An in vitro model of epithelial cell growth stimulation in the rodent mammary gland

Abstract. Mouse mammary epithelial cell cultures previously described bring about extensive proliferation and a cell population with the appropriate markers for luminal ductal epithelial cells, and also the ability to form normal tissue after implantation into mice. This success may result from a culture environment that resembles certain aspects of the environment in the mammary gland. Mouse mammary epithelial cells, whose proliferation is limited when plated alone, can be stimulated to multiply by contact with lethally irradiated cells of the LA7 rat mammary tumour line. Most of the proliferative stimulus is imparted by direct cell contact between LA7 and mouse mammary cells. Junctions, including adherens junctions, form among all cells in the culture, much as junctions form in the mammary gland. LA7 cells secrete TGFα and bFGF, factors found in the mammary gland, and factors to which mouse mammary cells respond in culture. Mouse mammary cells express keratins 8 and 18, markers for luminal cells of the mammary duct. LA7 cells express keratin 14 and vimentin, markers for myoepithelial cells. These facts, taken together, fit a model of cell replacement in an epithelial tissue and also imitate the relationship between luminal ductal cells and myoepithelial cells in the mammary gland. This method of culturing cells is useful, not only for in vitro – in vivo carcinogenesis studies, but also for the study of mechanisms by which growth signals are imparted from one cell to another.     

A prospective observational study of community-acquired bacterial bloodstream infections in Metro Manila,the Philippines

Community-acquired bacterial bloodstream infections are caused by diverse pathogens with changing antimicrobial-resistance patterns. In low-middle income countries in Southeast Asia, where dengue fever is endemic and a leading cause of fever, limited information is available about bacterial bloodstream infections due to challenges of implementing a blood culture service. This study describes bacterial bloodstream pathogens and antimicrobial-resistance patterns in Metro Manila, the Philippines. We aimed to identify the proportion of patients with a positive blood culture, the bacteria isolated and their antimicrobial resistance patterns, and the clinical characteristics of these patients, in this dengue endemic area. We conducted a prospective observational study in a single hospital enrolling febrile patients clinically suspected of having a community-acquired bacterial bloodstream infection between 1^st July 2015 and 30^th June 2019. Each patient had a blood culture and additional diagnostic tests according to their clinical presentation. We enrolled 1315 patients and a significant positive blood culture was found in 77 (5.9%) including Staphylococcus aureus (n = 20), Salmonella enterica Typhi (n = 18), Escherichia coli (n = 16), Streptococcus pneumoniae (n = 3) and Burkholderia pseudomallei (n = 2). Thirty-four patients had meningococcal disease diagnosed by culture (n = 8) or blood PCR (n = 26). Additional confirmed diagnoses included leptospirosis (n = 177), dengue virus infection (n = 159) and respiratory diphtheria (n = 50). There were 79 (6.0%, 95%CI 4.8%−7.4%) patients who died within 28 days of enrollment. Patients with a positive blood culture were significantly more likely to die than patients with negative culture (15.2% vs 4.4%, P<0.01). Among S. aureus isolates, 11/20 (55%) were methicillin-resistant (MRSA) and ST30: USA1100 was dominant sequence type (88.9%). Antimicrobial-susceptibility was well preserved in S. enterica Typhi. Among hospitalized patients with clinically suspected community-acquired bacterial bloodstream infection in Metro Manila, the Philippines, 5.9% had a blood culture confirmed infection of whom 15.6% died. S. aureus, including a significant number of MRSA (USA1100 clones), S. enterica Typhi, E.coli and Neisseria meningitidis were frequently identified pathogens.     

Effect of storage time of pleural effusions on immunocytochemical cell surface analysis of tumor cells.

To determine how long tumor cells can be stored without losing their immunocytochemical reactivity, five malignant pleural effusions in EDTA-coated tubes were analyzed after different storage times at either 4 degrees C or room temperature. Only minor differences were observed between the cells from the two storage conditions. Though there was a considerable decrease in the number of tumor cells attached to the slides from day 0 to 1, the number of tumor cells was still sufficient to allow their clear detection with the monoclonal antibody HEA-125 even on day 4 of storage in all cases. Therefore, for routine purposes, pleural fluids in EDTA-coated tubes can be stored for at least one day prior to immunocytochemical staining if the cells are gently handled during preparation. Pleural fluid is a richly nutritious medium not only for keeping cells alive but also for preserving their immunoreactivity.     

