Modifications of S100-protein immunoreactivity in rat brain induced by tissue preparation

Immunocytochemistry using antibodies against various molecular forms of the Ca⁺⁺ and Zn⁺⁺-binding S100 proteins predominantly labelled astrocytes. However, especially in the neocortex the staining pattern is variable. Methods of tissue preparation have been evaluated with the aim to preserve as much S 100 immunoreactivity as possible. Optimal results were obtained after perfusion fixation with 4–5% aldehydes, 0.1M sodium cacodylate, 0.1% CaCl₂, pH 7.3. In such preparations, astrocytes were completely labelled including their lamellar compartments in large parts of the central nervous system. Ca⁺⁺-withdrawal had adverse affects on S100 immunoreactivity. Cryostat sections treated with EDTA-containing solutions before fixation showed that Ca⁺⁺-free S100 can apparently not be fixed to the tissue. Perfusion fixatives containing EDTA resulted in inhomogeneous loss of S100 staining, indicating a differential susceptibility of astrocytic subpopulations. A different type of reduction in S100 immunoreactivity occurred around large neocortical blood vessels. Perivascular defects in immunostaining occasionally appeared even after optimal fixation, but could be regularly provoked by mildly acidic fixation (pH 6.6) or prolonged barbiturate anaesthesia. These defects might be based on S100 release into the cerebrospinal fluid. Presumably under none of the conditions studied can the immunoreactivity of all S100-forms and-fractions be completely preserved in the tissue. However, recommendations are presented for optimizing tissue preparation, to the extent that premortal modifications affecting the stainability of astrocytes may be detected by S100 immunohistochemistry in fixed brain tissue.     

Determinants of the high-methionine trait in wild and exotic germplasm may have escaped selection during early cultivation of maize

The 18 kDa high-methionine δ-class zein gene from maize has been cloned, and its regulation, structure, and map position studied. These studies have shown that (i) zein genes may also contain tryptophan and lysine codons, (ii) the 18 kDa and the related 10 kDa zein gene are coordinately regulated, but their products accumulate to different levels in a genotype-dependent manner, (iii) the duplication of δ-zein genes probably involved unequal crossing over, (iv) no copy correction in either direction has occurred from teosinte to modern corn, and (v) the duplication of of the 18 kDa zein gene probably occurred before the tetraploidization of a progenitor chromosome. The work shows that important nutritional quality determinants like the high-methionine seed proteins are abundant in several exotic and wild corn varieties and low in most of the inbreds screened. The lack of a selectable phenotype for such quality traits during initial domestication and breeding of corn would have eliminated cis and trans regulatory determinants from the germplasm used in modern corn breeding. Examples of the high-methionine δ-class zeins shown here may be generally applicable in explaining the low nutritional quality of most present-day corn grown.     

C4 polymorphism in the dog: Molecular heterogeneity of the C4α and C4γ subunit chains

Using an immunoblotting technique and goat antihuman C4, we observed five distinct electrophoretic variants of C4 in a panel of 60 random dogs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated C4 showed that dog C4 is composed of three polypeptide subunit chains (, , and ) and that structural variability occurs within the - and -chain regions. Two distinct molecular weight forms of both the C4- ( _A and _B) and C4-( _A and _B) chain were detected. The variant forms of C4 and C4 were found in association with particular C4 allotypes.     

Amino acid sequence of the phosphorylation site of isocitrate dehydrogenase fromEscherichia coli

InEscherichia coli, NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) may undergo a phosphorylation catalyzed by a cAMP-independent protein kinase, with a concomitant decrease in catalytic activity. In this report, we describe the purification and amino acid sequence of a³²P-labeled peptide obtained from in vivo³²P-labeled isocitrate dehydrogenase. The³²P-labeled peptide was isolated from a tryptic digest and found to contain seven amino acids, including a single serine residue. Following automated Edman degradation and reversephase high-pressure liquid chromatography of the phenylthiohydantoin-amino acids, the sequence of this peptide was established to be-Ser(P)-Leu-Asn-Val-Ala-Leu-Arg.     

