Comparison of the structure of the immunoglobulins from horse serum

A study of the chemical structure of the horse immunoglobulins IgG and IgA(T) has shown that the amino acid contents of the peptide chains are very similar. These globulins differ most markedly in the products of papain digestion. IgG gives 3.5s products, whereas IgA(T) gives a 5s fraction and smaller components. This difference appears to be associated with the presence of an additional easily reducible disulphide bond in the Fd fragment of the heavy chain. There is two to three times as much carbohydrate in IgA(T) as in IgG. In both, this is in the heavy chain and in IgA(T) more than half is covalently bound to the Fd fragment. The differences in antigenic specificity between horse IgG and IgA(T) appear to be due to structural differences in the Fc fragment.     

Studies on the Endoplasmic Reticulum : IV. Its Form and Distribution during Mitosis in Cells of Onion Root Tip

Cells of onion and garlic root tips were examined under the electron and phase contrast microscopes after fixation in KMnO₄. Special attention was focused on the distribution and behavior of the endoplasmic reticulum (ER) during the several phases of mitosis. Slender profiles, recognized as sections through thin lamellar units of the ER (most prominent in KMnO₄-fixed material), are distributed more or less uniformly in the cytoplasm of interphase cells and show occasional continuity with the nuclear envelope. In late prophase the nuclear envelope breaks down and its remnants plus cytoplasmic elements of the ER, which are morphologically identical, surround the spindle in a zone from which mitochondria, etc., are excluded. During metaphase these ER elements persist and concentrate as two separate systems in the polar caps or zones of the spindle. At about this same time they begin to proliferate and to invade the ends of the spindle. The invading lamellar units form drape-like partitions between the anaphase chromosomes. In late anaphase, their advancing margins reach the middle zone of the spindle and begin to fray out. Finally, in telophase, while elements of the ER in the poles of the spindle coalesce around the chromosomes to form the new envelope, the advancing edges of those in the middle zone reticulate at the level of the equator to form a close lattice of tubular elements. Within this, which is identified as the phragmoplast, the earliest signs of the cell plate appear in the form of small vesicles. These subsequently grow and fuse to complete the separation of the two protoplasts. Other morphological units apparently participating in mitosis are described. Speculation is provided on the equal division or not of the nuclear envelope and the contribution the envelope fragments make to the ER of the new cell.     

Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.     

Relative nutritional value of ciliate protozoa and algae as food forDaphnia

The relative importance of autotrophic flagellates, desmids, cyanobacteria, and ciliates as food forDaphnia magna was examined using cohort life tables. Each cohort was fed a single food type at a given concentration, and comparisons among each type were made. Algal feeding treatments included three levels of young (7 to 14 days old)Chlamydomonas reinhardi (Chlorophyta, Chlamydomonadacae), two levels of senescent (> 14 days old)C. reinhardi, two levels ofCryptomonas sp. (Chlorophyta, Cryptomonadacae), two levels ofStaurastrum sp. (Chlorophyta, Desmidacae), four levels of young (7 to 15 days old) or senescent (> 15 days old)Microcystis aeruginosa (Cyanophyta, Chlorococcacae), and a no-food treatment. The ciliatesCyclidium sp. andParamecium caudatum were also presented at concentrations of 1 or 10² cells/ml, as well as mixtures ofC. reinhardi (10³/ml) andCyclidium (1/ml) orP. caudatum (1/ml).Daphnia growth, reproduction, and survivorship were highest whenC. reinhardi orCryptomonas were the food source, while those starved or fedM. aeruginosa had shorter survivorship and lower growth and reproduction.Daphnia grew and had high survivorship when fedP. caudatum, but even though eggs were produced, most were aborted after 2 or 3 days.Staurastrum andCyclidium produced intermediate growth and survivorship, but reproduction was seen only in the 10³ Staurastrum/ml treatment. Carbon and nitrogen content were general indicators of nutritional value. However, growth, reproduction, and survivorship were higher in some cohorts fed treatments containing relatively low levels of carbon and nitrogen. Other cohorts were short-lived and did not reproduce, despite being fed much higher levels of carbon and nitrogen. The results also suggest that green algae are nutritionally valuable forDaphnia, whereas cyanobacteria are not. As measured by life-table parameters, the nutritional value of ciliates was variable, with some being poor food sources. Thus, the potential of ciliates as a trophic link between microbial production and higher trophic levels may vary with the ciliate community structure. Our results suggest that ciliates alone were insufficient as a food source to supportDaphnia population growth.     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

Mosquitocidal toxins, genes and bacteria: the hit squad

Certain entomopathogenic species of bacilli and Clostridium produce one or more toxins that kill mosquito larvae even at concentrations in the picomolar range. Altogether, 19 distinct genes are known that encode mosquitocidal toxins, which vary in their potency, species specificity and mode of action. Unlike chemical insecticides, mosquitocidal bacilli used as larvicides are safe for animals and the environment, and do not affect non-pest insects. Mosquitocidal bacteria are effective to varying degrees against Culex, Anopheles and Aedes mosquito larvae, but their rapid sedimentation from the larval feeding zone, UV-light sensitivity and narrow host range have hampered their development. New genetic engineering approaches are being investigated that could overcome these limitations and allow stable expression of broad host range combinations of toxins in UV-resistant, buoyant recombinant bacteria, as discussed here by Alan Porter.     

Zooplankton community response to reservoir aging

Changes in zooplankton diversity and density in response to reservoir aging in Pawnee Reservoir were investigated. Zooplankton samples collected from April 1992 through April 1993, were compared to a similar study conducted after initial impoundment by Helzer (1971), in 1968–1970. Since this initial study, increases in turbidity and resulting changes in biotic interactions significantly altered the zooplankton community. A significant increase in total zooplankton density and a decrease in species richness were observed between study periods. Density increased from 24.6 to 95.4 individuals L^–1, while the number of taxa declined from fourteen to ten. During this time period, Cyclops vernalis became the dominant zooplankter in the reservoir. The density of this predatory copepod increased significantly, from 0.1 l^–1 in 1968–1970, to 44.3 l^–1 in 1992–1993, which accounted for most of the increase in total zooplankton density. Though a greater spring maximum of another dominant, Bosmina spp. was found during the 1992–1993 study period, the annual density of this cladoceran was not significantly different between study periods. Similar trends for Daphnia ambigua and D. parvula were also observed, as greater spring maxima levels were attained, however overall annual densities were not significantly different. The dominance of C. vernalis (46% of annual density) and Bosmina spp. (33%), indicate that these two zooplankters were tolerant of changes in physical conditions resulting from reservoir aging and biotic interactions that followed in the reservoir during the 22 years between study periods.