Protein differences in tau mutant hamsters: candidate clock proteins.

In the tau mutant hamster, the period of the circadian rhythm is shortened from about 24 h to about 22 h in heterozygotes and to about 20 h in homozygotes. Understanding the biochemical basis of the period changes in the tau mutant may elucidate the regulation of the vertebrate pacemaker. Using two-dimensional gel electrophoresis, we have found two sets of proteins that differ between the different genotypes. P33tau (about 33 kDa; pI 6.5) was found in all gels from wild type and heterozygous animals, but was absent in gels from all except one of the homozygous mutant animals. P32tau (about 32 kDa; pI 4.8) was a chain of spots, which showed a striking difference in pattern between gels from wild type animals and from mutant animals. P33tau was greatly enriched in soluble cellular fractions, whereas P32tau was found only in insoluble fractions. These differences between P33tau and P32tau were apparent in gels from both SCN and cortical tissue, suggesting that both proteins are distributed throughout the brain. These proteins should be useful as new tools to explore the biochemistry of circadian pacemakers.

Characterization of a human high density lipoprotein-associated protein, NA1/NA2. Identity with SP-40,40, an inhibitor of complement-mediated cytolysis.

Two peptides, NA1 and NA2, which we previously suggested to be associated with high density lipoproteins (HDLs), have been purified. Polyclonal antibodies against each peptide and a monoclonal antibody against NA2 have been used to further characterize them and their association with HDL. Immunoblotting studies revealed that the peptides form a complex of molecular mass of approximately 80 kd. Agarose gel filtration showed coelution of NA1/NA2 and apolipoprotein (apo) A-I, the structural protein of HDL. This was confirmed by fast protein liquid chromatography, which further indicated that up to 60% of NA1/NA2 was located within the lower density range of the HDL spectrum. Complementary studies with anti-apo A-I immunoaffinity columns provided evidence that at least 40% of NA1/NA2 was associated with HDL, an association easily disrupted by ultracentrifugal manipulation. Finally, partial amino acid sequences showed virtually complete homology with a recently identified protein, SP-40,40, or cytolysis inhibitor. The protein is suggested to have a powerful inhibitory effect on complement-mediated cell lysis. Our results could thus furnish an explanation for the previously observed modulating influence of HDL on complement activity.

Preparative isoelectrofocusing and high resolution 2-dimensional gel electrophoresis for concentration and purification of proteins.

We have analysed the potential of liquid phase isoelectrofocusing allied to high resolution 2-dimensional gel electrophoresis for concentrating and purifying in particular plasma proteins present in low concentrations. The primary fractionation step, liquid phase isoelectrofocusing, offers several advantages. Firstly, it allows protein profiles to be examined within defined pH ranges. Additionally, it confines the bulk plasma proteins to their respective pI ranges, allowing larger quantities of protein to be employed for the second fractionation phase. Concurrently, it concentrates proteins present at low concentrations, also by restricting them to their pI ranges. Yields are such that detailed characterisation studies of the proteins can be undertaken, providing the means of generating comprehensive data banks to complement plasma protein maps. The procedure should be applicable to a wide range of biological fluids and tissues.

From biopsy to automatic diagnosis.

High resolution two-dimensional gel electrophoresis is a very powerful biochemical tool for analysis of complex protein mixtures. In well defined situations, protein maps, obtained from tissue biopsies or biological fluids by this technique, can be automatically analyzed by computer. Some polypeptide patterns are the fingerprints of diseases. Applying clustering algorithm and learning techniques, the prototype expert system MELANIE recognized patterns and associated the correct diagnosis to the specific pattern.

A novel high-density lipoprotein particle and associated protein in rat plasma.

This report describes the characterization of a novel rat apolipoprotein, which, as partial sequencing suggests, does not correspond to any described protein. The protein (termed PX) has an estimated molecular mass of 19.5 kDa and pI in the range 5.5-5.8. Monoclonal antibodies were obtained against protein PX and results on distribution among rat lipoproteins show it to be associated mainly with high-density lipoproteins (HDL), but also with VLDL. Immunoaffinity chromatography of total HDL shows protein PX to be included in a distinct lipoprotein particle, particularly enriched in free cholesterol, with which only traces of other apolipoproteins are associated. Immunologically crossreacting entities are found in the plasma of several species, including man. Retention of the epitope carried by the protein PX would suggest that it is of particular structural or functional importance. It remains to be established whether its function is associated with lipid metabolism.

Methods for increasing the resolution of two-dimensional protein electrophoresis.

A two-dimensional gel elctrophoresis protocol has been developed which provides for a 1.5-to 3-fold increase in the resolution of proteins compared to other frequently used methods. The major variations from previous protocols include increased pore size in the isoelectric focusing gels; cholamidopropyldimethylhydroxypropanesulfonate, a zwitterionic detergent, replaces most of the Nonidet P-40, a nonionic detergent, in the isoelectric focusing gels; no equilibration step is employed between the first and second dimensional separation. The use of a stacking gel in the second dimension has been eliminated; a more efficient and evenly distributed cooling system has been designed for the molecular mass separation, allowing faster migration with higher current. Finally, the crosslinker diacrylylpiperazine is employed which improves protein separation and detection with ammoniacal silver staining. Silver-stained two-dimensional gel electrophoretograms of human plasma and hamster brain tissues and autoradiographs of rat liver cells are compared to the results obtained from previous methods.

HDL particle associated proteins in plasma and cerebrospinal fluid: identification and partial sequencing.

The proteins from plasma HDL particles isolated by immunoaffinity chromatography on anti-apolipoprotein A-1 affinity columns have been analysed and purified by high resolution two dimensional gel electrophoresis. Two of the lipoprotein-associated proteins found in the HDL plasma fraction, previously referred to as NA1 and NA2, have also been found in cerebrospinal fluid. After separation by 2DGE, these two proteins were transferred to PVDF membranes, stained and cut out for N-terminal sequencing. The partial sequences (11 and 13 amino acids) obtained for the two HDL particle associated proteins do not match any of those included in the December 1987 National Biomedical Research Foundation (NBRF) database, and there are no significant sequence similarities.

Hyperinsulinemia of preobese and obese fa/fa rats is partly vagus nerve mediated.

In vivo glucose-induced insulin secretion was greater in preweaned preobese 17-day-old Zucker rats than in the corresponding controls. This hypersecretion of insulin was reversed to normal by acute pretreatment with atropine. A short-lived (30 s) electrical stimulation of the vagus nerve preceding a glucose load potentiated the in vivo glucose-induced insulin release in adult animals (6-9 wk) and more so in obese Zucker (fa/fa) than in lean rats. This suggested the existence of enhanced sensitivity and/or responsiveness of the B cells of obese animals to the parasympathetic system. That the parasympathetic tone was increased in adult obese Zucker (fa/fa) rats was corroborated by the observation that acute vagotomy of these animals resulted in a significant decrease in glucose-induced insulin secretion, whereas no such effect was seen in lean rats. Also, perfused pancreases from adult obese (fa/fa) rats oversecreted insulin during a stimulation by arginine when compared with controls, an oversecretion that was restored toward normal by superimposed infusion of atropine. It is concluded that a) the increased insulin secretion of preobese Zucker fa/fa rats is an early abnormality that is mediated by the vagus nerve, and b) increased secretion of insulin in adult obese fa/fa rats continues to be partly vagus-nerve mediated, although a decreased sympathetic tone and other unknown defects could conceivably play a role as well.