Influence of Endogenous Factors of Food Matrices on Avidin–Biotin Immunoassays for the Detection of Bacitracin and Colistin in Food

(Strept)avidin–biotin technology is frequently used in immunoassay systems to improve their analytical properties. It is known from clinical practice that many (strept)avidin–biotin-based tests provide false results when analyzing patient samples with a high content of endogenous biotin. No specific investigation has been carried out regarding possible interferences from avidin (AVI) and biotin (B7) contained in food matrices in (strept)avidin–biotin-based immunoanalytical systems for food safety. Two kinds of competitive ELISAs for bacitracin (BT) and colistin (COL) determination in food matrices were developed based on conventional hapten–protein coating conjugates and biotinylated BT and COL bound to immobilized streptavidin (SAV). Coating SAV–B7–BT and SAV–B7–COL complexes-based ELISAs provided 2- and 15-times better sensitivity in BT and COL determination, corresponding to 0.6 and 0.3 ng/mL, respectively. Simultaneously with the determination of the main analytes, these kinds of tests were used as competitive assays for the assessment of AVI or B7 content up to 10 and 1 ng/mL, respectively, in food matrices (egg, infant milk formulas enriched with B7, chicken and beef liver). Matrix-free experiments with AVI/B7-enriched solutions showed distortion of the standard curves, indicating that these ingredients interfere with the adequate quantification of analytes. Summarizing the experience of the present study, it is recommended to avoid immunoassays based on avidin–biotin interactions when analyzing biosamples containing these endogenous factors or enriched with B7.

Localization and activity of recombinant streptavidin in cell fractions of Escherichia coli

The authors provide a genetic construct for obtaining recombinant biologically active wild type streptavidin with high yield. Addition of leader peptide and oligohistidine sequence to the streptavidin sequence makes it possible to isolate soluble streptavidin from the culture medium and from the soluble fraction. Temperature conditions for inducing of protein synthesis were found to have a significant effect on the distribution profile of streptavidin between fractions. The obtained protein product is not contaminated with endogenous biotin. The provided method excludes denaturation and renaturation steps which are necessary for isolation of the desired product from inclusion bodies but substantially reduce the yield. Thus, the provided approach allows to increase the yield of biologically highly active strepatividin which is capable of binding biotin and biotin-containing compounds and can be used for various practical purposes.

Mitonucleons formed during differentiation of Ishikawa endometrial cells generate vacuoles that elevate monolayer syncytia: Differentiation of Ishikawa domes, Part 1

In 1998, we published a paper (Fleming et.al, 1998) describing some aspects of Ishikawa endometrial epithelial cell differentiation from monolayer cells into cells forming fluid-filled hemispheres called domes. The process begins with the dissolution of membranes within discrete regions of the monolayer. Nuclei from fused cells aggregate and endogenous biotin in particulate structures assumed to be mitochondria increase throughout the resulting syncytium. Endogenous biotin is also the distinguishing feature of a membrane that surrounds aggregates of multiple nuclei in a structure called a mitonucleon. The current paper includes additional observations on structural changes accompanying Ishikawa differentiation. Vacuoles form in the heterochromatin of the mitonucleon and within the biotin-containing double membrane surrounding heterochromatin. With the formation of vacuoles, the mitonucleon can be seen to rise along with the apical membrane of the syncytium in which it formed. The small vacuoles that form within the heterochromatin result in structures similar to “cells with optically clear nuclei” found in some cancers. The second larger vacuole that forms within the membrane surrounding the heterochromatin transforms the cell profile to one that resembles “signet ring” cells also observed in some cancers. Eventually the membrane surrounding the massed heterochromatin, generated three to four hours earlier, is breached and previously aggregated nuclei disaggregate. During this process heterochromatin in the mitonucleons undergoes changes usually ascribed to cells undergoing programmed cell death such as pyknosis and DNA fragmentation (Fleming, 2016b). The cells do not die, instead chromatin filaments appear to coalesce into a chromatin mass that gives rise to dome-filling nuclei by amitosis during the final three to four hours of the 20 hour differentiation (Fleming, 2016c).

