Cryopreserving human peripheral blood progenitor cells

High-dose chemotherapy followed by autologous peripheral blood progenitor cell (PBPC) transplantation is used in the treatment of chemosensitive malignancies. Cryopreservation of PBPC in 10% dimethyl sulfoxide (DMSO) has been the standard procedure in most institutions. Infusion of PBPC cryopreserved with DMSO can be associated with toxic reactions such as vomiting, cardiac dysfunction, anaphylaxia and acute renal failure. The grade of toxicity experienced by patients is related to the amount of DMSO present in the PBPC. Cryopreservation with lower DMSO concentrations would be expected to reduce the toxicity. In recent studies done with PBPC cells cryopreserved with 5%, 4% and 2% DMSO, using 10% DMSO as a reference control, CD34+ cells were investigated for preservation of viability, apoptosis, and necrosis. Also preservation of mature colony-forming (CFU) cells, specifically mature myeloid, erythroid progenitors, CFU-megakaryocytes and long-term culture-initiating cells (LTC-ICs) were investigated, using 5% and 10% DMSO as cryoprotectant. All samples were frozen in a rate-controlled programmed freezer and stored in the vapor phase of liquid nitrogen until used. Conclusion: 5% DMSO is the optimal concentration for cryopreserving human PBPC in vitro. Consequently, some hospitals have started using 5% DMSO as cryoprotectant for the autologous PBPC as a standard procedure.     

Isolation of an Ethylene-induced Putative Nucleotide Laccase in Miniature Roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis> L.)

Using differential display we isolated five ethylene-responsive cDNAs from Rosa hybrida L. and identified for the first time an ethylene-induced cDNA homologous to a laccase gene. Three cDNAs were isolated from petioles and two cDNAs from pedicels. Expression levels of all cDNAs in pedicels were higher than in petioles. The laccase homolog cDNA was termed the RhLAC (Rosa hybrida Laccase) gene. The RhLAC gene encodes for a putative protein of 573 amino acids containing three conserved domains characteristic of the multicopper oxidase family. Southern blot hybridization analyses indicated that there are multiple copies of the RhLAC gene in the Rosa species. Comparison of the relative expression of isolated RhLAC in various organs showed that it was highly induced in the leaf abscission zone of petioles and the bud abscission zone of floral bud pedicels, whereas it was low in both leaf blades and petioles. These results suggest that RhLAC may play an important role in senescence and abscission in roses.     

A comparison of the reproduction of Brevicoryne brassicae and Myzus persicae in relation to soluble nitrogen concentration and leaf age (leaf position) in the brussels sprout plant

Wide variations in total soluble nitrogen occurred in different leaves of Brussels sprout plants. These variations were partly the result of leaf age (leaf position) and partly the result of different fertiliser application to the plants. The fecundity of Brevicoryne brassicae and Myzus persicae caged on such leaves also varied greatly and was correlated with total soluble nitrogen within leaves of the same age, but not between leaves of different ages. The value of total soluble nitrogen concentrations, based on analysis of leaf tissue, for indicating plant suitability for aphids is discussed as well as the difference in response of the two aphid species to experimental conditions favouring proteolysis in the leaf.

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Ein vergleich der vermehrung von Brevicoryne brassicae und Myzus persicae in beziehung zum gehalt löslichen stickstoffs und dem blattalter von rosenkohlpflanzen

Zusammenfassung Brevicoryne brassicae (L.) und Myzus persicae (Sulz.) wurden an Kohlblättern gekäfigt, die infolge verschiedener physiologischer Ursachen sehr unterschiedliche Mengen löslichen Stickstoffs enthielten. Den Pflanzen wurden zwei Düngerstoffe ((NH₄)₂SO₄ und KCL) verabreicht und die Blattläuse auf jungen, mittelalten und alten Blättern gekäfigt. Dadurch wurde der Gehalt löslichen Stickstoffs in den Blättern zwischen 0,543% und 1,286% variiert. Die Nachkommenzahl der Blattläuse bei diesen Düngungen und auf verschiedenaltrigen Blättern war sehr unterschiedlich (zwischen 7,3 und 30,2 bei B. brassicae; 17,6 und 59,4 bei M. persicae) und konnte in jedem Blattalter mit dem Stickstoffgehalt korreliert werden. Der Befund, daß die Korrelationen zwischen Blattlausnachkommenzahl und Stickstoffgehalt nicht unabhängig vom Blattalter waren, deutet darauf hin, daß der Stickstoffgehalt in physiologisch aktiven Blattgeweben nicht relativ mit dem der Siebröhren übereinstimmt. M. persicae vermochte in höherem Maße als B. brassicae Blätter auszunützen, in denen der Eiweißabbau durch Düngung oder Alter begünstigt war. Dieser Unterschied zwischen den beiden Arten steht möglicherweise mit ihren verschiedenen Nährstoffbedürfnissen in Zusammenhang.

