Dianthus barbatus-A New Host of Stolbur Phytoplasma in Serbia.

Sweet William (Dianthus barbatus, Caryophyllaceae) is a biennial or short-lived perennial plant native to southern Europe, from the Pyrenees to the Carpathians and the Balkans. During the summers of 2012 and 2013, phytoplasma-like symptoms were observed on D. barbatus plants on a Serbian plantation (Pancevo, 44°51'49″ N, 20°39'33″ E, 80 m ASL). Only seven symptomatic plants were observed in the summer of 2012. Disease incidence in 2013 was estimated to be less than 1% but increased during 2014 to 4%. Affected plants, showing symptoms of leaf reddening, malformation, and proliferation; flower bud deficiency; and abnormal shoot production, were tested for phytoplasmas. Samples were collected from seven symptomatic and three symptomless plants each year (20 samples), and total nucleic acid was extracted from midrib tissue using a method that includes a phytoplasma enrichment step and DNA purification by chloroform/phenol (3). Oligonucleotide primers specific to the phytoplasma 16S to 23S rRNA intergenic spacer region were used in polymerase chain reaction (PCR) assays on DNA extracted from Sweet William plants (1,3). Using phytoplasma universal primer pairs P1/P7 and P1/16S-Sr, phytoplasma-specific 1.8- and 1.5-kb amplicons were obtained from four and six symptomatic plants collected in 2012 and 2013, respectively. Nested PCR with R16F2n/R2 primers yielded ~1.2-kb amplicons from DNAs of all symptomatic plants (1). No amplicon was generated in PCRs conducted with DNA templates from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of amplified 1.2-kb fragments was performed using four endonucleases (AluI, Tru1I, HhaI, and HpaII). Comparative analysis was done using RFLP patterns of Stolbur (Stol), Aster Yellows (AY), Flavescence Doree-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas. PCR-RFLP patterns from tested samples were identical to those of the Stol reference strain, indicating that diseased Sweet William was affected by phytoplasma belonging to the 16SrXII-A (Stolbur) group. The sequence of a 1.2-kb rDNA PCR product derived from sample Tk9 (deposited under accession number KM401436 in NCBI GenBank) showed the closest identity (100%) to those of Bulgarian corn (KF907506.1), Iranian 'Bois Noir' (KJ637208.1), and two Serbian phytoplasmas (KJ174507.1 from Calendula officinalis and KF614623.1 from Paeonia tenuifolia), all belonging to the 'Candidatus Phytoplasma solani' Stolbur subgroup. Previously, Aster Yellows Phytoplasma (16SrI) had been detected in two Dianthus species: D. barbatus (Sweet William) and D. caryophyllus (carnation) (2). This is the first record of the 16SrXII-A phytoplasma subgroup being associated with yellowing and reddening of D. barbatus in Serbia. The Stolbur phytoplasma occurrence on Sweet William is significant for the management of the disease in Serbia. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) P. Northover et al. http://www.umanitoba.ca/faculties/afs/MAC_proceedings/proceedings/ 2007/Philip_Northover.pdf , 2007. (3) J. P. Prince et al. Phytopathology 83:1130, 1993.

First Report of Pseudomonas syringae pv. syringae Causing Bacterial Leaf Spots of Oil Pumpkin (Cucurbita pepo) in Serbia.

