Cell-free ring expansion of penicillin N to deacetoxycephalosporin C by Cephalosporium acremonium CW-19 and its mutants.

To examine microbiological ring expansion of penicillin N to a cephalosporin, we obtained five mutants of Cephalosporium acremonium blocked in beta-lactam antibiotic biosynthesis from 2500 survivors of mutagenesis. In submerged fermentation, mutants M-0198, M-0199, and M-2351 produced no beta-lactam antibiotic (type A), whereas mutants M-1443 and M-1836 formed penicillin N but not cephalosporin C (type B). Cell-free extracts of type A mutants converted penicillin N to a cephalosporin; those of type B mutants did not. The product of the cell-free reaction was identified as deacetoxycephalosporin C by thin-layer chromatography, paper chromatography, paper electrophoresis, and enzyme tests. These data strongly support our hypothesis that penicillin N is an intermediate of cephalosporin biosynthesis.     

Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G

Many medically useful semisynthetic cephalosporins are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA), which has been traditionally made by the polluting chemical method. Here, a whole-cell biocatalytic process based on an engineered Escherichia coli strain expressing 2-oxoglutarate–dependent deacetoxycephalosporin C synthase (DAOCS) for converting penicillin G to G-7-ADCA is developed. The major engineering strategy is to reconstitute the tricarboxylic acid (TCA) cycle of E. coli to force the metabolic flux to go through DAOCS catalyzed reaction for 2-oxoglutarate to succinate conversion. Then the glyoxylate bypass was disrupted to eliminate metabolic flux that may circumvent the reconstituted TCA cycle. Additional engineering steps were taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. These steps include engineering strategies to reduce acetate accumulation in the biocatalytic process and to knock out a host β-lactamase involved in the degradation of penicillin G and G-7-ADCA. By combining these manipulations in an engineered strain, the yield of G-7-ADCA was increased from 2.50 ± 0.79 mM (0.89 ± 0.28 g/L, 0.07 ± 0.02 g/gDCW) to 29.01 ± 1.27 mM (10.31 ± 0.46 g/L, 0.77 ± 0.03 g/gDCW) with a conversion rate of 29.01 mol%, representing an 11-fold increase compared with the starting strain (2.50 mol%).Most clinically important semisynthetic cephalosporins are manufactured from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). The traditional chemical process to produce 7-ADCA by ring expansion of penicillin G is expensive and pollutive (1, 2); thus, a bioconversion process is highly desirable. To this end, a process based on fermentation of Penicillium chrysogenum transformants expressing cefE (encoding deacetoxycephalosporin C synthase from Streptomyces clavuligerus, scDAOCS) in culture medium supplemented with adipate was first developed to produce adipyl 7-ADCA (3), which can be further converted to 7-ADCA by glutaryl acylase (3). A second process is based on the expression of the cefE gene from S. clavuligerus in a cefEF-disrupted Acremonium chrysogenum; the engineered strain can convert penicillin N accumulated in the cefEF disrupted mutant into deacetoxycephalosporin C (DAOC), which can be deacylated to 7-ADCA (1). Alternatively, a bioconversion process that directly converts penicillin G to G-7-ADCA by DAOCS had also been considered and investigated (4). This process holds promise to be the most commercially viable route to produce 7-ADCA. Imperative to the success of this strategy is the development of mutant DAOCS enzymes that can efficiently catalyze the conversion of the cheap substrate, penicillin G, to G-7-ADCA.The scDAOCS from S. clavuligerus (also known as penicillin expandase) is the key enzyme that catalyzes the ring expansion of penicillin substrates into the corresponding cephalosporins (5, 6). It is a Fe (II)- and 2-oxoglutarate (2OG)-dependent oxidase (Fig. 1A). Several structures of DAOCS had been reported (7, 8). Presteady-state kinetics and binding studies of DAOCS (9) suggested that DAOCS employs a mechanism similar to other 2OG-dependent oxygenases, where the binding of 2OG occurs first with subsequent formation of a ternary DAOCS·Fe(II)·2OG·penicillin substrate complex (9). The natural substrate of DAOCS is penicillin N, and its activity toward other penicillin analogs, such as penicillin G, is low (4). To apply DAOCS in a bioconversion process, great efforts had been devoted to improving the catalytic activity of DAOCS toward penicillin G (10). These works led to the discoveries of many improved mutants with increased activity toward penicillin G (10). In this study, we chose a scDAOCS mutant (H7) (2) with significantly increased activity toward penicillin G (22.48-fold for specific activity and 80.74-fold for k_cat/K_m), as the biocatalyst to develop a bioconversion process of penicillin G in Escherichia coli. Previously, Cho et al. (4) reported successful bioconversion of penicillin G to G-7-ADCA using resting cells and extracts of S. clavuligerus NP1. Further studies revealed that the addition of ethanol or catalase stimulated the bioconversion of penicillin G (11, 12). Heterologous expression of DAOCS in Streptomyces lividans W25 resulted in significantly increased bioconversion of penicillin G (0.24 g/L after 24 h at the optimized condition, with 10 g/L penicillin G) (13). Whole-cell bioconversion is preferred because it does not require the exogenous addition of cosubstrates, such as, 2OG. In this work, we considered E. coli as a whole-cell catalyst of DAOCS catalyzed reaction because it is a well-characterized host with a clean genetic background.Open in a separate windowFig. 1.Reconstitution of TCA cycle using DAOCS catalyzed reaction. (A) DAOCS catalyzed reaction to convert penicillin G to G-7-ADCA, which is coupled with the conversion of 2OG to succinate to compensate the disrupted TCA cycle. (B) The manipulations to disrupt the TCA cycle and glyoxylate bypass.Because DAOCS catalyzed reaction needs 2OG as a cosubstrate, which is a key intermediate of the tricarboxylic acid (TCA) cycle, the efficiency of this reaction depends on the amount of 2OG that can be diverted from the TCA cycle. In the TCA cycle (Fig. 1B), 2OG is converted to succinyl-CoA and CO₂ by 2OG dehydrogenase multienzyme complex (SucAB in E. coli), then to succinate by succinyl-CoA synthetase (SucCD). We envision that if the TCA cycle were disrupted at the 2OG to the succinate step, the TCA cycle can then be forced to go through a DAOCS catalyzed reaction. Such an outcome is a major goal of this work: that is, to reconstitute the TCA cycle with a DAOCS catalyzed reaction.     

