Cytochemical examination of the compartments involved in the transcellular transport of horseradish peroxidase in rat hepatocytes

Horseradish peroxidase (HRP, 10 mg/100 g body weight) was intravenously injected into rats in order to investigate the nature of the compartments involved in the transcellular transport of the protein through hepatocytes into bile. Double cytochemistry for HRP and the marker enzymes for cytoplasmic organelles was used. HRP was shown to be taken up by hepatocytes via vesicles at the sinusoidal surface, some of which were positive for 5'-nucleotidase activity. HRP was then found in the smooth-surfaced vesicles and tubules which were negative in 5'-nucleotidase, glucose 6-phosphatase, thiamine pyrophosphatase and acid phosphatase activity, suggesting that the tubular structures are neither the endoplasmic reticulum, the Golgi apparatus nor lysosomes. Biochemical studies revealed that the lead procedures used for the double cytochemistry did not inhibit the peroxidatic activity of HRP, and conversely that HRP did not interfere with the marker enzyme activity. Such cytochemical observations seemed to be supported by the observation that administration of monensin (3.5 mg/100 g) and chloroquine (5 mg/100 g), which markedly altered the structure of the Golgi apparatus and lysosomes, respectively, slightly altered the biliary excretion of HRP but not to a significant extent.     

Translocation of hepatocyte lysosomes following partial hepatectomy and its inhibition by colchicine

Hepatocyte microtubules were studied during the translocation of lysosomes which follows partial hepatectomy. In hepatocytes of normal rat liver the lysosomes are seen mainly along the bile canaliculi. After partial hepatectomy, these lysosomes lose their pericanalicular arrangement and move towards large inclusions (‘protein droplets’) which result from the marked pinocytosis induced by the removal of about two-thirds of the liver mass. The lysosomes fuse with the inclusions, and by 4 h after partial hepatectomy most of the inclusions demonstrate acid phosphatase activity histochemically. Many microtubules are found in close proximity to the lysosomes. When the rats are treated with colchicine prior to the partial hepatectomy, events are markedly different. The lysosomes change their location but do not hit the cytoplasmic inclusions, and the inclusions remain negative for acid phosphatase activity even 4 h post-hepatectomy. Quantitative ultrastructural study shows that the volume density of microtubules in the hepatocytes decreases to one-third of that in control hepatocytes. This strongly suggests an involvement of microtubules in giving orientation to the translocation of hepatocyte lysosomes under these conditions.     

Proliferation-associated increase in sensitivity of mammary epithelial cells to inositol-1,4,5-trisphosphate

Injection of D -myo-inositol-1,4,5-trisphosphate (IP3) was found to induce a transient increase of intracellular Ca²⁺ concentration in cancerous mammary cells (MMT060562) and in normal mammary cells treated with epidermal growth factor. Responses to injection of either D -myo-inositol-1,4-bisphosphate (IP2) or D -myo-inositol-1,3,4,5-tetrakisphosphate (IP4) were small or absent. Furthermore, normal mammary cells cultivated with low-protein serum replacement alone or in the presence of differentiation-inducing hormones (insulin + cortisol + prolactin) were less sensitive to IP3. Thapsigargin induced a transient increase of Ca²⁺ due to the release of Ca²⁺ from an intracellular pool. There was no difference in the peak heights of the thapsigargin-induced Ca²⁺ increase when mammary cells were cultivated in the presence or absence of epidermal growth factor or insulin + cortisol + prolactin. These findings suggest that the releasable intracellular Ca²⁺ pool remained unchanged whereas sensitivity to IP3 increases during the proliferation stage. Mechanical stimulus of a mammary cell induces an increase of intracellular Ca²⁺ in the stimulated cell. A certain stimulating factor is released from the mechanically stimulated cell into the extracellular space, and it induces an increase of Ca²⁺ in surrounding cells.¹⁸ In contrast, the IP3-induced Ca²⁺ increase in both cancerous and epidermal growth factor-treated normal mammary cells did not spread to adjacent cells. Therefore, increase of Ca²⁺ is not sufficient to account for the release of stimulating substances from mammary cells in the mechanically-induced spreading response.     

A Combined Enrichment and Aptamer Pulldown Assay for Francisella tularensis Detection in Food and Environmental Matrices

Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1–10⁶ CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology.     

Insect Feeding Inhibitors in Plants

Shiromodiol-diacetate, shiromool, and shiromodiol-monoacetate are insect feeding inhibitors isolated from Parabenzoin trilobum Nakai. On the bases of chemical and spectral evidence we deduced that these compounds have the structure shown in I, XVIII, and XI, respectively.     

Isolation and Characterization of PDE-I and II,the Inhibitors of Cyclic Adenosine-3′,5′-monophosphate Phosphodiesterase

In the screening for inhibitors of cyclic adenosine-3′,5′-monophosphate phosphodiesterase, two compounds, PDE-I (C₁₃H₁₃N₃O₅) and PDE-II (C₁₄H₁₄N₂O₅), were isolated from culture filtrates of a Streptomyces. Concentrations for 50% inhibitions of PDE-I and PDE-II against the high Km enzyme were 15 µm and 13 µm, and those against the low Km enzyme were 65 µm and 130 µm, respectively. Production, isolation and characterization of these compounds are described.     

Total synthesis of aurachins C,D, and L,and a structurally simplified analog of aurachin C

The quinoline antibiotics aurachins C, D, and L, and a structurally simplified analog of aurachin C were synthesized from 1-(2-nitrophenyl)butane-1,3-dione via reductive cyclizations of δ-nitro ketone intermediates, with zinc or iron as key steps. The results of antimicrobial tests indicate that the N-hydroxyquinolone nucleus mimics the electron carrier in the respiratory chain more strongly than the quinoline N-oxide nucleus.     

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M1 and M4 muscarinic acetylcholine receptors agonists

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M₁ and M₄ muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M₁ and M₄ agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.