Biochemical comparison of two proteolytic enzymes from Carica candamarcensis: Structural motifs underlying resistance to cystatin inhibition

The lattices of Carica candamarcensis and Carica papaya, members of the Caricaceae family, contain isoforms of cysteine proteinases that help protect these plants against injury. In a prior study, we fractionated 14 discrete proteinaceous components from C. candamarcensis, two of them displaying mitogenic activity in mammalian cells. In this study, we compared the kinetic parameters of one of the mitogenic proteinases (CMS2MS2) with one of the isoforms displaying the highest enzyme activity of this group (CMS1MS2). Both enzymes display a similar Km value with either BAPNA (Benzoyl-Arg-pNA) or PFLPNA (Pyr-Phe-Leu-pNA), but the kcat of CMS1MS2 is about 14-fold higher for BAPNA and 129-fold higher with PFLPNA. While both enzymes are inhibited by E-64 and iodoacetamide, chicken cystatin fully inhibits CMS1MS2, but scarcely affects activity of CMS2MS2. Based on the structure of these proteins and other enzymes from the Caricaceae family whose structures have been resolved, it is proposed that Arg¹⁸⁰ located in the cleft at the active site in CMS2MS2 is responsible for its resistance to cystatin.     

Self-organization of trajectory formation

 Most studies examining the stability and change of patterns in biological coordination have focused on identifying generic bifurcation mechanisms in an already active set of components (see Kelso 1994). A less well understood phenomenon is the process by which previously quiescent degrees of freedom (df ) are spontaneously recruited and active df suppressed. To examine such behavior, in part I we study a single limb system composed of three joints (wrist, elbow, and shoulder) performing the kinematically redundant task of tracing a sequence of two-dimensional arcs of monotonically varying curvature, κ. Arcs were displayed on a computer screen in a decreasing and increasing κ sequence, and subjects rhythmically traced the arcs with the right hand in the sagittal plane at a fixed frequency (1.0 Hz), with motion restricted to flexion-extension of the wrist, elbow, and shoulder. Only a few coordinative patterns among the three joints were stably produced, e.g., in-phase (flexion-extension of one joint coordinated with flexion-extension of another joint) and antiphase (flexion-extension coordinated with extension-flexion). As κ was systematically increased and decreased, switching between relative phase patterns was observed around critical curvature values, κ_c. A serendipitous finding was a strong 2:1 frequency ratio between the shoulder and elbow that occurred across all curvature values for some subjects, regardless of the wrist-elbow relative phase pattern. Transitions from 1:1 to 2:1 frequency entrainment and vice versa were also observed. The results indicate that both amplitude modulation and relative phase change are utilized to stabilize the end-effector trajectory. In part II, a theoretical model is derived from three coupled nonlinear oscillators, in which the relative phases (φ) between the components and the relative joint amplitudes (ρ) are treated as collective variables with arc curvature as a control parameter. Received: 2 February 1996/Accepted in revised form: 13 December 1996     

Direct procedure for the determination of the number of replication forks and the reinitiation fraction in bacteria

A direct method for calculating the average number of replicationforks per chromosome in an exponentially growing bacterial cultureand the fraction of reinitiation after an inductive treatmentof the initiation step is presented. This method has allowedthe development of REPLJCON, a computer program designed forthe resolution of the algorithm and simulation of the bacterialchromosome replication. Using REPLJCON the following parameterscan be obtained: average number of replication forks per chromosome,time required for the complete replication cycle, average amountof DNA per nucleotide, gene frequency of any chromosomal locusand reinitiation fraction. The use of this analysis also permitsthe determination of the uni- or bidirectionality of replication. Received on September 30, 1987; accepted on May 10, 1988     

Phytochemical variation inCondalia microphylla (Rhamnaceae)

Seed oil composition (sterol esters and sterols) and seed protein profiles ofCondalia microphylla were investigated. The chemical differences observed may be attributed to genotypic changes and could support the existence of infraspecific taxa.     

