Structural basis of the polymorphism of human complement components C4A and C4B: gene size, reactivity and antigenicity.

The human complement components C4A and C4B are highly homologous proteins, but they show markedly different, class-specific, chemical reactivities. They also differ serologically in that C4A generally expresses the Rodgers (Rg) blood group antigens while C4B generally expresses the Chido (Ch) blood group antigens. C4A 1 and C4B 5 are exceptional variants which possess their class-specific chemical reactivities, but express essentially the reversed antigenicities. The genes encoding the typical Rg-positive C4A 3a and Ch-positive C4B 3 allotypes and the interesting variants C4A 1 and C4B 5 have been cloned. Characterization of the cloned DNA has revealed that the genes encoding the A 3a, A 1 and B 3 allotypes are 22 kb long, but that encoding B 5 is only 16 kb long. Comparison of derived amino acid sequences of the polymorphic C4d fragment has shown that C4A and C4B can be defined by only four isotypic amino acid differences at position 1101-1106. Over this region C4A has the sequence PCPVLD while C4B has the sequence LSPVIH, and this presumably is the cause of their different chemical reactivities. Moreover, the probable locations of the two Rg and the six Ch antigenic determinants have been deduced. Our structural data on the C4A and C4B polymorphism pattern suggests a gene conversion-like mechanism is operating in mixing the generally discrete serological phenotypes between C4A and C4B.     

Rapid screening for metabolite overproduction in fermentor broths, using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks

Binary mixtures of model systems consisting of the antibiotic ampicillin with either Escherichia coli or Staphylococcus auresu were subjected to pyrolysis mass spectrometry (PyMS). To deconvolute the pyrolysis mass spectra, so as to obtain quantitative information on the concentration of ampicilin in the mixtures, partial least squares regression (PLS), principal components regression (PCR), and fully interconnected feedforward artificial neural networks (ANNs) were studied. In the latter case, the weights were modified using the standard backpropagation algorithm, and the nodes used a sigmoidal squsahing funciton. It was found that each of the methods could be used to provide calibration models which gave excellent predictions for the concentrations of ampicillin in samples on which they had not been trained. Furthermore, ANNs trained to predict the amount of ampicilin in E. coli were able to generalise so as to predict the concentration of ampicillin in a S. aureus background, illustrating the robustness of ANNs to rather substantial variations in the biological background. The PyMS of the complex mixture of ampicilin in bacteria could not be expressed simply in terms of additive combinations of the spectra describing the pure components of the mixtures and their relative concentrations. Intermolecular reactions took place in the pyrolysate, leading to a lack of superposition of the spectral components and to a dependence of the normalized mass spectrum on sample size. Samples from fermentations of a single organism in a complex production medium were also analyzed quantitatively for a drug of commercial interest. The drug could also be quantified in a variety of mutant-producing strains cultivated in the same medium. The combination of PyMS and ANNs constitutes a novel, rapid, and convenient method for exploitation in strain improvement screening programs. (c) 1994 John Wiley & Sons, Inc.     

Mutational analysis of U1 function in Schizosaccharomyces pombe: pre-mRNAs differ in the extent and nature of their requirements for this snRNA in vivo.

The U1 snRNP is known to play a critical role in spliceosome assembly, at least in part through base pairing of its RNA moiety to the substrate, but many details remain to be elucidated. To further dissect U1 snRNA function, we have analyzed 14 single point mutations in the six nucleotides complementary to the 5' splice site for their effects on growth and splicing in the fission yeast Schizosaccharomyces pombe. Three of the four alleles previously found to support growth of Saccharomyces cerevisiae are lethal in S. pombe, implying a more critical role for the 5' end of U1 in fission yeast. Furthermore, a comparison of phenotypes for individual nucleotide substitutions suggests that the two yeasts use different strategies to modulate the extent of pairing between U1 and the 5' splice site. The importance of U1 function in S. pombe is further underscored by the lethality of several single point mutants not examined previously in S. cerevisiae. In total, only three alleles complement the U1 gene disruption, and these strains are temperature-sensitive for growth. Each viable mutant was tested for impaired splicing of three different S. pombe introns. Among these, only the second intron of the cdc2 gene (cdc2-I2) showed dramatic accumulation of linear precursor. Notably, cdc2-I2 is spliced inefficiently even in cells containing wild-type U1, at least in part due to the presence of a stable hairpin encompassing its 5' splice site. Although point mutations at the 5' end of U1 have no discernible effect on splicing of pre-U6, significant accumulation of unspliced RNA is observed in a metabolic depletion experiment. Taken together, these observations indicate that the repertoire of U1 activities is used to varying extents for splicing of different pre-mRNAs in fission yeast.     

