Assignment of genes encoding a unique cytokine (IL12) composed of two unrelated subunits to chromosomes 3 and 5.

IL12 (formerly NKSF or CLMF) is a unique cytokine composed of two unrelated disulfide-linked subunits. The larger 40-kDa subunit (p40) is a member of the cytokine receptor family, and the smaller 35-kDa subunit (p35) is related to IL6 and GCSF. The chromosomal localization of these two subunits was determined by PCR analysis of DNA from rodent-human hybrids. More refined mapping was obtained by PCR analysis of hybrids containing translocation chromosomes and for p40, by analysis of radiation hybrids. The subunits map to different chromosomes: p40 (IL12B) to 5q31-q33 and p35 (IL12A) to 3p12-3q13.2.     

A detailed study of the regulation and evolution of the two classes of patatin genes in Solanum tuberosum L.

The class-specific expression of patatin genes was investigated by analysing four new patatin genes. A class I patatin gene from cv. Berolina as well as a class I and two class II patatin genes from the monohaploid cultivar AM 80/5793 were isolated and partially sequenced. Sequence comparison indicates rearrangements as the major source for the generation of diversity between the different members of the classes. The expression of single genes was studied in potato plants transformed with chimaeric genes where the putative patatin promoters were fused to the GUS reporter gene. A detailed histochemical analysis reveals that both class I genes are expressed as the previously described class I patatin gene B33 from cv. Berolina [1], i.e. in the starch-containing cells of potato tubers and in sucrose-induced leaves. The class II gene pgT12 shows the same pattern as the previously described class II gene pgT2 [2], i.e. expression in root tips and in the vascular tissue of tubers, whereas no activity was detectable for pgT4. Thus the expression pattern of both classes of genes seems to be stable at least within or even between different cultivars.     

A quartz crystal biosensor for measurement in liquids.

The detection of anti-human immunodeficiency virus (HIV) antibodies by means of synthetic HIV peptide immobilized on a piezoelectric quartz sensor is demonstrated. The measurement set-up consists of an oscillator circuit, a suitably modified AT-cut thickness-shear-mode quartz crystal with gold electrodes, which is housed in a special reaction vessel, and a computer-controlled frequency counter for the registration of the measured frequency values. The quartz crystal is adapted for a steady operation in liquids at a frequency of 20 MHz. In phosphate-buffered saline solution the oscillator reaches a stability of about 0.5 Hz within a few seconds, of about 2 Hz within 10 min and about 30 Hz within 1 h. The frequency shift due to the adsorption of various proteins to the uncoated sensor surface has been investigated. It can be shown that a stable adsorptive binding of proteins to an oscillating gold surface is feasible and can be used for the immobilization of a receptor layer (e.g. HIV peptide). Specific binding of the anti-HIV monoclonal antibody to the HIV peptide immobilized on the quartz sensor is demonstrated. Control experiments show, however, additional unspecific binding. According to the experiments, the Sauerbrey formula gives a sufficiently accurate value for the decrease of the resonant frequency due to adsorption or binding of macromolecular proteins on the quartz crystal surface.     

Potentiation and suppression of mouse liver cytochrome P-450 isozymes during the acute-phase response induced by bacterial endotoxin

Infection and inflammation are known to affect the metabolism and disposition of drugs and carcinogens. We report a detailed study of the effects of bacterial endotoxin on the constitutive and inducible expression and activities of cytochrome P-450 isozymes from families P-450I, P-450IIB, P-450IIC and P-450III. In general high doses of high endotoxin caused very marked suppression of P-450 isozymes and associated activities. However, this effect was differential, the expression of certain isozymes being only slightly reduced whereas others were suppressed to almost undetectable levels. Low doses of endotoxin also gave differential effects on cytochrome P-450 expression. Of particular interest was the very marked potentiation of the inductive effect of both 3-methylcholanthrene and phenobarbital. In the case of 3-methylcholanthrene the 10-fold induction of activity was increased to 24-fold by concomitant endotoxin administration. In this regard it was interesting that 3-methylcholanthrene was an effective inducer of a wide variety of acute-phase proteins including metallothionein, serum amyloid A, fibrinogen and hemopexin. These data show that endotoxin, and therefore bacterial infection and inflammation, can have profound and differential effects on components of the cytochrome-P-450 monooxygenase system which could result in significant changes in susceptibility to the effects of drugs, chemical toxins and carcinogens.     

