Structures and Functions of Calcium Channel β Subunits

Calcium channel subunits have profound effects on how ₁ subunits perform. In this article we summarize our present knowledge of the primary structures of subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with ₁ subunits, the effects of subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by subunits, such as long-lasting prepulse facilitation of _1C (absent in _1E) and inhibition by G protein dimer of _1E, absent in _1C. One subunit, a brain 2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of ₁ in oocytes requires a subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca²⁺ channels to account for ₁ alone channels seen in cells with limited subunit expression. In one model, dissociates from the mature ₁ after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of would then have subunit composition ₁. In the other model, the chaperoning remains associated with the mature channel and ₁ alone channels would in fact be ₁ channels. Upon co-expression of high levels of the regulated channels would have composition [₁].     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Substrate specificity and distribution of acid β-galactosidase activities in seizure-susceptible and non-susceptible strains of mice

The properties and distribution of -galactosidase were studied in the mouse brain using the artificial substrate methylumbelliferyl--galactoside. Enzyme activities were compared between an audiogenic seizure-susceptible mouse strain (DBA/2) and three non-susceptible strains of mice (BALB/c, C3H/He and Swiss A2G). At all ages, DBA/2 mice have significantly lower -galactosidase activity compared with the three other mouse strains: this is attributed to the different alleles present at the Bgs locus. The low activity of -galactosidase is also evident when the natural substrate GMI-ganglioside is hydrolyzed. In contrast to this low GMI-ganglioside--galactosidase activity, there is no difference in the activity of the second form of acid -galactosidase, galactosylceramidase, in DBA/2 mice at 7 and 14 days. However, at 21 and 28 days the activity is significantly lower in DBA/2 mice compared with the other strains of mice. These results on -galactosidase activity in the brain of seizure-susceptible and non-susceptible mice are discussed in relation to published levels of GMI-ganglioside and galactosylceramide present in the developing mouse brain.Dedicated to Henry McIlwain.     

Translocation and the control of photosynthesis in sugar beet

Summary Evidence is presented that the size and activity of sinks for the products of photosynthesis exert a large measure of control over carbohydrate levels in the leaves of the sugar beet plant. Further data is presented to show that a correlation exists between the level carbohydrate in the leaf and the rate of photosynthesis. The rate of photosynthesis can thus be linked directly to the sinks of the plant by their ability to control rates of translocation from leaves.     

Interpretation of metameric architecture in dominant shrubs of the Chilean matorral

Summary The seasonal progression of phenophases in 21 shrub species of the Chilean matorral was analyzed. Five modules or basic units that are responsible for the aboveground architecture of the plants were characterized. These modules appear to be organized in seven different spatial arrangements. In drought-deciduous species a module type with an absolute short shoot with limited apical growth, leafy or spiny, predominated. In evergreen species long shoot and temporal leafy short shoot module types were more frequent. The spatial arrangement of morphologically different modules and the temporal sequence of their formation allow a dynamic interpretation of the modular architecture of the plants.     

MHC class II sequences of an HLA-DR2 narcoleptic

Narcolepsy has a 98% association with the DR2-Dw2/DQw1 haplotype. To establish if a disease-specific allele is present in narcolepsy, a cDNA library was made from a B-cell line from a DR2,4/DQw1,3 narcoleptic. Clones encoding the two expressed DR2 chains, along with DQw1 and chains, were isolated and completely sequenced. The coding regions of these four genes were similar to published nucleotide and protein sequences from corresponding healthy controls, with some minor exceptions. The 3 untranslated region of one of the DR2 genes in the narcoleptic was extended by 42 bp. Complete sequences were not available for DQw1.2 or from healthy individuals, but first domain nucleotide sequences showed only a single nonproductive difference in DQ. Partial protein sequences of both DQ and from published data were identical. Although the effects of minor differences cannot be ruled out completely, it is concluded that there are probably no narcolepsy-specific DR or DQ / sequences, and that the alleles found in narcolepsy are representative of those found in the healthy population.     

Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli

Summary The and subunit of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme. To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed. This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control. When the structural genes encoding the components of core RNA polymerase (, and ) or holoenzyme (, , and ⁷⁰) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase. The induction of RNA polymerase overproduction is characterized by an initial large burst of synthesis followed by a gradual decrease as the concentration of RNA polymerase increases. Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of synthesis off the chromosome. These results indicate that RNA polymerase feedback regulation controls synthesis in vivo.     

A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes

A novel approach was used to compare male and female recombination rates in wheat. Doubled haploid lines were developed from an F₁ using two distinct approaches: the anther-culture technique and the Hordeum bulbosum system, from which sets of lines were developed from male and female meioses, respectively. The genotype of the lines was established at RFLP and isozyme markers polymorphic on chromosomes of homoeologous groups 6 and 7, and male and female linkage maps were calculated using this information. The markers in one segment of chromosome 6B exhibited disturbed segregation frequencies in the anther-culture population. The male and female maps differed significantly in recombination frequency between some markers on two chromosomes, and these were consistent in direction within chromosomes and inconsistent in direction between chromosomes. In two of the four chromosomes studied the male map was much longer than the female map. These results suggest that significant differences may exist in male and female recombination frequencies in bread wheat which are specific to certain chromosomal segments but are inconsistent in direction between chromosomes. Other factors, such as environmental influences, may also be important in creating differences.     

Hämatologische beobachtungen an regenbogenforellen (Salmo gairdneriRich.) VII. Färbeindex (FI), absoluter Hämoglobingehalt des Einzelerytrozyten (HbE) and mittlere Hämoglobinkonzentration des Einzelerythrozyten (MCHC)

180 rainbow trouts (Salmo gairdneriRich.), aged from 1 to 3 years, were examined for fluctuations, caused by age and season, by means of colour index (CI), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).CI and MCH behave similarly. Both are increasing until the 2nd year and stay relatively constant thereafter. If the gender is not considered — there are no significant differences in the values of males and females — the CI increases from 1,4 in the first year over 1,6 to 1,7 in the age of 3 years, and the MCH increases from 44,4 over 52,6 , 56,8 , 58,1 to 55,5 .A seasonal periodicity of both indices could not be indicated on not-matured animals (F₂) which were two summers of age. Only, the january values appeared increased — CI: 2, MCH: 68,3 — otherwise the CI varies between 1,8 and 1,7 and the MCH between 53,3 and 59,1 .The MCHC-values of the age groups examined vary between 24,4% and 27,3%. The values of the yearlings form an exception (19,8%). These values certainly are inexact and too low because of the small number of individuals checked (3).

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Mit finanzieller Unterstützung durch die DFG.Institut für Siedlungswasserbau und Wassergütewirtschaft der Universitat Stuttgart Fischtoxikologische Arbeitsgruppe     

In vitro meristem cloning of Eucalyptus tereticornis Sm.

Rapid multiplication of axillary meristems and direct shoot development occurred from nodal explants of mature Eucalyptus tereticornis Sm. with 5.3 M NAA, 1.1 M IAA and 4.4 M BA in Murashige-Skoog medium. Repeated subcultures of the second generation shoot cultures into low cytokinin-auxin containing media (0.44–0.88 M BA+0.1 M NAA) yielded axillary microshoots in large numbers. Half-strength MS liquid medium with 4.9 M IBA, 5.5 M IAA and 5.3 M NAA for four days, half-strength semi-solid hormonefree MS medium with charcoal, and MS liquid medium without charcoal and hormones, in sequence, induced rooting of shoots in the dark. This system is suitable for the mass propagation of this difficult-to-root eucalypt.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - IBA -indole-3-butyric acid - 2-iP isopentyl adenine - Kn kinetin - MS Murashige-Skoog - NAA -naphthalene acetic acid - PVP polyvinylpyrrolidone     