Can Nocodazole,an Inhibitor of Microtubule Formation,Be Used to Synchronize Mammalian Cells?

Abstract Nocodazole, a temporary inhibitor of microtubule formation, has been used to partly synchronize Ehrlich ascites tumour cells growing in suspension. the gradual entry of cells into mitosis and into the next cell cycle without division during drug treatment has been studied by flow cytometric determination of mitotic cells, analysing red and green fluorescence after low pH treatment and acridine orange staining. Determination of the mitotic index (MI) by this method has been combined with DNA distribution analysis to measure cell-cycle phase durations in asynchronous populations growing in the presence of the drug. With synchronized cells, it was shown that in the concentration range 0.4–4.0 μg/l, cells could only be arrested in mitosis for about 7 hr and at 0.04 μg/ml, for about 5 hr. After these time intervals, the DNA content in nocodazole-blocked cells was found to be increased, and, in parallel, the ratio of red and green fluorescence was found to have changed, showing entry of cells into a next cell cycle without division (polyploidization). It was therefore only possible to partially synchronize an asynchronous population by nocodazole. However, a presynchronized population, e.g. selected G₁ cells or metabolically blocked G₁/S cells, were readily and without harmful effect resynchronized in M phase by a short treatment (0.4 μg/ml, 3–4 hr) with nocodazole; after removal of the drug, cells divided and progressed in a highly synchronized fashion through the next cell cycle.     

The tibial thread-hairs ofAcheta domesticus L. (Saltatoria,Gryllidae)

Summary The ultrastructure of the thread-like hairs (sensilla) on the tibia of the front leg ofAcheta domesticus (Gryllidae) Saltatoria was examined by serial sectioning. The presence of a tubular body indicates that these sensilla are mechanosensitive; electrophysiological measurements also confirmed this. The opposing forces on the articulating apparatus of single hairs and the sensitivity of the single receptor cell were measured after deflection of the hair in different directions. The articulating apparatus is characterized by three cuticular elements: a joint membrane, suspension fibers, and a socket septum. These elements form the basis for a structural bilateral symmetry along whose plane of symmetry the direction line of both the minimum receptor sensitivity and the minimum opposing forces lie. The tubular body embedded in the tip of the socket septum is attached to the base of the hair shaft. The hair provides the leverage for displacing the tubular body and the socket septum limits the extent to which it may be laterally displaced.These investigations have been supported by the Deutsche Forschungsgemeinschaft     

Glycogen synthase kinases. Distribution in mammalian tissues of forms that are independent of cyclic AMP.

Extracts of rat tissues contain kinases which catalyze the conversion of glycogen synthease from the glucose 6-phosphate-independent (I) form to the glucose 6-phosphatate-dependent (D) form. These kinases were stimulated by adenosine 3':5' monophosphate (cyclic AMP). The glycogen synthase kinase activity ratio (activity in the absence of cyclic AMP divided by activity in the presence of cyclic AMP) varied from 0.28 to 0.97. The activity ratio for histone kinase in the same extracts ranged from 0.11 to 0.29. The levels of glycogen synthase kinase varied by a factor of 80 in the following rat tissues (given in order of decreasing enzyme activity): kidney, liver, stomach mucosa, lung, brain, heart, skeletal muscle, and adipose tissue. In the same tissues the levels of histone kinase varied by only a factor of 6 and did not correlate with the levels of glycogen synthase kinase. A modification of the method of Walsh et al. ((1971) J. Biol. Chem. 246, 1977-1985) was developed for purification of the heat-stable inhibitor of cyclic AMP-dependent protein kinases (inhibitor). The modified procedure resulted in good yields of highly purified inhibitor and was much simpler than the previously described procedure. This inhibitor completely inhibited cyclic AMP-dependent histone kinase activity of the extracts but much of the glycogen synthase kinase activity was not inhibited. The portion of glycogen synthase kinase that was insensitive to the inhibitor was: stomach mucosa, 95%; brain, 90%; liver, 82%; kidney, 81%; lung, 68%; adipose tissue, 65%; skeletal muscle, 63%; and heart, 54%. This histone kinase activity in the extracts and hte ratio of glycogen synthase kinase to histone kinase activity of purified catalytic subunit of the cyclic AMP-dependent protein kinase was used to calculate for each extract the glycogen synthase kinase activity contributed by the cyclic AMP-dependent protein kinase. Based on these calculations, the portion of the glycogen synthase kinase which was due to kinases independent of cyclic AMP was: kidney, 97%; liver, 91%; lung, 89%; brain, 87%, heart, 85%; stomach mucosa, 84%; adipose tissue, 38%; and skeletal muscle, 33%. A significant portion of the glycogen synthase kinase activity, but virtually none of the cyclic AMP-dependent histone kinase activity, of these extracts could be adsorbed to phosphocellulose columns. Liver extracts contained, in addition, a form of glycogen synthase kinase which was not adsorbed to phosphocellulose and which could be separated from the cyclic AMP-dependent protein kinase by additional chromatography. These studies demonstrate that kinases independent of cyclic AMP account for most of the glycogen synthase kinase activity of many tissues. The widespread distribution and high concentrations of these enzymes suggest that they are of physiological importance.     

