Steven B. Barlow

Pim-1 Kinase Protects Mitochondrial Integrity in Cardiomyocytes

Rationale: Cardioprotective signaling mediates antiapoptotic actions through multiple mechanisms including maintenance of mitochondrial integrity. Pim-1 kinase is an essential downstream effector of AKT-mediated cardioprotection but the mechanistic basis for maintenance of mitochondrial integrity by Pim-1 remains unexplored. This study details antiapoptotic actions responsible for enhanced cell survival in cardiomyocytes with elevated Pim-1 activity. Objective: The purpose of this study is to demonstrate that the cardioprotective kinase Pim-1 acts to inhibit cell death by preserving mitochondrial integrity in cardiomyocytes. Methods and Results: A combination of biochemical, molecular, and microscopic analyses demonstrate beneficial effects of Pim-1 on mitochondrial integrity. Pim-1 protein level increases in the mitochondrial fraction with a corresponding decrease in the cytosolic fraction of myocardial lysates from hearts subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Cardiac-specific overexpression of Pim-1 results in higher levels of antiapoptotic Bcl-XL and Bcl-2 compared to samples from normal hearts. In response to oxidative stress challenge, Pim-1 preserves the inner mitochondrial membrane potential. Ultrastructure of the mitochondria is maintained by Pim-1 activity, which prevents swelling induced by calcium overload. Finally, mitochondria isolated from hearts created with cardiac-specific overexpression of Pim-1 show inhibition of cytochrome c release triggered by a truncated form of proapoptotic Bid. Conclusion: Cardioprotective action of Pim-1 kinase includes preservation of mitochondrial integrity during cardiomyopathic challenge conditions, thereby raising the potential for Pim-1 kinase activation as a therapeutic interventional approach to inhibit cell death by antagonizing proapoptotic Bcl-2 family members that regulate the intrinsic apoptotic pathway.

Chattonella marina (Raphidophyceae), a potentially toxic alga in the Salton Sea, California

Chattonella marina was found in high abundance in the phytoplankton of the Salton Sea from April to November, 1997. Maximum mean density was over 600 cells ml−1 mid-lake. It was not detected from January 1997 to March 1997 or in January and February 1998. Transmission electron microscope studies show the typical Chattonella features: a teardrop-shaped nucleus, numerous radially arranged chloroplasts and lack of a cell wall. Gills of fish collected at the height of the Chattonella bloom showed damage to the primary and secondary lamellae and increased numbers of mucus cells. To our knowledge this is the first report of a species of Chattonella in a salt lake.

Penetration of the blood-brain barrier by Staphylococcus aureus: contribution of membrane-anchored lipoteichoic acid

Staphylococcus aureus is one of the most prevalent organisms responsible for nosocomial infections, and cases of community-acquired S. aureus infection have continued to increase despite widespread preventative measures. Pathologies attributed to S. aureus infection are diverse; ranging from dermal lesions to bacteremia, abscesses, and endocarditis. Reported cases of S. aureus-associated meningitis and brain abscesses have also increased in recent years, however, the precise mechanism whereby S. aureus leave the bloodstream and gain access to the central nervous system (CNS) are not known. Here we demonstrate for the first time that S. aureus efficiently adheres to and invades human brain microvascular endothelial cells (hBMEC), the single-cell layer which constitutes the blood–brain barrier (BBB). The addition of cytochalasin D, an actin microfilament aggregation inhibitor, strongly reduced bacterial invasion, suggesting an active hBMEC process is required for efficient staphylococcal uptake. Furthermore, mice injected with S. aureus exhibited significant levels of brain bacterial counts and histopathologic evidence of meningeal inflammation and brain abscess formation, indicating that S. aureus was able to breech the BBB in an experimental model of hematogenous meningitis. We found that a YpfP-deficient mutant, defective in lipoteichoic acid (LTA) membrane anchoring, exhibited a decreased ability to invade hBMEC and correlated to a reduced risk for the development of meningitis in vivo. Our results demonstrate that LTA-mediated penetration of the BBB may be a primary step in the pathogenesis of staphylococcal CNS disease.