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.     

A review of UVA-mediated photosensitivity disorders

A number of skin conditions are characterised by photosensitivity to UVA. Some of these are exclusively UVA-mediated conditions, while others include UVA in the action spectrum which also include UVB and/or visible light. This review aims to describe this diverse range of conditions for non-dermatologist scientists with an interest in this topic. As such, clinical details, including treatments, are brief and succinct. Recent advances in understanding the pathogenesis of these conditions is highlighted.     

Life history variation within a parthenogenetic population of Daphnia parvula (Crustacea: Cladocera)

Summary Evidence for genetically determined life history variability within a population or a species is rare. In this three year experimental examination of a parthenogenetically reproducing population of the planktonic crustacean Daphnia parvula, we found evidence for a succession of clones or groups of clones that exhibited distinctive body size and reproductive differences that were maintained after numerous generations under standardized conditions in the laboratory. The D. parvula population reached maximum density in the fall and maintained relatively high densities through the winter and spring. Isolates from this fall-winter-spring period all had a larger body size at death and higher fecundity when compared with summer isolates under natural food and temperature conditions. These differences could not be accounted for by differences in temperature and food abundance among the seasons. An additional difference in these experiments was a shift in reproductive effort by the summer isolate which produced a higher proportion of its offspring in the first two broods. The shift in life history characteristics and a summer decline of the Daphnia parvula population was correlated with both an increase in inedible and perhaps toxic blue-green algae and an increase in a dipteran predator Chaoborus. Comparison of the survivorship curves for all of the seasonal life history experiments indicated that D. parvula survivorship was not lower during the summer discounting a toxic effect from blue-green algae. Positive population growth on natural food in the laboratory at this time indicated food was not limiting and that predation was the probable cause of the population decline.Laboratory life history experiments under standardized food and temperature conditions were run with D. parvula isolates from the spring and summer plankton. Genetically based differences as determined in these experiments were smaller body size, lower fecundity, smaller brood size, and shorter life span for the summer animals relative to spring animals. Thirty seven percent of the summer animals also reproduced at an earlier age under standardized conditions. The shift in reproductive effort to earlier broods by summer animals rnder natural conditions appeared to be a phenotypic response as the summer isolate did not produce a higher proportion of its offspring in early broods under standardized conditions.When estimates of predatory mortality were added to the life tables of the standardized experiments, the earlier reproduction of some of the summer animals allowed a population increase under a regime of intense predation. Life tables for the spring animals predicted a population decline under these circumstances. Predictable seasonal changes in biotic factors such as predation suggest a mechanism whereby diverse life history patterns with corresponding differences in r may be maintained within a population.     

Alpha-actinin localization in the cleavage furrow during cytokinesis

We used antibodies against alpha-actinin and myosin labeled directly with contrasting fluorochromes to localize these contractile proteins simultaneously in dividing chick embryo cells. During mitosis anti-alpha-actinin stains diffusely the entire cytoplasm including the mitotic spindle, while in the same cells intense antimyosin staining delineates the spindle. During cytokinesis both antibodies stain the cleavage furrow intensely, and until the midbody forms the two staining patterns in the same cell are identical at the resolution of the light microscope. Thereafter the anti-alpha-actinin staining of the furrow remains strong, but the antimyosin staining diminishes. These observations suggest that alpha-actinin participates along with actin and myosin in the membrane movements associated with cytokinesis.     

Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery

BACKGROUND: The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs). METHODS: A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery. RESULTS: Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation. CONCLUSION: These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans.