Crystallization and preliminary X-ray analysis of gamma-aminobutyric acid transaminase

gamma-Aminobutyric acid transaminase from pig liver, an alpha 2 dimeric enzyme of Mr 110,100, has been crystallized by the vapour diffusion method with polyethylene glycol as precipitant. The crystals are monoclinic, space group P2(1), unit cell dimensions a = 82.1 A, b = 230.0 A, c = 70.3 A, beta = 123.9 degrees and diffract to 2.5 A resolution. There are two dimers per asymmetric unit.     

Evidence for the inheritance of silver-stained nucleolus organizer regions

Summary The inheritance of nucleolus organizer regions (NORs) was investigated by examining the degree of silver-staining in individual acrocentric chromosomes in two successive generations. The study was undertaken in six Down's syndrome children and their respective parents. Quinacrine fluorescent polymorphisms were used to identify individual acrocentrics and to determine which of the child's acrocentrics were informative as to parental homologue of origin. Of the 66 acrocentrics in the six children, 31 were informative. The correlation between the degree of silver-staining in the child's chromosomes and the respective parental chromosomes of origin was highly significant (P<0.001), with a correlation coefficient of 0.90. The results suggest that the degree of Ag-AS staining is characteristic for a particular chromosome and that this characteristic is an inherited property.     

Myoglobin with chlorophyllous chromophores: influence on protein stability

The stabilities of myoglobin, apo-myoglobin, and of two myoglobins with chlorophyllous chromophores (Zn-pheophorbide a and Zn-bacteriopheophorbide a), have been studied by thermal and chemical denaturation. With guanidinium chloride, the stability order is myoglobin>Zn-pheophorbide-myoglobin>Zn-bacteriopheophorbide-myoglobin approximately apo-myoglobin. The thermal behavior is more complex. The transition temperature of thermal unfolding of the apoprotein (62.4 degrees C) is increased by Zn-pheophorbide a (83.9 degrees C) and Zn-bacteriopheophorbide a (82.6 degrees C) to a similar degree as by the native chromophore, heme (83.5 degrees C). The recovery with Zn-pheophorbide (92-98%) is even higher than with heme (74-76%), while with Zn-bacteriopheophorbide (40%) it is as low as with the apoprotein (42%). Recovery also depends on the rates of heating, and in particular the time spent at high temperatures. It is concluded that irreversibility of unfolding is related to loss of the chromophores, which are required for proper re-folding.     

In vitro introduction of healthy and virus-infected genotypes of native Croatian grapevine cultivars

We evaluated the response of eight economically important Croatian grapevine cultivars and studied the impact of their sanitary status on in vitro introduction, by comparing the response of healthy and virus-infected genotypes of one cultivar. Nodal explant survival on three media, M1 (half-strength MS), M2 (full-strength MS) or M3 (full-strength MS with 4.4 µM L^?1 benzylaminopurine) was measured after 2 weeks and regrowth after 8 weeks. After 8 weeks, average shoot length and node number were significantly higher on M2 compared to M1 and M3. M3 induced significantly shorter average internode length, compared to M1 and M2. Survival of one healthy and of five cultivar Plavac mali genotypes infected with GFLV, GLRaV-1, GLRaV-3, GLRaV-3+GVA and GLRaV-1+GLRaV-3 was 97.5 and 82.8–87.5%, respectively. Regrowth of the healthy genotype reached 95.5%, but dropped to 5.5–31.4% in infected ones. The healthy genotype showed significantly higher shoot length (6.3 cm) and node number (7.3) compared to infected genotypes, with shoot length between 1.2–2.6 cm and node number between 1.2–3.0. By contrast, internode length was not significantly different between the healthy and the infected genotypes. The present work represents the first successful in vitro introduction for three of the eight native Croatian cultivars studied.     

