(AGCTG) (AGCTG) (AGCTG) (GGGTG) as the primordial sequence of intergenic spacers: the role in immunoglobulin class switch

The means employed for immunoglobulin heavy chain class switch appears to be no different from that by which meiotic intergenic crossing-overs at accomplished. As with other intergenic spacers, the 5' noncoding sequence of each Ig CH (immunoglobulin heavy chain constant region) gene apparently undergoes unconstrained sequence changes due to randomly sustained base substitutions, deletions, and duplications. Yet, there remains sufficient regional sequence homology between the Ig Cmu 5' noncoding sequence and those of its somatic recombination partners, e.g., Ig C gamma 1, Ig C gamma 2b, Ig C alpha, because each of these 5' noncoding sequences is made of multiple copies in various stages of degeneracy of one primordial 20 base pair-long sequence: (AGCTG) (AGCTG) (AGCTG) (GGGTG).     

Metabolism of non-phenolic diarylpropane lignin substructure model compound by Coriolus versicolor

Abstract A lignin substructure model, 1-(4-ethoxy-3,5-dimethoxyphenyl)-2-(4-ethoxy-3-methoxyphenyl)-propane-1,3-diol(I), was actively metabolized by a white-rot fungus Coriolus versicolor in low nitrogen stationary cultures favouring the ligninolytic activity in the fungus. Cleavage of the dimer I between C_α and C_β of the propanoid side chain was the major degradative reaction by the fungus.     

The involvement of a G-protein in phytochrome-regulated, Ca2+-dependent swelling of etiolated wheat protoplasts

The red light (R)-induced swelling of mesophyll protoplasts, isolated from dark-grown wheat ( Triticum aestivum L. cv. Arminda) leaves, was inhibited by guanosine-5'-0-(2-thiodiphosphate) (GDP-β-S). In darkness or after control irradiation with far-red light (FR), guanosine-5'-O-(3-thiotriphosphate) (GTP-γ-S) induced swelling to the same extent as after R. Both GDP-β-S and GTP-γ-S were introduced into the cytoplasm by means of electroporation. The possibility of R-induced activation of the phosphatidylinositol pathway of transmembrane signalling was investigated. Neomycin, Li⁺ and l-(5-isoquinolinesulfonyl)-2-methylpiperazine (H₇) inhibited the R-induced swelling. Phorbol 12-myristate 13-acetate (PMA) induced swelling after control irradiation with FR. Neomycin and Li⁺ also inhibited GTP-γ-S-induced swelling. These results suggest that a GTP-binding protein is involved in the phytochrome-regulated swelling response. Addition of N⁶, 2'-0-dibutyryladenosine 3':5'-cyclic monophosphate (DB-cAMP) induced swelling to the same extent as R-irradiation. The calmodulin antagonist N-(6-aminohexyl)5-chloro-l-naphthalenesulfonamide (W₇) induced swelling after FR, while R-induced swelling was not affected. The less active analogue N-(6-aminohexyl)-l-naphthalenesulfonamide (W₅) induced no swelling after FR. It is speculated that the protoplast volume is correlated with the cytoplasmic concentration of free Ca²⁺.     

Stimulation of Guanosine 5'-O-(3-[35S]Thiotriphosphate) Binding by Cholinergic Muscarinic Receptors in Membranes of Rat Olfactory Bulb

Abstract: In membranes of rat olfactory bulb, a brain region in which muscarinic agonists increase cyclic AMP formation, the muscarinic stimulation of guanosine 5'- O -(3-[³⁵S]thiotriphosphate) ([³⁵S]GTPγS) binding was used as a tool to investigate the receptor interaction with the guanine nucleotide-binding regulatory proteins (G proteins). The stimulation of the radioligand binding by carbachol (CCh) was optimal (threefold increase) in the presence of micromolar concentrations of GDP and 100 m M NaCl. Exposure to N -ethylmaleimide and pertussis toxin markedly inhibited the CCh effect, whereas it increased the relative stimulation of [³⁵S]GTPγS binding elicited by pituitary adenylate cyclase-activating polypeptide (PACAP). On the other hand, membrane treatment with cholera toxin curtailed the PACAP stimulation of [³⁵S]GTPγS binding but did not affect the response to CCh. Like CCh, a number of cholinergic agonists stimulated [³⁵S]GTPγS binding in a concentration-dependent and saturable manner. The antagonist profile of the muscarinic stimulation of [³⁵S]GTPγS binding was highly correlated with that displayed by the muscarinic stimulation of adenylyl cyclase. These data indicate that the olfactory bulb muscarinic receptors couple to G_i/G_o, but not to G_s, and support the possibility that activation of G_i/G_o mediates the stimulatory effect on adenylyl cyclase activity.     