Pyknotic chromatin in mitonucleons elevating in syncytia undergo karyorhhexis and karyolysis before coalescing into an irregular chromatin mass: Differentiation of Ishikawa Domes, Part 2

Pyknosis, karyorrhexis and karyolysis, harbingers of programmed cell death in many systems, appear to be driving forces that transform Ishikawa monolayer epithelial cells into differentiated dome cells. The heterochromatin affected by these process is contained in multiple nuclei aggregated in the syncytia that form when Ishikawa monolayers are stimulated to differentiate (Fleming, 2016a). The nuclear aggregates are enveloped in a double membrane staining for the endogenous biotin in mitochondrial carboxylases. The structure called a mitonucleon becomes vacuolated, along with the heterochromatin it envelops, and this structure elevates with the apical membrane of the syncytium 6 to 8 hours into the 20 hour differentiation, becoming increasingly pyknotic. This phase of the differentiation comes to an end when the mitonucleon membranes are breached and nuclei emerging from the aggregated state can be seen to fragment explosively. Fragmented DNA associates with an array of microtubules, filling the large central clearing of the predome. Some chromatin remains unfragmented and can be seen of the edges of the predome clearing. Cell death does not occur. Instead, the fragmented DNA coalesces into an irregular mass within the apical and basal membranes of the predome under which fluid has been accumulating. From the chromatin sheet, nuclei emerge amitotically as described in Part 3 of this series (Fleming, 2016c).

Pyknotic chromatin in mitonucleons elevating in syncytia undergo karyorhhexis and karyolysis before coalescing into an irregular chromatin mass: Differentiation of Ishikawa Domes, Part 2

Pyknosis, karyorrhexis and karyolysis, harbingers of programmed cell death in many systems, appear to be driving forces that transform Ishikawa monolayer epithelial cells into differentiated dome cells. The heterochromatin affected by these process is contained in multiple nuclei aggregated in the syncytia that form when Ishikawa monolayers are stimulated to differentiate (Fleming, 2016a). The nuclear aggregates are enveloped in a double membrane staining for the endogenous biotin in mitochondrial carboxylases. The structure called a mitonucleon becomes vacuolated, along with the heterochromatin it envelops, and this structure elevates with the apical membrane of the syncytium 6 to 8 hours into the 20 hour differentiation, becoming increasingly pyknotic. This phase of the differentiation comes to an end when the mitonucleon membranes are breached and nuclei emerging from the aggregated state can be seen to fragment explosively. Fragmented DNA associates with an array of microtubules, filling the large central clearing of the predome. Some chromatin remains unfragmented and can be seen of the edges of the predome clearing. Cell death does not occur. Instead, the fragmented DNA coalesces into an irregular mass within the apical and basal membranes of the predome under which fluid has been accumulating. From the chromatin sheet, nuclei emerge amitotically as described in Part 3 of this series (Fleming, 2016c).

Mitonucleons formed during differentiation of Ishikawa endometrial cells generate vacuoles that elevate monolayer syncytia: Differentiation of Ishikawa domes, Part 1

In 1998, we published a paper (Fleming et.al, 1998) describing some aspects of Ishikawa endometrial epithelial cell differentiation from monolayer cells into cells forming fluid-filled hemispheres called domes. The process begins with the dissolution of membranes within discrete regions of the monolayer. Nuclei from fused cells aggregate and endogenous biotin in particulate structures assumed to be mitochondria increase throughout the resulting syncytium. Endogenous biotin is also the distinguishing feature of a membrane that surrounds aggregates of multiple nuclei in a structure called a mitonucleon. The current paper includes additional observations on structural changes accompanying Ishikawa differentiation. Vacuoles form in the heterochromatin of the mitonucleon and within the biotin-containing double membrane surrounding heterochromatin. With the formation of vacuoles, the mitonucleon can be seen to rise along with the apical membrane of the syncytium in which it formed. The small vacuoles that form within the heterochromatin result in structures similar to “cells with optically clear nuclei” found in some cancers. The second larger vacuole that forms within the membrane surrounding the heterochromatin transforms the cell profile to one that resembles “signet ring” cells also observed in some cancers. Eventually the membrane surrounding the massed heterochromatin, generated three to four hours earlier, is breached and previously aggregated nuclei disaggregate. During this process heterochromatin in the mitonucleons undergoes changes usually ascribed to cells undergoing programmed cell death such as pyknosis and DNA fragmentation (Fleming, 2016b). The cells do not die, instead chromatin filaments appear to coalesce into a chromatin mass that gives rise to dome-filling nuclei by amitosis during the final three to four hours of the 20 hour differentiation (Fleming, 2016c).