13C/31P NMR studies of glucose transport in human skeletal muscle

The muscle intracellular (IC) free glucose concentration and the rate of muscle glycogen synthesis were measured by using in vivo ¹³C and ³¹P NMR spectroscopy in normal volunteers under hyperinsulinemic (≈300 pM) clamp conditions at the following three plasma glucose levels: euglycemia (≈6 mM), mild (≈10 mM), and high (≈16 mM) hyperglycemia. In keeping with biopsy studies, muscle IC free glucose concentration at euglycemia (−0.03 ± 0.03 mmol/kg of muscle, mean ± SEM, n = 10) was not statistically different from zero. A small but statistically significant amount of IC free glucose was observed during mild and high hyperglycemia: 0.15 ± 0.08 (n = 5) and 0.43 ± 0.20 mmol/kg of muscle (n = 5), respectively. Muscle glycogen synthesis rate, in mmol per kg of muscle per min, was 111 ± 11 at euglycemia (n = 10), 263 ± 29 during mild hyperglycemia (n = 5), and 338 ± 42 during high hyperglycemia (n = 5), these three rates being significantly different from each other. As previous in vitro and in vivo studies, these rates suggest a K_m (concentration at which unidirectional glucose transport reaches half-maximal rate) of the muscle glucose transport system in the 15–25 mM range under hyperinsulinemic conditions. The low concentrations of muscle IC free glucose observed under hyperinsulinemic conditions were interpreted, with this estimate and in the framework of metabolic control theory, as glucose transport being the predominant step controlling muscle glucose flux not only at euglycemia but also during hyperglycemia.     

