The differential effects of the carcinogen dimethylnitrosamine on isocitrate and 6-phosphogluconate metabolism in single intact cells

A microspectrofluorimetric study is made of the influence of dimethylnitrosamine on NADP reduction, following sequential microinjections into the same L cell, of two substrates: (1) isocitrate, with activity of isocitrate dehydrogenase both in the extramitochondrial and intramitochondrial compartments, (2) 6-phosphogluconate, with activity of the dehydrogenase in the extramitochondrial compartment. In control L cells a two-step reduction of NAD(P) is obtained followed by relatively slow reoxidation. In the minutes which follow addition of carcinogen, e.g., dimethylnitrosamine, to the cell medium the isocitrate and 6-phosphogluconate-induced transient NADP reoxidation is decreased in magnitude compared to control, while the rate constant of NADPH reoxidation is considerably accelerated, possibly due to requirements at the level of the microsomal metabolizing system. Observations within the first hour of carcinogen addition suggest an interesting system for evaluating the immediate actions of carcinogens at extranuclear sites: i.e., a comparative study of NADP reduction-reoxidation rate constants via injection of substrates for extra- vs. intramitochondrial pathways.     

Complement Factor H-Related Protein 3 Serum Levels Are Low Compared to Factor H and Mainly Determined by Gene Copy Number Variation in CFHR3

The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein.     

Acid proteases from the yolk and the yolk-sac of the hen's egg. Purification, properties and identification as cathepsin D

Two acid proteases from the yolk and one from the yolk-sac of the hen's egg were purified and characterized. They belong all three to the class of cathepsin D. Mostly, minor differences were found in their properties. Possible endogenous substrates, as some egg-proteins and some other yolk-enzymes, were hydrolysed with variable intensity, in vitro.     

Photoaffinity probes for serotonin and histamine receptors. Synthesis and characterization of two azide analogues of ketanserin

Two azide analogues of ketanserin (6- and 7-azido-3-[2- [4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-2, 4(1H,3H)-quinazolinedione) were synthesized and tested as possible photoaffinity probes for serotonin-S2 and histamine-H1 receptors. In reversible binding experiments, the azides showed high affinity for both receptor types. When membrane preparations were incubated with nanomolar concentrations of 7-azidoketanserin and subsequently irradiated with UV light, both serotonin and histamine receptors became irreversibly blocked. This irreversible binding was dependent on azide concentrations and time of irradiation and did not change in the presence of the scavenger p-aminobenzoic acid. In contrast, irreversible blockade at low concentrations of 6-azidoketanserin was only obtained for histamine receptors. However, this blockade was abolished by addition of the scavenger p-aminobenzoic acid indicating that it was not due to a real photoaffinity mechanism. In the rat prefrontal cortex, irreversible blocking of serotonin receptors with 7-azidoketanserin could be inhibited by serotonin agonists or antagonists but not by histaminergic compounds. On the contrary, in the guinea pig cerebellum, inactivation of histamine receptors could be inhibited by histamine antagonists and histamine itself but not by serotonergic compounds. This provides a way for differential photolabeling of either of these receptors.     

Persistence of the pBR 322 plasmid in Escherichia coli K 12 grown in chemostat cultures

Populations of a Escherichia coli K 12 strain, containing the vector plasmid p BR 322, were grown in chemostat culture under glucose- and phosphatelimited conditions. Resistance to tetracycline and ampicillin were lost after prolonged cultivation, resulting in the production of apparent plasmid-free populations which were more competitive than the original population. This competitiveness between plasmid-free and plasmid-containing populations was greatest in environments where the nutrient restriction was severe. Also during sequential subcultivation in batch cultures loss of plasmid was observed.     

Construction and characterization of mutants impaired in the biosynthesis of the K88ab antigen

Plasmid pFM205 contains the genetic determinant for the K88ab antigen and is composed of a 4.3-megadalton DNA fragment derived from wild-type K88ab plasmid pRI8801 and cloning vehicle pBR322. The K88 NA of pFM205 contains five genes, which code for polypeptides with apparent molecular weights of 17,000, 26,000 (the K88ab subunit), 27,000 27,500, and 81,000. All five polypeptides were synthesized as precursors approximately 2,000 daltons larger than the mature polypeptides, indicating that they are transported across the cytoplasmic membrane by means of a signal sequence. A set of deletion derivatives of pFM205 was constructed, each containing a deletion in one of the five genes. In strains harboring derivatives of pFM205 containing a deletion in the gene for the 17,000- or 81,000-dalton polypeptide, the K88ab subunit was synthesized and transported to the outside of the cell. However, these strains did not adhere to brushborders or guinea pig erythrocytes, suggesting that the K88ab subunits were not assembled into normal fimbriae. Strains harboring plasmids containing a deletion in the gene for the 27,500-dalton polypeptide still adhered to brush borders and guinea pig erythrocytes, although very little K88ab antigen could be detected with an immunological assay. In strains harboring plasmids containing a deletion in the gene for the 27,000-dalton polypeptide, the K88ab subunit was synthesized but was probably subsequently degraded rapidly.     

Isolation and characterization of lipoteichoic acid, a cell envelope component involved in preventing phage adsorption, from Lactococcus lactis subsp. cremoris SK110.

The cell envelope of the phage-resistant Lactococcus lactis subsp. cremoris SK110 differed from its phage-sensitive variant by the presence of a galactosyl-containing component. This component was present in material obtained from SK110 by a mild alkali treatment. In a similar fraction extracted from SK112, no galactosyl-containing components were detected. With respect to gel permeation chromatography and electrophoretic mobility, identical characteristics of the alkali-extracted material and purified lipoteichoic acid (LTA) were measured. Chemical analysis of the latter component showed the absence of galactose in LTA isolated from SK112, whereas it was present in LTA obtained from SK110. In this paper, we propose that galactosyl-containing LTA is involved in preventing phage adsorption to L. lactis subsp. cremoris SK110.