Cloning, mutagenesis, and characterization of the microalga Parietochloris incisa acetohydroxyacid synthase, and its possible use as an endogenous selection marker

Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic-resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched-chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the wild-type and mutant proteins were characterized in the AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wild-type and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild-type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type.     

Direct closure of round skin defects: a four-step technique with multiple subcutaneous and cutaneous "figure-of-8" sutures alleviating dog-ears

Excisional biopsies of rounded lesions are performed daily by surgeons. The objective of this article is to establish a surgical technique for direct closure of circular and elliptical defects without the need for additional excision of dog-ears. A four-step technique based on multiple cutaneous and subcutaneous "figure-of-8" sutures is presented. When correctly placed, these sutures can equally distribute the excess tissue along the scar and alleviate dog-ears. The results of the 65 presented cases show that this technique yields short scars and saves healthy skin. A significant reduction of the length-to-width ratio and the arc-to-scar length ratio is obtained. Direct closure of a circular or elliptical defect without stipulating a 3:1 length-to-width ratio is feasible. Among the advantages of the technique are that no excessive healthy tissue is removed and that the scar length is reduced. The long-term outcome is a scar that is thin, linear, flat, and concealed in the body structure.     

Gas exchange and metabolite fluctuations in green and yellow bands of variegated leaves of the monocotyledonous CAM species Agave americana

The variegated leaves of the Crassulacean acid metabolism (CAM) species Agave americana have a large central longitudinal green band with narrow yellow bands on either side. The yellow bands had 97% less pigment content, 84% lower ribulose‐1,5‐bisphosphate carboxylase/oxygenase activity, but only 20% lower phosphoenolpyruvate carboxylase activity than the green band. The green bands exhibited gas exchange typical of CAM plants, with most CO₂ uptake occurring at night, leading to a daily net CO₂ uptake of 127 mmol m⁻² day⁻¹. The yellow bands had some nighttime net CO₂ uptake but a larger loss during the daytime, indicating that they were sink tissues. Nocturnal citrate and malate accumulations for the yellow bands were 65 and 75%, respectively, of those of the green bands; sucrose supported 64‐83% of their nocturnal acid accumulation. This is the first evidence that agaves, which are malic‐enzyme‐type CAM plants, use sucrose as the carbon source for nocturnal acid accumulation. About 44% of the carbon demand of the yellow bands can be supplied by sucrose diffusing via the symplast from the adjacent green band, about 25% from fructose and glucose diffusion, and some via the apoplast.     

Transfer of Ho endonuclease and Ufo1 to the proteasome by the UbL-UbA shuttle protein, Ddi1, analysed by complex formation in vitro

The F-box protein, Ufo1, recruits Ho endonuclease to the SCF(Ufo1) complex for ubiquitylation. Both ubiquitylated Ho and Ufo1 are transferred by the UbL-UbA protein, Ddi1, to the 19S Regulatory Particle (RP) of the proteasome for degradation. The Ddi1-UbL domain binds Rpn1 of the 19S RP, the Ddi1-UbA domain binds ubiquitin chains on the degradation substrate. Here we used complex reconstitution in vitro to identify stages in the transfer of Ho and Ufo1 from the SCF(Ufo1) complex to the proteasome. We report SCF(Ufo1) complex at the proteasome formed in the presence of Ho. Subsequently Ddi1 is recruited to this complex by interaction between the Ddi1-UbL domain and Ufo1. The core of Ddi1 binds both Ufo1 and Rpn1; this interaction confers specificity of SCF(Ufo1) for Ddi1. The substrate-shield model predicts that Ho would protect Ufo1 from degradation and we find that Ddi1 binds Ho, Ufo1, and Rpn1 simultaneously forming a complex for transfer of Ho to the 19S RP. In contrast, in the absence of Ho, Rpn1 displaces Ufo1 from Ddi1 indicating a higher affinity of the Ddi1-UbL for the 19S RP. However, at high Rpn1 levels there is synergistic binding of Ufo1 to Ddi1 that is dependent on the Ddi1-UbA domain. Our interpretation is that in the absence of substrate, the Ddi1-UbL binds Rpn1 while the Ddi1-UbA binds ubiquitin chains on Ufo1. This would promote degradation of Ufo1 and disassembly of SCF(Ufo1) complexes.     