RIBONUCLEASE V OF ESCHERICHIA COLI, I. DEPENDENCE ON RIBOSOMES AND TRANSLOCATION

A new RNase activity, tentatively named RNase V, was found in cell-free extracts of E. coli. This activity requires ribosomes, G and T factors, tRNA, K⁺ or NH₄⁺, Mg²⁺, GTP, and a sulfhydryl compound to degrade poly U, poly A, T4 phage mRNA, or E. coli mRNA. RNase V is specific for mRNA; it does not attack ribosomal RNA. It is inhibited by antibiotics that decrease breakdown of mRNA in vivo, such as chloramphenicol and streptomycin, and by such agents as 5′-β, γ-methylene-guanosine triphosphate, and fusidic acid, which inhibit ribosome-dependent GTPase and translocation of ribosomes along mRNA. The evidence suggests that RNase V is either an integral part of the ribosome or is tightly associated with it, and that it selectively degrades mRNA in intact cells.     

Interaction between the intermediary electron acceptor (pheophytin) and a possible plastoquinone-iron complex in photosystem II reaction centers

Photoreduction of the intermediary electron acceptor, pheophytin (Pheo), in photosystem II reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and redox potential E_h = -450 mV produces an EPR doublet centered at g = 2.00 with a splitting of 52 G at 7 K in addition to a narrow signal attributed to Pheo^[unk] (g = 2.0033, ΔH ≈ 13 G). The doublet is eliminated after extraction of lyophilized TSF-II with hexane containing 0.13-0.16% methanol but is restored by reconstitution with plastoquinone A (alone or with β-carotene) although not with vitamin K₁. TSF-II and TSF-IIa are found to contain ≈2 nonheme Fe atoms per reaction center. Incubation with 0.55 M LiClO₄ plus 2.5 mM o-phenanthroline (but not with 0.55 M LiClO₄ alone) decreases this value to ≈0.6 and completely eliminates the EPR doublet, but photoreduction of Pheo is not significantly affected. Partial restoration of the doublet (about 25%) was achieved by subsequent incubation with 0.2 mM Fe²⁺, but not with either Mn²⁺ or Mg²⁺. The Fe removal results in the development of a photoinduced EPR signal (g = 2.0044 ± 0.0003, ΔH = 9.2 ± 0.5 G) at E_h = 50 mV, which is not observed after extraction with 0.16% methanol in hexane. It is ascribed to plastosemiquinone no longer coupled to Fe in photosystem II reaction centers. The results show that a complex of plastoquinone and Fe can act as the stable “primary” electron acceptor in photosystem II reaction centers and that the interaction of its singly reduced form with the reduced intermediary acceptor, Pheo^[unk], is responsible for the EPR doublet.     

Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport

Nitrate-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) contain a 45-kDa protein as a major protein in the cytoplasmic membrane but ammonium-grown cells lack it. A mutant (M45) was constructed by inactivating the gene encoding the 45-kDa protein. M45 did not grow under low concentrations of nitrate but high concentrations of nitrate could support its growth, with the optimal concentration being 40-70 mM. The growth rate of M45 was as high as that of the wild-type cells when ammonium was the nitrogen source. The 45-kDa protein was absent in M45 irrespective of the growth conditions. The activities of nitrate and nitrite reductases were higher in M45 than in wild type. The rate of nitrate-dependent O₂ evolution in wild type measured in the presence of L-methionine D,L-sulfoximine and D,L-glyceraldehyde showed saturation kinetics with respect to nitrate concentration in the external medium. The nitrate concentration required to produce half the maximal rate was 1 μM. In M45, the rate of nitrate-dependent O₂ evolution was nearly zero at nitrate concentrations <1 mM and was linearly increased as the concentration increased. The presumed absence of nitrate transport in M45 demonstrated by these results suggested that the 45-kDa protein is a nitrate transporter.     

Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer β-amyloid peptides

The excessive generation and accumulation of 40- and 42-aa β-amyloid peptides (Aβ₄₀/Aβ₄₂) in selectively vulnerable brain regions is a major neuropathological feature of Alzheimer’s disease. Aβ, derived by proteolytic cleavage from the β-amyloid precursor protein (βAPP), is normally secreted. However, recent evidence suggests that significant levels of Aβ also may remain inside cells. Here, we have investigated the subcellular compartments within which distinct amyloid species are generated and the compartments from which they are secreted. Three experimental approaches were used: (i) immunofluorescence performed in intact cortical neurons; (ii) sucrose gradient fractionation performed with mouse neuroblastoma cells stably expressing wild-type βAPP₆₉₅ (N2a₆₉₅); and (iii) cell-free reconstitution of Aβ generation and trafficking from N2a₆₉₅ cells. These studies demonstrate that: (i) Aβ₄₀ (Aβ_1–40 plus Aβ_x-40, where x is an NH₂-terminal truncation) is generated exclusively within the trans-Golgi Network (TGN) and packaged into post-TGN secretory vesicles; (ii) Aβ_x-42 is made and retained within the endoplasmic reticulum in an insoluble state; (iii) Aβ₄₂ (Aβ_1–42 plus Aβ_x-42) is made in the TGN and packaged into secretory vesicles; and (iv) the amyloid peptides formed in the TGN consist of two pools (a soluble population extractable with detergents and a detergent-insoluble form). The identification of the organelles in which distinct forms of Aβ are generated and from which they are secreted should facilitate the identification of the proteolytic enzymes responsible for their formation.     

Non-Thermal Plasma Reduction of Ag+ Ions into Silver Nanoparticles in Open Atmosphere under Statistically Optimized Conditions for Biological and Photocatalytic Applications

An environmentally friendly non-thermal DC plasma reduction route was adopted to reduce Ag⁺ ions at the plasma–liquid interface into silver nanoparticles (AgNPs) under statistically optimized conditions for biological and photocatalytic applications. The efficiency and reactivity of AgNPs were improved by statistically optimizing the reaction parameters with a Box–Behnken Design (BBD). The size of the AgNPs was chosen as a statistical response parameter, while the concentration of the stabilizer, the concentration of the silver salt, and the plasma reaction time were chosen as independent factors. The optimized parameters for the plasma production of AgNPs were estimated using a response surface methodology and a significant model p < 0.05. The AgNPs, prepared under optimized conditions, were characterized and then tested for their antibacterial, antioxidant, and photocatalytic potentials. The optimal conditions for these three activities were 3 mM of stabilizing agent, 5 mM of AgNO₃, and 30 min of reaction time. Having particles size of 19 to 37 nm under optimized conditions, the AgNPs revealed a 82.3% degradation of methyl orange dye under UV light irradiation. The antibacterial response of the optimized AgNPs against S. aureus and E. coli strains revealed inhabitation zones of 15 mm and 12 mm, respectively, which demonstrate an antioxidant activity of 81.2%.     

Phytochemical Characterization of Olea europea Leaf Extracts and Assessment of Their Anti-Microbial and Anti-HSV-1 Activity

Owing to the richness of bioactive compounds, Olea europea leaf extracts exhibit a range of health effects. The present research evaluated the antibacterial and antiviral effect of leaf extracts obtained from Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) from Tunisia. LC-DAD-ESI-MS analysis allowed the identification of different compounds that contributed to the observed biological properties. Both OESA and OESY were active against Gram-positive bacteria (MIC values between 7.81 and 15.61 μg/mL and between 15.61 and 31.25 μg/mL against Staphylococcus aureus ATCC 6538 for OESY and OESA, respectively). The antiviral activity against the herpes simplex type 1 (HSV-1) was assessed on Vero cells. The results of cell viability indicated that Olea europea leaf extracts were not toxic to cultured Vero cells. The half maximal cytotoxic concentration (CC₅₀) values for OESA and OESY were 0.2 mg/mL and 0.82 mg/mL, respectively. Furthermore, both a plaque reduction assay and viral entry assay were used to demonstrate the antiviral activity. In conclusion, Olea europea leaf extracts demonstrated a bacteriostatic effect, as well as remarkable antiviral activity, which could provide an alternative treatment against resistant strains.     

