High sequence similarity within ras exons 1 and 2 in different mammalian species and phylogenetic divergence of the ras gene family

We have determined the canine and feline N-, K-, and H-ras gene sequences from position +23 to +270 covering exons I and II which contain the mutational hot spot codons 12, 13, and 61. The results were used to assess the degree of similarity between ras gene DNA regions containing the critical domains affected in neoplastic disorders in different mammalian species. The comparative analyses performed included human, canine, feline, murine, rattine, and, whenever possible, bovine, leporine (rabbit), porcelline (guinea pig), and mesocricetine (hamster) ras gene sequences within the region of interest. Comparison of feline and canine nucleotide sequences with the corresponding regions in human DNA revealed a sequence similarity greater than 85% to the human sequence. Contemporaneous analysis of previously published ras DNA sequences from other mammalian species showed a similar degree of homology to human DNA. Most nucleotide differences observed represented synonymous changes without effect on the amino acid sequence of the respective proteins. For assessment of the phylogenetic evolution of ras gene family, a maximum parsimony dendrogram based on multiple sequence alignment of the common region of exons I and II in the N-, K-, and H-ras genes was constructed. Interestingly, a higher substitution rate among the H-ras genes became apparent, indicating accelerated sequence evolution within this particular clade. The most parsimonious tree clearly shows that the duplications giving rise to the three ras genes must have occurred before the mammalian radiation. Received: 23 July 1997 / Accepted: 30 October 1997     

Nucleolus organizer regions and heterochromatin in the zebu (Bos indicus L.)

Summary Ag-NOR staining and a counterstain enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI-staining = CDD-method) and G-banding, respectively, have been applied to the zebu (Bos indicus L.) chromosomes. The nucleolus organizer regions (NORs) were found in the telomeric regions of chromosomes nos. 2, 3, 4, 11, and 28. CDD staining led to a well-defined R-banding pattern along the chromosome arms and to the visualization of centric heterochromatic bands of variable sizes.     

Molecular structure of the bilin binding protein (BBP) from Pieris brassicae after refinement at 2.0 A resolution

The bilin binding protein (BBP) from the insect Pieris brassicae has been analysed for amino acid sequence, spectral properties and three-dimensional structure. The crystal structure that had been determined by isomorphous replacement has been refined at 2.0 A (1 A = 0.1 nm) resolution to an R-value of 0.20. The asymmetric unit contains four independent subunits of BBP. The co-ordinate differences are 0.25 A, in accord with the estimated error in co-ordinates. The polypeptide chain fold is characterized by an eight-stranded barrel. The connecting loops splay out at the upper end of the barrel and open it, whilst the lower end is closed. The overall shape resembles a calyx. The biliverdin IX gamma chromophore is located in a central cleft at the upper end of the barrel. The bilatriene moiety is in cyclic helical geometry with configuration Z,Z,Z and conformation syn,syn,syn. The geometry is in accord with the spectral properties and permits a correlation between sign of the circular dichroism bands and sense of the bilatriene helices. The fold of BBP is related to retinol binding protein (RBP), as had been recognized in the preliminary analysis, although the amino acid sequences of RBP and BBP show only 10% homology. There are large differences in the loops at the upper end of the barrel, whilst the segments of the centre and the lower end of the barrel superimpose closely. The ligands of BBP and RBP, biliverdin and retinol, respectively, are also similarly located.     

Genetic studies on nucleolus organizer regions (NORs) in cattle

Experimentally produced monozygotic twins, natural opposite sex blood chimeras (freemartins), and several pedigrees were used to evaluate the genetic influences on the nucleolus organizer region (NOR) patterns in cattle. In monozygotic twins, the NOR patterns of both twins are extremely similar. In chimeras, NOR patterns of genetically identical, peripheral blood lymphocytes (PBL) from the two partners resemble each other. In contrast, genetically different PBL (sib organ) differ significantly in the same environment. A high heritability of the individual NOR patterns is also demonstrated in our 23 pedigrees. In conclusion, our data demonstrate that variation in NOR expression is predominantly due to genetic factors.     

Relation of phosphatidylinositol metabolism to glycolytic pathway in skeletal muscle membranes