     

Hypertension and Genetic Variation in Endothelial-Specific Genes

Genome-wide association (GWA) studies usually detect common genetic variants with low-to-medium effect sizes. Many contributing variants are not revealed, since they fail to reach significance after strong correction for multiple comparisons. The WTCCC study for hypertension, for example, failed to identify genome-wide significant associations. We hypothesized that genetic variation in genes expressed specifically in the endothelium may be important for hypertension development. Results from the WTCCC study were combined with previously published gene expression data from mice to specifically investigate SNPs located within endothelial-specific genes, bypassing the requirement for genome-wide significance. Six SNPs from the WTCCC study were selected for independent replication in 5205 hypertensive patients and 5320 population-based controls, and successively in a cohort of 16537 individuals. A common variant (rs10860812) in the DRAM (damage-regulated autophagy modulator) locus showed association with hypertension (P = 0.008) in the replication study. The minor allele (A) had a protective effect (OR = 0.93; 95% CI 0.88–0.98 per A-allele), which replicates the association in the WTCCC GWA study. However, a second follow-up, in the larger cohort, failed to reveal an association with blood pressure. We further tested the endothelial-specific genes for co-localization with a panel of newly discovered SNPs from large meta-GWAS on hypertension or blood pressure. There was no significant overlap between those genes and hypertension or blood pressure loci. The result does not support the hypothesis that genetic variation in genes expressed in endothelium plays an important role for hypertension development. Moreover, the discordant association of rs10860812 with blood pressure in the case control study versus the larger Malmö Preventive Project–study highlights the importance of rigorous replication in multiple large independent studies.     

Induction of adventitious shoots in vitro in Campanula carpatica

Efficient protocols have been developed to induce adventitious shoots in different types of explants of Campanula carpatica Jacq. More than five shoots per explant developed on hypocotyls of 5-week-old seedlings after 2 weeks of culture. Hypocotyls produced twice as many shoots as the cotyledons. TDZ proved to be about 6 times more efficient than BA. NAA had to be added to the regeneration medium to obtain the optimal balance of auxin and cytokinin to induce shoot regeneration. Significant differences were noted between different growth regulator concentrations in their effects on shoot organogenesis. BA induced double the number of callus clumps as TDZ. Incubation of explants in the dark produced about 6 shoots per explant while those in the light produced about 2 shoots per explant. Explants derived from 5-week-old seedlings were five times more regenerative compared to those derived from 15-week-old seedlings. Explants from cv. White Uniform were more organogenic than those from cv. Blue Clip. Root segments were also found to form shoots when treated with CPPU.     

Description of Citricoccus nitrophenolicus sp. nov., a para-nitrophenol degrading actinobacterium isolated from a wastewater treatment plant and emended description of the genus Citricoccus Altenburger et al. 2002

A novel actinobacterium, designated PNP1(T), was isolated from a wastewater treatment plant at a pesticide factory by selective enrichment with para-nitrophenol. The strictly aerobic strain PNP1(T) grew with para-nitrophenol as the sole carbon and energy source. Metabolism of para-nitrophenol resulted in the stoichiometric release of nitrite. When incubated with both para-nitrophenol and acetate, para-nitrophenol was degraded and utilized as growth substrate prior to acetate. When grown on acetate (in the absence of ammonium) both nitrite and nitrate served as nitrogen sources, nitrate being quantitatively reduced to nitrite which accumulated in cultures during aerobic growth. Cells were coccoid and stained Gram-positive, were non-motile and did not form endospores. Colonies of strain PNP1(T) on agar medium were bright yellow, circular and smooth. The dominant menaquinone was MK-8(H(2)) (54%) and the major cellular fatty acid was anteiso C15:0 (75%). Strain PNP1(T) grew optimally at 27°C, at pH 8-8.5, at salinities 3% (w/v) NaCl, yet exhibited a substantial halotolerance with growth occurring at salinities up to 17% (w/v) NaCl. In addition to para-nitrophenol, a range of sugars, short chain fatty acids and alcohols served as electron donors for growth. The DNA G + C mol% was 68%. The genotypic and phenotypic properties suggest that strain PNP1(T) represents a novel species of the actinobacterial genus Citricoccus for which the name Citricoccus nitrophenolicus is proposed. It is the first member of this genus that has been reported to hydrolyze and grow on para-nitrophenol. The type strain is PNP1(T) (=DSM 23311(T) = CCUG 59571(T)).     

Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma

Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin-like substances found in lizard saliva or snake venom. Human CRISP-3 is present in exocrine secretions and in secretory granules of neutrophilic granulocytes and is believed to play a role in innate immunity. On the basis of the relatively high content of CRISP-3 in human plasma and the small size of the protein (28 kDa), we hypothesized that CRISP-3 in plasma was bound to another component. This was supported by size-exclusion chromatography and immunoprecipitation of plasma proteins. The binding partner was identified by mass spectrometry as alpha(1)B-glycoprotein (A1BG), which is a known plasma protein of unknown function and a member of the immunoglobulin superfamily. We demonstrate that CRISP-3 is a specific and high-affinity ligand of A1BG with a dissociation constant in the nanomolar range as evidenced by surface plasmon resonance. The A1BG-CRISP-3 complex is noncovalent with a 1:1 stoichiometry and is held together by strong electrostatic forces. Similar complexes have been described between toxins from snake venom and A1BG-like plasma proteins from opossum species. In these cases, complex formation inhibits the toxic effect of snake venom metalloproteinases or myotoxins and protects the animal from envenomation. We suggest that the A1BG-CRISP-3 complex displays a similar function in protecting the circulation from a potentially harmful effect of free CRISP-3.     

Succession of cable bacteria and electric currents in marine sediment

Filamentous Desulfobulbaceae have been reported to conduct electrons over centimetre-long distances, thereby coupling oxygen reduction at the surface of marine sediment to sulphide oxidation in sub-surface layers. To understand how these ‘cable bacteria'' establish and sustain electric conductivity, we followed a population for 53 days after exposing sulphidic sediment with initially no detectable filaments to oxygen. After 10 days, cable bacteria and electric currents were established throughout the top 15 mm of the sediment, and after 21 days the filament density peaked with a total length of 2 km cm⁻². Cells elongated and divided at all depths with doubling times over the first 10 days of <20 h. Active, oriented movement must have occurred to explain the separation of O₂ and H₂S by 15 mm. Filament diameters varied from 0.4–1.7 μm, with a general increase over time and depth, and yet they shared 16S rRNA sequence identity of >98%. Comparison of the increase in biovolume and electric current density suggested high cellular growth efficiency. While the vertical expansion of filaments continued over time and reached 30 mm, the electric current density and biomass declined after 13 and 21 days, respectively. This might reflect a breakdown of short filaments as their solid sulphide sources became depleted in the top layers of the anoxic zone. In conclusion, cable bacteria combine rapid and efficient growth with oriented movement to establish and exploit the spatially separated half-reactions of sulphide oxidation and oxygen consumption.     

Linkage analysis of juvenile myoclonic epilepsy and microsatellite loci spanning 61 cM of human chromosome 6p in 19 nuclear pedigrees provides no evidence for a susceptibility locus in this region.

Linkage analysis in separately ascertained families of probands with juvenile myoclonic epilepsy (JME) has previously provided evidence both for and against the existence of a locus (designated "EJM1"), on chromosome 6p, predisposing to a trait defined as either clinical JME, its associated electroencephalographic abnormality, or idiopathic generalized epilepsy. Linkage analysis was performed in 19 families in which a proband and at least one first- or two second-degree relatives have clinical JME. Family members were typed for seven highly polymorphic microsatellite markers on chromosome 6p: D6S260, D6S276, D6S291, D6S271, D6S465, D6S257, and D6S254. Pairwise and multipoint linkage analysis was carried out under the assumptions of autosomal dominant inheritance at 70% and 50% penetrance and autosomal recessive inheritance at 70% and 50% penetrance. No significant evidence in favor of linkage to the clinical trait of JME was obtained for any locus. The region formally excluded (LOD score < -2) by using multipoint analysis varies depending on the assumptions made concerning inheritance parameters and the proportion of linked families, alpha-that is, the degree of locus heterogeneity. Further analysis either classifying all unaffected individuals as unknown or excluding a subset of four families in which pyknoleptic absence seizures were present in one or more individuals did not alter these conclusions.