A Comparison of Pigment-Protein Complexes among Normal, Chlorophyll-Deficient and Senescent Soybean Genotypes

Pigment-protein complexes were isolated from chloroplasts of normal green and several types of chlorophyll-deficient soybeans. The complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and comparisons were made between normal and chlorophyll-deficient genotypes of the relative amounts of chlorophyll associated with Photosystem I (PSI), Photosystem II (PSII), light-harvesting, and free pigment complexes.

Chlorophyll-deficient genotypes, compared to normal green genotypes, have fewer light-harvesting complexes and a higher ratio of PSII to PSI complexes. Chlorophyll associated with PSII in yellow genotypes is in relatively higher amounts in spite of the fact that these genotypes have much less grana stacking than normal green genotypes. Although PSII activity has been associated with appressed regions of grana in normal plants, our work shows that the association does not always hold true.

     

Rational design of a receptor super-antagonist of human interleukin-6.

Interleukin-6 (IL-6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post-menopausal osteoporosis. IL-6, a four-helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low-affinity IL-6 receptor (IL-6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL-6R alpha. These mutants behave as full and selective IL-6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL-6 super-antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL-6R alpha. The IL-6 super-antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild-type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL-6 suggests that it could be possible to design super-antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.     

Liver-specific alpha 2 interferon gene expression results in protection from induced hepatitis

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-alpha). However, systemic delivery of r-hIFN-alpha is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-alpha antiviral efficacy, we have explored the therapeutic potential of murine IFN-alpha2 (mIFNalpha2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-alpha2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-alpha2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-alpha2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-alpha2.     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

The endangered white-clawed crayfish <Emphasis Type="Italic">Austropotamobius pallipes</Emphasis> (Decapoda,Astacidae) east and west of the Maritime Alps: a result of human translocation?

The genetic variability among Italian populations of the white-clawed crayfish (Austropotamobius pallipes) was examined to determine their phylogeography and to assess their conservation status as a management unit. A fragment of the mitochondrial DNA COI gene of 107 specimens from ten populations was sequenced, and the phylogenetic relationships were established. Two out of three haplotypes sampled in two French populations from the Rhône basin were shared with Italian populations. Despite a moderate level of genetic variability within the Italian populations of A. pallipes, no genetic structure was revealed. It has been suggested that there have been translocation events throughout the Alpine barrier between the North-Western Italian basins and the Rhône basin. Genetic exchangeability of the French and Italian populations was demonstrated in this study, and a shift of conservation efforts towards the native, congeneric Austropotamobius italicus is recommended.     

Combining geometric morphometrics with molecular genetics to investigate a putative hybrid complex: a case study with barbels Barbus spp. (Teleostei: Cyprinidae)

This integrative study examined the morphological and genetic affinities of three endemic barbel species from Italy (brook barbel Barbus caninus, Italian barbel Barbus plebejus and horse barbel Barbus tyberinus) and of putative hybrid specimens to their species of origin. Two of the species frequently occur together with the non‐native barbel Barbus barbus. DNA barcoding indicates that mitochondrial (mt) haplotypes often do not match the species expected from morphology. Linear distance measurements and meristics are not informative for discrimination of the species and putative hybrids, but a discriminant analysis of principal components (DAPC) of geometric landmark data produces reassignments largely in congruence with mt and nuclear genetic data. Cyto‐nuclear conflicts confirm the presence of hybridization in B. plebejus and B. tyberinus and identify additional introgressed specimens. A comparison between mixed genotypes and their morphology‐based assignment reveals no predictable pattern. The finding that most individuals of the morphologically similar B. plebejus and B. tyberinus have very high assignment probabilities to their respective species suggests that the presented approach may serve as a valuable tool to distinguish morphologically very similar taxa.     

Estimates of DNA and protein sequence divergence: an examination of some assumptions

Some of the assumptions underlying estimates of DNA and protein sequence divergence are examined. A solution for the variance of these estimates that allows for different mutation rates and different population sizes in each species and for an arbitrary structure in the initial population is obtained. It is shown that these conditions do not strongly affect estimates of divergence. In general, they cause the variance of divergence to be smaller than a binomial variance. Thus, the binomial variance that is usually assumed for these estimates is safely conservative. It is shown that variability in the mutation rate among sites can have an effect as large as or larger than variability in the mutation rate among bases. Variability in the mutation rate among bases and among sites causes the number of substitutions between two sequences to be underestimated. Protein and DNA sequences from several species are collected to estimate the variability in mutation rates among sites. When many homologous sequences are known, standard methods to estimate this variability can be used. The estimates of this variability show that this factor is important when considering the spectrum of spontaneous mutations and is strongly reflected in the divergence of sequences. Smaller variability is found for the third position of codons than for the first and second codon positions. This may be because of less selective constraints on this position or because the third position has been saturated with mutations for the sequences examined.      

Adeno-cosmid cloning vectors for regulated gene expression

Background

Adenovectors are widely used for efficient delivery of genes into a variety of cell types and organisms. However, the construction of the desired vector/genes combination, especially if it involves the cloning of several gene cassettes, can be laborious due to the large size of these vectors. New methods are needed to simplify the construction of complex combinations of gene cassettes into adenovectors.

Methods

Using simple cloning techniques and exploiting the λ‐phage packaging system, we devised efficient methods for the ‘selection’ of the desired vector constructs. Thus we generated a series of cosmids containing the adeno helper dependent (HD) backbone in which we inserted cis‐ and trans‐acting tetracycline (tet) elements for the regulation of any gene of interest. One of these cosmids has been used to produce an HD adenovirus carrying a tetracycline‐regulated gene expressing β‐galactosidase.

Results

We have demonstrated that the adeno‐cosmid system allows rapid and efficient cloning of genes of interest in helper dependent vectors, and described a prototype ‘ready‐to‐use’ vector in which any gene of interest can be easily expressed under the control of the tet system. The HD viruses produced with this novel methodology can be grown at high titers, can be easily separated from the helper adenovirus, and allow delivery and regulated gene expression in a variety of tissues.

Conclusions

Exploiting the λ‐packaging system, complex adeno constructs can be generated with a simple and reproducible protocol, which allows selection of the desired size construct, counterselecting for the frequently observed intramolecular recombinations and deletions. Copyright © 2002 John Wiley & Sons, Ltd.