this work, we tested several predictions or models resulting from the observation that the gonococcus is polyploid. We confirmed that the chromosomes exist as separate molecules and show that the gonococcal nucleoids reside in discrete cellular regions. We confirm that these bacteria are genetically haploid, suggesting that chromosomal segregation mechanisms ensure a homozygous population. Finally, we show that the other pathogenic Neisseria species, N. meningitidis, is also polyploid, while a commensal Neisseria species, N. lactamica, is not. These studies show that polyploidy is correlated with Neisseria pathogenesis and suggest that this property has evolved to allow diploid chromosomes while maintaining the haploid status of these obligate human pathogens.     

Using NMR Metabolomics to Investigate Tricarboxylic Acid Cycle-dependent Signal Transduction in Staphylococcus epidermidis

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to “sense” environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is “sensing” disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are “sensed” by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.     

Parallel processing in two transmitter microenvironments at the cone photoreceptor synapse

A cone photoreceptor releases glutamate at ribbons located atop narrow membrane invaginations that empty onto a terminal base. The unique shape of the cone terminal suggests that there are two transmitter microenvironments: within invaginations, where concentrations are high and exposures are brief; and at the base, where concentrations are low and exposure is smoothed by diffusion. Using multicell voltage-clamp recording, we show that different subtypes of Off bipolar cells sample transmitter in two microenvironments. The dendrites of an AMPA receptor-containing cell insert into invaginations and sense rapid fluctuations in glutamate concentration that can lead to transient responses. The dendrites of kainate receptor-containing cells make basal contacts and respond to a smoothed flow of glutamate that produces sustained responses. Signaling at the cone to Off bipolar cell synapse illustrates how transmitter spillover and synapse architecture can combine to produce distinct signals in postsynaptic neurons.     

Cancer progression by non-clonal chromosome aberrations

The establishment of the correct conceptual framework is vital to any scientific discipline including cancer research. Influenced by hematologic cancer studies, the current cancer concept focuses on the stepwise patterns of progression as defined by specific recurrent genetic aberrations. This concept has faced a tough challenge as the majority of cancer cases follow non-linear patterns and display stochastic progression. In light of the recent discovery that genomic instability is directly linked to stochastic non-clonal chromosome aberrations (NCCAs), and that cancer progression can be characterized as a dynamic relationship between NCCAs and recurrent clonal chromosome aberrations (CCAs), we propose that the dynamics of NCCAs is a key element for karyotypic evolution in solid tumors. To support this viewpoint, we briefly discuss various basic elements responsible for cancer initiation and progression within an evolutionary context. We argue that even though stochastic changes can be detected at various levels of genetic organization, such as at the gene level and epigenetic level, it is primarily detected at the chromosomal or genome level. Thus, NCCA-mediated genomic variation plays a dominant role in cancer progression. To further illustrate the involvement of NCCA/CCA cycles in the pattern of cancer evolution, four cancer evolutionary models have been proposed based on the comparative analysis of karyotype patterns of various types of cancer.     

Expression of the mechanosensitive 2PK+ channel TREK-1 in human osteoblasts

TREK-1 is a mechanosensitive member of the two-pore domain potassium channel family (2PK+) that is also sensitive to lipids, free fatty acids (including arachidonic acid), temperature, intracellular pH, and a range of clinically relevant compounds including volatile anaesthetics. TREK-1 is known to be expressed at high levels in excitable tissues, such as the nervous system, the heart and smooth muscle, where it is believed to play a prominent role in controlling resting cell membrane potential and electrical excitability. In this report, we use RT-PCR, Western blotting and immunohistochemistry to confirm that human derived osteoblasts and MG63 cells express TREK-1 mRNA and protein. In addition, we show gene expression of TREK2c and TRAAK channels. Furthermore, whole cell patch clamp electrophysiology demonstrates that these cells express a spontaneously active, outwardly rectifying potassium "background leak" current that shares many similarities to TREK-1. The outward current is largely insensitive to TEA and Ba2+, and is sensitive to application of lysophosphatidylcholine (LPC). In addition, blocking TREK-1 channel activity is shown to upregulate bone cell proliferation. It is concluded that human osteoblasts functionally express TREK-1 and that these channels contribute, at least in part, to the resting membrane potential of human osteoblast cells. We hypothesise a possible role for TREK-1 in mechanotransduction, leading to bone remodelling.     

