Mendelian inheritance of variations in β-galactosidase activities in the house mouse

A genetic test of differences in -galactosidase activities in three mouse tissues, liver, kidney, and spleen, is demonstrated. Activities fall in three distinct categories in F ₂ crosses between the two inbred strains C57BL/K1 and DBA/2/K1. C57 mice consistently show high activities in all three tissues, and DBA mice show low activities except for some male kidneys. F ₁ mice are intermediate to the parental strains. There seems to be a simple mendelian ratio 1:2:1, between the numbers of animals belonging to the three activity classes in F ₂ crosses and a 1:1 ratio in backcrosses. Thus it is suggested that one single locus is responsible for most of the differences seen in this system.This work was supported by the Nilsson-Ehle fund and the Marcus Borgström fund.     

Control of neural stem cell survival by electroactive polymer substrates

Stem cell function is regulated by intrinsic as well as microenvironmental factors, including chemical and mechanical signals. Conducting polymer-based cell culture substrates provide a powerful tool to control both chemical and physical stimuli sensed by stem cells. Here we show that polypyrrole (PPy), a commonly used conducting polymer, can be tailored to modulate survival and maintenance of rat fetal neural stem cells (NSCs). NSCs cultured on PPy substrates containing different counter ions, dodecylbenzenesulfonate (DBS), tosylate (TsO), perchlorate (ClO(4)) and chloride (Cl), showed a distinct correlation between PPy counter ion and cell viability. Specifically, NSC viability was high on PPy(DBS) but low on PPy containing TsO, ClO(4) and Cl. On PPy(DBS), NSC proliferation and differentiation was comparable to standard NSC culture on tissue culture polystyrene. Electrical reduction of PPy(DBS) created a switch for neural stem cell viability, with widespread cell death upon polymer reduction. Coating the PPy(DBS) films with a gel layer composed of a basement membrane matrix efficiently prevented loss of cell viability upon polymer reduction. Here we have defined conditions for the biocompatibility of PPy substrates with NSC culture, critical for the development of devices based on conducting polymers interfacing with NSCs.     

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

Acetate as the major by-product in industrial-scale bioprocesses with Escherichia coli is found to decrease process efficiency as well as to be toxic to cells, which has several effects like a significant induction of cellular stress responses. However, the underlying phenomena are poorly explored. Therefore, we studied time-resolved population heterogeneity of the E. coli growth reporter strain MG1655/pGS20PrrnBGFPAAV expressing destabilized green fluorescent protein during batch growth on acetate and glucose as sole carbon sources. Additionally, we applied five fluorescent stains targeting different cellular properties (viability as well as metabolic and respiratory activity). Quantitative analysis of flow cytometry data verified that bacterial populations in the bioreactor are more heterogeneous in growth as well as stronger metabolically challenged during growth on acetate as sole carbon source, compared to growth on glucose or acetate after diauxic shift. Interestingly, with acetate as sole carbon source, significant subpopulations were found with some cells that seem to be more robust than the rest of the population. In conclusion, following batch cultures population heterogeneity was evident in all measured parameters. Our approach enabled a deeper study of heterogeneity during growth on the favored substrate glucose as well as on the toxic by-product acetate. Using a combination of activity fluorescent dyes proved to be an accurate and fast alternative as well as a supplement to the use of a reporter strain. However, the choice of combination of stains should be well considered depending on which population traits to aim for.     

Concomitant Tumor Expression of EGFR and TATI/SPINK1 Associates with Better Prognosis in Colorectal Cancer

Background

Epidermal growth factor receptor (EGFR) activation plays a role in colorectal cancer (CRC) carcinogenesis, and anti-EGFR drugs are used in treatment of advanced CRC. One of the EGFR ligands is tumor-associated trypsinogen inhibitor TATI, also called serine protease inhibitor Kazal type1 (SPINK 1), which we recently showed to be an independent prognostic marker in CRC.

Methods

We studied the prognostic value of immunohistochemical expression of EGFR and concomitant expression of EGFR and TATI/SPINK1 in a series of 619 colorectal cancer patients.

Results

Of the samples, 92% were positive for EGFR. EGFR+/TATI+ was seen in 62.8%, EGFR+/TATI− in 29.5%, EGFR−/TATI+ in 4.9%, and EGFR−/TATI− in 2.7% of patients. EGFR expression correlated with WHO grade (p = 0.040). In univariate analysis, EGFR expression correlated with favourable survival (p = 0.006). EGFR+/TATI+ patients showed better survival than did those with other combinations (p<0.001). In multivariate analysis, EGFR+/TATI+ was an independent prognostic factor of favourable prognosis (p<0.001).

Conclusion

Concomitant positivity of EGFR and TATI/SPINK1 predicts favourable prognosis in CRC.     

Comparison of Methods for Extraction of Bacterial Adenine Nucleotides Determined by Firefly Assay

Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP, and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view.     

C-Glycosylflavones from Zea mays that inhibit insect development

A new C-glycosylflavone isolated from corn silk inhibits the growth and development of the corn earworm, Heliothis zea. This new compound was shown to be a 2″-O-α-l-rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl)luteolin. Also found co-occurring in corn silk were minor amounts of the corresponding 6-C-glycosylated analogs of chrysoeriol and apigenin.     

Self-assembling supramolecular complexes by single-stranded extension from plasmid DNA

Self-assembling supramolecular complexes are of great interest for bottom-up research like nanotechnology. DNA is an inexpensive building block with sequence-specific self-assembling capabilities through Watson-Crick and/or Hoogsteen base pairing and could be used for applications in surface chemistry, material science, nanomechanics, nanoelectronics, nanorobotics, and of course in biology. The starting point is usually single-stranded DNA, which is rather easily accessible for base pairing and duplex formation. When long stretches of double-stranded DNA are desirable, serving either as genetic codes or electrical wires, bacterial expansion of plasmids is an inexpensive approach with scale-up properties. Here, we present a method for using double-stranded DNA of any sequence for generating simple structures, such as junctions and DNA lattices. It is known that supercoiled plasmids are strand-invaded by certain DNA analogs. Here we add to the complexity by using "Self-assembling UNiversal (SUN) anchors" formed by DNA analog oligonucleotides, synthesized with an extension, a "sticky-end" that can be used for further base pairing with single-stranded DNA. We show here how the same set of SUN anchors can be utilized for gene therapy, plasmid purification, junction for lattices, and plasmid dimerization through Watson-Crick base pairing. Using atomic force microscopy, it has been possible to characterize and quantify individual components of such supra-molecular complexes.     

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G₀ and G₁ phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.     

Use of structural phylogenetic networks for classification of the ferritin-like superfamily

In the postgenomic era, bioinformatic analysis of sequence similarity is an immensely powerful tool to gain insight into evolution and protein function. Over long evolutionary distances, however, sequence-based methods fail as the similarities become too low for phylogenetic analysis. Macromolecular structure generally appears better conserved than sequence, but clear models for how structure evolves over time are lacking. The exponential growth of three-dimensional structural information may allow novel structure-based methods to drastically extend the evolutionary time scales amenable to phylogenetics and functional classification of proteins. To this end, we analyzed 80 structures from the functionally diverse ferritin-like superfamily. Using evolutionary networks, we demonstrate that structural comparisons can delineate and discover groups of proteins beyond the "twilight zone" where sequence similarity does not allow evolutionary analysis, suggesting that considerable and useful evolutionary signal is preserved in three-dimensional structures.