Growth rate and production of a microcin by Salmonella typhimurium LT2 (strain M799-0) in continuous culture

The production of a bacteriostatic microcin, of molecular weight 520 kD by Salmonella typhimurium LT2, strain M799-0, was studied in continuous culture. The effect of specific growth rate (mu) on microcin production was measured under aerobic and anaerobic conditions. In aerobiosis the microcin specific production rate (qp) was found to be strictly associated with cell growth. Thus, it is constitutively synthesized though at low production rate. As growth conditions become limited (i.e. anaerobiosis) its production is clearly induced and the highest yields are reached. Under the latter conditions microcin production follows a kinetic partial growth-linked process.     

Polymorphonuclear leukocyte migration through human amnion membrane

A new in vitro model has been developed for studying migration of human polymorphonuclear leukocytes (PMN) through living native cellular and matrix barriers. Human amnion membrane consists of a single layer of epithelium bound to a continuous basement membrane interfacing an avascular collagenous stroma. Living amnion was placed in plastic chambers with separate compartments on each side of the membrane. PMN were introduced on the epithelial side of the amnion, and a Millipore filter (Millipore Corp., Bedford, Mass.) was placed against the stromal side. In response to N-formylmethionyl-leucyl- phenylanlanine (FMLP) chemoattractant, PMN penetrated the full thickness of the amnion and were collected and counted on the filter. The rate of PMN traversal of the amnion was dependent on the concentration of FMLP (optimal at 10(-8)M) as well as the slope of the FMLP gradient across the amnion. The route of PMN migration was studied by transmission electron microscopy. PMN first attached to the epithelial surface, then infiltrated between intercellular junctions. PMN migrated around or through tight junction and hemidesmosome attachments. The PMN then penetrated the basement membrane and migrated through the dense collagenous stroma. The present amnion migration system has characteristics of the in vivo inflammatory state not described in any previous method for monitoring PMN migration in vitro. Prior methods have not used native epithelium, whole basement membrane, or collagenous stroma. PMN penetration of these barriers occurs in the normal inflammatory response and probably involves biochemical mechanisms not required for simple migration through the pores of an artificial filter. The amnion system can be useful for future biochemical and morphological studies of PMN penetration of these barriers and possible repair processes that may follow.     

Vergleichende Untersuchungen zum Kontaktverhalten verschiedener Arten der Gattung Tilapia (Cichlidae,Pisces) und ihrer Bastarde

• 1 Tilapia tholloni (Substratbrüter), T. nilotica (weiblicher Maulbrüter) und T. heudeloti macrocephala (männlicher Maulbrüter) wurden künstlich erbrütet und ihre angeborenen Kontaktreaktionen in standardisierten Attrappenversuchen untersucht.
• 2 Das Kontaktverhalten muß während einer kritischen Phase (bei T. nilotica unter den angegebenen Versuchsbedingungen bis ungefähr zum 21. Tag nach dem Ablaichen) aktiviert werden, wenn es längere Zeit andauern soll (= Reaktionsphase). Die Reaktionsphase kann bei T. nilotica mehrere Wochen dauern. In ihr nimmt die Reaktionsstärke (Anzahl und Dauer der Kontakte) zunächst rasch zu, erreicht ein Maximum und nimmt dann allmählich wieder ab. Anstieg, Maximum und Abnahme sind an bestimmte Entwicklungsabschnitte gebunden, weitgehend unabhängig davon, ob die Tiere zuvor schon Kontaktverhalten geäußert haben oder nicht. Die Kontaktreaktionen unterscheiden sich u. a. durch die Dauer der Kontakte: tholloni = 0,7 Sek., nilotica = 86,5 Sek., heudeloti m. = 1,5 Sek. je Tag und Tier (Maximalwerte bei bestimmten, für alle Arten gleichen Versuchsbedingungen).
• 3 Von der Aufzuchttemperatur hängt es ab, in welchem Entwicklungsabschnitt die Reaktionsphase liegt. Das Reaktionsmaximum junger T. nilotica lag bei 24° C am 9., bei 29° C am Tag nach der Eiablage.
• 4 Geblendete nilotica-Jungfische zeigten nur zu Beginn der Reaktionsphase schwaches Kontaktverhalten, normale Tiere äußerten gegenüber einer durchsichtigen Glasattrappe abgeschwächtes, nur kurze Zeit dauerndes Kontaktverhalten. Das Kontaktverhalten wird durch mechanische (Strömung) und optische Reize ausgelöter und gesteuert.
• 5 Junge Maulbrüter aus kleinen Eiern erreichten eine längere Kontaktdauer als solche aus größeren. Nach künstlicher Reduktion der Dottermenge um 10–20%) erhöhte sich die Kontaktdauer bei jungen nilotica um 23,1%, die Zahl der Kontakte nahm gleichzeitig um 7,6% ab. Die ♀♀ der substratbrütenden T. mariae legen große Eier, die Jungen zeigten gegenüber Attrappen intensive Kontaktreaktionen. Es wird die Frage diskutiert, ob das Erreichen einer bestimmten Eigröße eine Voraussetzung für das Entstehen des Kontaktverhaltens gewesen sein könnte.
• 6 Die Kurzkontakte junger T. tholloni werden aus ihrer Orientierungsreaktion abgeleitet und als Vorstufe des Kontaktverhaltens gedeutet.
• 7 7. Mehrere Tilapia-Arten wurden künstlich gekreuzt. Bei Verwendung von tholloni-Sperma wiesen die Bastarde eine erhöhte Sterblichkeit auf. Aus der Kreuzung T. tholloni ♀ ～ T. nilotica ♂ (Substratbrüter ～ weiblicher Maulbrüter) gingen nur ♀♀ hervor. Die Kreuzung T. heudeloti macrocephala ～ T. nilotica (männlicher ～ weiblicher Maulbrüter) erbrachte fertile F₁- und F₂-Generationen sowie alle vier möglichen Rückkreuzungen. Bei der Vererbung des Kontaktverhaltens (gemessen an der Dauer der Kontakte) scheinen relativ wenig Erbfaktoren mitzuwirken, T. heudeloti m. erwies sich gegenüber nilotica als praevalent. Ein Teil der Bastarde aus der Kreuzung T. tholloni ♀ ～ T. nilotica ♂ (F₁) (Substratbrüter ～ weiblicher Maulbrüter) lag auf der Merkmalsskala zwischen den Ausgangsarten, der Rest verteilte sich sowohl auf den Bereich von tholloni als auch auf den Bereich von nilotica.