An attempt to distinguish between the actions of neuromuscular blocking drugs on the acetylcholine receptor and on its associated ionic channel

The effects of lobeline and tubocurarine on the voltage-clamped endplates of frog sartorius and cutaneous pectoris muscles were examined at room temperature (20-23°C). Like tubocurarine, lobeline causes nondepolarizing neuromuscular blockade. The half-time of decay (t_½) of endplate currents (e.p.c.s) recorded at a holding potential (V_m) of -90 mV was significantly shorter in endplates treated with lobeline (50 μM; mean t_½ ± SEM = 0.41 ± 0.02 ms) or tubocurarine (11.4 μM; t_½ = 0.64 ± 0.04 ms) than in those treated with Mg²⁺ (13 mM; t_½ = 1.39 ± 0.11 ms) or a low concentration of tubocurarine (3 μM; t_½ = 0.87 ± 0.05 ms). Similarly, lobeline (10 μM) shortened the t_½ of untreated miniature e.p.c.s by 35%; tubocurarine, however, abolished miniature e.p.c.s at the concentration required to observe its actions on e.p.c. decay kinetics. The t_½ of e.p.c.s recorded from preparations treated with Mg²⁺ (13 mM), tubocurarine at low concentrations (3 μM), or untreated miniature e.p.c.s was logarithmically related to V_m, being slower at more hyperpolarized values. By contrast, the t_½s of e.p.c.s recorded in either lobeline (50 μM) or tubocurarine (11.4 μM) were independent of voltage in the range -150 to -80 mV. The ability of lobeline to shorten t_½ and to remove the voltage dependence of t_½ was partially antagonized by Mg²⁺ (13 mM). As expected, when lobeline or tubocurarine was removed from the bath or when acetylcholine release from the motor nerve terminals was increased by 4-aminopyridine (20 μM) and Ca²⁺ (10 mM) (in the presence of lobeline or tubocurarine), the amplitude of e.p.c.s increased as a function of time. However, the t_½ of the decay phase of the e.p.c.s remained shortened (i.e., unaltered from the earlier treatment). These results suggest that both tubocurarine and lobeline have at least two distinct postjunctional actions including: (i) a block of the acetylcholine receptor and (ii) a block of the ionic channel associated with the acetylcholine receptor.     

Gelsolin mediates calcium-dependent disassembly of Listeria actin tails

The role of intracellular Ca²⁺ in the regulation of actin filament assembly and disassembly has not been clearly defined. We show that reduction of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to <40 nM in Listeria monocytogenes-infected, EGFP–actin-transfected Madin–Darby canine kidney cells results in a 3-fold lengthening of actin filament tails. This increase in tail length is the consequence of marked slowing of the actin filament disassembly rate, without a significant change in assembly rate. The Ca²⁺-sensitive actin-severing protein gelsolin concentrates in the Listeria rocket tails at normal resting [Ca²⁺]_i and disassociates from the tails when [Ca²⁺]_i is lowered. Reduction in [Ca²⁺]_i also blocks the severing activity of gelsolin, but not actin-depolymerizing factor (ADF)/cofilin microinjected into Listeria-infected cells. In Xenopus extracts, Listeria tail lengths are also calcium-sensitive, markedly shortening on addition of calcium. Immunodepletion of gelsolin, but not Xenopus ADF/cofilin, eliminates calcium-sensitive actin-filament shortening. Listeria tail length is also calcium-insensitive in gelsolin-null mouse embryo fibroblasts. We conclude that gelsolin is the primary Ca²⁺-sensitive actin filament recycling protein in the cell and is capable of enhancing Listeria actin tail disassembly at normal resting [Ca²⁺]_i (145 nM). These experiments illustrate the unique and complementary functions of gelsolin and ADF/cofilin in the recycling of actin filaments.     