Oil pumpkin (Cucurbita pepo L.) is commonly used for oil production, mainly in central and eastern Europe (1). In Serbia, it grows only in the north (Vojvodina Province), up to 1,500 ha. In June 2008, typical bacterial spot symptoms (dark green, water-soaked, transparent and greasy spots with yellow margins) were observed for the first time, cultivated at the experimental fields near Backi Petrovac. Since then, bacterial spots were regularly observed on oil pumpkin in the beginning of the growing seasons and during rainy weather, with disease incidence ranging from 5 to 20%. Bacteria isolated from 40 diseased leaves formed white, round, convex, and mucoid colonies on nutrient sucrose agar (NSA). Eight representative strains were aerobic, gram-negative, non-spore-forming rods. All strains produced fluorescent pigment and catalase. In levan-oxidase-potato rot-arginine dihydrolase-tobacco hypersensitivity (LOPAT) tests (3), they induced a hypersensitive reaction in tobacco leaves, did not cause soft rot of potato tubers, and were positive for levan and negative for oxidase and arginine dihydrolase. According to the LOPAT profile, they were classified in the Ia subgroup of pseudomonads (3). Strains hydrolyzed aesculin, but were unable to hydrolyze starch or reduce nitrates to nitrites. Negative reactions were obtained with hydrogen sulfide and indole. Reactions were identical to those of reference strain Pseudomonas syringae pv. syringae CFBP 1582, which was included in all biochemical, physiological, and molecular tests for comparison. To identify the pathogen, PCR and DNA sequencing were employed. Fragments of 752 bp for the syrB gene and 1,040 bp for the syrD gene were amplified from all strains, using B1/B2 and SyD1/SyD2 primer sets, respectively (2). The pathogenicity was tested on seeds and seedlings of oil pumpkin cv. Olinka. Strains were grown for 48 h on nutrient broth (NB) at 28°C and bacterial suspensions of ~108 CFU ml-1 were used for inoculations. Sterile water was used as negative control. Seeds (at the BBCH-1-0 stage) allowed to imbibe water were wounded by needle, immersed in the bacterial suspensions, and maintained in humid petri dishes to allow symptom development. The cotyledons of seedlings at the BBCH-10 stage were inoculated by hypodermic needle and potted plants were maintained at 25 ± 1°C and 75% relative humidity. Symptoms, including dark green, water-soaked spots, appeared 5 to 7 days after inoculation of both seeds and seedlings. The bacterium was re-isolated from spots of all seeds and seedlings tested, fulfilling Koch's postulates (the identity of re-isolated strains was confirmed by pathogenicity, morphology, and biochemical features). No symptoms were observed on controls. 16S rDNA amplicons obtained from representative strain Tk21 and re-isolated strain Tk21R with fD1/rD1 primers (4) were sequenced and deposited in GenBank under accession nos. KF305578 and KF735064, respectively. The sequences showed 100% similarity to each other and P. syringae pv. syringae from pepper (KC816630.1) (China), Ficus carica (JQ071937) (Serbia), and culture-collection ICMP:3023 (HM190217). On the basis of the symptoms, biochemical tests, and 16S rDNA sequence homology, the pathogen was identified as P. syringae pv. syringae. To our knowledge, this is the first report of P. syringae pv. syringae causing bacterial leaf spot on oil pumpkin in Serbia. References: (1) J. Berenji et al. Oil pumpkin Cucurbita pepo. Monography. IFVC, Novi Sad, 2011. (2) K. Gasic et al. Pestic. Phytomed. 27:219, 2012. (3) R. A. Lelliott et al. J. Appl. Bact. 29:470, 1966. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

First Report of 16SrXII-A Subgroup Phytoplasma (Stolbur) Associated with Reddening of Oenothera biennis in Serbia.

Evening primrose (Oenothera biennis L.) is a biennial medicinal, edible, and ornamental plant species. It has attracted great interest for its seed oil that contains gamma linolenic acid, thus distinguishing this plant as a main commercial source of this essential fatty acid (4). This species has been grown as a permanent member of a medicinal plant collection established near Backi Petrovac (northern Serbia) for 22 years. The first disease symptoms were recognized as red spots on leaf rosette in July 2011, spreading gradually during vegetative growth and covering 1/3 to 1/2 of the leaf surface. Symptoms, observed on 16% of the plants (32 of 200) in the second half of May 2012 and on 23% (69 of 300) at the beginning of May 2013, appeared as reddening of lower leaves of flower-bearing stems. Affected plants exhibited stunted growth, while reddening spread over other leaves of flower-bearing stems. In severely affected plants, the flower-bearing stems were poorly developed, frequently forming witches' brooms. For that reason, 30 reddened and 20 symptomless leaves (2 leaves per plant) were sampled in both July 2012 and 2013 and total nucleic acids were extracted. Direct PCR assays were performed using phytoplasma universal primer pair P1/P7 (2) to amplify 1,800-bp fragments (the 16S rRNA gene, the 16S-23S intergenic spacer region, and a part of the 5' region of the 23S rRNA gene). PCR products were used in nested PCR with primers R16F2n/R2 (2) to amplify 1,200-bp fragments. The identification of phytoplasmas was done using RFLP (restriction fragments length polymorphisms) analyses of R16F2n/R2 amplicons digested with AluI, Kpn I, HpaII, TruI1, or HhaI endonucleases (Thermo Scientific, Lithuania) (2). RFLP patterns were identical to that of STOL reference strain of the 16SrXII-A subgroup, indicating that symptomatic plants were infected with phytoplasma (2). The 16S rDNA nucleotide sequence of representative strain E7 was deposited in GenBank under accession number KF850526. The BLASTn search showed 100% homology to an Iranian strain (KF263684.1) from peach and Serbian strains JQ730742.1 and JQ730750 from valerian and corn, respectively, all belonging to 'Candidatus Phytoplasma solani' (Stolbur). Sequencing data confirmed the association of Stolbur phytoplasma with affected O. biennis plants. It has already been reported that phytoplasma infection caused yellows disease of O. biennis (1). Also, the virescence of O. hookeri was associated with phytoplasma strain OAY from aster yellows (AY) group (subgroups 16SrI-B), and selected as the reference strain for the novel taxon 'Ca. P. asteris' (3). Here we provide the first report of naturally occurring Stolbur phytoplasma disease of O. biennis in Serbia. References: (1) S. F. Hwang et al. Z. Pflanzenkr. Pflanzenschutz 105:64, 1998. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (4) E. Small and P. M. Catling. Canadian Medicinal Crops. NRC Research Press, Ottawa, Ontario, Canada, 1999.

First Report of Stolbur Phytoplasma Affecting Cichorium intybus in Serbia.

Chicory (Cichorium intybus, Asteraceae) is a typical Mediterranean plant indigenous to Europe, western Asia, Egypt, and North America (3). It is commonly consumed as a fresh vegetable in salads. In rural areas of Serbia it grows as a weed in crops, but it is used in folk medicine to treat skin disorders due to its antihepatotoxic activity (3). Methanol extracts of chicory leaves showed moderate antibacterial activity against enteric bacteria (3). A phytoplasma-like disease, expressed as proliferation of chicory shoots and flowers, was observed on wild plants for the first time in Obrenovac vicinity (44°40' N, 20°20' E) in July 2012. A flattening of the stem with a large number of filamentous leaves, contortion and abnormal growth of flowers on the stem (typical fasciation symptoms) were observed. Diseased plants did not produce seeds. Total DNA was extracted from the leaf midveins of 15 symptomatic and five symptomless plants (4). PCR amplification of 1.5-kb 16S rDNA fragment was performed using DreamTaq Green master mix (Thermo Scientific, Lithuania) and phytoplasma universal primer pairs P1/16S-Sr (1). Products of nested PCR (1.2 kb) were obtained using primer pair R16F2n/R2 (1). Both amplicons were detected in all diseased samples; however, DNA from symptomless samples yielded no amplicons. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 PCR products was performed in independent reactions using four endonucleases (AluI, TruI1, HhaI and HpaII). RFLP patterns from chicory samples were compared to those of Stolbur (STOL), Aster Yellows (AY), Flavescence Dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (1). All RFLP profiles from the chicory samples were identical to STOL reference strain, indicating that diseased chicory was affected by a phytoplasma that belongs to 'Candidatus Phytoplasma solani' (16SrXII-A group). The 16S rDNA sequence of representative sample from symptomatic plant (Vp4) was deposited under accession number KF661322 in NCBI GenBank. It showed 100% identity to KF263684.1 from Iranian peach, JQ730742.1 from Serbian valerian, and JQ730750 from Serbian corn, all belonging to the 'Ca. P. solani' taxon. Puna chicory disease on C. intybus associated with a subgroup 16SrV-B of phytoplasma was detected in China (2). This is the first report of the Stolbur phytoplasma associated with fasciation of C. intybus in Serbia and worldwide. References: (1) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 56:1593, 2006. (2) Z. N. Li et al. Can. J. Plant Pathol. 34:34, 2012. (3) J. Petrovic et al. Fitoterapia 75:737, 2004. (4) J. P. Prince. Phytopathology 83:1130, 1993.