     

Using functional groups to investigate community response to environmental changes: two grassland case studies

Functional groups (FG) are an useful generalization to investigate environmental change effects on biotic communities. Assigning species to FGs is a contextual task and carries an arbitrary element, regardless of whether the grouping is obtained a priori or by sophisticated numerical methods. Using two grassland community case studies, we show that even simple FG allocation based on growth form, architecture and longevity (plants and mosses), or foraging characteristics (above-ground invertebrates) can be useful to increase our understanding of community processes. For example, the sensitivity of organisms to climate change increases with trophic rank and is higher in disturbed than in undisturbed communities. Complexity of interaction webs (in terms of web connectance), however, is larger in undisturbed than in disturbed communities. A significant and important relationship is likely to exist between anthropogenic disturbances, community complexity and the ecosystem effects of climate change. Trophic interactions may be disrupted much easier by climate changes in disturbed than in undisturbed communities where complexity may be buffering these effects.     

Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways

There are several pathways for the incorporation of sugars into glycosphingolipids (GSL). Sugars can be added to ceramide that contains sphinganine (dihydrosphingosine) synthesized de novo (pathway 1), to ceramide synthesized from sphingoid bases produced by hydrolysis of sphingolipids (pathway 2), and into GSL recycling from the endosomal pathway through the Golgi (pathway 3). We reported previously the surprising observation that SW13 cells, a human adrenal carcinoma cell line, synthesize most of their GSL in pathway 2. We now present data on the synthesis of GSL in four additional cell lines. Approximately 90% of sugar incorporation took place in pathway 2, and 10% or less in pathway 1, in human foreskin fibroblasts and NB41A3 neuroblastoma cells. In contrast, approximately 50-90% of sugar incorporation took place in pathway 1 in C2C12 myoblasts. The C2C12 cells divide more rapidly and synthesize 10-14 times as much GSL as the other three cell lines. In C6 glioma cells, approximately 30% of sugar incorporation occurred in pathway 1 and 60% in pathway 2. There was no relation between the utilization of pathways for GSL and sphingomyelin synthesis in foreskin fibroblasts and C2C12 cells. In both cells pathways 1 and 2 each accounted for 50% of incorporation of choline into sphingomyelin. In five of the six cell lines that we have studied, most GSL synthesis takes place in pathway 2. We suggest that when the need for synthesis is relatively low, as in slowly dividing cells, GSL are synthesized predominantly from sphingoid bases salvaged from the hydrolytic pathway. When cells are dividing more rapidly, the need for increased synthesis is met by upregulating the de novo pathway.      

Isolation and Genotyping of Acanthamoeba Strains from Soil Sources from Jamaica,West Indies

Acanthamoeba spp. are opportunistic pathogens that are ubiquitous in nature. Many species of this genus are responsible for a fatal encephalitis and keratitis in humans and other animals. Seventy‐two soil samples were collected from the parishes across Jamaica and assessed for the presence of Acanthamoeba spp. Cultivation was carried out on non‐nutrient agar plates seeded with heat killed Escherichia coli. PCR and sequencing of the DF3 region were carried out in order to genotype the isolated strains of Acanthamoeba. Thermotolerance and osmotolerance assays were utilized to investigate the pathogenic potential of the Acanthamoeba isolates. Acanthamoeba spp. was isolated from 63.9% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T4, T5, and T11. T4 genotype was most frequently isolated. Most isolates were thermotolerant or both thermotolerant and osmotolerant, indicating that they may present the potential to cause disease in humans and other animals.     

