Cardiolipin is the membrane receptor for mitochondrial creatine phosphokinase

Treatment of rat heart mitochondria with phosphate or mersalyl releases a number of proteins, including the mitochondrial creatine kinase (mt-CK). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the released proteins showed that phosphate is more selective than mersalyl in releasing mt-CK. The rebinding of mt-CK to mitochondria was selectively inhibited by adriamycin, which complexes membrane-bound cardiolipin. mt-CK activity and binding experiments have shown that intact mitochondria are able to bind approximately twice the amount of mt-CK they originally contain. Liver mitochondria bound heart mitochondria mt-CK to the same extent as creatine kinase-depleted heart mitochondria. mt-CK was bound by liposomes but only if they contained cardiolipin. The binding of mt-CK to cardiolipin-containing liposomes was inhibited by adriamycin. Phosphatidylcholine liposomes reconstituted with the purified ADP/ATP translocator failed to bind mt-CK.     

Idiotypic analysis of polyclonal and monoclonal anti-p-azophenylarsonate antibodies of BALB/c mice expressing the major cross-reactive idiotype of the A/J strain

The idiotypic cascade allows the induction of silent idiotypes, and as such, the immune system can be reprogrammed towards predetermined goals. To understand the genetic origin of silent idiotypes, we have used a system in which detailed structural and genetic information is available. The major cross-reactive idiotype (CRIA) of A/J mice (positive strain) immunized with arsonate coupled to a carrier can be regularly induced in BALB/c mice (negative strain) by anti-idiotypic treatment with or without subsequent antigen immunization. By using a panel of monoclonal anti-idiotypic antibodies, we have found that the germline-encoded CRIA displays a mosaic of at least five idiotopes. Polyclonal and monoclonal anti-arsonate antibodies prepared from idiotypically manipulated BALB/c mice have been studied. Four germline idiotopes are shared between the CRIA of the A/J strain and the CRIA-like idiotype induced in BALB/c mice. Furthermore, CRIA-like antibodies can appear "spontaneously" in some BALB/c mice immunized with antigen only. The data suggest that anti-idiotypic treatment in BALB/c mice selects a preexisting subset of antibodies. From the serological analysis, it is predicted that CRIA molecules from A/J and CRIA-like molecules from BALB/c employ different VH subgroups but share some components of the hypervariable regions. These predictions are tested in a forthcoming paper that describes the amino acid sequences of BALB/c monoclonal antibodies displaying the major cross-reactive idiotype of the A/J strain.     

On the methodology of dissolved oxygen saturation methods

Biotechnology Letters -     

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.     

Subcellular distribution of protein kinase C (pKC) in erythrocytes and concentration ofd-myo-inositol-1,4,5-trisphosphate (IP3) in platelets and monocytes of force-fed zinc-deficient rats

The purpose of the present study was to investigate whether alimentary zinc (Zn) deficiency affects the activities of the Zn metalloenzymes protein kinase C (pKC) and the phosphatidylinositol-specific phospholipase C (PLC) in force-fed Zn-deficient rats. The in vivo activity of pKC was determined by measuring the subcellular distribution of the enzyme between the cytosolic and the particulate fraction of erythrocytes, whereas the activity of PLC was measured indirectly through the concentration of its metabolite inositol-1,4,5-trisphosphate (IP₃) in platelets and monocytes. For this purpose, 24 male Sprague-Dawley rats with an average live mass of 126 g were divided into 2 groups of 12 animals each. The Zn-deficient and the control rats received a semisynthetic casein diet with a Zn content of 1.2 and 24.1 ppm, respectively. All animals were fed the same amount of the diet (10.8 g dry matter [DM]/d and rat) four times daily by gastric tube. After 12 d, the depleted rats were in a state of severe Zn deficiency, as demonstrated by a 70% lower Zn concentration and a 66% reduction in the serum activity of alkaline phosphatase. The radioimmunologically determined concentration of IP₃ was reduced by a significant 55% in the platelets of the Zn-deficient rats (8.4 pmol IP₃/5·10⁸) as compared with the control rats (18.8 pmol IP₃/5·10⁸), whereas the IP₃ concentration in the monocytes was not affected by the alimentary Zn supply (1.4 vs 1.2 pmol IP₃/10⁶), nor was there any difference between the Zn-deficient and the control rats with regard to the radioenzymatically determined specific activity of pKC, either in the cytosolic fraction (32.7 vs 32.5 pmol P/min/mg protein) or in the particulate fraction (38.1 vs 36.5 pmol P/min/mg protein) of the erythrocytes.     

Immunologic correlates of protection against rotavirus challenge after intramuscular immunization of mice.

The capacity of intramuscular (i.m.) inoculation of mice with homologous or heterologous host rotaviruses to induce protection from challenge was evaluated. i.m. inoculation with live, wild-type rotavirus (murine strain EDIM) induced complete protection from viral shedding after challenge for at least 6 weeks after inoculation; protection was correlated with production of virus-specific immunoglobulin A (IgA) by lamina propria (LP) lymphocytes. i.m. inoculation with inactivated EDIM, cell culture-adapted EDIM, or simian strain RRV was associated with partial protection, characterized by reduced viral shedding after challenge. Partial protection after challenge was not associated with production of virus-specific IgA by LP lymphocytes. The mechanisms by which i.m. inoculation induces virus-specific humoral immune responses in the small intestinal LP were examined.     

Quantitative colorimetric and gas chromatoographic determination of arecaidine propargyl ester

Arecaidine propargyl ester (APE) is a potent muscarinic agonist often used in pharmacological studies. To date, no sensitive quantitative analytical method for APE has been published. In this study, two methods for the quantitative determination of APE are compared: a colorimetric assay, based on the formation of the corresponding ferric(III)-hydroxamic acid complex, and a direct gas chromatographic method, using arecoline as the internal standard. The latter method was found to be more precise. The utility of the gas chromatographic assay was further demonstrated in a stability study of the drug in the biological fluid aqueous humor of rabbits.