Expression of lysosomal enzymes in human mutant fibroblast-chick erythrocyte heterokaryons

The generation of enzymes located in lysosomes, in cytosol or in endoplasmatic reticulum/Golgi complex is studied in heterokaryons in which chick erythrocyte nuclei are reactivated. The lysosomal enzymes, alpha-glucosidase (alpha-glu) and beta-galactosidase (beta-gal), are synthesized in heterokaryons obtained after fusion of chick erythrocytes with human fibroblasts of patients with Pompe's disease (alpha-glu-deficient) and GM1-gangliosidosis (beta-gal-deficient), respectively. The enzymes appear to be of chick origin and their activities can be detected at first around 4 days after fusion, i.e., at a time when the nucleoli in the erythrocyte nuclei have been reactivated. Maximal activities are reached around 15 days after fusion. No generation of the lysosomal enzyme beta-hexosaminidase is detected in the heterokaryons up to 23 days after fusion of chick erythrocyte with either beta-hexosaminidase A- and B-deficient fibroblasts (Sandhoff's disease) or beta-hexosaminidase A-deficient fibroblasts (Tay-Sachs disease). Similarly no expression of the cytosol enzyme glucose-6-phosphate dehydrogenase (G6PD) is fond up to 30 days after fusion, when chick erythrocytes are fused with fibroblasts from two different G6PD-deficient cell strains (residual activities of 4 and 20% respectively). Indirectly we examined N-acetyl-glucosamine-1-phosphate transferase activity, an enzyme located in the endoplasmic reticulum/Golgi region. This enzyme is needed for the phosphorylation of the lysosomal hydrolases and absence of its activity is the cause of the multiple lysosomal enzyme deficiencies in patients with I-cell disease. The retention of both, chick and human beta-galactosidase in the experiments in which I-cell fibroblasts were fused with chick erythrocytes indicates a reactivation of the gene coding for this phosphorylating enzyme. It also implies that this step in the processing of human lysosomal enzymes is not species-specific.     

Optimal growth of Streptococcus cremoris HP in milk is related to β- and ϰ-casein degradation

Summary Initiation of growth and the growth rate of Streptococcus cremoris HP in a complete synthetic medium supplemented with an enzymatic digest of casein appeared to be inhibited by (di)hydrogen phosphate. The inhibitory effect of the inorganic phosphate fraction was counteracted by -glycerophosphate.Growth experiments involving different caseins or combinations of caseins as the only source of nitrogen (and essential amino acids) were performed in an adapted medium in which optimal growth was expected to depend only on the type of nitrogen source. Maximal growth occurred on a combination of -casein and a relatively low concentration of . It approximated the growth on milk added to the medium. These results and those showing the capacity of the organism to grow on milk-derived fractions suggest that in milk it is mainly the soluble (-) casein fraction together with the easily accessible hydrophilic part of micellar , which maintain optimal growth.     

Correction to: First report of a comparative patient-oriented perspective on the use of non-vitamin-K oral anticoagulants or vitamin-K antagonists in atrial fibrillation: patients’ experiences,side-effects and practical problems leading to non-adherence

Netherlands Heart Journal - A Correction to this paper has been published: https://doi.org/10.1007/s12471-019-01331-x     

Cost-Effectiveness of Treatment Strategies for BRAF-Mutated Metastatic Melanoma

Purpose

Genetically-targeted therapies are both promising and costly advances in the field of oncology. Several treatments for metastatic melanoma with a mutation in the BRAF gene have been approved. They extend life but are more expensive than the previous standard of care (dacarbazine). Vemurafenib, the first drug in this class, costs $13,000 per month ($207,000 for a patient with median survival). Patients failing vemurafenib are often given ipilimumab, an immunomodulator, at $150,000 per course. Assessment of cost-effectiveness is a valuable tool to help navigate the transition toward targeted cancer therapy.

Methods

We performed a cost-utility analysis to compare three strategies for patients with BRAF+ metastatic melanoma using a deterministic expected-value decision tree model to calculate the present value of lifetime costs and quality-adjusted life years (QALYs) for each strategy. We performed sensitivity analyses on all variables.

Results

In the base case, the incremental cost-effectiveness ratio (ICER) for vemurafenib compared with dacarbazine was $353,993 per QALY gained (0.42 QALYs added, $156,831 added). The ICER for vemurafenib followed by ipilimumab compared with vemurafenib alone was $158,139. In sensitivity analysis, treatment cost had the largest influence on results: the ICER for vemurafenib versus dacarbazine dropped to $100,000 per QALY gained with a treatment cost of $3600 per month.

Conclusion

The cost per QALY gained for treatment of BRAF+ metastatic melanoma with vemurafenib alone or in combination exceeds widely-cited thresholds for cost-effectiveness. These strategies may become cost-effective with lower drug prices or confirmation of a durable response without continued treatment.     