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — energy-dependent switching between two different active states of the proton-translocation adenosine triphosphatase

Single-turnover flash-induced ATP synthesis coupled to natural cyclic electron flow in Photosystem I-enriched subchloroplast vesicles (from spinach) was continuously followed by the luciferin-luciferase luminescence. The ATP yield per flash was maximal (1 ATP per s per 1000 Chl) around a flash frequency of 0.5–2 Hz. It decreased both at lower and higher flash frequencies. The decrease at high flash frequency was due to limitation by the electron-transfer rate, while the decrease at low flash frequency was directly due to intrinsic properties of the ATPase itself. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) decreased the yield at low frequency more than at high frequency. The same behaviour was observed if electron transfer was artificially mediated by pyocyanin. If the ADP concentration was increased from 40 to at least 80 μM, or if the vesicles were preincubated with 5 mM dithiothreitol (DTT), the decrease of the yield at flash frequencies below 0.5 Hz was no longer observed. Incubation with DTT increased the rates of ATP hydrolysis and synthesis at any flash frequency. The decrease of the yield could be elicited again by addition of 50 nM FCCP. It is concluded that at low levels of the protonmotive force (Δ gm_H⁺), the ATPase is converted into an active ATP-hydrolyzing state in which ATP synthesis activity is decreased due to a decreased affinity towards ADP and/or to a decreased release of newly synthesized ATP, that can be cancelled by increasing the ADP concentration or by addition of DTT in the absence of uncoupler.     

Causes of nondiffusing lipid in the plasma membrane of mammalian spermatozoa

In the plasma membranes of most mammalian somatic cells, lipid is nearly completely free to diffuse laterally in the plane of the membrane. In mammalian spermatozoa and certain other highly polarized mammalian cells, a significant fraction of the plasma membrane lipid is not free to diffuse laterally. Using the technique of fluorescence recovery after photobleaching, we have demonstrated that a variety of fluorescent lipid analogues exhibit a nondiffusing fraction in the plasma membrane of the anterior region of the ram sperm head. The possible causes of this nondiffusing fraction were investigated. The nondiffusing lipid fraction is not the result of lipid oxidation during handling, and it is not released by extensive enzymatic digestion of the membrane surface proteins or the "bleeding" of the membrane by hypoosmotic shock. When lipid bilayers were prepared from protein-free lipid extracts of the plasma membranes of spermatozoa, most of the nondiffusing fraction was retained. These results suggest that the nondiffusing lipid fraction results from lipid factors such as lateral phase separations, which can cause such a nondiffusing fraction in model systems.     

Binding of spin-labeled clofibrate to lipoproteins

The binding of spin-labeled clofibrate to native and partially delipidated lipoproteins is a rapid, linear and non-saturable process observed up to the critical micellar concentration of the drug. Low-density lipoproteins (LDL) display a lower affinity for the drug than very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) relative to their respective specific volume. Unlike various lipophilic drugs, uptake of spin-labeled clofibrate does not correlate with lipoprotein lipid volume. Spin-labeled clofibrate binding to LDL is enhanced when the temperature increases above 25 degrees C. The binding to HDL and VLDL is less temperature-sensitive. The simulation of the ESR spectra has shown that two types of motion should be superimposed for the spin-labeled clofibrate in HDL, in LDL or in partially delipidated LDL. From 40 down to 25 degrees C for HDL and LDL, a fast anisotropic motion is observed. From 25 degrees C down to 5 degrees C, a two-component motion takes place, including a slow isotropic motion of the probe tumbling in a highly hydrophobic environment. Interactions of spin-labeled clofibrate with the apolipoproteins in HDL and LDL are assumed from the emergence of this strongly immobilized component observed when the temperature decreases. In contrast, for spin-labeled clofibrate inserted in the apolar core of VLDL, ESR shows only one component in the whole temperature range (5-40 degrees C). The location of the spin-labeled drug inside the various lipoprotein particles is discussed as a function of temperature.     

Postsynaptic densities contain a subtype of protein kinase C

Protein kinase C or an isoenzyme thereof appears to be a significant component of postsynaptic densities (PSDs) from rat brain. This cytoskeletal organelle binds 4 beta-phorbol 12,13-dibutyrate (PDBu) with a Bmax of about 20 pmol/mg protein and an apparent Kd of 3.3 nM. Ca2+ and phosphatidyl serine (PS) stimulated the endogenous phosphorylation of a subset of PSD polypeptides with Mr values between 16,000 and 22,000. Finally, a monospecific protein kinase C antibody reacted with a Mr 70,000 PSD polypeptide which migrated on SDS-PAGE slightly ahead of the Mr 77,000 purified enzyme. These data suggest that protein kinase C or a similar enzyme can be integrated into a cytoskeletal system and may play an important role in postsynaptic function.