First Report on Natural Infection of Paeonia tenuifolia by 'Candidatus Phytoplasma solani' in Serbia.

Peony (Paeonia tenuifolia L.) is a herbaceous perennial plant known for its beautiful and showy flowers. In Serbia it is native to the Deliblato Sands and is used as an ornamental and medicinal plant in folk medicine. This plant species has become a rarity and for that reason peony was introduced into a botanical collection near Backi Petrovac (northern Serbia), where it has been maintained since 1988. Reddening of lower leaves observed on 10% of plants (5 of 50) in the collection at flowering in May 2012 gradually progressed throughout affected plants by the seed maturation stage. Five leaves from each of three reddened and three symptomless plants were sampled at the end of July 2012. Total nucleic acid was extracted separately from individual leaves (30 samples) using the CTAB (cetyltrimethylammonium bromide) method (2). A nested PCR assay using universal primer pairs P1/P7, followed by R16F2n/R16R2 (4), amplified 16S rDNA fragments of 1.8 and 1.2 kb, respectively. DNA from all three reddened plants (15 samples) yielded 1.2-kb amplicons after nested PCRs. Restriction fragment length polymorphism (RFLP) patterns obtained by digestion of nested products with endonucleases AluI, TruI, HpaII, or HhaI (Thermo Scientific, Lithuania) (4) were identical to those of the STOL reference strain included for comparative purposes, indicating that symptoms were consistently associated with plant infection by 'Ca. Phytoplasma solani' (Stolbur) phytoplasma. The 16S rDNA amplicons from two peony plants (1.2 kb from B15 and 1.8 from B18) were sequenced (GenBank Accession No. KC960487 and KF614623, respectively). BLAST analysis revealed a 100% identity between the sequences and GenBank sequences of Stolbur phytoplasma, subgroup 16SrXII-A phytoplasma, previously detected in maize (JQ730750) in Serbia and red clover (EU814644.1) in the Czech Republic. Phytoplasma associated diseases of other species of the genus Paeonia (P. lactiflora Pall. and P. suffruticosa Andrews) have been described elsewhere. Disease symptoms on P. lactiflora from Chile were associated with the phytoplasma that belongs to the ribosomal subgroup 16SrVII-A ('Ca. Phytoplasma fraxini') (1). Also, Stolbur phytoplasma from the 16SrXII group was detected on P. suffruticosa plants in China, manifesting yellowing symptoms (3). To our knowledge, this is the first report of naturally occurring Stolbur phytoplasma disease of P. tenuifolia L. in Serbia. References: (1) N. Arismendi et al. Bull. Insectol. 64:S95, 2011. (2) X. Daire et al. Eur. J. Plant Pathol. 103:507, 1997. (3) Y. Gao et al. J. Phytopathol. 161:197, 2013. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

The First Report of Stolbur Phytoplasma Associated with Phyllody of Calendula officinalis in Serbia.

