The Why and How of Species

The biological species concept deals both with the meaning of the sexual species as a harmonious gene pool and with its protection against deleterious outbreeding (effected by isolating mechanisms). According to the Darwin-Muller-Mayr theory isolating mechanisms are acquired by incipient species during alloparty. Isolating mechanisms are not the result of ad hoc selection, but of a change of function of properties acquired during the preceding isolation of the incipient species. The role of behavioral properties (recognition) among the isolating mechanisms has long been recognized and described by naturalists but was rejected as basis of a species definition for a number of valid reasons.     

The quaternary structure of four crustacean two-hexameric hemocyanins: immunocorrelation,stoichiometry, reassembly and topology of individual subunits

Summary Two-hexameric (2×6) hemocyanins from the brachyuran crabsCancer pagurus andCallinectes sapidus, the freshwater crayfishAstacus leptodactylus and the lobsterHomarus americanus were isolated and dissociated into native subunits.The subunits of each hemocyanin were analyzed by electrophoresis and immunology. Three immunologically distinct subunit types, which were termed, and, could be identified in each case. They were isolated preparatively, and interspecifically correlated. Subunit is subdivided into several electrophoretically distinct isoforms which are immunologically closely related (Astacus) or identical (other species). InAstacus andCancer one of these isoforms was shown to dimerize and to act as inter-hexamer bridge. It represents a fourth subunit type termed. A fifth, diffuse component, which in PAGE migrated at the position of a dimer, was identified in the crossed immunoelectrophoretic patterns as denatured hemocyanin.A common feature of the four hemocyanins is the presence of 4 copies of and 8 copies of/ within the 2×6 particles. The: ratio is 4:4 in the two Astacidea and 6:2 in the two Brachyura. exists in 2 copies inAstacus andCancer which means that a single dimer- is present in a two-hexamer. This leaves 2 monomeric copies inAstacus and 4 inCancer.Every subunit from the four species except ofAstacus - was capable to form hexamers in reassembly experiments. If subunit combinations were tested, hetero-hexamers were formed preferentially. Two-hexamers were reconstituted only in the presence of all subunit types and the native subunit stoichiometry was required to obtain twohexamers in considerable yields. Factors limiting 2×6 reassembly are discussed.Authentic 2×6 molecules ofAstacus, Homarus andCancer hemocyanin were immunolabeled with subunit-specific antibody fragments (F_ab) or IgG molecules, and the resulting immuno complexes were studied in the electron microscope. A topological model of the quaternary structure of decapod 2×6 hemocyanins is derived, showing the position of each copy of the four subunit types. In this model, the inter-hexamer bridge- is surrounded by two and two subunits forming the central core of the dodecamer. Two additional and two additional subunits form the periphery together with one subunit occupying the peripheral short edges of each hexameric half structure. The model is discussed with respect to the current literature.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This paper is decicated to Professor Dr. Bernt LinzenPreliminary accounts of this work have been presented in the proceedings of a symposium at Tutzing 1985. Linzen B (ed) (1986) Invertebrate oxygen carriers. Springer, Berlin Heidelberg New York. This also includes: Stöcker et al. 1986; Markl et al. 1986) and in a review article (Markl 1986)     

Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release from Cultured Mouse Brain

The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 0.1 M NaCl at 0 degrees C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 microM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high-affinity SP binding site belongs to the SP-P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 +/- 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax = 31.3 +/- 3.56 fmol/mg protein, KD = 0.82 nM). Gel filtration analysis showed that the detergent-SP binding protein complex has two calculated molecular weights of greater than 1,000,000 and 55,000-60,000 (a corresponding Stokes radius of 35.5 nm).     

Photoperiod control of poplar bark storage protein accumulation

Bark storage proteins (BSPs) accumulate in the inner bark parenchyma of many woody plants during autumn and winter. We investigated the effect of a short-day (SD) photoperiod on the accumulation of the 32-kilodalton bark storage protein of poplar (Populus deltoides Bart. ex Marsh.) under controlled environmental and natural growing conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein gel blot analysis revealed that 10 days of SD exposure (8 hours of light) resulted in a 20% increase in the relative abundance of the 32-kilodalton bark storage protein of poplar. After 17 days of SD exposure, the 32-kilodalton bark storage protein accounted for nearly one-half of the soluble bark proteins. In natural field conditions, accumulation of the 32-kilodalton bark storage protein was observed to start by August 18 (daylength 14.1 hours). Immunoprecipitation of in vitro translation products with anti-BSP serum revealed that the SD protein accumulation was correlated with changes in the pool of translatable mRNA. A survey of poplar clones from different geographic origins revealed the presence of the 32-kilodalton BSP in the dormant bark of all the clones tested. These results demonstrate that a SD photoperiod induces, whether directly or indirectly, rapid changes in woody plant gene expression, leading to the accumulation of BSP.     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Expression of membrane activation antigens on murine B lymphocytes stimulated with lipopolysaccharide

The expression of two membrane glycoproteins, RL388 antigen and transferrin receptor (TfR), was examined on murine B cells stimulated with lipopolysaccharide (LPS) in vitro. Immunofluorescent staining with monoclonal antibodies and flow cytofluorometric analysis were used to monitor the expression of these markers as a function of the time in culture, the state of membrane Ia antigen expression, the position in cell cycle, and the degree of B-cell differentiation. Freshly explanted splenic B cells expressed low levels of RL388 antigen and TfR. Following LPS stimulation, increased expression of RL388 antigen was detectable by 8 to 12 hr of culture, a time span characterized by increased Ia antigen expression, blast transformation, and G0 to G1 phase transition. The increased expression of TfR was apparent later and correlated with entry into late G1 phase and the onset of S phase. LPS-stimulated cell cultures treated with actinomycin D (G0/G1 block) exhibited increased expression of Ia antigen, but neither RL388 antigen nor TfR, whereas hydroxyurea treatment (G1/S block) allowed expression of all three markers. These results indicate that hyperexpression of RL388 antigen and TfR occurs during G1 phase and that these events are subsequent to Ia antigen hyperexpression. Finally, B cells in late G1 through M phase of the cell cycle simultaneously express high levels of RL388 antigen and TfR. These findings suggest that the expression patterns of RL388 antigen and TfR might be useful parameters for defining compartments of the murine B-cell cycle.     

Prostaglandin binding activity and myoblast fusion in aggregates of avian myoblasts

Myoblast aggregates provide a system for studying cell interactions which have several advantages over standard, stationary cultures. In gyrotory rotation, aggregate size can be controlled and is independent of cell migration. In muscle aggregates, fibroblasts are excluded, yet myoblast differentiation and fusion occur in a highly synchronous fashion. Specific PG binding occurs in chick or quail myoblast aggregates: in chick the peak of binding is at 35-36 hr. Aggregation is complete 16 hr before PG binding activity appears. This suggests either that gyrotory aggregation is not identical to myoblast recognition, or that PG binding activity occurs subsequent to myoblast recognition. Myoblast aggregates begin to release PG before 18 hr. The amount detected remains constant until binding begins at 34 hr when PG binding to the aggregates begins. Thus, both the release of PG and PG receptor activity are characteristics of the myoblasts and release of prostaglandin precedes appearance of the binding activity. As a first step in identifying the PG receptor and determining its appearance on the myoblast cell surface, we have prepared antisera against myoblast surfaces which blocks receptor-ligand interaction and have absorbed it against both peripheral and intrinsic membrane fractions. The results indicate that the PG receptor is a myoblast peripheral membrane macromolecule.     

Sequence homologies of glucose-dehydrogenases of Bacillus megaterium and Bacillus subtilis

The sequence homologies of the glucose dehydrogenase subunits of B. megaterium and B. subtilis are compared. From the known B. megaterium aminoacid sequence and the base sequence of the cloned B. subtilis structural gene we predict the B. megaterium structural glucose dehydrogenase gene. Assuming the minimal mutational changes to convert one gene into the other 23 transitions, 30 transversions, 1 inversion, 3 insertion-deletions, but no frameshifts are postulated necessary to interconvert the structural genes. The homology of both enzyme subunits of 85% reflects the close evolutionary distance between B. subtilis and B. megaterium.     

Immunoreactivity and ouabain-dependent phosphorylation of (Na+ + K+)-adenosinetriphosphatase catalytic subunit doublets

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 6% polyacrylamide was used to resolve the 100-kDa catalytic (alpha subunit) polypeptide of (Na+ + K+)-adenosinetriphosphatase from various tissues. The catalytic subunit was identified on immunoblots with antisera against mouse brain catalytic subunit and lamb kidney holoenzyme. Immunoblots and Coomassie Blue-stained companion gels showed double species of the 100-kDa subunit in sucrose gradient fractions of mouse brain and kidney, bovine grey and white matter, purified lamb kidney and duck salt gland holoenzyme, electroplax microsomes, and NaI-extracted microsomes of goldfish and rat brain. The apparent molecular mass differences between the two species in each tissue all ranged between 5 and 8 kDa. Both forms in rat brain and lamb kidney enzyme contain common epitopes reactive with antibodies immunoaffinity-purified on either species from mouse brain. In addition, ouabain-dependent acid-stable inorganic phosphate incorporation was tested with mouse brain, lamb kidney, and electroplax enzyme. Ouabain-dependent phosphorylation was demonstrated in both species in lamb kidney and electroplax and in the larger of the two forms in mouse brain. These results suggest that double species of the phosphorylatable subunit are present generally in epithelial as well as excitable tissues and in fish and avian as well as mammalian species. Work is needed to elucidate their qualitative and quantitative characteristics in different tissues.     

Constant Phycobilisome Size in Chromatically Adapted Cells of the Cyanobacterium Tolypothrix tenuis, and Variation in Nostoc sp

Phycobilisomes of Tolypothrix tenuis, a cyanobacterium capable of complete chromatic adaptation, were studied from cells grown in red and green light, and in darkness. The phycobilisome size remained constant irrespective of the light quality. The hemidiscoidal phycobilisomes had an average diameter of about 52 nanometers and height of about 33 nanometers, by negative staining. The thickness was equivalent to a phycocyanin molecule (about 10 nanometers). The molar ratio of allophycocyanin, relative to other phycobiliproteins always remained at about 1:3. Phycobilisomes from red light grown cells and cells grown heterotrophically in darkness were indistinguishable in their pigment composition, polypeptide pattern, and size. Eight polypeptides were resolved in the phycobilin region (17.5 to 23.5 kilodaltons) by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Half of these were invariable, while others were variable in green and red light. It is inferred that phycoerythrin synthesis in green light resulted in a one for one substitution of phycocyanin, thus retaining a constant phycobilisome size. Tolypothrix appears to be one of the best examples of phycobiliprotein regulation with wavelength. By contrast, in Nostoc sp., the decrease in phycoerythrin in red light cells was accompanied by a decrease in phycobilisome size but not a regulated substitution.