Factor Associated With Neutral Sphingomyelinase Activation and Its Role in Cardiac Cell Death

Generation of proapoptotic sphingolipids by neutral sphingomyelinase activation is an early response to hypoxia/reoxygenation (HR) in cardiomyocytes. F actor a ssociated with n eutral sphingomyelinase activation (FAN) mediates activation of sphingomyelinase and subsequent apoptosis. However, the participation of FAN in HR-induced cardiomyocyte cell death has not been elucidated. We therefore investigated the expression and role of FAN in rat cardiomyocytes. A cDNA was isolated from rat heart encoding putative rat FAN. Reverse transcriptase–polymerase chain reaction, immunoelectron microscopy, and immunofluorescence demonstrated for the first time the expression of FAN specifically in rat cardiomyocytes. FAN expression was confirmed by the finding that expression of a dominant-negative FAN almost completely abrogated HR-induced cell death, whereas overexpression of wild-type FAN led to an increase. Treatment of FAN and dominant-negative FAN–expressing cells with C2-ceramide produced substantial cell death, indicating dominant-negative FAN exerts its protective action by interfering with the activation of the sphingolipid cascade. Taking these results together, we conclude that FAN is a previously undescribed and important HR signaling component in the heart and that inhibition of FAN may provide a novel intervention point for reducing ischemia/reperfusion injury.

Pleurochrysis pseudoroscoffensis (Prymnesiophyceae) blooms on the surface of the Salton Sea, California

Dense populations of the coccolithophore Pleurochrysis pseudoroscoffensis were found in surface films at several locations around the Salton Sea in February–August, 1999. An unidentified coccolithophorid was also found in low densities in earlier studies of the lake (1955–1956). To our knowledge, this is the first record of this widespread marine species in any lake. Samples taken from surface films typically contained high densities of one or two other phytoplankton species as well as high densities of the coccolithophore. Presence or absence of specific algal pigments was used to validate direct cell counts. In a preliminary screen using a brine shrimp lethality assay, samples showed moderate activity. Extracts were then submitted to a mouse bioassay, and no toxic activity was observed. These results indicate that blooms of P. pseudoroscoffensis are probably not toxic to vertebrates and do not contribute to the various mortality events of birds and fish that occur in the Salton Sea.

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae).

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.

SILICEOUS SCALE PRODUCTION IN CHRYSOPHYTE AND SYNUROPHYTE ALGAE. I. EFFECTS OF SILICA‐LIMITED GROWTH ON CELL SILICA CONTENT, SCALE MORPHOLOGY, AND THE CONSTRUCTION OF THE SCALE LAYER OF SYNURA PETERSENII1

The cells of synurophyte flagellates (algal class Synurophyceae, formerly included in the Chrysophyceae) are enclosed within a regularly imbricate layer of ornamented siliceous scales. Scale morphology is of critical taxonomic importance within this group of algae, and the scales are valuable indicator microfossils in paleolimnological studies. The data presented here demonstrate that scale morphology and the integrity of the scale layer can exhibit extreme variability in culture as a function of the cellular quota of silica under silica‐limited growth. Silica‐limited, steady‐state populations of the colonial flagellate Synura petersenii Korsh. were maintained over a range of specific growth rates (μ= 0.11–0.69 days−1) and silica cell quotas (Qsi= 0.13–2.40 pmoles Si · cell1). Scale morphology and the organization of the scale layer became increasingly aberrant as silica stress increased. Under severe stress, scale deposition was completely suppressed so that cells appeared scale‐free. This depression of scale deposition was reversible; populations of silica‐starved, scale‐free cells rapidly regenerated new scale layers when placed in batch culture and spiked with dissolved silica. During recovery from silica stress, cell division was repressed for 24 h while mean cell silica quota increased 25‐fold. The first new scales appeared within 2 h after the silica addition, and development of the new scale layer proceeded in an approximately synchronous manner, residting in normal scale layers on virtually all cells after 48 h of recovery in Sirich medium. Silica content of silica‐replete Synura cells is comparable to freshwater diatoms of siynilar size, but Synura has much greater potential quota variability than diatoms and no apparent threshold silica requirement. Silica‐limited growth kinetics and competition between diatoms and Synura for silica are discussed.