Skeletal muscle triads are possessing the whole set of enzymes of the phosphatidylinositol (PI)-linked signal generating pathway, PI-kinase, PI(4)P-kinase, and PI(4,5)P₂-phospholipase C (PLC). The activities of these enzymes are comparable to those found in other cell types for which a functional role of the PI-pathway in intracellular signal transduction has been established. For skeletal muscle an unequivocal function and an initiating signal for Ins(1,4,5)P₃-liberation is still unknown. However, the observed Ca-dependency of PLC activity suggests that here Ins(1,4,5)P₃ production is a consequence rather than a cause of increasing cytosolic Ca²⁺. Recently, the glycolytic enzyme aldolase, whose activity can be modulated by inositol polyphosphates, has been localized in the triadic structure. The enzyme which has a high affinity to Ins(1,4)P₂, Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄, seems to be compartmentalized to the junctional foot structure from which it is released upon binding of these molecules. This phenomenon could reflect a capability for regulation of the glycolytic flux even for aldolase, especially if a non steady-state situation in the junctional gap is considered. Meanwhile we have accumulated evidence for the operation of a partial glycolytic sequence in the junctional region established by the enzymes aldolase, glyceraldehyde-3-P (GAP) dehydrogenase and phosphoglycerate kinase. This system is able to produce ATP upon oxidation of GAP and could be, because of the inositol polyphosphate-sensing abilities of aldolase, a target for the membrane associated PI-pathway. The ATP production is however transient which indicates the coupling to an ATP hydrolyzing reaction. Thus, it appears that the ATP produced by the membrane associated system is effectively utilized by an ATP consuming membrane localized system like PI-metabolism or protein kinases. There are indications that exogeneously added ATP does not equilibrate with the ATP synthesized in the junctional region which suggests an effective structural or kinetical compartmentalization of this system. Therefore it is hypothesized that the ATP synthesized by the membrane associated glycolytic sequence is utilized in membrane localized reactions.     

A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.

A novel complexometric dye- and transition-metal-based post-column detection system for polyanions, called 'metal-dye detection' has been developed. This technique, combined with a new h.p.l.c. separation protocol, permits a direct highly-isomer-selective determination of bis- to poly-phosphorylated non-radioactively labelled compounds in the picomolar range, a sensitivity hitherto unknown for these substances. The application of the technique in the quantitative microanalysis of inositol polyphosphates from milligram amounts of cells or tissue specimens is described. The technique promises to answer hitherto unresolved questions about the role of inositol phosphates, especially those in intact tissues, which are not readily amenable to analysis by radioisotopic techniques.     

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 refined 2.3 A crystal structure of human leukocyte elastase in a complex with a valine chloromethyl ketone inhibitor

The stoichiometric complex formed between human leukocyte elastase and a synthetic MeO-Suc-Ala-Ala-Pro-Val chloromethyl ketone inhibitor was co-crystallized and its X-ray structure determined, using Patterson search methods. Its structure has been crystallographically refined to a final R value of 0.145 (8.0 and 2.3 A). The enzyme structure is very similar to that recently observed in a complex formed with the ovomucoid third domain from turkey [(1986) EMBO J. 5,2453-2458]. The rms deviation of all alpha-carbon atoms is 0.32 A. The peptidic inhibitor is bound in a similar overall conformation as the ovomucoid binding segment. Covalent bonds are formed between Val-P1 of the inhibitor and His-57 NE2 and Ser-195 OG of the enzyme. The carbonyl carbon is tetrahedrally deformed to a hemiketal. The valine side chain is arranged in the S1 pocket in the g-conformation.     

Mathematical models for mixing in deep-jet bioreactors: Calculation of parameters

The macroscopic mathematical model based on compartments with ideal mixing zones and tanks-in series was evaluated. Based on the experimental data obtained in a 300 dm³ pilot reactor and the dependence of mixing time on the volume of liquid phase, we have found mathematical relations between the ratio of vessel diameter to liquid level, adjustable parameters of model and the mixing time.List of Symbols V dm³ total volume of bioreactor - V _g dm³ total volume of liquid - V ₁ dm³ volume of ideally mixed zone in the vessel - V ₂ dm³ volume of macromixer in inner circulation flows - V ₃ dm³ volume of liquid phase in the pump - V ₄ dm³ volume of liquid phase in the pipe between the vessel and the pump - V ₅ dm³ volume of liquid phase in the pipe between the pump and air input system included falling jet - V _LT dm³ volume of liquid in the tank - V _LC dm³ volume of liquid in the circulation system - F _E dm³/s inner volumetric circulation flow rate across the macromixers - F _cir dm³/s external volumetric circulation flow rate, pumping capacity - t _A s time interval of the pulse application - t _AA s time point of the pulse application related to the free choosen starting point of the experiment - t _m s mixing time - t _c s circulation time - t _end s end time of simulation - C _*,* kg/m³ concentration of tracer in the indicated compartment - C ₀ kg/m³ concentration of the tracer before the injection - C _t kg/m³ concentration of the tracer at the indicated time - C kg/m³ theoretical concentration of the full mixed tracer - C _sim kg/m³ calculated concentration of tracer during numerical integration method - i index of an arbitrary tank - D _T m diameter of bioreactor - D 1/s dilution rate - H _L m level of liquid in the unaerated vessel - vector of inhomogenities     

Crystals of the NC1 domain of human type IV collagen

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.