Native phytochrome: Inhibition of proteolysis yields a homogeneous monomer of 124 kilodaltons from Avena

Phytochrome purified from Avena as the red-absorbing form, Pr, by an established immunoaffinity column procedure is heterogeneous. Two major polypeptides and one minor polypeptide with apparent molecular masses of 118, 114, and 112 kilodaltons (kDal), respectively, are observed on NaDodSO₄/polyacrylamide gel electrophoresis. In contrast, only a single band of 124 kDal is obtained when phytochrome is rapidly immunoprecipitated after extraction either (i) as the far-red absorbing form, Pfr, in detergent-free buffer or (ii) in either spectral form in a 100°C NaDodSO₄-containing buffer. On two-dimensional gel electrophoresis the three column-purified species have pIs of 5.8, 6.0, and 6.0, whereas 124-kDal phytochrome is a single spot with a pI of 5.9. Incubation as Pr in extracts causes progressive conversion of the 124-kDal polypeptide to the 118- and 114-kDal species. This process is inhibited by phenylmethylsulfonyl fluoride, suggesting that Pr is susceptible and Pfr resistant to limited proteolysis during extraction. These data, and the fact that the cell-free translation product of phytochrome mRNA is also 124 kDal [Bolton, G. W. & Quail, P. H. (1982) Planta, in press], indicate that the native monomer from Avena is a single species of 124 kDal. Thus the heterogeneous preparations of slightly lower molecular weight (“large” or “120-kilodalton” phytochrome) previously extensively characterized appear to have consisted of a mixture of partially degraded molecules that have undergone limited proteolysis during purification as Pr, as is established practice. A reexamination of the molecular properties of phytochrome appears necessary.     

ADP-ribosylation factor and phosphatidic acid levels in Golgi membranes during budding of coatomer-coated vesicles

The finding that ADP-ribosylation factor (ARF) can activate phospholipase D has led to debate as to whether ARF recruits coat proteins through direct binding or indirectly by catalytically increasing phosphatidic acid production. Here we test critical aspects of these hypotheses. We find that Golgi membrane phosphatidic acid levels do not rise—in fact they decline—during cell-free budding reactions. We confirm that the level of membrane-bound ARF can be substantially reduced without compromising coat assembly [Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C. & Roth, M. G. (1996) J. Cell Biol. 134, 295–306], but find that under all conditions, ARF is present on the Golgi membrane in molar excess over bound coatomer. These results do not support the possibility that the activation of coat assembly by ARF is purely catalytic, and they are consistent with ARF forming direct interactions with coatomer. We suggest that ARF, like many other G proteins, is a multifunctional protein with roles in trafficking and phospholipid signaling.     

The γ134.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1α to dephosphorylate the α subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase

In human cells infected with herpes simplex virus 1 the double-stranded RNA-dependent protein kinase (PKR) is activated but phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2) and total shutoff of protein synthesis is observed only in cells infected with γ₁z34.5⁻ mutants. The carboxyl-terminal 64 aa of γ₁34.5 protein are homologous to the corresponding domain of MyD116, the murine growth arrest and DNA damage gene 34 (GADD34) protein and the two domains are functionally interchangeable in infected cells. This report shows that (i) the carboxyl terminus of MyD116 interacts with protein phosphatase 1α in yeast, and both MyD116 and γ₁34.5 interact with protein phosphatase 1α in vitro; (ii) protein synthesis in infected cells is strongly inhibited by okadaic acid, a phosphatase 1 inhibitor; and (iii) the α subunit in purified eIF-2 phosphorylated in vitro is specifically dephosphorylated by S10 fractions of wild-type infected cells at a rate 3000 times that of mock-infected cells, whereas the eIF-2α-P phosphatase activity of γ₁34.5⁻ virus infected cells is lower than that of mock-infected cells. The eIF-2α-P phosphatase activities are sensitive to inhibitor 2. In contrast to eIF-2α-P phosphatase activity, extracts of mock-infected cells exhibit a 2-fold higher phosphatase activity on [³²P]phosphorylase than extracts of infected cells. These results indicate that in infected cells, γ₁34.5 interacts with and redirects phosphatase to dephosphorylate eIF-2α to enable continued protein synthesis despite the presence of activated PKR. The GADD34 protein may have a similar function in eukaryotic cells. The proposed mechanism for maintenance of protein synthesis in the face of double-stranded RNA accumulation is different from that described for viruses examined to date.     

