Single-step,rapid separation of acidic and basic isoenzymes from commercial horseradish peroxidase

A simple and sensitive high-performance liquid chromatography method with electrochemical detector is described for the determination of free 3-methoxy-4-hydroxyphenylacetic acid (HVA) in human plasma. The method does not involve any extraction, is specific and reproducible, and has the potential to measure serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) simultaneously. The plasma concentration of free HVA in eight normal, healthy adult volunteers was 10.9 ± 4.6 (mean ± SD). In a preliminary study, in one schizophrenic patient the plasma HVA increased twofold after neuroleptic treatment.     

Dormancy in peach (Prunus persica L.) flower buds

Morphological studies were carried out with peach flower buds collected monthly in 1989 and 1990, from two months before leaf fall (7 March) until two to three weeks before bloom (7/8 August). Chilled (2–4°C for 30 days) and unchilled buds were exposed to 20 to 25°C, 100% RH and continuous light. Gibberellin A₃ (3 ng or 30 ng) was applied to some of the non-chilled cuttings at three days intervals. Then, 12, 19, and 26 days after they were planted, the buds were sampled and processed for histological studies. Cultured flower buds (chilled or unchilled) had accelerated anther and gynoecium morphogenesis after 12 days under controlled conditions, compared to buds processed immediately after collection from the field. Chilling treatment augmented the bud culture effect, while Gibberellin A₃ applications to the excised buds retarded bud morphogenesis to a stage comparable to that of buds collected directly from the field. This, suggests that the comparatively high levels of Gibberellin A_1/3 we previously found in mid winter [15, 18] could be at least one of the factors that controls floral bud dormancy by retarding anther and gynoecium development.     

Inhibition by insulin of glucagon-dependent phospholipid methyltransferase phosphorylation in rat hepatocytes

Addition of insulin to isolated rat hepatocytes prelabeled with [32P]phosphate inhibited glucagon-dependent phospholipid methyltransferase phosphorylation and activation. Insulin alone had no effect on either the phosphorylation of the enzyme or on its activity. The effect of insulin on glucagon-dependent phospholipid methyltransferase phosphorylation was dose-dependent and occurred at physiological doses of the hormone (10(-11)-10(-10) M). Analysis of 32P-labeled peptides after digestion with trypsin revealed only one site of phosphorylation regulated by glucagon (10(-8) M) in isolated rat hepatocytes. This site, as analyzed by HPLC and thin-layer chromatography, coincided with that phosphorylated by the cAMP-dependent protein kinase using purified rat liver phospholipid methyltransferase.     

Oxidation of Ferrous Iron and Elemental Sulfur by Thiobacillus ferrooxidans

The oxidation of ferrous iron and elemental sulfur by Thiobacillus ferrooxidans that was absorbed and unabsorbed onto the surface of sulfur prills was studied. Unadsorbed sulfur-grown cells oxidized ferrous iron at a rate that was 3 to 7 times slower than that of ferrous iron-grown cells, but sulfur-grown cells were able to reach the oxidation rate of the ferrous iron-adapted cells after only 1.5 generations in a medium containing ferrous iron. Bacteria that were adsorbed to sulfur prills oxidized ferrous iron at a rate similar to that of unadsorbed sulfur-grown bacteria. They also showed the enhancement of ferrous iron oxidation activity in the presence of ferrous iron, even though sulfur continued to be available to the bacteria in this case. An increase in the level of rusticyanin together with the enhancement of the ferrous iron oxidation rate were observed in both sulfur-adsorbed and unadsorbed cells. On the other hand, sulfur oxidation by the adsorbed bacteria was not affected by the presence of ferrous iron in the medium. When bacteria that were adsorbed to sulfur prills were grown at a higher pH (ca. 2.5) in the presence of ferrous iron, they rapidly lost both ferrous iron and sulfur oxidation capacities and became inactive, apparently because of the deposition of a jarosite-like precipitate onto the surface to which they were attached.     

Phospholipid methyltransferase phosphorylation by intact hepatocytes: effect of glucagon

We have obtained a rabbit antiserum that specifically immunoprecipitates the 50K and 25K proteins of rat liver phospholipid methyltransferase. Exposure of intact rat hepatocytes preincubated with [32P]phosphate to glucagon induces a time-dependent phosphorylation of the 50K protein of phospholipid methyltransferase. The incorporation of 32P into the 50K protein was only on phosphoserine. These data support the concept that the activation of rat liver phospholipid methyltransferase by glucagon is mediated by phosphorylation of the enzyme.     

An attempt to differentiate further between microglia and fluorescent granular perithelial (FGP) cells by their capacity to incorporate exogenous protein

It seems established that under pathological conditions, microglia and blood monocytes (invading the cerebral parenchyma) behave as histiocytic cells in the central nervous system. However, it has not been clear whether or not phagocytic cells are present in normal cerebral tissue. Recently, we found a new type of cell having an uptake capacity for exogenous substance at the bifurcations of small cerebral vessels except for capillaries. According to Imamoto et al. (1982), ameboid microglia, a kind of precursor of microglia, appear at a perinatal stage and can incorporate exogenous material. In the present paper, the developmental sequences of ameboid microglia and the unique cells laden with fluorescent granules are compared at a light and electron-microscopic level. From this study, it is clear that ameboid microglia are already present in the corpus callosum at 5 days after birth and are potent in their uptake capacity for horseradish peroxidase (HRP). However, at 2 weeks, they transform into star cells and the capacity for incorporation diminishes markedly. The finding is also supported by the quantitative analysis of transformation of ameboid microglia. At 3 months, glial cells do not take the administered HRP under the present conditions. On the other hand, fluorescent granular perithelial (FGP) cells arise from a leptomeningeal tissue (pia mater) and become situated in the perivascular spaces. They are not clearly defined at 5 days, and their uptake capacity for HRP has not yet developed. At 2 weeks, the FGP cells take definite forms with several inclusion bodies, and their uptake capacity for HRP attains a certain degree. Often, they are located at bifurcations of small blood vessels. At 3 months, the FGP cells differentiate completely in appearance, and their pinocytotic capacity reaches a high level. Consequently, the FGP cells belong to a different type of cell from that of ameboid microglia in their developmental sequences and assume a principal role of scavenging waste products in normal cerebral tissue.     

Monoclonal antibodies to epitopes on different regions of the 200 00 dalton neurofilament protein : Probes for the geometry of the filament

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.