Minor contribution of presenilin 2 for γ-secretase activity in mouse embryonic fibroblasts and adult mouse brain

γ-Secretase plays an important function in the development of Alzheimer disease, since it participates in the production of the toxic amyloid β-peptide (Aβ) from the amyloid precursor protein (APP). Besides APP, γ-secretase cleaves many other substrates resulting in adverse side effects when γ-secretase inhibitors are used in clinical trials. γ-Secretase is a membrane bound protein complex consisting of at least four subunits, presenilin (PS), nicastrin, Aph-1 and Pen-2. PS and Aph-1 exist as different homologs (PS1/PS2 and Aph-1a/Aph-1b, respectively), which generates a variation in complex composition. PS1 and PS2 appears to have distinct roles since PS1 is essential during embryonic development whereas PS2 deficient mice are viable with a mild phenotype. The molecular mechanism behind this diversity is, however, largely unknown. In order to investigate whether PS1 and PS2 show different substrate specificity, we used PS1 or PS2 deficient mouse embryonic fibroblasts to study the processing on the γ-secretase substrates APP, Notch, N-cadherin, and ephrinB. We found that whereas depletion of PS1 severely affected the cleavage of all substrates, the effect of PS2 depletion was minor. In addition, less PS2 was found in active γ-secretase complexes. We also studied the effect of PS2 depletion in adult mouse brain and, in concordance with the results from the mouse embryonic fibroblasts, PS2 deficiency did not alter the cleavage of the two most important substrates, APP and Notch. In summary, this study shows that the contribution of PS2 on γ-secretase activity is of less importance, explaining the mild phenotype of PS2-deficient mice.     

Separation and properties of a “Neutral” hexosaminidase from embryonic chicken brain

• 1.1. A “neutral” hexosaminidase has been separated from other hexosaminidase forms (I and II) by DEAE-cellulose chromatography and characterized in embryonic (16-days old) and 1-day old chicken brains.
• 2.2. Its properties differ from those of the forms I and II. It has optimum activity at about pH 6.0 and can be eluted from DEAE-cellulose with 0.25 M KCl only.
• 3.3. It has no N-acetylgalactosaminidase activity and cannot be successfully detected after isoelectric focusing since it is very acidic and completely unstable below pH 5.0.
• 4.4. “Neutral” hexosaminidase is heat-stable at pH 6.0 and is inhibited by chloride.
• 5.5. These properties, very different from those of forms I and II, suggest that this “neutral” form of hexosaminidase would be very similar to known hexosaminidase C separated from other materials.
• 6.6. We have found no significant differences for the above-mentioned three forms in chick embryos (16-days old) in comparison with those from 1-day old chicken.

     

Inhibition of androgen metabolism and binding by a liposterolic extract of “serenoa repens B” in human foreskin fibroblasts

We previously suggested [Steroids33, (1979) 3; Steroids37, (1981) 6] that cultured genital skin fibroblasts should prove useful for screening of potential antiandrogens in human and living target cells. “Serenoa repens” lipidic extract (S.R.E.) was recently reported (Br. J. Pharmacoi., in press) to inhibit androgen action in animals. The present investigation was designed to study the antiandrogenicity of this compound in human cells: we therefore analyzed the effects of S.R.E. on the intracellular conversion of testosterone (T) to 5α-reduced derivatives, and we investigated interaction of S.R.E. with the intracellular androgen-receptor complex. Since the chemical structure of the active component of S.R.E. is still unknown, results are expressed in U/ml (one unit is defined as the amount of S.R.E. required to inhibit 50% of the specific binding (IC₅₀) of [³H]1881 to rat prostate cytosol).S.R.E. at different dilutions (5.7 to 28.6 U/ml) is added to culture media containing [³H]T or [³H]dihydrotestosterone (DHT) and incubated at 37°C with cultured fibroblasts. 28.6 U/ml S.R.E. significantly alters the formation of DHT and strongly inhibits 3 ketosteroid reductase mediated conversion of DHT to 5α-androstane-3α, 17β-diol, characterized radiochemically by thin-layer chromatography. S.R.E. is a good competitor for the whole cell androgen receptor: 7.1 U/ml S.R.E. gives 50% inhibition of the binding of 2 × 10⁻⁹ M [³H]DHT to its receptor. Competitive binding assays after cell fractionation indicate that S.R.E. is less potent in nuclear than in cytosol receptors. Sucrose gradient centrifugation of the radioactive cell lysate of fibroblasts demonstrates that 28.6 U/ml S.R.E. abolishes 70% of the 3.6 S receptor-complex radioactive peak.The present studies show that S.R.E. inhibits 5α-reductase, 3-ketosteroid reductase and receptor binding of androgens in cultured human foreskin fibroblasts. As the search for the ideal antiandrogen continues, S.R.E. appears to be a new type of antiandrogenic compound as therapeutics for the treatment of benign prostatic hypertrophy, hirsutism and so forth.     