Bioelectricity production from wastewater treatment in dual chambered microbial fuel cell (MFC) using selectively enriched mixed microflora: Effect of catholyte

The performance of aerated and ferricyanide catholytes on the bioelectricity production was evaluated in dual chambered microbial fuel cell (MFC) (mediatroless anode; graphite electrodes) employing selectively enriched H(2) producing mixed consortia as anodic inoculum. Two MFCs with aerated catholyte (MFC(AC)) and ferricyanide catholyte (MFC(FC)) were operated separately to elucidate the difference in power generation potential and carbon removal efficiency under similar operating conditions [ambient pressure; room temperature (28+/-2 degrees C); acidophilic microenvironment (pH 6)]. The experimental data demonstrated the feasibility of in situ bioelectricity generation along with wastewater treatment. Effective power generation and substrate removal efficiency was documented in the fuel cell operated with ferricyanide catholyte (586 mV; 2.37 mA; 0.559 kg COD/m(3) day) than aerated catholyte (572 mV; 1.68 mA; 0.464 kg COD/m(3) day). Maximum power yield (0.635 W/kg COD(R) and 0.440 W/kg COD(R)) and current density (222.59 mA/m(2) and 190.28 mA/m(2)) was observed at 100 Omega resistor with ferricyanide and aerated catholytes, respectively. The study documented both wastewater treatment and electricity production through direct conversion of H(2) in a single system.     

Isolation of Potentially Pathogenic Escherichia coli O157:H7 from the Ganges River

Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx₁, stx₂, and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.     

Efficient Implementation of Consecutive Reactions by Peptidases at the Periphery of the Streptococcus cremoris Membrane

Activities detectable in Streptococcus cremoris with the chymotrypsin substrate N-glutaryl-l-phenylalanine-4-nitroanilide and formerly designated endopeptidases P37 and P50 (F. A. Exterkate, Appl. Environ. Microbiol. 47:177-183, 1984) are both coupled peptidase reactions. These coupled reactions involve a membrane-bound, restricted l-alpha-glutamyl aminopeptidase which is responsible for the initial release of the glutaryl moiety. The subsequent reaction is catalyzed by either a so-called low-temperature or a high-temperature phenylalanyl aminopeptidase activity, both located at the outside surface of the membrane. Altered microenvironmental conditions created by the membrane-perturbing action of n-butanol or obtained by solubilization resulted in the removal of a restriction on the activity of l-alpha-glutamyl aminopeptidase and in a less efficient functioning of the coupled reactions; a long transient phase occurred before the steady state was reached. The results suggest that the in situ spatial organization is conducive to an efficient attuning of at least three peptidases which are located at the outer membrane surface and in the membrane. The possibility that peptidases in these locations exist as a cluster with physiological significance is discussed in relation to growth of S. cremoris in milk.     

Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine alpha(s1)-, beta-, and kappa-Casein

Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine alpha(s1)-, beta-, and kappa-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM(1), was concluded. The action of the HP-type proteinase P(1) (also detectable in strains Wg(2), C(13), and TR) was established by electrophoretic methods to be directed preferentially towards beta-casein. The AM(1)-type proteinase P(III) (also detectable in strain SK(11)) was also able to degrade beta-casein, but at the same time split alpha(s1)- and kappa-casein more extensively than did P(I). Strain FD(27) exhibited mainly P(I) activity but also detectable P(III) degradation characteristics. The cell wall proteinase preparation of strain E(8) showed low P(I) as well as low P(III) activity. All proteinase preparations produced from kappa-casein positively charged degradation products with electrophoretic mobilities similar to those of degradation products released by the action of the milk-clotting enzyme chymosin. The differences between P(I) and P(III) in mode of action, as detected by gel electrophoresis and thin-layer chromatography, were reflected by the courses of the initial degradation of methyl-C-labeled beta-casein and by the effect of alpha(s1)- plus kappa-casein on these degradations. The results are discussed in the light of previous comparative studies of cell wall proteinases in strains of S. cremoris and with respect to the growth of this organism in milk.     

Eutrophication and mussel culture in the western Dutch Wadden Sea: Impact on the benthic ecosystem; a hypothesis

Since 1950, two large-scale changes have taken place in the western Dutch Wadden Sea, namely the eutrophication of the area and the introduction of an extensive mussel culture. Although eutrophication in the fresh waters started already around 1950, nutrient concentrations in the western Wadden Sea remained fairly constant until about 1970, due to the retention of nutrients in Lake IJssel, the main source. From 1970–1980 concentrations increased strongly, and during the last years the situation has stabilized. Mussel culture was introduced in 1950 and expanded during the next decade to an area of 70 km², all situated in the sublittoral area. From 1960 the area of mussel culture remained about constant with fluctuating yields of between 35 and 120 millions of kg fresh weight. Due to a lack of data for the period until 1970 the impact of eutrophication and mussel culture on the ecosystem cannot be assessed. From 1970 onwards an increased biomass and production of the macrofauna in the intertidal zone has been observed, which is attributed to eutrophication. The hypothesis is postulated that the introduction of mussel culture between 1950 and 1960 has resulted in an increased food competition in the area, leading to a decreased stock of the macrofauna in the intertidal. Eutrophication from about 1970 onwards has improved the food conditions and as a result both the macrofauna in the intertidal and the mussel in the sublittoral area would have increased in biomass, allowing higher maximum yields of the mussel culture. The importance of monitoring programs is stressed to follow these trends in the near future and to check the above hypothesis in areas where it is decided to introduce or intensify mussel culture. Presented at the VI International Wadden Sea Symposium (Biologische Anstalt Helgoland, Wattenmeerstation Sylt, D-2282 List, FRG, 1–4 November 1988) Publication No. 30 of the project “Ecological Research of the North Sea and Wadden Sea” (EON)