William L. Stahl

The Na,K-ATPase of nervous tissue

The Na,K-ATPase has been only partially purified from nervous tissue, yet it is clear that two forms (and ? +) of the catalytic subunit are present. ? is a component subunit of the glial Na,K-ATPase, which has a relatively low affinity for binding cardiac glycosides and ? + has been identified as a subunit of the Na,K-ATPase which has relatively high affinity for cardiac glycosides. The ? + form may also be sensitive to indirect modulation by neurotransmitters or hormones. The ratio of ? + /? changes in the nervous system during development, and ? + appears to be the predominant species in adult neurones. Changes in Na,K-ATPase activity have been associated with several abnormalities in the nervous system, including epilepsy and altered nerve conduction velocity, but a causal relationship has not been definitively established. Although the Na,K-ATPase has a pivotal role in Na(+) and K(+) transport in the nervous system, a special role for the glial Na,K-ATPase in clearing extracellular K(+) remains controversial.

Biochemical abnormalities in Huntington's chorea brains

The enzymes glutamic acid decarboxylase, choline acetylase, and succinate dehydrogenase were reduced by 74 to 93 percent in striatum of brains from three patients who died with long-standing Huntingtons chorea. Little change was found in acetylcholinesterase or monoamine oxidase activities. In an early case of this disease in a fourth patient, there were no enzymatic changes. A high-molecular-weight soluble protein was present in the striatum of the advanced cases but membrane protein patterns did not differ from those of controls.

Expression of Oncogenic Epidermal Growth Factor Receptor Family Kinases Induces Paclitaxel Resistance and Alters β-Tubulin Isotype Expression

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII andHER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of β-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed β-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total β-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2.95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both β-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.

Plasma membrane Ca2+-ATPase isoforms: distribution of mRNAs in rat brain by in situ hybridization

Several mRNAs which encode for isoforms of the plasma membrane Ca(2+)-transport ATPase (PMCA) are present in adult rat brain. Using in situ hybridization with antisense oligonucleotide probes we found complex patterns of specific hybridization for three isoforms (PMCA1-3). Each rat brain region studied exhibited a distinct pattern of expression of isoforms. PMCA1 mRNA, which is widely distributed in rat tissues, was highest in CA1 pyramidal cells of hippocampus and very low in hypothalamic nuclei, cerebellum and choroid plexus. PMCA2 mRNA was highest in Purkinje cells of cerebellum and low in caudate-putamen, hypothalamic nuclei, habenula and choroid plexus. The highest levels of PMCA3 mRNA were found in habenula and choroid plexus. The PMCA1-3 isoforms appeared to be expressed primarily in neurons since hybridization was detected neither in white matter nor in regions rich in astrocytes. In different regions, different levels of expression of each PMCA mRNA may underlie specialized requirements for calcium homeostasis in specific neurons.

Uptake of calcium ions by synaptosomes from rat brain

Abstract Rat brain synaptosomes incubated in modified Krebs-Ringer media accumulated 45 Ca in amounts dependent upon the medium calcium concentration. At 37 °C, 45 Ca uptake was increased by 19% upon the application of electrical pulses. Uptake was effectively inhibited by ruthenium red, uncouplers of oxidative phosphorylation, antimycin A and rotenone, and fluorocitrate while oligomycin and ouabain increased 45 Ca uptake. Transfer to a medium low in Na + led to marked increase in 45 Ca uptake and this effect was also found in the presence of rotenone and arsenate. The results indicate the requirements of metabolic energy for a component of calcium uptake by synaptosomes. Effects of low Na + incubation and electrical stimulation may be due to events at the outer synaptosomal membrane though the possibility of direct effects at the mitochondrion have not been excluded.

Role of phospholipids in the Na+,K+-stimulated adenosine triphosphatase system of brain microsomes

Abstract Removal of phospholipids from brain microsomes using a purified, protease-free phospholipase C preparation led to proportional losses of net Na + ,K + -stimulated adenosine triphosphatase, K + -stimulated p -nitrophenylphosphatase, and Na + -stimulated ADP-ATP exchange activities. These enzymatic activities were restored to 60–100% of control values by the addition of a variety of purified phospholipids, but not by detergents or EGTA. These findings support the concept of a general phospholipid requirement for this enzyme system. This work further suggests that phospholipids are important both for formation and decomposition of the phosphorylated intermediate (s) which probably participate in the net reaction.