Pot marigold (Calendula officinalis L.) is native to southern Europe. Compounds of marigold flowers exhibit anti-inflammatory, anti-tumor-promoting, and cytotoxic activities (4). In Serbia, pot marigold is cultivated as an important medicinal and ornamental plant. Typical phyllody, virescence, proliferation of axillary buds, and witches' broom symptoms were sporadically observed in 2011 in Pancevo plantation, Serbia (44°51'49″ N, 20°39'33″ E, 80 m above sea level). Until 2013, the number of uniformly distributed affected pot marigold plants reached 20% in the field. Due to the lack of seed production, profitability of the cultivation was seriously affected. Leaf samples from 10 symptomatic and 4 symptomless marigold plants were collected and total nucleic acid was extracted from midrib tissue (3). Direct PCR and nested PCR were carried out with primer pairs P1/16S-SR and R16F2n/R16R2n, respectively (3). Amplicons 1.5 and 1.2 kb in length, specific for the 16S rRNA gene, were amplified in all symptomatic plants. No PCR products were obtained when DNA isolated from symptomless plants was used. Restriction fragment length polymorphism (RFLP) patterns of the 1.2-kb fragments of 16S rDNA were determined by digestion with four endonucleases separately (TruI1, AluI, HpaII, and HhaI) and compared with those of Stolbur (Stol), Aster Yellows (AY), Flavescence dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (2). RFLP patterns from all symptomatic pot marigold plants were identical to the Stol pattern, indicating Stolbur phytoplasma presence in affected plants. The 1.2-kb amplicon of representative Nv8 strain was sequenced and the data were submitted to GenBank (accession no. KJ174507). BLASTn analysis of the sequence was compared with sequences available in GenBank, showing 100% identity with 16S rRNA gene of strains from Paeonia tenuifolia (KF614623) and corn (JQ730750) from Serbia, and peach (KF263684) from Iran. All of these are members of the 16SrXII 'Candidatus Phytoplasma solani' group, subgroup A (Stolbur). Phytoplasmas belonging to aster yellows (16SrI) (Italy and Canada) and peanut witches' broom related phytoplasma (16SrII) group (Iran) have been identified in diseased pot marigold plants (1). To our knowledge, this is the first report of natural infection of pot marigold by Stolbur phytoplasma in Serbia. References: (1) S. A. Esmailzadeh-Hosseini et al. Bull. Insectol. 64:S109, 2011. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) J. P. Prince. Phytopathology 83:1130, 1993. (4) M. Ukiya et al. J. Nat. Prod. 69:1692, 2006.

First Report of Blueberry Reddening Disease in Serbia Associated with 16SrXII-A (Stolbur) Phytoplasma.

Blueberries (Vaccinium corymbosum) are among the healthiest fruits due to their high antioxidant content. The total growing area of blueberries in Serbia ranges from 80 to 90 ha. A phytoplasma-like disease was observed for the first time during July 2009 in three blueberry cultivars (Bluecrop, Duke, and Spartan) grown in central Serbia, locality Kopljare (44°20'10.9″ N, 20°38'39.3″ E). Symptoms of yellowing and reddening were observed on the upper leaves and proliferating shoots, similar to those already described on blueberries (4). There was uneven ripening of the fruits on affected plants. Incidence of affected plants within a single field was estimated to be greater than 20% in 2009 and 50% in 2010. Blueberry leaves, together with petioles, were collected during two seasons, 2009 and 2010, and six samples from diseased plants and one from symptomless plants from each cultivar, resulting in 42 samples in total. For phytoplasma detection, total DNA was extracted from the veins of symptomatic and asymptomatic leaves of V. corymbosum using the protocol of Angelini et al. (1). Universal oligonucleotide primers P1/P7 were used to amplify a 1.8-kb DNA fragment containing the 16S rRNA gene, the 16S-23S spacer region, and the 5' end of the 23S rRNA gene. Subsequently, a 1.2-kb fragment of the 16S rRNA gene was amplified by nested PCR with the R16F2n/R16R2 primers. Reactions were performed in a volume of 50 µl using Dream Taq Green master mix (Thermo Scientific, Lithuania). PCR reaction conditions were as reported (3), except for R16F2n/R2 primers set (annealing for 30 s at 58°C). PCR products were obtained only from the DNA of symptomatic plants. Fragments of 1.2 kb were further characterized by the PCR-RFLP analysis, using AluI, HpaII, HhaI, and Tru1I restriction enzymes (Thermo Scientific, Lithuania), as recommended by the manufacturer. The products of restriction enzyme digestion were separated by electrophoresis on 2.5% agarose gel. All R16F2n/R2 amplicons showed identical RFLP patterns corresponding to the profile of the Stolbur phytoplasma (subgroup 16SrXII-A). The results were confirmed by sequencing the nested PCR product from the representative strain Br1. The sequence was deposited in NCBI GenBank database under accession number KC960486. Phylogenetic analysis showed maximal similarities with SH1 isolate from Vitis vinifera, Jordan (KC835139.1), Bushehr (Iran) eggplant big bud phytoplasma (JX483703.1), BA strain isolated from insect in Italy (JQ868436.1), and also with several plants from Serbia: Arnica montana L. (JX891383.1), corn (JQ730750.1), Hypericum perforatum (JQ033928.1), tobacco (JQ730740.1), etc. In conclusion, our results demonstrate that leaf discoloration of V. corymbosum was associated with a phytoplasma belonging to the 16SrXII-A subgroup. The wild European blueberry (Vaccinium myrtillus L.) is already detected as a host plant of 16SrIII-F phytoplasma in Germany, North America, and Lithuania (4). The main vector of the Stolbur phytoplasma, Hyalesthes obsoletus Signoret, was already detected in Serbia (2). The first report of Stolbur phytoplasma occurrence on blueberry in Serbia is significant for the management of the pathogen spreading in blueberry fields. Since the cultivation of blueberry has a great economic potential in the region, it is important to identify emerging disease concerns in order to ensure sustainable production. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) J. Jovic et al. Phytopathology 99:1053, 2009. (3) S. Pavlovic et al. J. Med. Plants Res. 6:906, 2012. (4) D. Valiunas et al. J. Plant Pathol. 86:135, 2004.

