Retinotopic map refinement requires spontaneous retinal waves during a brief critical period of development

During retinocollicular map development, spontaneous waves of action potentials spread across the retina, correlating activity among neighboring retinal ganglion cells (RGCs). To address the role of retinal waves in topographic map development, we examined wave dynamics and retinocollicular projections in mice lacking the beta2 subunit of the nicotinic acetylcholine receptor. beta2(-/-) mice lack waves during the first postnatal week, but RGCs have high levels of uncorrelated firing. By P8, the wild-type retinocollicular projection remodels into a refined map characterized by axons of neighboring RGCs forming focal termination zones (TZs) of overlapping arbors. In contrast, in P8 beta2(-/-) mice, neighboring RGC axons form large TZs characterized by broadly distributed arbors. At P8, glutamatergic retinal waves appear in beta2(-/-) mice, and later, visually patterned activity appears, but the diffuse TZs fail to remodel. Thus, spontaneous retinal waves that correlate RGC activity are required for retinotopic map remodeling during a brief early critical period.     

The precursor of a psychrophilic alpha-amylase: structural characterization and insights into cold adaptation

The alpha-amylase precursor from the bacterium Pseudoalteromonas haloplanktis possesses a propeptide at the C-terminus possibly responsible for outer membrane translocation. Unlike the predicted beta-barrel of autotransporters, this C-terminal propeptide displays a noticeable alpha-helix content. It is connected to the enzyme by a disordered linker and has no significant interaction with the catalytic domain. The microcalorimetric pattern of the precursor also demonstrates that the stability of protein domains may evolve differently.     

Ovarian follicular activity and hormonal profile during estrous cycle in cows: the development of 2 versus 3 waves

Most estrous cycles in cows consist of 2 or 3 waves of follicular activity. Waves of ovarian follicular development comprise the growth of dominant follicles some of which become ovulatory and the others are anovulatory. Ovarian follicular activity in cows during estrous cycle was studied with a special reference to follicular waves and the circulating concentrations of estradiol and progesterone. Transrectal ultrasound examination was carried out during 14 interovulatory intervals in 7 cows. Ovarian follicular activity was recorded together with assessment of serum estradiol and progesterone concentrations. Three-wave versus two-wave interovulatory intervals was observed in 71.4% of cows. The 3-wave interovulatory intervals differed from 2-wave intervals in: 1) earlier emergence of the dominant follicles, 2) longer in length, and 3) shorter interval from emergence to ovulation. There was a progressive increase in follicular size and estradiol production during growth phase of each wave. A drop in estradiol concentration was observed during the static phase of dominant anovulatory follicles. The size of the ovulatory follicle was always greater and produced higher estradiol compared with the anovulatory follicle. In conclusion, there was a predominance of 3-wave follicular activity that was associated with an increase in length of interovulatory intervals. A dominant anovulatory follicle during its static phase may initiate the emergence of a subsequent wave. Follicular size and estradiol concentration may have an important role in controlling follicular development and in determining whether an estrous cycle will have 2 or 3-waves.     

Effects of hydrogen bonding within a damaged base pair on the activity of wild type and DNA-intercalating mutants of human alkyladenine DNA glycosylase

Human alkyladenine DNA glycosylase "flips" damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted into the DNA helix in place of the damaged base and may assist in nucleotide flipping by "pushing" it. Mutating this DNA-intercalating Tyr to Ser reduces the DNA binding and base excision activities of alkyladenine DNA glycosylase to undetectable levels demonstrating that Tyr-162 is critical for both activities. Mutation of Tyr-162 to Phe reduces the single turnover excision rate of hypoxanthine by a factor of 4 when paired with thymine. Interestingly, when the base pairing partner for hypoxanthine is changed to difluorotoluene, which cannot hydrogen bond to hypoxanthine, single turnover excision rates increase by a factor of 2 for the wild type enzyme and about 3 to 4 for the Phe mutant. In assays with DNA substrates containing 1,N(6)-ethenoadenine, which does not form hydrogen bonds with either thymine or difluorotoluene, base excision rates for both the wild type and Phe mutant were unaffected. These results are consistent with a role for Tyr-162 in pushing the damaged base to assist in nucleotide flipping and indicate that a nucleotide flipping step may be rate-limiting for excision of hypoxanthine.     