Controls for Immunohistochemistry: The Histochemical Society’s Standards of Practice for Validation of Immunohistochemical Assays

Immunohistochemistry is widely used in biomedical research to localize specific epitopes of molecules in cells and tissues. The validity of interpretations based on immunohistochemistry requires appropriate positive and negative controls that are often not reported in publications. This omission may lead to incorrect interpretations and irreproducible results in the literature and contribute to wasted time, effort, and resources as well as erosion of confidence in scientific investigation by the general public, legislative bodies and funding agencies. The present article summarizes essential controls required for validation of immunohistochemical findings and represents a standard of practice for the use of immunohistochemistry in research and diagnostic investigations. Adherence to the guidelines described in the present article can be cited by authors as support for the validity of interpretations of the immunohistochemistry reported in their publications.

Developmental and regional differences in the localization of Na,K-ATPase activity in the rabbit hippocampus

Regional differences in Na,K-ATPase activity, and development of Na,K-ATPase activity were examined in rabbit hippocampus using a histochemical marker of enzyme activity. Stratum lucidum of CA3/CA2, corresponding to the mossy fiber terminal field, showed high Na,K-ATPase activity compared to stratum radiatum of CA1. A significant increase in Na,K-ATPase activity was found between 8 and 15 days postnatal. Tissues with limited Na,K-ATPase activity (immature hippocampus, the mature CA1 region) appear particularly prone to seizure-like abnormalities, perhaps reflecting an inability to regulate extracellular potassium.

Phospholipase C purification and specificity with respect to individual phospholipids and brain microsomal membrane phospholipids

Abstract Phospholipase C was purified from a crude preparation derived from Cl. perfringens utilizing a one-step polypreparative electrophoresis procedure. The purified enzyme has a molecular weight of 46,500 ± 500 and is essentially free of proteolytic and phospholipase A enzymatic activities. It exhibited the following substrate specificity: PC ≥ SM > PS > PI, lyso PC. PE was hydrolyzed when PC was present. Treatment of brain microsomes with purified phospholipase C reduced membrane phospholipids by 69%. All phospholipids were attacked including PE. PC was reduced to 4% and all other phospholipids to 23–43% of their control levels. Total fatty acid composition of brain microsomes was not affected by phospholipase C action.

Localization of the plasma membrane Ca2+-ATPase isoform PMCA3 in rat cerebellum, choroid plexus and hippocampus

mRNA encoding rat plasma membrane Ca(2+)-ATPase isoform PMCA3 was localized in the granule cell layer of the cerebellum and in choroid plexus by in situ hybridization with an 35S-labelled oligodeoxynucleotide probe. In order to examine whether this isoform is expressed as a protein in brain, polyclonal antibodies were raised against a peptide corresponding to a C-terminal 18 amino acid sequence of PMCA3 which had been conjugated to bovine serum albumin. Using immunoblot analysis with affinity-purified antibodies, PMCA3 protein was found in rat brain microsomes and cultured neurons. The translated protein had an observed molecular mass of approximately 135 kDa, as predicted from molecular cloning studies. The pattern of localization of PMCA3 in brain using anti-peptide antibodies was consistent with findings from in situ hybridization. PMCA3-like immunoreactive sites were found in the granule cell and molecular layers of rat cerebellum and in choroid plexus, and the pattern of staining suggests that immunoreactive sites are associated with granule cell processes. This conclusion was supported by the finding that growth-associated protein-43, a protein known to be present in axons and nerve terminals, had a pattern of distribution similar to PMCA3 in the molecular layer of cerebellum. Very low levels of PMCA3-like immunoreactivity were associated with Purkinje cell soma or processes, consistent with the low levels of PMCA3 mRNA found in these neurons. PMCA3-like immunoreactivity was lower in hippocampus than in cerebellum; hippocampal CA1 region immunoreactivity was primarily associated with dendritic fields rather than with pyramidal cell bodies. The results demonstrate that a PMCA3-like protein is expressed in neurons of rat brain and is localized primarily in cell processes.