Differential engagement of modules 1 and 4 of vascular cell adhesion molecule-1 (CD106) by integrins alpha4beta1 (CD49d/29) and alphaMbeta2 (CD11b/18) of eosinophils

We have studied adhesion of eosinophils to various forms of vascular cell adhesion molecule 1 (VCAM-1, CD106), an integrin counter-receptor implicated in eosinophil recruitment to the airway in asthma. Full-length 7d-VCAM-1, with seven immunoglobulin-like modules, contains integrin-binding sites in modules 1 and 4. The alternatively spliced six-module protein, 6d-VCAM-1, lacks module 4. In static assays, unactivated purified human blood eosinophils adhered similarly to recombinant soluble human 6d-VCAM-1 and 7d-VCAM-1 coated onto polystyrene microtiter wells. Further experiments, however, revealed differences in recognition of modules 1 and 4. Antibody blocking indicated that eosinophil adhesion to 6d-VCAM-1 or a VCAM-1 construct containing only modules 1-3, 1-3VCAM-1, is mediated by alpha4beta1 (CD49d/29), whereas adhesion to a construct containing modules 4-7, 4-7VCAM-1, is mediated by bothalpha4beta1 andalphaMbeta2 (CD11b/18). Inhibitors of phosphoinositide 3-kinase, which block adhesion of eosinophils mediated by alphaMbeta2, blocked adhesion to 4-7VCAM-1 but had no effect on adhesion to 6d-VCAM-1. Consistent with the antibody and pharmacological blocking experiments, eosinophilic leukemic cell lines lacking alphaMbeta2 did not adhere to 4-7VCAM-1 but did adhere to 6d-VCAM-1 or 1-3VCAM-1. Activation of eosinophils by interleukin (IL)-5 enhanced static adhesion to 6d-VCAM-1, 7d-VCAM-1, or 4-7VCAM-1; IL-5-enhanced adhesion to all 3 constructs was blocked by anti-alphaMbeta2. Adhesion of unstimulated eosinophils to 7d-VCAM-1 under flow conditions was inhibited by anti-alpha4 or anti-alphaM. IL-5 treatment decreased eosinophil adhesion to 7d-VCAM-1 under flow, and anti-alphaM had the paradoxical effect of increasing adhesion. These results demonstrate that alphaMbeta2 modulatesalpha4beta1-mediated eosinophil adhesion to VCAM-1 under both static and flow conditions.     

Swimming in the sea hare Aplysia brasiliana: Cost of transport, parapodial morphometry, and swimming behavior

The energetics and behavior of the parapodial-swimming Aplysia brasiliana were investigated in order to compare net cost of transport (COT_net) between swimming and crawling, and to compare transport costs with other swimmers. Oxygen consumption (V_O2) increased with increasing animal mass for resting, crawling, and swimming animals. Slopes of the regressions of log V_O2 on log mass were 0.90, 0.91, and 0.89 for resting, crawling, and swimming, respectively. The regression for resting V_O2 on mass was significantly lower than regressions of crawling and swimming on mass, which fell into a statistically homogenous subgroup. During 4-h swimming bouts, parapodial beat frequency dropped by less than 10% of starting values after 2 h and then stabilized for the remainder of the trial, whereas velocity steadily decreased to about 70% of starting values over the 4-h period. Initial beat frequency (at the start of a swimming bout) was negatively related to body mass, varying from 1.1 beat s^− 1 for a 34 g individual to 0.7 beats s^− 1 for a 500 g individual. Final beat frequency (at the end of a swimming bout) was also negatively related to body mass, but had a significantly lower intercept than initial beat frequency. Neither initial swimming velocity nor final swimming velocity was related to mass, but final velocity was significantly lower than initial velocity. A 250 g A. brasiliana swam at 345 m h^− 1 and crawled at 7 m h^− 1. Swimming COT_net (0.1 ml O₂ kg^− 1 m^− 1) for a 250 g A. brasiliana was 50 times less than crawling COT_net (5.3 ml O₂ kg^− 1 m^− 1). While the crawling COT_net for A. brasiliana fell within the range of other marine gastropods, swimming COT_net was less than that of swimming crustaceans, and much less than another gastropod, Melibe leonina, that uses lateral bending to swim.     

Habitat use, movement, and growth of young-of-the-year Fundulus spp. in southern New Jersey salt marshes: Comparisons based on tag/recapture

We examined habitat use, movement, and growth of young-of-the-year (YOY) Fundulus heteroclitus and Fundulus luciae with a tag/recapture experiment in tide-dominated salt marshes to determine if movements from Spartina marsh surface can account for the occurrence of larger, older individuals in other habitats. Evaluation of the tagging techniques in laboratory experiments with YOY F. heteroclitus (15-35 mm TL) found that coded wire tags were retained at least up to 77 days. The high rates of recapture in the field also indicate that the tagging approach generally worked well. Of a total of 5748 YOY F. heteroclitus (14-40 mm TL) and 133 YOY F. luciae (17-40 mm TL) tagged, 56.0% and 74.4% were recaptured, respectively. Most (44%) YOY F. heteroclitus recaptured occurred at or near (0-5 m) the release site, but some were captured up to 299 m away up to 166 days after tagging. By comparison, movement of F. luciae was very limited, with 99% of recaptures occurring at the exact site of release after up to 66 days at liberty. These different movement patterns by YOY Fundulus indicate that species-specific behavior plays an important role in habitat selection. In addition, it appears that dispersal of YOY F. heteroclitus can help to explain the occurrence of larger individuals of this species in Phragmites-dominated marshes even though there is little evidence of use of this habitat by small YOY.