Unusual characteristics of opaque Ishikawa endometrial cells include the envelopment of chromosomes with material containing endogenous biotin in the latter stages of cytokinesis

We have identified a small dynamic population of opaque cells in Ishikawa endometrial cultures whose unusual characteristics include the fact that chromosomes become enveloped during the final stages of cytokinesis by material staining for endogenous biotin. Endogenous biotin, ultimately shown to be due to mitochondrial carboxylases, was detected in a membrane that wraps around aggregated nuclei in syncytia that develop as part of the differentiation of domes in Ishikawa cells. (Fleming H et al. 1998). The “wrapped chromosomes” in individual opaque Ishikawa cells stain similarly suggesting a similar origin. We were able to show that opaque cells form from transparent monolayer cells, can be polyploid, and often appear to be detaching from the colony and from the underlying substrate. We were also able to show an opaque cell fissioning asymmetrically, to give rise to a monolayer cell whose nucleus appeared to be wrapped. We believe that the cycle of differentiation of monolayer cells into opaque, polyploid cells and depolyploidization back into monolayer cells is involved in the spatial extension of cells as they develop from discrete colonies into a confluent monolayer. Wrapping of chromosomes may ensure that complete genomes are inherited by daughter cells during depolyploidization.

Unusual characteristics of opaque Ishikawa endometrial cells include the envelopment of chromosomes with material containing endogenous biotin in the latter stages of cytokinesis

We have identified a small dynamic population of opaque cells in Ishikawa endometrial cultures whose unusual characteristics include the fact that chromosomes become enveloped during the final stages of cytokinesis by material staining for endogenous biotin. Endogenous biotin, ultimately shown to be due to mitochondrial carboxylases, was detected in a membrane that wraps around aggregated nuclei in syncytia that develop as part of the differentiation of domes in Ishikawa cells. (Fleming H et al. 1998). The “wrapped chromosomes” in individual opaque Ishikawa cells stain similarly suggesting a similar origin. We were able to show that opaque cells form from transparent monolayer cells, can be polyploid, and often appear to be detaching from the colony and from the underlying substrate. We were also able to show an opaque cell fissioning asymmetrically, to give rise to a monolayer cell whose nucleus appeared to be wrapped. We believe that the cycle of differentiation of monolayer cells into opaque, polyploid cells and depolyploidization back into monolayer cells is involved in the spatial extension of cells as they develop from discrete colonies into a confluent monolayer. Wrapping of chromosomes may ensure that complete genomes are inherited by daughter cells during depolyploidization.

Endogenous biotin expression in renal and testicular tumors and literature review

Introduction: The aim of this study was to examine endogenous biotin levels in tumour specimens collected from patients with renal and testicular tumours and compare them to the surrounding non-neoplastic surgical margin.Methods: Frozen samples were obtained from the Ontario Tumour Bank. Renal and testicular tumour tissue were included in this study. Normal tissue from the negative surgical margins of each tumour served as a control. Biotin detection in tissue specimens was determined using immunohistochemistry (IHC).Results: Specimens collected from 56 patients (36 men and 20 women) were included in this study. Histopathology of the 52 renal tumours included 31 (60%) conventional type RCC, 5 (10%) chromophobe RCC, 5 (10%) papillary RCC, 1 (2%) oncocytoma and 10 (19%) upper tract urothelial carcinoma (UC). The 4 testicular tumours included 1 seminomatous (25%) germ cell tumour and 3 (75%) non-seminomatous germ cell tumours.Conclusion: No biotin signal was perceived in all tested tumour samples. Endogenous biotin expression was detected in the matching non-neoplastic surgical margin of tested renal tissues. This lack of staining may prove to be a valuable tool in future studies.