Revision of Mecinus heydenii species complex (Curculionidae): integrative taxonomy reveals multiple species exhibiting host specialization

A combined taxonomic, morphological, molecular and biological study revealed that the species presently named Mecinus heydenii is actually composed of five different species: M. heydenii Wencker, 1866; M. raphaelis Baviera & Caldara sp. n., M. laeviceps Tournier, 1873; M. peterharrisi To?evski & Caldara sp. n. and M. bulgaricus Angelov, 1971. These species can be distinguished from each other by a few subtle characteristics, mainly in the shape of the rostrum and body of the penis, and the colour of the integument. The first four species live on different species of Linaria plants, respectively, L. vulgaris (L.) P. Mill., L. purpurea (L.) P. Mill. L. genistifolia (L.) P. Mill. and L. dalmatica (L.) P. Mill., whereas the host plant of M. bulgaricus is still unknown. An analysis of mtCOII gene sequence data revealed high genetic divergence among these species, with uncorrected pairwise distances of 9% between M. heydenii and M. raphaelis, 11.5% between M. laeviceps, M. heydenii and M. raphaelis, while M. laeviceps and M. peterharrisi are approximately 6.3% divergent from each other. Mecinus bulgaricus exhibits even greater divergence from all these species and is more closely related to M. dorsalis Aubé, 1850. Sampled populations of M. laeviceps form three geographical subspecies: M. laeviceps laeviceps, M. laeviceps meridionalis To?evski & Jovi? and M. laeviceps corifoliae To?evski & Jovi?. These subspecies show clear genetic clustering with uncorrected mtDNA COII divergences of approximately 1.4% from each other.     

Monitoring the presence of genetically modified food on the market of the Republic of Croatia

From the beginning of the human race people have been applying different methods to change the genetic material of either plants or animals in order to increase their yield as well as to improve the quality and quantity of food. Genetically modified organism (GMO) means an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination. Analysing the presence of GMO in food is done by detecting the presence of either specific DNA sequences inserted in the genome of transgenic organism, or detecting proteins as a result of the expression of the inserted DNA. In this work food testing for the presence of genetically modified organisms was conducted during the period from 2004 to 2007 in the GMO laboratory of the Croatian National Institute of Public Health. According to the regulations, among the samples in which the presence of GMO was detected, all those which had more than 0.9% of GMO content were either rejected from the border or removed from the market, because such GM food has to be appropriately labelled. Among the food samples which were analysed in 2004: 127 (2.37%) of a total of 1226 samples contained more than 0.9% of GMOs; in 2005 there was only one in 512 (0.20%) samples in total; in 2006 there were 4 out of 404 samples (0.99%), and in 2007: 7 of a total of 655 samples (1.07%) had GMO content above the allowed threshold of 0.9%.     

Molecular Characterization of a Novel Bacteriocin and an Unusually Large Aggregation Factor of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis> BGSJ2-8, a Natural Isolate from Homemade Cheese

Screening the collection of natural isolates from semi-hard homemade cheese resulted in isolation and characterization of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8. The strain BGSJ2-8 harbors several important phenotypes, such as bacteriocin production, aggregation phenomenon, and production of proteinase. Bacteriocin SJ was purified by three-step chromatography. Mass spectrometry established molecular mass of the active peptide at 5372 Da. The auto-aggregation phenotype of wild-type (WT) strain was mediated by secreted aggregation-promoting factor (protein of molecular mass > 200 kDa), probably acting in cooperation with other cell surface protein(s). Comparative study of WT and its spontaneous nonaggregating derivative revealed that aggregation factor was responsible for the observed differences in the bacteriocin and proteinase activities. Bacteriocin SJ activity and resistance to different stresses were higher in the presence of aggregating factor. In contrast, proteinase activity was stronger in the nonaggregating derivative.     

Changes in L-phenylalanine ammonia-lyase activity and isoflavone phytoalexins accumulation in soybean seedlings infected with Sclerotinia sclerotiorum

Soybean [Glycine max (L.) Merr.] cultivars (Meli, Alisa, Sava and 1511/99) were grown up to V1 phase (first trifoliate and one node above unifoliate) and then inoculated with Sclerotinia sclerotiorum (Lib.) de Bary under controlled conditions. Changes in L-phenylalanine ammonia-lyase (PAL) activity and isoflavone phytoalexins were recorded 12, 24, 48 and 72 h after the inoculation. Results showed an increase in PAL activity in all four examined soybean cultivars 48 h after the inoculation, being the highest in Alisa (2-fold higher). Different contents of total daidzein, genistein, glycitein and coumestrol were detected in all samples. Alisa and Sava increased their total isoflavone content (33.9% and 6.2% higher than control, respectively) as well as 1511/99, although 48 h after the inoculation its content decreased significantly. Meli exhibited the highest rate of coumestrol biosynthesis (72 h after the inoculation) and PAL activity (48 h after the inoculation). All investigated cultivars are invariably susceptible to this pathogen. Recorded changes could point to possible differences in mechanisms of tolerance among them.