Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8-1%, in which approximately 20-50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.     

Orientation and ingestion rates of larval Anopheles albimanus in response to floating particles

Response of fourth-instar larvae of Anopheles albimanus Wiedemann (Diptera: Culicidae) to food and inert particles floating at the water surface was studied. In a choice test, larvae aggregated at powdered organic materials (blood meal, liver powder alfalfa flour and wheat flour) but not at inert materials (kaolin, chalk or charcoal). Larvae responded positively to proteins as well as some carbohydrates, but not to cellulose. Retention of larvae at food sources found by random locomotion was found to be responsible for larval aggregation. Larvae ingested food particles 6–9 times faster than insert particles. The significance of Anopheline feeding behavior in the development of formulations of stomach toxins (bacterial agents) used in larval control is discussed.

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Zusammenfassung Die vorliegende Studie befasst sich mit Verhaltensreaktionen von Anopheles albimanus Viertlarven auf an der Wasseroberfläche schwimmende Partikel. Verteilung und Orientierung der Larven wurde in einer Wahlapparatur quantifiziert. Nach Auftrag von Alfalfamehl, Weizenmehl, Stärke, Blutmehl, Leberpulver und Fischmehl wurde Aggregation der Larven in den beköderten Fächern der Apparatur beobachtet. Sowohl Proteine (Casein) als auch einige Kohlehydrate (Amylose, Amylopectin) lösten Aggregationen der Larven aus. Im Unterschied dazu führte Auftrag von Kreide, Kaolin, Polyaethylenpulver, Talcum oder Cellulose nicht zu Aggregationen. Zur Beschreibung der Entstehung larvaler Aggregationen bei Futterstoffen wurden die Schwimmbewegungen der Larven in Anwesenheit von Weizenmehl als Ködersubstanz quantifiziert. Da keine Attraktion der Larven im Sinne einer gerichteten Schwimmbewegung beobachtet werden konnte, wird geschlussfolgert, dass sofortige Beendung der Suchaktivität der Larven bei zufällig gefundenen Futterquellen für die beobachteten Aggregationen bei organischen Substanzen verantwortlich ist.Die Fressraten der Larven bei Angebot verschiedener Substanzen im Überschuss wurde bestimmt. Larven fülten drei von insgesamt sechs Darmabschnitten innerhalb von 15–30 min bei Angebot von Futtersubstanzen, während die Füllung von nur zwei Darmabschnitten mit inerten Materialien erst nach 90–120 min zu beobachten war. Die Resultate werden in Bezug auf wasseroberflächengebundene Formulierungen von Frassgiften diskutiert. Inerte Trägersubstanzen werden wahrscheinlich wesentlich langsamer aufgenommen als Futtersubstanzen. Da An. albimanus Larven nicht von Futterquellen angezogen werden, ist eine rasche und wirksame Giftaufnahme besonders dann zu erwarten, wenn die gesamte Oberfläche der Brutgewässer mit toxinhaltigen Trägerpartikeln bedeckt werden kann.

     

Immunological characterization of cytochrome H-450 in rat tissues

Rabbit antiserum produced against rat liver cytochrome H-450 was specific for cytochrome H-450. The antiserum did not react with hemolysate, microsomal and mitochondrial fractions of liver, and tissue extracts from heart, lung skeletal muscle, and testis of rat. With the monospecific antiserum, a rocket immunoelectrophoretic assay method was developed for the quantitation of the antigen with a sensitivity of 25 ng. By using rocket immunoelectrophoresis, the total amounts of the antigen found in liver, kidney, and brain of 20 rats were 33.6, 3.6, and 1.3 mg, respectively. It appears that the antigens in liver, kidney, and brain are immunologically identical. From immunological studies with subcellular fractions of rat liver, the antigen was found only in the postmicrosomal fraction. This indicates that the antigen is not a precursor or a proteolytic product of known cytochromes in mitochondria or microsomes. Therefore, cytochrome H-450 is a unique cytosolic protein found in brain, kidney, and liver.     

Novel Method for Detection of Butanolides in Streptomyces coelicolor Culture Broth, Using a His-Tagged Receptor (ScbR) and Mass Spectrometry

γ-Butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.     

Nonthermal Plasma‐Induced Degradation of Morin and Enhancement of Biological Activities

In the present study, non‐thermal dielectric barrier discharge (DBD) plasma of induced structural changes of morin resulted in the isolation of one previously undescribed benzofuranone derivative, along with two known compounds. The chemical structures of these degradation products were elucidated by UV, NMR and FAB‐MS spectroscopic analyses. The isolated three compounds showed potent antioxidative activities in two different tests, with IC₅₀ values in the range of 12.9–41.8 μm in the 2,2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS⁺) radical scavenging activity, 19.0–71.9 μm for hydroxyl radical scavenging activity test. Furthermore, the new methoxylated benzofuranone exhibited enhancement of inhibitory effects against pancreatic lipase with an IC₅₀ value of 90.7±1.6 μm , when compared to the parent morin. These results suggested that the degradation products isolated from plasma exposed morin might be beneficial for prevention of obesity and related diseases.     

Stabilizing ER Ca2+ Channel Function as an Early Preventative Strategy for Alzheimer’s Disease

Alzheimer’s disease (AD) is a devastating neurodegenerative condition with no known cure. While current therapies target late-stage amyloid formation and cholinergic tone, to date, these strategies have proven ineffective at preventing disease progression. The reasons for this may be varied, and could reflect late intervention, or, that earlier pathogenic mechanisms have been overlooked and permitted to accelerate the disease process. One such example would include synaptic pathology, the disease component strongly associated with cognitive impairment. Dysregulated Ca²⁺ homeostasis may be one of the critical factors driving synaptic dysfunction. One of the earliest pathophysiological indicators in mutant presenilin (PS) AD mice is increased intracellular Ca²⁺ signaling, predominantly through the ER-localized inositol triphosphate (IP₃) and ryanodine receptors (RyR). In particular, the RyR-mediated Ca²⁺ upregulation within synaptic compartments is associated with altered synaptic homeostasis and network depression at early (presymptomatic) AD stages. Here, we offer an alternative approach to AD therapeutics by stabilizing early pathogenic mechanisms associated with synaptic abnormalities. We targeted the RyR as a means to prevent disease progression, and sub-chronically treated AD mouse models (4-weeks) with a novel formulation of the RyR inhibitor, dantrolene. Using 2-photon Ca²⁺ imaging and patch clamp recordings, we demonstrate that dantrolene treatment fully normalizes ER Ca²⁺ signaling within somatic and dendritic compartments in early and later-stage AD mice in hippocampal slices. Additionally, the elevated RyR2 levels in AD mice are restored to control levels with dantrolene treatment, as are synaptic transmission and synaptic plasticity. Aβ deposition within the cortex and hippocampus is also reduced in dantrolene-treated AD mice. In this study, we highlight the pivotal role of Ca²⁺ aberrations in AD, and propose a novel strategy to preserve synaptic function, and thereby cognitive function, in early AD patients.