Nitrogen metabolism and remobilization during senescence

Senescence is a highly organized and well-regulated process. As much as 75% of total cellular nitrogen may be located in mesophyll chloroplasts of C(3)-plants. Proteolysis of chloroplast proteins begins in an early phase of senescence and the liberated amino acids can be exported to growing parts of the plant (e.g. maturing fruits). Rubisco and other stromal enzymes can be degraded in isolated chloroplasts, implying the involvement of plastidial peptide hydrolases. Whether or not ATP is required and if stromal proteins are modified (e.g. by reactive oxygen species) prior to their degradation are questions still under debate. Several proteins, in particular cysteine proteases, have been demonstrated to be specifically expressed during senescence. Their contribution to the general degradation of chloroplast proteins is unclear. The accumulation in intact cells of peptide fragments and inhibitor studies suggest that multiple degradation pathways may exist for stromal proteins and that vacuolar endopeptidases might also be involved under certain conditions. The breakdown of chlorophyll-binding proteins associated with the thylakoid membrane is less well investigated. The degradation of these proteins requires the simultaneous catabolism of chlorophylls. The breakdown of chlorophylls has been elucidated during the last decade. Interestingly, nitrogen present in chlorophyll is not exported from senescencing leaves, but remains within the cells in the form of linear tetrapyrrolic catabolites that accumulate in the vacuole. The degradation pathways for chlorophylls and chloroplast proteins are partially interconnected.     

Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase

α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.Psychrophilic enzymes possess specific properties, such as high activity at low temperatures and weak thermal stability, which promise to allow the use of these enzymes as industrial biocatalysts, as biotechnological tools, or for fundamental research (6, 8, 11). For instance, substantial energy savings can be obtained if heating is not required during large-scale processes which take advantage of the efficient catalytic capacity of cold-adapted enzymes in the range 0 to 20°C. The pronounced heat lability of psychrophilic enzymes also allows their selective inactivation in a complex mixture, as illustrated by an antarctic bacterial alkaline phosphatase which is available for molecular biology research (7). Finally, psychrophilic enzymes represent the lower natural limit of protein stability (3) and are useful tools for studies in the field of protein folding.Large-scale fermentation of psychrophilic microorganisms suffers from two main drawbacks, however: the low production levels of wild strains and the prohibitive cost of growing wild strains at low temperatures. A possible alternative is to overexpress the gene coding for a psychrophilic protein in a mesophilic host for which efficient expression systems have been designed. In this context, two crucial questions remain to be solved: (i) what is the folding state of an enzyme normally synthesized at 0°C when it is expressed by the mesophilic genetic machinery at higher temperatures, and (ii) is there a temperature at which a compromise can be reached between the stability of the psychrophilic enzyme and the mesophilic growth rate? To address these questions, the heat-labile α-amylase from the antarctic psychrophile Alteromonas haloplanktis (2, 4) was expressed in Escherichia coli at various temperatures.

Construction of the expression vector and α-amylase production.

The α-amylase gene (2) was cloned downstream from the lacZ promoter in pUC12 by ligating the SmaI site of the polylinker to the HpaI site located 60 nucleotides upstream from the formylmethionine codon. This construction is devoid of the C-terminal peptide cleaved by the wild strain following α-amylase secretion. The recombinant enzyme was expressed in E. coli RR1 with the constitutive assistance of lacZ (without IPTG [isopropyl-β-d-thiogalactopyranoside] induction) in a medium containing 16 g of bactotryptone, 16 g of yeast extract, 5 g of NaCl, 2.5 g of K₂HPO₄, 0.1 μM CaCl₂, and 100 mg of ampicillin per liter. The effect of the culture temperature on α-amylase production by E. coli is illustrated in Fig. ​Fig.1.1. Within the range of temperatures used, maximal enzyme production was reached below 18°C, whereas higher temperatures induced a gradual decrease of α-amylase activity in cultures. Three independent cultures were pooled for the purification of the recombinant enzymes produced at 18 and 25°C. Open in a separate windowFIG. 1Temperature dependence of α-amylase production by E. coli. Results are expressed as percent mean maximal activity recorded at 18°C.