Organelle differentiation in the chick blastoderm during hypoblast formation

Summary The ultrastructure of the chick blastoderm was examined at three developmental stages, from an unincubated single-layered system through hypoblast advancement to full hypoblast formation.With the onset of incubation the nucleolus changes from a loose network of intermingled pars fibrosa and pars granulosa into a compact body with a definite matrix material.The endoplasmic reticulum, mitochondria, and Golgi complex increase in complexity and volume. In blastoderms with a fully developed hypoblast a special asymmetrical endoplasmic reticulum becomes abundant. These data are analysed in relation to similar structural differentiation of the nucleolus, endoplasmic reticulum, Golgi complex and mitochondria in the embryonic development of other vertebrate groups.The above changes in organelle structure are noted in both the epi- and the hypoblast, although these organelles become more abundant in the former. In the intermediate stage no differences are noted between epiblast cells underlined by hypoblast and those of the anterior single-layered region. The above changes in the epiblast must therefore be related to age and not to contact with the advancing hypoblast.Previous studies mentioned in the text seem to indicate that the inducing effect of the hypoblast on the epiblast is exerted after its complete formation and not during its advancement. Our results in the organelle differentiation during hypoblast formation are in accordance with this hypothesis.     

Genetic analysis of a streptomycin-resistant Escherichia coli mutant temperature-sensitive for nonsense suppression

Summary Continuing the genetic and biochemical characterization of the streptomycin-resistant Escherichia coli mutant LD1, we confirmed that LD1 is temperature-sensitive for suppression of nonsense codons, and that this phenotype of the mutant and its streptomycin-resistance are genetically linked and are probably caused by a single mutation, strA (LD1). We also isolated a spontaneous revertant, called LD1-R, which partially relieves the restriction of nonsense suppression caused by the strA (LD1) mutation. LD1-R is derived by an additional mutation (revA) which is closely linked to strA (LD1). We further demonstrate that the weak suppression of a lacZ_UGA mutation in a suppressor-free strain, which probably takes place by normal tRNA^Trp, can be detected by the use of the chromagenic substance x-gal (5-Bromo-4-chloro-3-indolyl--d-Galactopyranoside).     

Umbilical transposition in TRAM flap: a simple horizontal translocation

A simple method of umbilical repositioning by incising the anterior rectus sheath and rectus abdominis muscle is reported for cases of unilateral abdominal wall plication during the TRAM flap operation. This method keeps the umbilicus stable and nonstenotic, and it avoids hypertrophic scars, which result from other techniques such as direct suturing of the stalk to the skin. Although this method might weaken contralateral muscle activity, the patients we operated on maintained their ability to perform sit-ups, and no periumbilical weakening was noticed.     

Two novel biological active modified peptides from the cyanobacterium Microcystis sp.

Two novel biologically active short peptides, aeruginosins KY642, KY608 and the known aeruginosin 98A were isolated from the cyanobacterium Microcystis sp. strain (IL-323), which was collected from a water reservoir near Kfar-Yehoshua, Valley of Armageddon, Israel. Aeruginosins KY642, KY608 and 98A are linear modified peptides containing four building blocks, one of which is the modified amino acid, Choi (2-carboxy-6-hydroxyoctahydroindole). Aeruginosin KY642 and aeruginosin KY608 inhibit the activity of the photolytic enzyme trypsin with IC₅₀ of 1.4 and 1.7 μg/mL, respectively.     

Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.     

Oxygen availability as a possible limiting factor in LDL oxidation

Kinetic studies of copper-induced LDL peroxidation commonly assume that the availability of molecular oxygen in the reaction media is not a limiting factor. The present study reveals that this assumption is valid only at low LDL concentrations. At high LDL concentrations, accumulation of oxidation products, as monitored spectroscopically under conditions of various oxygen concentrations in the medium, comes to a halt when the oxygen concentration in the solution, as measured by an oxygen electrode, decreases to near zero levels. Bubbling of the oxygen into the solution results in resumption of peroxidation. These results are important with respect to the ex vivo assaying of lipoprotein peroxidation because many previous studies have been conducted with LDL concentrations that corresponded to polyunsaturated fatty acid concentrations in access of the concentration of molecular oxygen. The possible pathophysiological significance of the results of this study has yet to be evaluated.