Potential vessel collisions with Southern Hemisphere humpback whales wintering off Pacific Panama

Vessel collision is a threat to many whale species, and the risk has increased with expanding maritime traffic. This compromises international conservation efforts and requires urgent attention from the world's maritime industry. Humpback whales (Megaptera novaeangliae) are at the top of the death toll, and although Central America is a wintering area for populations from both the Northern and Southern Hemispheres, existing efforts to reduce ship‐whale collisions are meager. Herein, we evaluated the potential collisions between vessels and humpback whales wintering off Pacific Panama by following the movements of 15 whales tagged with satellite transmitters and comparing these data with tracks plotted using AIS real‐time latitude‐longitude points from nearly 1,000 commercial vessels. Movements of whales (adults and calves) in the Gulf of Panama coincide with major commercial maritime routes. AIS vessel data analyzed for individual whale satellite tracks showed that 53% (8 whales) of whales had 98 encounters within 200 m with 81 different vessels in just 11 d. We suggest implementing a 65 nmi Traffic Separation Scheme and a 10 kn maximum speed for vessel routing into the Gulf of Panama during the wintering season. In so doing, the area for potential whale‐vessel collisions could be reduced by 93%.     

Distribution patterns of the genus Pacifigorgia (Octocorallia: Gorgoniidae): track compatibility analysis and parsimony analysis of endemicity

Aim We analysed the distribution patterns of the eastern Pacific octocoral genus Pacifigorgia and deduced its ancestral distribution to determine why Pacifigorgia is absent from the Gulf of Mexico, the Caribbean of central America, and the Antilles. We also examined the current patterns of endemism for Pacifigorgia to look for congruence between hot spots of endemism in the genus and generally recognized areas of endemism for the eastern Pacific. Location The tropical eastern Pacific and western Atlantic, America. Methods We used track compatibility analysis (TCA) and parsimony analysis of endemicity (PAE) to derive ancestral distribution patterns and hot spots of endemism, respectively. Distributional data for Pacifigorgia were gathered from several museum collections and from fieldwork, particularly in the Pacific of Costa Rica and Panama. Results A single generalized track joined the three main continental eastern Pacific biogeographical provinces and the western Atlantic. This track can be included within a larger eastern Atlantic–eastern Pacific transoceanic track that may be the oldest transoceanic track occurring in the region. PAE results designate previously recognized eastern Pacific biogeographical provinces as Pacifigorgia hot spots of endemism. The number of endemic species, which for other taxonomic groups is similar among the eastern Pacific provinces, is higher in the Panamic province for Pacifigorgia. Main conclusions We propose that the absence of Pacifigorgia from the Gulf of Mexico, the Caribbean of central America, and the Antilles is the result of an ancient absence of the genus from these areas rather than the consequence of a major, recent, extinction episode. The Cortez province and the Mexican province appear together as a result of either non‐response to vicariance or dispersal across the Sinaloan Gap. We posit that the Central American Gap acts as a barrier that separates the Panamic province from the northern Cortez–Mexican province.     

Novel Immunomodulators from Hard Ticks Selectively Reprogramme Human Dendritic Cell Responses

Hard ticks subvert the immune responses of their vertebrate hosts in order to feed for much longer periods than other blood-feeding ectoparasites; this may be one reason why they transmit perhaps the greatest diversity of pathogens of any arthropod vector. Tick-induced immunomodulation is mediated by salivary components, some of which neutralise elements of innate immunity or inhibit the development of adaptive immunity. As dendritic cells (DC) trigger and help to regulate adaptive immunity, they are an ideal target for immunomodulation. However, previously described immunoactive components of tick saliva are either highly promiscuous in their cellular and molecular targets or have limited effects on DC. Here we address the question of whether the largest and globally most important group of ticks (the ixodid metastriates) produce salivary molecules that specifically modulate DC activity. We used chromatography to isolate a salivary gland protein (Japanin) from Rhipicephalus appendiculatus ticks. Japanin was cloned, and recombinant protein was produced in a baculoviral expression system. We found that Japanin specifically reprogrammes DC responses to a wide variety of stimuli in vitro, radically altering their expression of co-stimulatory and co-inhibitory transmembrane molecules (measured by flow cytometry) and their secretion of pro-inflammatory, anti-inflammatory and T cell polarising cytokines (assessed by Luminex multiplex assays); it also inhibits the differentiation of DC from monocytes. Sequence alignments and enzymatic deglycosylation revealed Japanin to be a 17.7 kDa, N-glycosylated lipocalin. Using molecular cloning and database searches, we have identified a group of homologous proteins in R. appendiculatus and related species, three of which we have expressed and shown to possess DC-modulatory activity. All data were obtained using DC generated from at least four human blood donors, with rigorous statistical analysis. Our results suggest a previously unknown mechanism for parasite-induced subversion of adaptive immunity, one which may also facilitate pathogen transmission.