Phylogenetic Relationships of the Seven Coat Protein Subunits of the Coatomer Complex, and Comparative Sequence Analysis of Murine Xenin and Proxenin

The coatomer complex is involved in intracellular protein transport and comprises an assembly of seven polypeptide subunits designated , , , , , , and COP. Rooted phylogenetic trees constructed from the full-length cDNA and amino acid sequences of 49 COP entities in different eukaryotes from yeast to man generally revealed striking conservation of each subunit through evolution. Both nucleotide and protein trees displayed close relationships between and subunits, between and subunits, and between and subunits, implying evolution from common ancestors as well as functional similarity. Interestingly, although 6 out of 7 -COP genes appeared to be grouped and related to the -COP genes, 4 out of 7 -COP gene products clustered with other groups of other COP subunit proteins. A 5 coding segment of the murine -COP gene was amplified by RT-PCR and cycle-sequenced. The partial predicted amino acid sequence of this murine homolog was exactly identical to the human and bovine counterparts. Of particular significance was the complete identity of the first 25 and 35 N-terminal residues which constitute the gastrointestinal hormone xenin and its precursor proxenin, thus emphasizing their strict evolutionary conservation and alluding to their physiological importance.     

Nonspecific (“pseudo-plasmal”) dye-binding in the Feulgen nuclear stain and its blocking by azocarmin G

Summary In Feulgen nuclear staining nonspecific dye-binding due to the pseudo-plasmal reaction is intensified in isolated cells with intact cytoplasm, and cannot be eliminated by the post-irradiation method. Fluorescence intensity in the cytoplasm sometimes exceeds that of specific nuclear fluorescence, especially in brain and heart muscle cells, and it was almost impossible to perform cytofluorometric DNA quantification on such specimens. Various kinds of aldehyde-blocking agents such as sodium borohydride, 2,4-dinitrophenylhydrazine, aniline, and sodium pyrosulfite were effective in reducing the pseudo-plasmal reaction. But the blocking effects were not complete because of additional release of reactive aldehyde groups during subsequent Feulgen hydrolysis. Acidic azocarmin G produces a complete block of all pseudo-plasmal reaction in acriflavine-Feulgen nuclear staining, allowing accurate DNA-cytofluorometry to be carried out.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

Cajaflavanone and cajanone released from Cajanus cajan (L. Millsp.) roots induce nod genes of Bradyrhizobium sp.

A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium sp (F-4) nod DABC-lacZ translation fusion was constructed and used to monitor nod gene expression in response to pigeonpea root exudate. Two nod-inducing compounds were isolated and identified. Spectral analysis using ultraviolet absorption, infrared spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 5,4-dihydroxy-6-(3-methyl-2-butenyl)-2, 2-dimethyl pyrano-[5, 6:7, 8]-flavanone (cajaflavanone) and 2,4,5-trihydroxy-5-isopentenyl-6, 7-dimethylchromene iso-flavanone (cajanone). When pEA2-21 was introduced into Rhizobium trifolii and R. meliloti cajanone and cajaflavanone did not induce nod gene indicating that specificity of induction appears to be influenced by the host-strain genome.     

Distribution of the extended family of protein kinase C isoenzymes in fetal organs of mice: an immunohistochemical study