Human initiation protein Orc4 prefers triple stranded DNA.

In higher eukaryotes mechanism of DNA replication origin recognition and binding by origin recognition complex (ORC) is still unknown. Origin transfer studies have shown that origin sites are genetically determined, containing functionally interchangeable modules. One of such modules from the human lamin B2 origin of replication has the ability to adopt unorthodox structure partly composed of intramolecular triplex. Sequences involved in triplex formation coincide with ORC binding sites both in vitro and in vivo. To explore potential significance of unorthodox DNA structures in origin recognition by ORC, we tested DNA binding properties of human ORC subunit 4 (HsOrc4) which has independent DNA binding activity in vitro and similar binding characteristics as ORC holocomplex. Our results demonstrated that DNA binding activity of HsOrc4 depends on length and structure of DNA with triplex being the protein's preferred binding target. Such feature could play part in origin selection through directing ORC to DNA sequence prone to adopt unorthodox structure.

Characterization of human skeletal muscle Ankrd2.

Human Ankrd2 transcript encodes a 37-kDa protein that is similar to mouse Ankrd2 recently shown to be involved in hypertrophy of skeletal muscle. These novel ankyrin-rich proteins are related to C-193/CARP/MARP, a cardiac protein involved in the control of cardiac hypertrophy. A human genomic region of 14,300 bp was sequenced revealing a gene organization similar to mouse Ankrd2 with nine exons, four of which encode ankyrin repeats. The intracellular localization of Ankrd2 was unknown since no protein studies had been reported. In this paper we studied the intracellular localization of the protein and its expression on differentiation using polyclonal and monoclonal antibodies produced to human Ankrd2. In adult skeletal muscle Ankrd2 is found in slow fibers; however, not all of the slow fibers express Ankrd2 at the same level. This is particularly evident in dystrophic muscles, where the expression of Ankrd2 in slow fibers seems to be severely reduced.

[Specificity of lipid and lipoprotein status in healthy students at the University of Novi Sad]. / Specificnosti lipidskog i lipoproteinskog statusa zdravih studenata Novosadskog Univerziteta.

INTRODUCTION: The aim of this study was to determine the frequency of hyperlipoproteinemias and normolipidemic dyslipoproteinemias, and distribution of desirable, borderline and high-risk values of certain lipid status parameters in healthy young individuals. MATERIAL AND METHODS: In this investigation we examined 213 students of the University of Novi Sad of both genders, 20-30 years of age. Standard biochemical methods were used to determine values of total serum cholesterol, triglycerides, HDL cholesterol, and lipoproteins by cellulose acetate electrophoresis. The level of LDL cholesterol and LDL/HDL cholesterol and total/HDL cholesterol ratios were calculated. RESULTS: In this group hyperlipoproteinemia was established in 42.3% of cases and normolipidemic dyslipoproteinemia in 65.3%. Total serum cholesterol was minimally elevated in 39.0% of tested students, elevated with high risk in 3.3% and triglycerides were minimally elevated in 1.0%. Presence of elevated LDL cholesterol (24.4% minimally and 13.2% with high risk) is remarkably significant. HDL cholesterol is minimally decreased in 54.0% of tested students, and severely in 3.3%. DISCUSSION: The tested parameters deviate from desirable levels with an alarmingly high frequency, given the fact that this is a group of healthy young individuals with no previous history of lipid and lipoprotein metabolism disorders. It can be hypothesized that a joint hyper Lp(a)-lipoproteinemia can exist with a significant occurrence. These results could be associated with similar disorders in families of tested students, unhealthy food habits and lifestyle, use of oral contraceptives and smoking. CONCLUSION: Our results point to the need for performing gradual laboratory diagnostic procedures for routine check-ups in students.