An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

Conventional DNA ladder assay has certain shortcomings such as loss of DNA fragments during sample processing, involvement of multiple steps and requirement of expensive reagents. The present study demonstrates a rapid, easy-to-perform cost-effective method for detection of apoptotic DNA fragments with considerable improvement in the sensitivity by avoiding loss of DNA fragments. It involves a few minutes of procedure involving direct lysis of cells with dimethyl sulphoxide (DMSO), brief vortexing, addition of 2% SDS–TE buffer, and a single step of centrifugation. This cost- and time-efficient method reduces the assay time considerably and can be used for a large number of samples with excellent sensitivity.     

Interpretive conundrum on the exclusion criterion of “transplantation with xenografts” for tissue and cell donation

In the context of the EU Directives for human tissues and cells (2004/23/EC, 2006/17/EC and 2006/86/EC) further interest has arisen on the practical application of a few clauses. One such aspect, for the evaluation phase of a potential donor, is the interpretation of the exclusion criterion "transplantation with xenografts." This article outlines the consensus viewpoints regarding the earlier evaluation of the risks related to xenotransplantation and describes the current status of the terminology and recommendations/laws in several healthcare sectors. The application of uniform terminology is encouraged within the healthcare sectors at the international level.     

Evidence for a stromal-epithelial “lactate shuttle” in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts

Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.Key words: caveolin-1, oxidative stress, pseudohypoxia, lactate shuttle, MCT1, MCT4, metabolic coupling, tumor stroma, predictive biomarker, SLC16A1, SLC16A3, monocarboxylic acid transporter     

“Agouti NOD”: identification of a CBA-derived Idd locus on Chromosome 7 and its use for chimera production with NOD embryonic stem cells

Penetrance of the complex of genes predisposing the nonobese diabetic (NOD) mouse to autoimmune diabetes is affected by the maternal environment. NOD.CBALs-Tyr⁺/Lt is an agouti-pigmented Chromosome 7 congenic stock of NOD/Lt mice produced as a resource for embryo transfer experiments to provide the necessary maternal factors and allow the easy identification of NOD (albino) embryo donor phenotype. CBcNO6/Lt, a recombinant congenic agouti stock already containing approximately 50% NOD genome, was used as the donor source of a wild-type CBA tyrosinase allele. When the incidence of diabetes was assessed after nine generations of backcrossing and one generation of sib-sib mating, significant reduction in diabetes development was observed. No difference in diabetes development was observed in Tyr/Tyr^c heterozygotes, showing that protection was recessive. Analysis of diabetes progression in another NOD stock congenic for C57BL/6 alleles on Chromosome 7 linked to the glucose phosphate isomerase (Gpi1^b) locus provided no protection, indicating that the diabetes resistance (Idd) gene was distal to 34 cM (D7Mit346). Approximately 5 cM of the distal congenic region overlaps a region from C57L previously associated with protection when homozygous. The delayed onset and reduced frequency of diabetes in the NOD.CBALs-Tyr⁺/Lt stock is an advantage when females of this stock are used as surrogate mothers in studies involving hysterectomy or embryo transfers. Indeed, a newly developed NOD embryonic stem (ES) cell line injected into NOD.CBALs- Tyr⁺/Lt blastocysts produced approximately 50% live-born mice, of which approximately 11% were chimeric. Presumably because of high genomic instability, no germline transmission was observed.     

Evidence for a “Wattle and Daub” Model of the Cyst Wall of Entamoeba

The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a “wattle and daub” model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).     

“Addition” and “multiplication” theorems for the inputs of two neurons converging on a third

The output of a neuron innervated by two other neurons which, in turn, are subjected to two independent Poisson showers of stimuli, is derived as a function of the frequencies of the Poisson showers under two distinct assumptions, 1) where either of the two neurons can fire the third, and 2) where the stimuli from both neurons must impinge within a certain time interval to fire the third. For very small frequencies, the output of the third neuron is very nearly the sum of the input frequencies in the first case and proportional to the product of the input frequencies in the second case. Hence the designation “addition” and “multiplication” theorems. This treatment is a generalization of a previous treatment where the Poisson shower was assumed identical for the two outer neurons.     

