Production of monoclonal antibodies to the prion protein and their characterization

Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25–36 of PrP corresponds to one antibody, and epitopes located in region 222–229, to the other two. The antibodies to fragment 222–229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.     

Antitoxic properties of monoclonal antibodies against diphtheria toxin

Antitoxin properties of monoclonal antibodies to diphtheria toxin were studied in chick fibroblast culture and during the dermonecrotic tests in guinea pigs. Antitoxic properties were shown for antibodies specific to B subunit of the toxin but not for the ones specific to a subunit. The mixture of monoclonal B subunit specific antibodies reveals a higher neutralizing activity as compared with the activity produced by any single monoclonal antibody tested. The protective activity demonstrated by antibodies is connected with the preventing of toxin molecules fixation on the cellular receptors.     

Photo‐identification matches of humpback whales (Megaptera novaeangliae) from feeding areas in Russian Far East seas and breeding grounds in the North Pacific

Humpback whales migrate seasonally from high latitude feeding areas to lower latitude breeding areas for mating and calving. In 2004–2006, a North Pacific basin‐wide study called SPLASH was conducted as an international collaboration among various groups of researchers. The Russian Far East consists of multiple high latitude feeding areas and during SPLASH, 102 whales were identified and compared to catalogs from breeding areas. Our goal in this study was to further investigate the migratory destinations of whales from the Russian Far East using a total of 1,459 photographs of whales identified from 2004 to 2014. We compared the latest Russian catalog with the SPLASH catalog from wintering areas (2004–2006) and with two additional regional catalogs from Okinawa (1989–2006) and the northern Philippines (2000–2006). We found a total of 152 matches: 106 with Asian, 35 with Hawaiian, and 11 with Mexican breeding grounds. The match rate was higher in mainland Kamchatka and consisted mostly of whales from the Asian breeding ground. In the Commander Islands, the proportion of whales from Asia was twice that of Hawaii and six times higher than Mexico. The total match rate was low, supporting the hypothesis of some undiscovered humpback whale breeding location in the North Pacific.     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

SSR based genetic diversity of pigmented and aromatic rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes of the western Himalayan region of India

A set of 24 of SSR markers were used to estimate the genetic diversity in 16 rice genotypes found in Western Himalayas of Kashmir and Himachal Pradesh, India. The level of polymorphism among the genotypes of rice was evaluated from the number of alleles and PIC value for each of the 24 SSR loci. A total of 68 alleles were detected across the 16 genotypes through the use of these 24 SSR markers The number of alleles per locus generated varied from 2 (RM 338, RM 452, RM 171) to 6 (RM 585, RM 249, RM 481, RM 162). The PIC values varied from 0.36 (RM 1) to 0.86 (RM 249) with an average of 0.62 per locus. Based on information generated, the genotypes got separated in six different clusters. Cluster 1 comprised of 4 genotypes viz; Zag 1, Zag 13, Pusa sugandh 3, and Zag 14, separated from each other at a similarity value of 0.40. Cluster second comprised of 3 landraces viz; Zag 2. Zag 4 and Zag10 separated from each other at a similarity value of 0.45. Cluster third comprised of 3 genotypes viz; Grey rice, Mushk budji and Kamad separated from each other at a similarity value of 0.46. Cluster fourth had 2 landraces viz; Kawa kreed and Loual anzul, and was not sub clustered. Fifth cluster had 3 genotypes viz; Zag 12, Purple rice and Jhelum separated from each other at a similarity value of 0.28. Cluster 6 comprised of a single popular variety i.e. Shalimar rice 1 with independent lineage.     

Production of the antibiotic-algicide cyanobacterin LU-2 by a filamentous cyanobacterium Nostoc sp.]

A strain of cyanobacterium of Nostoc has been isolated, and found to produce a new antibiotic cyanobacterin LU-2. The antibiotic is synthesized by the cyanobnacterium under intensive cultivation conditions in a liquid mineral medium. Cyanobacterin LU-2 is an exometabolite; its maximum accumulation in the medium is achieved at 34 degrees. Cyanobacterin LU-2 is active against many cyanobacteria tested, including those of Microcystis and Aphanizomenon which are principals to give rise to blooms in fresh water supplies. It is poorly active against green algae and inactive against fungi and bacteria. The antibiotic hinders cell division in Synechococcus sp. R-2 (PCC 7942). It causes compression of the cytoplasm and exfoliation of the cell contents from cell wall; the distance between tylacoids is increased and their destruction is observed. The antibiotic hinders markedly light-dependent oxygen evolution. Cyanobacterin LU-2 is substance of a phenolic nature containing amino-sugar.     

Slipped-strand mispairing: a major mechanism for DNA sequence evolution

Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following several features characterize such sequences: (1) they typically consist of a variety of repeated motifs of 1-10 bases--but may include much larger repeats as well; (2) larger repeat units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped-strand mispairing events, in concert with unequal crossing- over, can readily account for all of these features. The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to result from nonrandom patterns of nucleotide substitution. We argue that the intrahelical process of slipped-strand mispairing is much more likely to be the major factor in the initial expansion of short repeated motifs and that, after initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossing-over or other interhelical events because of their propensity to mispair. Evidence is presented that single-base repeats (the shortest possible motifs) are represented by longer runs in mammalian introns than would be expected on a random basis, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Simple repetitive sequences may therefore represent a natural ground state of DNA unselected for coding functions.      

Complete sequences of the rRNA genes of Drosophila melanogaster

In this, the first of three papers, we present the sequence of the ribosomal RNA (rRNA) genes of Drosophila melanogaster. The gene regions of D. melanogaster rDNA encode four individual rRNAs: 18S (1,995 nt), 5.8S (123 nt), 2S (30 nt), and 28S (3,945 nt). The ribosomal DNA (rDNA) repeat of D. melanogaster is AT rich (65.9% overall), with the spacers being particularly AT rich. Analysis of DNA simplicity reveals that, in contrast to the intergenic spacer (IGS) and the external transcribed spacer (ETS), most of the rRNA gene regions have been refractory to the action of slippage-like events, with the exception of the 28S rRNA gene expansion segments. It would seem that the 28S rRNA can accommodate the products of slippage-like events without loss of activity. In the following two papers we analyze the effects of sequence divergence on the evolution of (1) the 28S gene "expansion segments" and (2) the 28S and 18S rRNA secondary structures among eukaryotic species, respectively. Our detailed analyses reveal, in addition to unequal crossing-over, (1) the involvement of slippage and biased mutation in the evolution of the rDNA multigene family and (2) the molecular coevolution of both expansion segments and the nucleotides involved with compensatory changes required to maintain secondary structures of RNA.      

Granular preparations ofAzotobacter containing clay minerals

Interaction ofAzotobacter chroococcum 20 with clay minerals increased their cell viability at supraoptimal temperatures. Therefore, clay minerals were used to develop granular bacterial preparations with high viable cell counts and stable compositions during long-term storage. The titers of viable bacteria in the preparations remained 60–70% of the initial level after 12 months of storage.