Ly-6C regulates endothelial adhesion and homing of CD8+ T cells by activating integrin-dependent adhesion pathways

Ly-6C belongs to the Ly-6 family of glycosyl phosphatidylinositol-anchored surface glycoproteins and is expressed on a subset of mature CD8⁺ T cells. Ly-6C ligation can mediate T cell activation and causes interleukin 2 secretion in cytolytic T cell clones. We characterize herein a new mAb 1G7.G10 against Ly-6C that recognizes an epitope involved in lymphocyte adhesion and in lymphocyte homing. Pretreatment of lymph node lymphocytes and of purified CD8⁺ T cells (but not of lymphocytes depleted of CD8⁺ T cells) with 1G7.G10 reduced their in vitro binding to lymph node high endothelial venules by 28% and 34%, respectively. This effect was bypassed by cross-linking Ly-6C molecules with 1G7.G10 and a second-step antibody. The in vivo homing of (donor) CD8⁺ T lymphocytes to lymph nodes was reduced by Ly-6C blocking with 1G7.G10 (whole antibody) or with its fragments [F(ab) or F(ab)₂] by 20% or by 32% and 48%, respectively. Cross-linking of Ly-6C in vitro induced very late antigen-4 and lymphocyte function-associated antigen 1-mediated aggregation of CD8⁺ T cells, suggesting that ligand binding to Ly-6C leads to activation of integrins. This activation may facilitate homing of Ly-6C⁺ CD8⁺ T cells in vivo.     

Sucrose biosynthesis in a prokaryotic organism: Presence of two sucrose-phosphate synthases in Anabaena with remarkable differences compared with the plant enzymes

Biosynthesis of sucrose-6-P catalyzed by sucrose-phosphate synthase (SPS), and the presence of sucrose-phosphate phosphatase (SPP) leading to the formation of sucrose, have both been ascertained in a prokaryotic organism: Anabaena 7119, a filamentous heterocystic cyanobacterium. Two SPS activities (SPS-I and SPS-II) were isolated by ion-exchange chromatography and partially purified. Four remarkable differences between SPSs from Anabaena and those from higher plants were shown: substrate specificity, effect of divalent cations, native molecular mass, and oligomeric composition. Both SPS-I and SPS-II accept Fru-6-P (K_m for SPS-I = 0.8 ± 0.1 mM; K_m for SPS-II = 0.7 ± 0.1 mM) and UDP-Glc as substrates (K_m for SPS-I = 1.3 ± 0.4 mM; K_m for SPS-II = 4.6 ± 0.4 mM), but unlike higher plant enzymes, they are not specific for UDP-Glc. GDP-Glc and TDP-Glc are also SPS-I substrates (K_m for GDP-Glc = 1.2 ± 0.2 mM and K_m for TDP-Glc = 4.0 ± 0.4 mM), and ADP-Glc is used by SPS-II (K_m for ADP-Glc = 5.7 ± 0.7 mM). SPS-I has an absolute dependence toward divalent metal ions (Mg²⁺ or Mn²⁺) for catalytic activity, not found in plants. A strikingly smaller native molecular mass (between 45 and 47 kDa) was determined by gel filtration for both SPSs, which, when submitted to SDS/PAGE, showed a monomeric composition. Cyanobacteria are, as far as the authors know, the most primitive organisms that are able to biosynthesize sucrose as higher plants do.     

Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation

Protein synthesis (PS) has been considered essential to sustain mammalian life, yet was found to be virtually arrested for weeks in brain and other organs of the hibernating ground squirrel, Spermophilus tridecemlineatus. PS, in vivo, was below the limit of autoradiographic detection in brain sections and, in brain extracts, was determined to be 0.04% of the average rate from active squirrels. Further, it was reduced 3-fold in cell-free extracts from hibernating brain at 37°C, eliminating hypothermia as the only cause for protein synthesis inhibition (active, 0.47 ± 0.08 pmol/mg protein per min; hibernator, 0.16 ± 0.05 pmol/mg protein per min, P < 0.001). PS suppression involved blocks of initiation and elongation, and its onset coincided with the early transition phase into hibernation. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2α are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a 3-fold increase in ribosomal mean transit times in cell-free extracts from hibernators (active, 2.4 ± 0.7 min; hibernator, 7.1 ± 1.4 min, P < 0.001). No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate “trickle” blood flow and reduced substrate and oxygen availability. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state.     

Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype

Despite the central role of human epidermal stem cells in tissue homeostasis, wound repair, and neoplasia, remarkably little is known about these cells, largely due to the absence of molecular markers that distinguish them from other proliferative cells within the germinative/basal layer. Epidermal stem cells can be distinguished from other cells in the basal layer by their quiescent nature in vivo and their greater overall proliferative capacity. In this study, we demonstrate enrichment and isolation of a subpopulation of basal epidermal cells from neonatal human foreskin based on cell surface phenotype, which satisfy these criteria. These putative stem cells are distinguished from other basal cells by their characteristic expression of high levels of the adhesion molecule α₆, a member of the integrin family (α₆^bri), and low levels of a proliferation-associated cell surface marker recognized by recently described mAb 10G7 (10G7^dim). We conclude that cells with the phenotype α₆^bri10G7^dim represent the epidermal stem cell population based on the demonstration that these cells (i) exhibit the greatest regenerative capacity of any basal cells, (ii) represent a minor subpopulation (≈10%) of immature epidermal cells, which (iii) are quiescent at the time of isolation from the epidermis, as determined by cell cycle analysis.     

In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca²⁺ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca²⁺ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca²⁺ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca²⁺ from ER, giving rise to a slow and small increase in [Ca²⁺]_i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca²⁺ release by 4-CEP, giving rise to Ca²⁺ spikes. In glucose-stimulated beta cells forskolin induced Ca²⁺ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY₂). We conclude that in situ activation of RY₂ in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.     

Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the ech42 (endochitinase-encoding) gene in Trichoderma harzianum

The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We have studied the molecular regulation of mycoparasitism in the host/mycoparasite system Botrytis cinerea/T. harzianum. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EMSAs) with two promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expression is triggered during mycoparasitic interaction [Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10903–10907]. All cell-free extracts formed high-molecular weight protein–DNA complexes, but those obtained from mycelia activated for mycoparasitic attack formed a complex with greater mobility. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Cre1, provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragments of the ech-42 promoter. The presence of two and three consensus sites for binding of Cre1 in the two ech-42 promoter fragments used is consistent with these findings. In contrast, the formation of the protein–DNA complex from mycoparasitic mycelia is unaffected by the addition of the competing oligonucleotides and hence does not involve Cre1. Addition of equal amounts of protein of cell-free extracts from nonmycoparasitic mycelia converted the mycoparasitic DNA–protein complex into the nonmycoparasitic complex. The addition of the purified Cre1::glutathione S-transferase protein to mycoparasitic cell-free extracts produced the same effect. These findings suggest that ech-42 expression in T. harzianum is regulated by (i) binding of Cre1 to two single sites in the ech-42 promoter, (ii) binding of a “mycoparasitic” protein–protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (iii) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the mycoparasitic protein–DNA complex.