Incubator concept

Comparison of incubator concepts for yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus), in Reinertsen H, Dahle L A, Jorensen L and Tvinnereim K (eds) Fish Farming Technology. Rotterdam Balkema, 51-56. 

By applying an extension of the clearance concept 30, 31), in vitro metabolism was used to predict in vivo toxin elimination. Hepatocytes were incubated with 0.5 to 10 pg unlabeled PbTx-3 containing 0.1 pg radiolabeled toxin as tracer. Disappearance of parent compound and the appearance of metabolites were measured by HPLC equipped with a Radiomatic isotope detector. (1.6 nmol/min/g liver)... 

Recent work in our laboratories has confirmed the existence of a similar pathway in the oxidation of vindoline in mammals (777). The availability of compounds such as 59 as analytical standards, along with published mass spectral and NMR spectral properties of this compound, served to facilitate identification of metabolites formed in mammalian liver microsome incubations. Two compounds are produced during incubations with mouse liver microsome preparations 17-deacetylvindoline, and the dihydrovindoline ether dimer 59. Both compounds were isolated and completely characterized by spectral comparison to authentic standards. This work emphasizes the prospective value of microbial and enzymatic transformation studies in predicting pathways of metabolism in mammalian systems. This work would also suggest the involvement of cytochrome P-450 enzyme system(s) in the oxidation process. Whether the first steps involve direct introduction of molecular oxygen at position 3 of vindoline or an initial abstraction of electrons, as in Scheme 15, remains unknown. The establishment of a metabolic pathway in mammals, identical to those found in Strep-tomycetes, with copper oxidases and peroxidases again confirms the prospective value of the microbial models of mammalian metabolism concept. 

Kinetics is one of the key issues of catalysis together with selectivity and catalyst stability. Chemical kinetics has been discussed in several dedicated works [1] and the readers will be aware of its basics [2], In the following sections several commonly used concepts are mentioned such as steady state approximation, rate-determining step, determination of selectivity, and a few points of particular interest to catalysis will be high-lighted such as incubation. 

Self (S4) first proposed the concept of noncompetitive assay for haptens utilizing an adequate combination of an a-type and a jS-type anti-idiotype antibody, in which he used the term, selective antibody for the a-type antibodies. Then, Barnard and Cohen (Bl) applied this assay principle for the determination of serum E2, naming the assay system an idiometric assay. Figure 12A illustrates the assay procedure of the idiometric assay of E2. The target hapten is captured by excess anti-E2 antibody immobilized on microtiter strips by incubation at room temperature for 1 h (step i). After washing the strips, the /3-type anti-idiotype antibody was added in order to saturate (or block) the unoccupied paratope of the anti-E2 antibody (incubation, room temperature for 30 min) (step ii). The a-type anti-idiotype antibody, which has been labeled with a europium chelate (H4), was then added to the plate and incubated at room temperature for a further 2 h (step iii). Finally, fluorescence intensity due to bound europium was measured with a time-resolved fluorometer. Because of large steric hindrance around the bound jS-type antibody (MW 150,000), the labeled a-type antibody would. 

The first suggestion of a practical form of antidotal therapy came in 1949 from Hestrin, who found that acetylcholinesterase (AChE) catalyzed the formation of acetohydroxamlc acid when incubated with sodium acetate and hydroxylamine. Critical in vitro studies in the next decade led to the development of a practical approach to therapy. The crucial concept in these studies was the recognition that the compound formed when AChE reacted with a phosphorus ester was a covalent phosphoryl-enzyme Intermediate similar to that formed in the hydrolysis of acetylcholine. 3 Wilson and colleagues, beginning in 1951, demonstrated that AChE inhibited by alkyl phosphate esters (tetraethyl pyrophosphate, TEPP) could be reactivated by water, but that free enzyme formed much more rapidly in the presence of hydroxylamine. 0 21 Similar results... 

In contrast to this work on whole brain, Kemp and Stoolmiller138 used cultured mouse-neuroblastoma cells for their study of incorporation of [ lH]2-acetamido-2-deox> -D-mannose into the sialic acid moiety of gangliosides. They were able to demonstrate clearly the precursor-product relationship among CMS, GM2, and GM1. They found that cultured NB41A cells incorporated [3H]2-acetamido-2-deoxy-D-man-nose into the sialic acid moiety of GM3 in less than 10 minutes. Labeled GM2 was not detected in cells incubated for less than 30 minutes, and measurable activity did not appear in G I1 until alter 60 to 90 minutes. These results further supported the concept that the pathway of synthesis of ganglio-tvpe gangliosides proceeds by way of GM3 —> GM2 — GM1. 

The incubation and birth of a scientific concept is interesting. Therefore, we shall trace, even if with a personal taste, the development of the zr-delocaliza-tion concept. Since much of the concept is connected with benzene, there will be a natural emphasis on this molecule. Subsequently, we shall describe the background related to zr-distortivity. 

A useful concept for entrepreneurs that may be especially applicable to pharmacist entrepreneurs is the incubator. This term describes an entity that exists to help get new businesses started. As stated earlier, entrepreneurs cannot be an expert in every area required ofentrepreneurship. Incubators bring all or many of the necessary areas together (e.g., accounting, business plan development, legal, capital acquisition, etc.) to help entrepreneurs bring their ideas to market. As discussed previously, investment early in the idea-development process costs the most. Incubators usually take significant equity of the business to get started. The... 

The concept that plasma membrane transport plays a key role in the regulation of intracellular thyroid hormone levels is supported by studies with a monoclonal antibody against an antigen exposed on rat liver cells [107], This antibody inhibited the uptake of different iodothyronines by rat hepatocytes under initial rate conditions as well as the metabolism of these compounds during prolonged incubations [107]. Uptake and metabolism of T4, T3 and rT3 were affected to the same extent, suggesting that a single system operates in the transport of different iodothyronines, which is opposite to the view advanced above. However, it is not excluded that the antibody interacts with a component of the plasma membrane and thereby affects multiple transport systems. 

The concept of MW enhancers was developed to increase polarization value changes caused by large size differences between substrate and product when small peptide substrates were used. The most frequently used approach is based on the attachment of a biotin molecule to the terminus of the peptide on the opposite site of the scissile bond to the fluorophore. After the incubation of the substrate with the protease, streptavidin is added to the assay. Streptavidin binds to biotin and enhances the masses of the substrate and the unlabeled product, thus causing an increase of the polarization... 

Analogous to cell viability measurements, many of the same basic concepts apply to the development of cell-based assays for apoptosis. The length of incubation of cells with the test compound is among the most important issues to address and optimize. The length of incubation is important because the markers of apoptosis may be present for relatively brief transient periods and subsequently disappear as the population of cells undergoes secondary necrosis. The induction of measurable caspase activity can occur in only a few minutes or can take days, depending on the model cell line, type of inducer, and effective concentration inside the cells. 

Rommelsbacher, in following the concept of Holtz that mammalian alkaloids are formed from chemically reactive biogenic amines and amino acids by reaction with endogenously formed ketonic substrates under physiological conditions, has extended this idea to include indole-3-acetaldehyde. It was found that tryptamine, on incubation with pig brain and bovine brain, but not with liver tissue, formed the thiazolidine carboxylic acid 60 shown in Fig. 20 Ml). 

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