Morphometric parameters of neurons in reticular nuclei of the brain stem in kittens

Three types of Golgi-stained neurons were discovered in brain stem reticular nuclei of 30-day-old kittens: sparsely branching reticular neurons, those with densely branching dendritic trees, and giant multipolar neurons (Leontovich's classification). Adopting computerized morphometric techniques enabled 23 different parameters to be measured in cells of these types. The measurements taken from the neuronal groups investigated revealed statistically significant differences between them for most parameters. It was concluded from this that each of the neuronal types distinguished has its own morphological identity (or stability). Characteristics of structural differences and properties of differing cell types in reticular nuclei are discussed in relation to their functional properties.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 399–409, July–August, 1991.     

A preliminary analysis of distribution patterns in a large, pantropical genus, Barleria L. (Acanthaceae)

Barleria L. (Acanthaceae) is a large, polymorphic, widespread genus of herbs and shrubs comprising about 300 species, occurring mainly in Africa and Asia but with one species, Barleria oenotheroides Dum.Cours., extending to the New World tropics. Recent completion of a monographic infra-generic classification of the genus (in which seven sections are recognised, and the names of four of these validated in this paper—see Appendix 1), has facilitated a comprehensive analysis of distribution patterns on a global scale. The richest representation of Barleria is in Africa where there are two centres of diversity, one in tropical East Africa (about eighty species) and the other in southern Africa (about seventy species). The number of species tails off rapidly to both the Far East and the West. Barleria shows a marked trans-Atlantic disjunction between West Africa and the Neotropics, with B. oenotheroides shared by these two regions. This type of disjunction, which is known in other genera of the family, cannot be adequately explained in Barleria on the basis of long-distance dispersal or past continental movements. There is a high degree of regional endemism (e.g. 75% for the Indian subcontinent) at both the species and sectional levels within this genus. The degree of similarity between regions is correspondingly low. The endemics in each region tend to belong to only one or a few of the sections. There are few truly widespread taxa within the genus. East and West Africa are the only regions in which all sections are represented. Sections Barleria and Prionitis C.B. Cl. are the most widespread in the genus; Sections Somalia (Oliv.) Lindau, Fissimura M. Balkwill and Stellatohirtae M. Balkwill are mainly restricted to Africa and Sections Chrysothrix M. Balkwill and Cavirostrata M. Balkwill are the most restricted, occurring mainly in India and Sri Lanka. On a local scale, many of the species show highly restricted, clumped distributions; this is apparently related to particular soil types and possibly to the short-distance, ballistic mode of seed dispersal. This account of the biogeography of Barleria is to be regarded as preliminary, as much taxonomic work at the species level remains to be done before a full-scale cladistic biogeographic account can be undertaken. Particular areas worthy of future investigation include establishing the centre of origin of the genus and investigating the basis for the high degree of endemism shown by many of the species.     

Simultaneous Display of Multiple Foreign Peptides in the FliD Capping and FliC Filament Proteins of the Escherichia coli Flagellum

The bacterial flagellum is composed of more than 20 different proteins. The filament, which constitutes the major extracellular part of the flagellum, is built up of approximately 20,000 FliC molecules that assemble at the growing distal end of the filament. A capping structure composed of five FliD molecules located at the tip of the filament promotes polymerization of FliC. Lack of FliD leads to release of the subunits into the growth medium. We show here that FliD can be successfully used in bacterial surface display. We tested various insertion sites in the capping protein, and the optimal region for display was at the variable region in FliD. Deletion and/or insertion at other sites resulted in decreased formation of flagella. We further developed the technique into a multihybrid display system in which three foreign peptides are simultaneously expressed within the same flagellum, i.e., D repeats of FnBPA from Staphylococcus aureus at the tip and fragments of YadA from Yersinia enterocolitica as well as SlpA from Lactobacillus crispatus along the filament. This technology can have biotechnological applications, e.g., in simultaneous delivery of several effector molecules.     

Spectral properties of cobalt carboxypeptidase A. Interaction of the metal atom with anions

At pH greater than 7 the absorption and magnetic circular dichroic spectra of cobalt carboxypeptidase A are insensitive to anions [Latt, S. A., & Vallee, B. L. (1971) Biochemistry 10, 4263-4270], but at pH less than 6 chloride and other anions perturb them in a manner specific for each anion. Lowering of the pH apparently facilitates the entry of an anion into the metal coordination sphere, suggesting that an acidic group normally stabilizes a metal-coordinated water molecule against displacement. The lack of sensitivity to anions at pHs between 7 and 9--when the enzyme is maximally active--and its evident abolition upon protonation of an active-site group are consistent with this interpretation. Selective modification of cobalt carboxypeptidase at Glu-270 using a carbodiimide affinity reagent generates sensitivity to anions at pH 7 very similar to that of the unmodified enzyme at pH approximately 5. This suggests that the group stabilizing the metal-coordinated water is the catalytically essential carboxylate of Glu-270. These and related results provide evidence for a mechanistically important interaction of Glu-270 with a metal-bound water molecule.     

Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme.     

Calcium-activated neutral protease inhibitor from rabbit erythrocytes lacks the N-terminal region of the liver inhibitor but retains three inhibitory units

Endogenous inhibitors for calcium-activated neutral protease (CANP) were purified from rabbit erythrocytes and liver. The purified inhibitors showed single bands but with significantly different mobilities on sodium dodecylsulfate-polyacrylamide gel electrophoresis. Peptide mapping and sequencing analyses have revealed that the erythrocyte inhibitor (429 residues) retains the C-terminal three repetitive units of the liver inhibitor (639 residues), which contains four potential repetitive units for inhibition of CANP. The erythrocyte and liver inhibitors inhibited 3 and 4 moles of CANP on the basis of the molecular weights of 46,000 and 68,000, respectively.     

Glycogen debranching enzyme: purification, antibody characterization, and immunoblot analyses of type III glycogen storage disease.

Type III glycogen storage disease is caused by a deficiency of glycogen debranching-enzyme activity. Many patients with this disease have both liver and muscle involvement, whereas others have only liver involvement without clinical or laboratory evidence of myopathy. To improve our understanding of the molecular basis of the disease, debranching enzyme was purified 238-fold from porcine skeletal muscle. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme gave a single band with a relative molecular weight of 160,000 that migrated to the same position as purified rabbit-muscle debranching enzyme. Antiserum against porcine debranching enzyme was prepared in rabbit. The antiserum reacted against porcine debranching enzyme with a single precipitin line and demonstrated a reaction having complete identity to those of both the enzyme present in crude muscle and the enzyme present in liver extracts. Incubation of antiserum with purified porcine debranching enzyme inhibited almost all enzyme activity, whereas such treatment with preimmune serum had little effect. The antiserum also inhibited debranching-enzyme activity in crude liver extracts from both pigs and humans to the same extent as was observed in muscle. Immunoblot analysis probed with anti-porcine-muscle debranching-enzyme antiserum showed that the antiserum can detect debranching enzyme in both human muscle and human liver. The bands detected in human samples by the antiserum were the same size as the one detected in porcine muscle. Five patients with Type III and six patients with other types of glycogen storage disease were subjected to immunoblot analysis. Although anti-porcine antiserum detected specific bands in all liver and muscle samples from patients with other types of glycogen storage disease (Types I, II, and IX), the antiserum detected no cross-reactive material in any of the liver or muscle samples from patients with Type III glycogen storage disease. These data indicate (1) immunochemical similarity of debranching enzyme in liver and muscle and (2) that deficiency of debranching-enzyme activity in Type III glycogen storage disease is due to absence of debrancher protein in the patients that we studied.