Microglia and reactive “M” cells of degenerating central nervous system: Does similar morphology and function imply a common origin?

Summary Penetration of the median eminence and ventrolateral walls of the III ventricle by intraventricularly injected horseradish peroxidase was studied in rats. Experimental times varied between 10 to 70 min (short-term experiments) and 12 hrs to 30 days (long-term experiments).In short-term experiments, the median eminence was found to be completely stained whereas the lateral walls of the III ventricle were penetrated only up to 1 mm in depth. Spreading of the tracer takes place predominantly through the extracellular space and cellular uptake and transport do not seem to play a role during the first 70 min following the injection.In long-term experiments, the tanycytes exhibit a variety of intracellular inclusions marked by HRP precipitate. Tanycytic perikarya contain dense bodies and lipofuscin-like aggregates. Lipoprotein granules are thought to arise from these and are interpreted as lysosomal residual bodies. In tanycytic processes and perivascular endfeet, accumulations of HRP-containing tubules and polymorphous granules are encountered suggesting a transport towards the blood vessels of the portal plexus or of the arcuate and ventromedial areas, respectively. Sporadic tanycytes which are completely and evenly stained from the ventricular surface to the end of their processes were observed in the arcuate and ventromedial area. Whether this appearance can be taken as a sign of normal cell function seems doubtful. Some possible routes of transport through the median eminence—extracellular and transcellular—are summarized in a schematic drawing, taking into account the present findings and those published elsewhere in the literature.We gratefully acknowledge an Anglo-German Collaboration Grant of the Wellcome Trust enabling stimulating discussions with other workers in the field.     

“Why throw away something useful?”: Attitudes and opinions of people treated for bipolar disorder and their relatives on organ and tissue donation

In regard to mental illness, brain donation is essential for the biological investigation of central pathology. Nevertheless, little is known about the thoughts of people with mental disorders on tissue donation for research. Here, our objective was to understand the attitudes and opinions of people treated for bipolar disorder and their relatives regarding donation in general, and particularly donation for research. This is a qualitative study that used in-depth interviews to determine the thoughts of participants regarding tissue donation for research. Theoretical sampling was used as a recruitment method. Grounded theory was used as a framework for content analyses of the interviews. A semi-structured interview guide was applied with the topics: donation in general; donation for research; mental health and body organs; opinion regarding donation; feelings aroused by the topic. Although all participants were aware of organ donation for transplant, they were surprised that tissue could be donated for research. Nevertheless, once they understood the concept they were usually in favor of the idea. Although participants demonstrated a general lack of knowledge on donation for research, they were willing to learn more and viewed it as a good thing, with altruistic reasons often cited as a motive for donation. We speculate that bridging this knowledge gap may be a fundamental step towards a more ethical postmortem tissue donation process.     

“Golden Rice”, a GMO-product for public good,and the consequences of GE-regulation

Compared to a non - genetically engineered (GE) variety, the deployment of Golden Rice suffers from a delay of more than 10 years. The cause for this delay is GE-regulation. Considering the potential impact of Golden Rice on the reduction in vitamin A-malnutrition, this delay is responsible for loss of numerous lives, mostly children and women. GE-regulation is also responsible for the fact that public institutions are prevented from delivering a public good GE-product with the consequence that we are faced with a de-facto monopoly in favour of a few potent industries. Considering the forgone benefits from putative public good GE-products, GE-regulation can be blamed of being responsible for millions of lives, all of them, of course, in developing countries. As there is no scientific justification for present GE-regulation and as it has, so far, not prevented any harm, our society has the responsibility to reconsider present regulation which is based on the concept of an extreme interpretation of the precautionary principle. It would be justified to change regulation to a science-base and to regulate traits instead of technology. This would make regulation cheaper and faster, without compromising safety. GE-technology has an unprecedented safety record and it is far more precise and predictable than any other “traditional” and unregulated breeding technology.     

β-Neo-endorphin,a new hypothalamic “big” Leu-enkephalin of porcine origin: Its purification and the complete amino acid sequence

A new hypothalamic “big” Leu-enkephalin of porcine origin, designated as β-neo-endorphin, has been isolated from a side fraction, obtained in our previous isolation of α-neo-endorphin and PH-8P (dynorphin[1–8]). The complete amino acid sequence has been elucidated to be : Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro. The sequence has been confirmed by the comparison of natural β-neo-endorphin with a synthetic peptide. In addition, β-neo-endorphin exhibits potent opioid activity in guinea-pig ileum assay.     