Occurrence of aminopropylcadaverine and its aminopropyl derivatives aminopentylnorspermidine and N, N' -bis(3-aminopropyl)cadaverine in Halococcus acetoinfaciens

Abstract Besides putrescine, cadaverine, spermidine, spermine and thermospermine, three novel polyamines were detected in a slightly halophilic eubacterium Halococcus acetoinfaciens (IAM 12094, ATCC 25861). These novel polyamines were found to be N -3-aminopropylcadaverine [NH₂(CH₂)₃NH(CH₂)₅NH₂] and its aminopropyl derivatives: aminopentylnorspermidine [NH₂(CH₂)₃NH(CH₂)₃NH(CH₂)₅NH₂] and N , N ' -bis(3-aminoprophyl)cadaverine [NH₂(CH₂)₃ NH(CH₂)₅NH(CH₂)₃NH₂]. Aminopropylcadaverine was also detected in two other species, Halococcus agglomeratus (IAM 12095, ATCC 25862) and Halococcus nondenitrificans (IAM 12096, ATCC 25863).     

Characterization of the Thyroid Hormone Transport System of Cerebrocortical Rat Neurons in Primary Culture

Abstract: The uptake of 3',3,5-triiodo- l -thyronine (T₃) and l -thyroxine (T₄) by primary cultures derived from rat brain hemispheres was studied under initial velocity conditions, at 25°C. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The K _m of T₃ uptake was very similar to that of T₄, and did not vary significantly from day 1 to 4 in culture (310–400 n M ). The maximal velocity ( V _max) of T₃ uptake nearly doubled between day 1 and 4 of culture (41 ± 3 vs. 70 ± 5 pmol/min/mg of DNA, respectively). The V _max of T₄ uptake did not change (28 ± 8 and 31 ± 4 pmol/min/mg of DNA on days 1 and 4, respectively). The rank order of unlabeled thyroid hormone analogues to compete with labeled T₃ or T₄ uptakes were the same (T₃ > T₄ > 3',5',3-triiodo- l -thyronine > 3',3,5-triiodo- d -thyronine > triiodothyroacetic acid), indicating that the transport system is stereospecific. Unlabeled T₄ was a stronger competitor of labeled T₄ uptake than of labeled T₃ uptake, whereas unlabeled T₃ had the same potency for both processes. These results suggest that T₃ and T₄ are transported either by two distinct carriers or by the same carrier bearing separate binding sites for each hormone. They also indicate that the efficiency of T₃ uptake increases during neuronal maturation.     

Is the large plasmid of 120 MDa in Shigella sonnei composed of two DNA segments—90 and 30 MDa?

Abstract The reversibility of adhesion of 3 representative strains of oral streptococci from a phosphate-buffered suspension onto 5 different solid substrata was studied.
Streptococcus mitis T9 (surface free energy γ_b= 39 mJ · m⁻²). Streptococcus sanguis CH3 (γ_b= 95 mJ · m⁻²) and Streptococcus mutans NS (γ_b= 117 mJ · m⁻²) were selected on basis of their surface free energy. Solid substrata were employed with a surface free energy γ_s ranging from 20 mJ · m⁻² for polytetrafluorethylene to 109 mJ · m⁻² for glass. Bacterial suspensions containing 2.5 × 10⁹ cells per ml were incubated with 2 samples of each substratum. After 1 h the number of adhering bacteria was evaluated on one sample, while the second sample was kept for another hour at a 10-fold lower bacterial concentration. Bacteria with a low surface free energy desorbed only from substrata with a high surface free energy, while bacteria with a high surface free energy desorbed from substrata with a low surface free energy. Thus low energy bacterial strains adhered reversibly to high energy substrata and vice versa. Similar observations were made with polystyrene particles. Calculation of the interfacial free energy of adhesion (Δ F _adh) for each bacterial strain as well as for the polystyrene particles showed that a reversible adhesion was associated with a positive Δ F _adh, denoting unfavourable adhesion conditions upon a thermodynamic basis.     

New butenolides from the photoconductivity screening of Streptomyces antibioticus (Waksman and Woodruff) Waksman and Henrici 1948

Abstract Streptomyces antibioticus strain TÜ 99, from which a wide variety of active compounds had been isolated previously, was reinvestigated using an HPLC photoconductivity screening system. Four new compounds were isolated, characterized and their constitutions determined. All four were α,β-unsaturated γ-lactones; the most abundant compound 3 (C₁₀H₁₆O₄), as well as compound 1 (C₉H₁₄O₄) had a hydroxy group at C(5) of the lactone ring. The four lactones showed antibiotic activity against Pseudomonas aeruginosa and also a weak inhibition of the chitinase from Serratia marcescens .     

The 44-kDa Regulatory Subunit of the Paramecium cAMP-Dependent Protein Kinase Lacks a Dimerization Domain and May Have a Unique Autophosphorylation Site Sequence

ABSTRACT. The 44-kDa regulatory subunit (R₄₄) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R₄₄ cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5' and 3' untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R₄₄ amino acid sequence had 31%–38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R₄₄ sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N -terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R₄₄ clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R₄₄ contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.     

Surface free energies of oral streptococci and their adhesion to solids

Abstract The adhesion of 3 strains of oral streptococci from a buffered suspension onto 3 different solid substrata was studied. Representative strains of streptococci were selected on the basis of their surface free energy ( γ _b), namely Streptococcus mitis L1 ( γ _b= 37 mJ·m⁻²), Streptococcus sanguis CH3 (95 mJ·m⁻²) and Streptococcus mutans NS (117 mJ·m⁻²). Solid substrata were also selected on basis of their surface free energy ( γ _s), and included polytetrafluorethylene ( γ _s= 20 mJ·m⁻²), polymethylmethacrylate (53 mJ·m⁻²) and glass (109 mJ·m⁻²). Bacterial adhesion was measured as the number of bacteria adhering per cm² at equilibrium. Equilibrium was usually obtained within 20 min. S. sanguis CH3, having an intermediate surface free energy did not show a clear preference for any of the 3 solids. S. mitis L1, however, the lowest surface free energy strain, adhered in highest numbers to the low energy solid PTFE, whereas the highest γ _b strain, S. mutans NS, adhered in highest numbers to the highest γ _s solid, glass. Calculation of the interfacial free energy of adhesion ( ΔF _adh) for each bacterial strain showed that this parameter was predictive of bacterial adhesion to solid substrata.     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Dopamine Receptor Stimulation Decreases Cytosolic γ Protein Kinase C Immunoreactivity in Rat Hippocampal Slices: Evidence for Increased Ca2+-Dependent Proteolysis

Abstract: The effect of dopamine (DA) receptor stimulation on the distribution of γ protein kinase C (γPKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic γPKC (56%) without altering membrane γPKC levels, resulting in decreased total γPKC immunoreactivity. The maximal decrease in cytosolic γPKC occurred at 20 min of incubation and was significantly blocked by the D₁ DA antagonist SCH 23390 (10⁻⁶ M ) but not by the D₂ antagonist sulpiride (10⁻⁵ M ). The D₁ agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 µ M and 1 n M , respectively. The D₂ agonist quinpirole had no effect on γPKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D₁-like receptor. DA had no effect on α, δ, or ζPKC isozyme immunoreactivity in the same hippocampal preparations. The DA-induced decrease in cytosolic γPKC immunoreactivity was blocked by the Ca²⁺-dependent protease inhibitor N -acetyl-Leu-Leu-norleucinal (100 µ M ) and by the inorganic Ca²⁺ channel blocker Co²⁺. The data suggest that DA stimulates a D₁-like DA receptor, which increases the influx of Ca²⁺ and activates the Ca²⁺-dependent proteolysis of γPKC.     

Regulation of Insulin Secretion by Phosphatidylinositol-4,5-Bisphosphate

The role of PIP₂ in pancreatic beta cell function was examined here using the beta cell line MIN6B1. Blocking PIP₂ with PH-PLC-GFP or PIP5KIγ RNAi did not impact on glucose-stimulated secretion although susceptibility to apoptosis was increased. Over-expression of PIP5KIγ improved cell survival and inhibited secretion with accumulation of endocytic vacuoles containing F-actin, PIP₂, transferrin receptor, caveolin 1, Arf6 and the insulin granule membrane protein phogrin but not insulin. Expression of constitutively active Arf6 Q67L also resulted in vacuole formation and inhibition of secretion, which was reversed by PH-PLC-GFP co-expression. PIP₂ co-localized with gelsolin and F-actin, and gelsolin co-expression partially reversed the secretory defect of PIP5KIγ-over-expressing cells. RhoA/ROCK inhibition increased actin depolymerization and secretion, which was prevented by over-expressing PIP5KIγ, while blocking PIP₂ reduced constitutively active RhoA V14-induced F-actin polymerization. In conclusion, although PIP₂ plays a pro-survival role in MIN6B1 cells, excessive PIP₂ production because of PIP5KIγ over-expression inhibits secretion because of both a defective Arf6/PIP5KIγ-dependent endocytic recycling of secretory membrane and secretory membrane components such as phogrin and the RhoA/ROCK/PIP5KIγ-dependent perturbation of F-actin cytoskeleton remodelling.     