α-Amylase purification.

The gram-negative A. haloplanktis was cultivated at 4°C, and α-amylase was purified from the culture supernatants by ion-exchange chromatography on DEAE-agarose followed by gel filtration on Sephadex G-100 and Ultrogel AcA54 as previously described (2, 4). The recombinant α-amylases were purified by the protocol developed for the wild-type enzyme except that concentration by ammonium sulfate precipitation at 70% saturation was required before the first chromatographic step. Recombinant enzyme production at 18 and 25°C ranged between 60 and 100 mg/liter of culture, which corresponds to a 10-fold improvement over production by the wild strain.

Characterization of the recombinant α-amylases.

N- and C-terminal amino acid sequences (determined on an Applied Biosystems Procise analyzer and by carboxypeptidase Y digestion, respectively) of α-amylase produced at 18 and 25°C indicated that the signal peptide is correctly cleaved in E. coli and that no additional posttranslational cleavage occurred. The isoelectric point (5.5) and the molecular mass (49,340 Da as determined from the sequence and 49,342 ± 8 Da as determined from electrospray mass spectroscopy measurements) were identical to the values recorded for the wild-type enzyme. Dynamic light scattering (DynaPro-801; DLS Instruments) also showed that the purified recombinant enzymes are homogeneous, without any evidence of aggregated forms.

Comparison of the wild-type and recombinant α-amylases.

Several properties of the wild-type enzyme produced at 4°C and the recombinant α-amylase expressed in E. coli at 18°C were compared (Table ​(Table1).1).

TABLE 1

Kinetic parameters, dissociation constants, and free thiol groups for the wild-type and recombinant α-amylases

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.     

Glucosylated N-acetyllactosamine O-antigen chain in the lipopolysaccharide from Helicobacter pylori strain UA861

The O-antigen chain from the lipopolysaccharide of Helicobacter pylori strain UA861 was determined to be composed of an elongated type 2 N - acetyllactosamine backbone, -[-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1- ]n-->, with approximately half of the GlcNAc units carrying a terminal alpha-d-Glc residue at the O -6 position. The O-chain of H.pylori UA861 was terminated by a N -acetyllactosamine [beta-D-Gal-(1-->4)-beta-D- GlcNAc] (LacNAc) epitope and did not express terminal Lewis X or Lewis Y blood-group determinants as previously found in other H.pylori strains. The absence of terminal Lewis X and Lewis Y blood-group epitopes and the replacement of Fuc by Glc as a side chain in the O- chain of H.pylori UA861 represents yet another type of lipopolysaccharide structure from H.pylori species. These structural differences in H.pylori lipopolysaccharide molecules carry implications with regard to possible different pathogenic events between strains and respective hosts.      

Nutrient accumulation and translocation in maturing wheat plants grown on waterlogged soil

Wheat plants (Triticum aestivum L., cv. Arina) growing in large pots (perforated at the bottom for controls, intact for flooding) were embedded in the field in spring. Waterlogging was initiated at anthesis and was maintained throughout the maturation period. Grain yield as well as potassium, phosphorus and magnesium contents in the shoot were decreased on flooded soil, while manganese and iron contents increased considerably. Total calcium and zinc contents per shoot remained comparable to those in controls. The reduction of potassium, phosphorus and magnesium contents by waterlogging was greatest in the grains, while manganese and iron accumulated mostly in the vegetative parts and the glumes. Zinc contents were also lowered in the grains during waterlogging due to an inhibited redistribution from the vegetative parts to the grains. Our results indicate that flooding caused not only an accumulation of manganese and iron in the shoot, but also affected the redistribution of macro- and micronutrients to the maturing gains.