FATZ, a filamin-, actinin-, and telethonin-binding protein of the Z-disc of skeletal muscle.

We report the identification and characterization of a novel 32-kDa protein expressed in skeletal muscle and located in the Z-disc of the sarcomere. We found that this protein binds to three other Z-disc proteins; therefore, we have named it FATZ, gamma-filamin/ABP-L, alpha-actinin and telethonin binding protein of the Z-disc. From yeast two-hybrid experiments we are able to show that the SR3-SR4 domains of alpha-actinin 2 are required to bind the COOH-terminal region of the FATZ as does gamma-filamin/ABP-L. Furthermore, by using a glutathione S-transferase overlay assay we find that FATZ also binds telethonin. The level of FATZ protein in muscle cells increases during differentiation, being clearly detectable before the onset of myosin. Although FATZ has no known interaction domains, it would appear to be involved in a complex network of interactions with other Z-band components. On the basis of the information known about its binding partners, we could envisage a central role for FATZ in the myofibrillogenesis. After screening our muscle expressed sequence tag data base and the public expressed sequence tag data bases, we were able to assemble two other muscle transcripts that show a high level of identity with FATZ in two different domains. Therefore, FATZ may be the first member of a small family of novel muscle proteins.

Den1, den2 and den3, ATP-inhibited deoxyribonucleases from Dropsophila embryonic nuclei.

Three Drosophila embryonic deoxyribonucleases, designated den1, den2 and den3, are identified in nuclear extracts separated by glycerol density gradient centrifugation. Den1, removes short products from the 5'-ends of single-stranded DNA or double-stranded DNA with either blunt or 5'-recessed termini. Den2 is inactive with single-stranded DNA and acts as 3'-exonuclease with double-stranded DNA possessing either blunt or 3'-recessed termini. Den3 preferentially uses partial duplex DNA containing single-stranded gap and it catalyzes hydrolysis, in 3'-5' direction, of one of the shorter strands that flank the gap. Nucleolytic activities of den1, den2 and den3 are inhibited with ATP.

Characterization of a helicase, DHEL III, from Drosophila melanogaster embryonic nuclei.

Nuclear extracts prepared from Drosophila preblastoderm embryos contain a nucleoside 5'-triphosphate-dependent helicase that unwinds circular or linear partial duplex DNA substrates and moves along the DNA in 3' to 5' direction. The helicase reaction is supported with both nucleoside and deoxynucleoside 5'-triphosphates and requires divalent cations. Optimum activity in vitro is achieved with dATP or ATP and with MgCl2. In glycerol density gradients, embryonic enzyme migrates as a single peak of around 90kDa and it is the most prominent DNA-unwinding activity detectable in nuclear extracts prepared from embryos 0-2 hours after egg laying. In embryos collected 2-4 hours after egg laying this DNA unwinding activity increases and then gradually decreases in embryos collected 4-8 and 8-16 hours after egg laying.

Novel DNA-binding protein from Drosophila embryos identified by binding site selection.

The DNA sequence C/AGAGCGC/AGA, related to binding sites for GAF and Zeste transcription factors, was selected from a pool of degenerate PCR fragments for binding to the cytoplasmic protein of Drosophila preblastoderm embryos. Identical DNA binding activity was also detected in embryonic nuclei. Based on several criteria, such as size, intracellular distribution, sensitivity to ATP and protein kinase inhibitor 6-DMAP, kinetics during development and lack of cross-reaction with rabbit anti-GAF serum, protein recognizing selected sequence was shown to differ from either Zeste or GAF.