STRUCTURAL FEATURES OF NUCLEAR GENES IN THE CENTRIC DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)

Thalassiosira weissflogii (Grun.) Fryxell et Hasle is one of the more commonly studied centric diatoms, and yet molecular studies of this organism are still in their infancy. The ability to identify open reading frames and thus distinguish between introns and exons, coding and noncoding sequence is essential to move from nuclear DNA sequences to predicted amino acid sequences. To facilitate the identification of open reading frames in T. weissflogii , two newly identified nuclear genes encoding β-tubulin and t  -complex polypeptide (TCP)-γ, along with six previously published nuclear DNA sequences, were examined for general structural features. The coding region of the nuclear open reading frames had a G + C content of about 49% and could readily be distinguished from noncoding sequence due to a significant difference in G + C content. The introns were uniformly small, about 100 base pairs in size. Furthermore, the 5' and 3' splice sites of introns displayed the canonical GT/AG sequence, further facilitating recognition of noncoding regions. Six of the nuclear open reading frames displayed relatively little bias in the use of synonymous codons, as exemplified by the cDNAs encoding β-tubulin and TCP-γ. Two open reading frames displayed strong bias in the use of particular codons (although the codons used were different), as exemplified by the cDNA encoding fucoxanthin chlorophyll a/c binding protein. Knowledge of codon bias should facilitate, for example, design of degenerate PCR primers and potential heterologous reporter gene constructs.     

Purification and characterization of an algal (Dunaliella tertiolecta) protein cross-reacting with an anti-Ga-common antibody

A 26-kDa and a 36-kDa protein that cross-reacted with anti-G_a-common and anti-G_β antibodies, respectively, were detected in Dunaliella cells. The 26-kDa protein was solubilized from a crude membrane fraction with deoxycholate and purified to homogeneity by DE52 and hydroxylapatite chromatography and DEAE-5PW high performance liquid chromatography (HPLC). The hydroxylapatite-purified preparation had GTPγS binding and GTPase activities, but the homogeneous 26-kDa protein had none. The sequence of the 28 N-terminal amino acids of the 26-kDa protein had no homology to any GTP binding protein thus far reported.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Influence of positive allosteric modulators on GABA(B) receptor coupling in rat brain: a scintillation proximity assay characterisation of G protein subtypes

Little is known concerning coupling of cerebral GABA_B receptors to G protein subtypes, and the influence of positive allosteric modulators (PAMs) has not been evaluated. These questions were addressed by an antibody-capture/scintillation proximity assay strategy. GABA concentration-dependently enhanced the magnitude of [³⁵S]GTPγS binding to Gαo and, less markedly, Gαi_1/3 in cortex, whereas Gq and Gs/olf were unaffected. ( R )-baclofen and SKF97581 likewise activated Gαo and Gαi_1/3, expressing their actions more potently than GABA. Similar findings were acquired in hippocampus and cerebellum, and the GABA_B antagonist, CGP55845A, abolished agonist-induced activation of Gαo and Gαi_1/3 in all structures. The PAMs, GS39783, CGP7930 and CGP13501, inactive alone, enhanced efficacy and potency of agonist-induced [³⁵S]GTPγS binding to Gαo in all regions, actions abolished by CGP55845A. In contrast, they did not modify efficacies at Gαi_1/3. Similarly, in human embryonic kidney cells expressing GABA_B(1a+2) or GABA_B(1b+2) receptors, allosteric modulators did not detectably enhance efficacy of GABA at Gαi_1/3, though they increased its potency. To summarise, GABA_B receptors coupled both to Gαo and to Gαi, but not Gq and Gs/olf, in rat brain. PAMs more markedly enhanced efficacy of coupling to Go versus Gi_1/3. It will be of interest to confirm these observations employing complementary techniques and to evaluate their potential therapeutic significance.     

Assembly and plasma membrane targeting of recombinant immunoglobulin chains in plants with a murine immunoglobulin transmembrane sequence

The cDNA encoding a full-length murine immunoglobulin 1 heavy chain with its native leader sequence, transmembrane and intracellular domains was introduced into transgenic plants. Transformed plants expressed the recombinant polypeptide, but, in contrast to plants expressing the heavy chain without transmembrane sequence, the protein appeared to be associated with a plant cell membrane. Extraction of the membrane-associated heavy chain required the presence of a non-ionic detergent, and immunofluorescence studies of protoplasts demonstrated surface expression of membrane Ig heavy chain on up to 40% of the cells from a transgenic leaf. In plants expressing both the membrane Ig heavy chain and its partner light chain, functional antibody was also localised to the plant cell membrane and retention of the heavy chain at this site appeared to have no effect on the efficiency of antibody assembly. This approach of localising and accumulating recombinant antibody in cell membranes may have a number of applications, including passive immunisation against plant pathogens.