Synthesis and characterization of 2′-azidoaminopterin as a potential photoaffinity label for folate-utilizing enzymes

A photoreactive analog of aminopterin, 2′-azidoaminopterin (VI), was synthesized and evaluated as a potential inhibitor and photoaffinity label of folate-utilizing enzymes. The compound was tightly bound to dihydrofolate reductase (DHFR) from escherichia coli (MB 1428) with K₁ equal to 3 × 10^?11M and to the enzyme from mouse (S-180) cells with K₁ approximately equal to 2 × 10^?10M. Dissociation constants measured by equilibrium dialysis using radioactive 2′-azidoaminopterin gave a value of K_D = 3.2 × 10^?9M for the bacterial enzyme. The presence of NADPH enhanced the affinity by more than an order of magnitude. Azidoaminopterin is also an inhibitor of thymidylate synthetase from Lactobacillus casei, competitive with methylene-tetrahydrofolate (K_i 7 × 10^?7M). Photolysis of the radioactive inhibitor in complex with DHFR from E. coli led to approximately 3% covalent incorporation of label into protein. The greater part of this attachment was nonspecific as shown by the lack of protection in the presence of methotrexate. Thymidylate synthetase from L. casei was not significantly inactivated upon photolysis in the presence of the inhibitor and deoxyuridylate. Model studies showed that photoreaction of the inhibitor led to covalent linkages with thiol, lysyl amino groups, and the hydroxyl groups of alcohols. Azidoaminopterin may be useful in labeling other enzymes of folate metabolism, although a minor photoproduct reacts nonspecifically with many proteins. The antifolate can be photoconjugated to polylysine as well as to proteins. The polylysine conjugates inhibit DHFR. Difference spectrum analysis of the photoproducts from the irradiation of the DHFR I complex indicates that water reacts efficiently with the enzyme-bound nitrene and must therefore have access to at least part of the bound p-aminobenzoyl group. This analysis suggests that azide analogs of protein ligands may be useful as reporter groups in probing the hydrophobicity of binding sites.     

Teuthiden aus dem Barrême der Insel Maio (Kapverdische Inseln)

The teuthids of the collection R. Stahlecker, 1929, from the Isle of Maio (Cape Verde Islands) are described.Neololigosepia stahleckeri n. gen. n. sp. andMaioteuthis morroensis n. gen. n. sp. are one of the first teuthids from the Lower Cretaceous (Barremian).     

Four guaianolides,a eudesmanolide and a germacranolide from Ursinia saxatilis

An investigation of Ursinia saxatilis afforded in addition to known compounds a new eudesmanolide derived from ursialpinolide, a germacranolide rel