Cryptomonads from the Salton Sea, California

Using freshly collected field samples and enrichment cultures, eight genera of cryptomonads from the Salton Sea are recorded for the first time. Comparative data from light and scanning electron microscopy were utilized to identify these genera and species. The genera included Chroomonas, Falcomonas, Hemiselmis, Plagioselmis, Pyrenomonas/Rhodomonas, Storeatula, Teleaulax, and the kathablepharid Leucocryptos. One putative genus remains unidentified and may represent a new taxon. SEM has not been conducted on this cryptomonad, but it has been isolated and is being maintained in culture. The genera and species identified from the Salton Sea are typical of marine rather then freshwater environments and may play an important role in primary productivity and as preferred food organisms for zooplankton.

Similarities between Exogenously- and Endogenously- Induced Envelope Stress: The Effects of a New Antibacterial Molecule, TPI1609-10

Antibiotics with novel and/or multiple targets are highly desirable in the face of the steady rise of clinical antibiotic resistance. We have screened and identified small molecules, typified by the compound TPI1609-10 (aka SM10), with antibiotic activity against both gram-positive and gram-negative bacteria. SM10 was screened in vitro to bind branched Holliday junction intermediates of homologous recombination and tyrosine recombinase-mediated recombination; thus, the cellular targets of the small molecules were expected to include the RuvABC Holliday junction resolvasome and the XerCD complex involved in proper segregation of replicated chromosomes to daughter cells. SM10 indeed induces DNA damage and filamentation in E. coli. However, SM10 also induces envelope stress and causes increased production of intracellular reactive oxygen species. In addition, SM10 has similar effects to endogenously-induced envelope stress via overproducing outer membrane proteins (OmpC and OmpF), which also induces the SOS response, chromosome fragmentation, and production of reactive oxygen species. The synergy between SM10, and cerulenin, a fatty acid synthesis inhibitor, together with the SM10 hypersensitivity of cpx and rpoE mutants, further support that SM10s mode of action damages membrane damage. The lethality of SM10 treatment and of OmpC overproduction are observed in both aerobically- and anaerobically-grown cells, and is accompanied by substantial DNA damage even anaerobically. Thus, only some DNA damage is due to reactive oxygen. We propose that membrane depolarization and the potential reduction in intracellular pH, leading to abasic site formation, cause a substantial amount of the DNA damage associated with both SM10 treatment and endogenous envelope stress. While it is difficult to completely exclude effects related to envelope damage as the sources of DNA damage, trapping intermediates associated with DNA repair and chromosome segregation pathways remains very likely. Thus SM10 may have distinct but synergistic modes of action.

Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea

An infestation of young tilapia, Oreochromis mossambicus Peters, by the flagellate Cryptobia branchialiswas observed at the Salton Sea, California, in September, 1997. This is the first report of C. branchialis in a highly saline water-body (43 g l−1). Ultrastructure of C. branchialis as well as its effect on the gills of tilapia were studied using the scanning and transmission electron microscopy. No direct effect of C. branchialis on the epithelial cells of fish gills was observed. However, alterations of gill general structure, such as deposition of copious mucus on the gill surface, swelling of filaments, reduction of respiratory lamellae and their transformation into short club-shaped structures were found in infected fish. This suggests mortality of young tilapia may arise from decreased gill function in response to Cryptobia infestation.