The “minimal boundary curve for endothermy” as a predictor of heterothermy in mammals and birds: a review

According to the concept of the “minimal boundary curve for endothermy”, mammals and birds with a basal metabolic rate (BMR) that falls below the curve are obligate heterotherms and must enter torpor. We examined the reliability of the boundary curve (on a double log plot transformed to a line) for predicting torpor as a function of body mass and BMR for birds and several groups of mammals. The boundary line correctly predicted heterothermy in 87.5% of marsupials (n = 64), 94% of bats (n = 85) and 82.3% of rodents (n = 157). Our analysis shows that the boundary line is not a reliable predictor for use of torpor. A discriminate analysis using body mass and BMR had a similar predictive power as the boundary line. However, there are sufficient exceptions to both methods of analysis to suggest that the relationship between body mass, BMR and heterothermy is not a causal one. Some homeothermic birds (e.g. silvereyes) and rodents (e.g. hopping mice) fall below the boundary line, and there are many examples of heterothermic species that fall above the boundary line. For marsupials and bats, but not for rodents, there was a highly significant phylogenetic pattern for heterothermy, suggesting that taxonomic affiliation is the biggest determinant of heterothermy for these mammalian groups. For rodents, heterothermic species had lower BMRs than homeothermic species. Low BMR and use of torpor both contribute to reducing energy expenditure and both physiological traits appear to be a response to the same selective pressure of fluctuating food supply, increasing fitness in endothermic species that are constrained by limited energy availability. Both the minimal boundary line and discriminate analysis were of little value for predicting the use of daily torpor or hibernation in heterotherms, presumably as both daily torpor and hibernation are precisely controlled processes, not an inability to thermoregulate.     

Preparation and Characterization of a Novel Decellularized Fibrocartilage “Book” Scaffold for Use in Tissue Engineering

At the tendon-to-bone insertion, there is a unique transitional structure: tendon, non-calcified fibrocartilage, calcified fibrocartilage, and bone. The reconstruction of this special graded structure after defects or damage is an important but challenging task in orthopedics. In particular, reconstruction of the fibrocartilage zone has yet to be successfully achieved. In this study, the development of a novel book-shape scaffold derived from the extracellular matrix of fibrocartilage was reported. Specifically, fibrocartilage from the pubic symphysis was obtained from rabbits and sliced into the shape of a book (dimensions: 10 mm × 3 mm × 1 mm) with 10 layers, each layer (akin to a page of a book) with a thickness of 100-μm. These fibrocartilage “book” scaffolds were decellularized using sequentially 3 freeze-thaw cycles, 0.1% Triton X-100 with 1.5 M KCl, 0.25% trypsin, and a nuclease. Histology and DNA quantification analysis confirmed substantial removal of cells from the fibrocartilage scaffolds. Furthermore, the quantities of DNA, collagen, and glycosaminoglycan in the fibrocartilage were markedly reduced following decellularization. Scanning electron microscopy confirmed that the intrinsic ultrastructure of the fibrocartilage tissue was well preserved. Therefore, the results of this study suggest that the novel “book” fibrocartilage scaffold could have potential applications in tissue engineering.     

The production of a “universal developer” for the immunological detection of human IgG and its application in immunodiagnostics

Summary This study describes the development of a bispecific monoclonal antibody capable of the simultaneous recognition of horseradish peroxidase (HRP) and human IgG. This antibody, coded McC2, has been applied in a novel manner as a universal developing reagent for the detection of human IgG. McC2 cross-reacts with all human IgG subtypes and was found to recognise an epitope on the Fe portion of human IgG. McC2 does not cross-react with human IgM or IgA. This bi-specific antibody belongs to the mouse IgG₁ subclass. McC2 was used for the detection of human IgG in a simple one step enzyme-linked immunosorbant assay (ELISA). Use of this bi-specific antibody in this assay resulted in an excellent signal to noise ratio with background in negative control wells virtually nonexistent. McC2 was also applied in a clinical diagnostic test for the detection of auto anti-nuclear antibodies in patient sera. McC2 was substituted, in a blind study, for a HRP-conjugated second antibody supplied with the test kit. All sera were tested both with the kit's second antibody and McC2. When using McC2, we obtained no false positive results whereas five false positives were obtained when using the kit's second antibody. However, one false negative result was obtained with the use of McC2 as a developing reagent while none were noted with the use of the kit's second antibody.This study demonstrates the potential use of bi-specific universal developers in a wide variety of immunobased techniques as well as the potential advantages for the production of a complete panel of bi-specific developing monoclonal antibodies against IgGs from a number of different species.