Improvements in obtaining New World Leishmania sp from mucosal lesions: Notes on isolating and stocking parasites

Tegumentary leishmaniasis is an endemic protozoan disease that, in Brazil, is caused by parasites from Viannia or Leishmania complex. The clinical forms of cutaneous disease comprise localized, disseminated, mucosal or mucocutaneous, and diffuse leishmaniasis. Viannia complex parasites are not easy to isolate from patient lesions, especially from mucosal lesions, and they are difficult to culture. The aim of the present study was to compare the efficiency of ex vivo (culture) and in vivo (IFNγ-deficient mice) parasite isolation methods to improve the isolation rate and storage of stocks of New World Leishmania sp that cause cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Biopsy fragments from cutaneous or mucosal lesions were inoculated into culture medium or mouse footpads. We evaluated 114 samples (86 CL, 28 ML) using both methods independently. Samples from CL patients had a higher isolation rate in ex vivo cultures than in mice (34.1% vs. 18.7%, P<0.05). Nevertheless, almost twice the number of isolates from ML lesions was isolated using the mouse model compared to ex vivo cultures (mouse, 6/25; culture, 3/27). The overall rates of isolation were 40.2% for CL samples and 29.6% for ML samples. Of the 43 isolations, we successfully stocked 35 isolates (81.4%; 27 CL, 8 ML). Contaminations were more frequently detected in cultures of ML than CL lesions. For comparison, the use of both methods simultaneously was performed in 74 samples of CL and 25 samples of ML, and similar results were obtained. Of the eight ML isolates, five were isolated only in mice, indicating the advantage of using the in vivo method to obtain ML parasites. All parasites obtained from in vivo isolation were cryopreserved, whereas only 68% of ex vivo isolations from CL lesions were stocked. In conclusion, the use of genetically modified mice can improve the isolation of parasites from ML. Isolation and stocking of New World Leishmania parasites, especially those from ML that are almost absent in laboratory stocks, are critical for evaluating parasite genetic diversity as well as studying host-parasite interactions to identify biological markers of Leishmania. In this paper, we also discuss some of the difficulties associated with isolating and stocking parasites.     

Synthetic menthyl α/β-(1→6)-diglucopyranosides-induced cell death in human leukemia cells is dependent on caspases

A series of alkyl α/β-(1→6)-diglucopyranosides 1-12 were synthesized and assessed for cytotoxicity against HL-60, U937, Molt-3 and MCF-7 cancer cell lines. The menthyl derivatives displayed strong cytotoxic properties showing IC(50) values between 6 and 16 μM. Furthermore, we demonstrated that the selected synthetic (+)-menthyl β-(1→6)-diglucopyranoside 5 induces apoptotic cell death in human leukemia cells through a mechanism that involves activation of multiple caspases. Cell death was completely prevented by the non-specific caspase inhibitor z-VAD-fmk and found to be associated with the release of cytochrome c, an increase in the expression of Bax levels and a decrease in the generation of reactive oxygen species.     

Vimang (Mangifera indica L. extract) induces permeability transition in isolated mitochondria, closely reproducing the effect of mangiferin, Vimang's main component

Mitochondrial permeability transition (MPT) is a Ca(2+)-dependent, cyclosporin A (CsA)-sensitive, non-selective inner membrane permeabilization process. It is often associated with apoptotic cell death, and is induced by a wide range of agents or conditions, usually involving reactive oxygen species (ROS). In this study, we demonstrated that Mangifera indica L. extract (Vimang), in the presence of 20 microM Ca(2+), induces MPT in isolated rat liver mitochondria, assessed as CsA-sensitive mitochondrial swelling, closely reproducing the same effect of mangiferin, the main component of the extract, as well as MPT-linked processes like oxidation of membrane protein thiols, mitochondrial membrane potential dissipation and Ca(2+) release from organelles. The flavonoid catechin, the second main component of Vimang, also induces MPT, although to a lesser extent; the minor, but still representative Vimang extract components, gallic and benzoic acids, show respectively, low and high MPT inducing abilities. Nevertheless, following exposure to H(2)O(2)/horseradish peroxidase, the visible spectra of these compounds does not present the same changes previously reported for mangiferin. It is concluded that Vimang-induced MPT closely reproduces mangiferin effects, and proposed that this xanthone is the main agent responsible for the extract's MPT inducing ability, by the action on mitochondrial membrane protein thiols of products arising as a consequence of the mangiferin's antioxidant activity. While this effect would oppose the beneficial effect of Vimang's antioxidant activity, it could nevertheless benefit cells exposed to over-production of ROS as occurring in cancer cells, in which triggering of MPT-mediated apoptosis may represent an important defense mechanism to their host.