Analysis of mitogenic activity of proteins after separation by gel electrophoresis

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rapid High Resolution Genotyping of Francisella tularensis by Whole Genome Sequence Comparison of Annotated Genes (“MLST+”)

The zoonotic disease tularemia is caused by the bacterium Francisella tularensis. This pathogen is considered as a category A select agent with potential to be misused in bioterrorism. Molecular typing based on DNA-sequence like canSNP-typing or MLVA has become the accepted standard for this organism. Due to the organism’s highly clonal nature, the current typing methods have reached their limit of discrimination for classifying closely related subpopulations within the subspecies F. tularensis ssp. holarctica. We introduce a new gene-by-gene approach, MLST⁺, based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains and apply this approach to investigate an epidemic of lethal tularemia among non-human primates in two animal facilities in Germany. Due to the high resolution of MLST⁺ we are able to demonstrate that three independent clones of this highly infectious pathogen were responsible for these spatially and temporally restricted outbreaks.     

ATP-Dependent Carboxylation of Acetophenone by a Novel Type of Carboxylase

Anaerobic ethylbenzene metabolism in the betaproteobacterium Aromatoleum aromaticum is initiated by anaerobic oxidation to acetophenone via (S)-1-phenylethanol. The subsequent carboxylation of acetophenone to benzoylacetate is catalyzed by an acetophenone-induced enzyme, which has been purified and studied. The same enzyme is involved in acetophenone metabolism in the absence of ethylbenzene. Acetophenone carboxylase consists of five subunits with molecular masses of 70, 15, 87, 75, and 34 kDa, whose genes (apcABCDE) form an apparent operon. The enzyme is synthesized at high levels in cells grown on ethylbenzene or acetophenone, but not in cells grown on benzoate. During purification, acetophenone carboxylase dissociates into inactive subcomplexes consisting of the 70-, 15-, 87-, and 75-kDa subunits (apcABCD gene products) and the 34-kDa subunit (apcE gene product), respectively. Acetophenone carboxylase activity was restored by mixing the purified subcomplexes. The enzyme contains 1 Zn²⁺ ion per αβγδ core complex and is dependent on the presence of Mg²⁺ or Mn²⁺. In spite of the presence of Zn in the enzyme, it is strongly inhibited by Zn²⁺ ions. Carboxylation of acetophenone is dependent on ATP hydrolysis to ADP and P_i, exhibiting a stoichiometry of 2 mol ATP per mol acetophenone carboxylated. The enzyme shows uncoupled ATPase activity with either bicarbonate or acetophenone in the absence of the second substrate. These observations indicate that both substrates may be phosphorylated, which is consistent with isotope exchange activity observed with deuterated acetophenone and inhibition by carbamoylphosphate, a structural analogue of carboxyphosphate. A potential mechanism of ATP-dependent acetophenone carboxylation is suggested.Ethylbenzene belongs to the BTEX (benzene, toluene, ethylbenzene, and xylene) group of petroleum-derived hydrocarbons with extensive industrial and ecological relevance. Anaerobic catabolism of ethylbenzene proceeds via different pathways in denitrifying and sulfate-reducing bacteria. The latter generate a succinate adduct of ethylbenzene as the first intermediate, probably by addition of fumarate to the methylene carbon atom (14). However, denitrifying bacteria are capable of oxygen-independent hydroxylation of the methylene group of ethylbenzene to yield (S)-1-phenylethanol (1, 12, 20), which is catalyzed by the molybdenum enzyme ethylbenzene dehydrogenase (11, 15, 21). The pathway continues with the oxidation of (S)-1-phenylethanol to acetophenone by an alcohol dehydrogenase (10, 16). Acetophenone may also be produced from (R)-1-phenylethanol or directly used as a substrate. The enzymes of further acetophenone catabolism are uncharacterized. From the observed CO₂ dependence of ethylbenzene and acetophenone degradation, acetophenone was proposed to be carboxylated to benzoylacetate, which is then activated to the coenzyme A (CoA) thioester and thiolytically cleaved to benzoyl-CoA and acetyl-CoA (1, 6, 20, 22). On the level of benzoyl-CoA, the pathway of anaerobic ethylbenzene degradation flows into that of anaerobic benzoate degradation (for reviews, see references 2, 8, and 9) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Proposed catabolic pathway of ethylbenzene, phenylethanol, and acetophenone in A. aromaticum strain EbN1.In this communication, we identify and characterize the postulated enzyme responsible for acetophenone carboxylation in Aromatoleum aromaticum strain EbN1. The enzyme is specifically induced in ethylbenzene- and acetophenone-grown cells. Acetophenone carboxylation is shown to be dependent on ATP hydrolysis, reminiscent of but distinct from the related carboxylation of acetone (25).     

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, β-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO^−/−) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO^−/− as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO^−/− testis. Occludin, N-cadherin and β-catenin levels were enhanced in SCCx43KO^−/− mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.     

Process parameter shifting: Part II. Biphasic cultivation-A tool for enhancing the volumetric productivity of batch processes using Epo-Fc expressing CHO cells

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization.     

TNF receptor-1 is required for the formation of splenic compartments during adult, but not embryonic life

Lymphotoxin β-receptor (LTβR) and TNF receptor-1 (TNFR1) are important for the development of secondary lymphoid organs during embryonic life. The significance of LTβR and TNFR1 for the formation of lymphoid tissue during adult life is not well understood. Immunohistochemistry, morphometry, flow cytometry, and laser microdissection were used to compare wild-type, LTβR(-/-), TNFR1(-/-) spleens with splenic tissue that has been newly formed 8 wk after avascular implantation into adult mice. During ontogeny, LTβR is sufficient to induce formation of the marginal zone, similar-sized T and B cell zones, and a mixed T/B cell zone that completely surrounded the T cell zone. Strikingly, in adult mice, the formation of splenic compartments required both LTβR and TNFR1 expression, demonstrating that the molecular requirements for lymphoid tissue formation are different during embryonic and adult life. Thus, interfering with the TNFR1 pathway offers the possibility to selectively block the formation of ectopic lymphoid tissue and at the same time to spare secondary lymphoid organs such as spleen and lymph nodes. This opens a new perspective for the treatment of autoimmune and inflammatory diseases.