Using isoenzyme-specific antisera, we have studied the distribution of protein kinase C isoforms in fetal mouse organs at the developmental age of 17 days. Two different sets of antibodies, produced by different manufacturers, were employed in this study. The specificity of the antisera was tested by immunoblotting experiments using whole fetal mouse extracts. Immunohistochemistry was carried out by means of an alkaline phosphatase-conjugated secondary antibody. Analysis of fetal mouse longitudinal cryostat sections stained with the antibodies demonstrated a distinct distribution of protein kinase C isoforms in the tissues. Protein kinase C- and C-I were present in all tissues examined, whereas the C-II isoform was absent in the lung and the liver. Protein kinase C- was identified in brain, spinal ganglia, and adrenal gland. The C- isoenzyme was abundantly expressed in spinal ganglia and in the smooth muscle cells of the bronchial wall. Antisera to C- and C- isoforms heavily stained liver, kidney, and spinal ganglia, whereas the C- isozyme was mainly detected in brain, stomach and kidney. Thus, protein kinase C-, C-I, C-II, C-, C- and C- were the isoforms present in many of the organs investigated. The two sets of antibodies gave slightly different results that might be ascribed to the different epitopes recognized by the antisera. One set of antisera was employed to investigate the distribution of the isoforms in selected organs from an earlier developmental age (15 days) and from adult animals. Both qualitative and quantitative differences were seen in comparison with the same organs from a 17-day fetus.     

Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida

Residual toxicities of registered and selected experimental pesticides used on citrus against Agistemus industani Gonzalez (Acari: Stigmaeidae) were compared. Pesticides considered highly toxic to A. industani were: abamectin 0.15 EC at 731ml/ha+FC 435-66 petroleum oil at 46.8l/ha, pyridaben 75WP at 469g/ha, ethion 4EC at 7.01l/ha+FC 435-66 petroleum oil at 46.8l/ha, propargite 6.55 EC at 3.51l/ha, chlorfenapyr 2SC at 1.46l/ha applied alone or in combination with FC 435-66 petroleum oil at 46.8l/ha, sulphur 80DF at 16.81kg/ha, dicofol 4EC at 7.01l/ha, fenbutatin oxide 50WP at 2.24kg/ha, benomyl 50WP at 2.24kg/ha, benomyl 50WP at 1.68kg/ha+ferbam 76 GF at 5.60kg/ha, ferbam 76GF at 11.21kg/ha, neem oil 90EC at 46.8l/ha, and copper hydroxide DF (40% metallic copper) at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha. Pesticides that were moderately to slightly toxic included: copper sulphate 98% at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha, fenbuconazole 2F at 280ml/ha+FC 435-66 petroleum oil at 46.8l/ha, FC 435-66 petroleum oil applied alone at 46.8l/ha or 23.4l/ha, and diflubenzuron 25WP at 1.40kg/ha. Pesticides that were non-toxic included: fenbuconazole 2F at 585ml/ha, malathion 57EC at 5.85l/ha, FC 435-66 petroleum oil at 46.8l/ha, carbaryl 80S at 3.36kg/ha, chlorpyrifos 4EC at 4.68l/ha, and formetanate 92SP at 1.12kg/ha. Understanding the toxic effects of field weathered pesticides against key predacious mite species is important for effective IPM. The results of this study provide a comparison of direct and indirect toxic effects of various pesticides to A. industani under field conditions.     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Synthesis of New 15-Ketosterol Analogues from Ergosterol

Ergosteryl acetate was converted through three stages into 3-acetoxy-24-methyl-5-cholesta-8(14),22-diene-15-one in 32% overall yield. The product was transformed to 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, and 24-methyl-5-cholesta-8(14),22-diene-3,15-dione. The compounds were characterized by ¹H and ¹³C NMR spectra.     

Interactions of protein kinase CK2β subunit within the holoenzyme and with other proteins

Protein kinase CK2 is a ubiquitous, highly conserved protein kinase with a tetrameric 22 structure. For the formation of this tetrameric complex a - dimer seems to be a prerequisite. Using the two-hybrid system and a series of CK2 deletion mutants, we mapped domains involved in - and - interactions. We also detected an intramolecular b interaction within the amino acid stretch 132-165.Using CK2 as a bait in a two-hybrid library screening several new putative cellular partners have been identified, among them the S6 kinase p90rsk, the putative tumor suppressor protein Doc-1, the Fas-associated protein FAF1, the mitochondrial translational initiation factor 2 and propionyl CoA carboxylase subunit.