Genetic basis for the polymorphism of rat liver cytosolic aldehyde dehydrogenase

Liver aldehyde dehydrogenase (ALDH), the enzyme involved in the oxidation of aldehydes such as acetaldehyde derived from ethanol, exists in multiple forms in most mammals. Up to five separable forms have been identified from the cytosolic fraction of Wistar rat liver. We investigated the genetic basis of a particular set of three enzyme forms by selective breeding and analysis of electrophoretic patterns of liver ALDH by isoelectric focusing. The forms of liver ALDH investigated were at pI 5.8 or 6.2, or a triple form with enzymes at pI 5.8, 6.0, and 6.2. There are two alleles found at the ALDH locus which encode in homozygotes for one of two electrophoretically separable ALDH forms. A rat heterozygous at the locus forms both ALDH types plus a hybrid. The alleles are expressed codominantly, found at an autosomal locus, and remain constant postpartum. The activities associated with the triplet enzyme form were statistically indistinguishable from a 1:2:1 ratio. This suggests that the enzymes hybridize to form a set of dimers or tetramers of the form A₂, AB, B₂ or A₄, A₂B₂, B₄, respectively.     

Oogenesis and chromosomal heterozygosity in the thelytokous midge,Lundstroemia parthenogenetica (Diptera,Chironomidae)

The thelytokous triploid midge Lundstroemia parthenogenetica (Diptera: Chironomidae:Chironominae), 3n = 9, is always found to be heterozygous for two inversions and a deletion. Parthenogenesis is maintained by an apomiotic restitutional oogenesis as found in the subfamily Orthocladiinae. This mechanism is compared with other groups exhibiting a restitutional or disordered first division. Possible modes of evolution of the apomictic restitutional system, origin of triploidy in apomictic forms, and chromosomal polymorphisms are discussed.     

Characterization of mitochondrial bioenergetic functions between two forms of Leishmania donovani – a comparative analysis

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and F_oF₁-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and F_oF₁-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by F_oF₁-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.     

Transversion of cell polarity from bi- to multipolarity is the mechanism determining multiple spore formation in Anaerobacter polyendosporus PS-1T

The number of spores formed in a single cell of Anaerobacter polyendosporus PS-1^T is significantly influenced by the composition of nutrient media. Depending on carbohydrate concentration in synthetic medium, the number of spores may vary from one or two to as many as five to seven. Investigation of spore formation process by fluorescence and electron microscopy revealed that on media with 0.5–1.0% glucose or galactose most of vegetative cells remained rod-shaped after cessation of cell division in the culture. The nucleoids of these cells were localized at cell poles close to the polar site of the cytoplasmic membrane. Fore-spores were formed at one or both of these poles. A satellite nucleoid (operator) was observed close to each forespore. In the variant with bipolar organization of mother cells, only one or two spores per cell were formed. In the second variant of culture development, when the cells were grown at low galactose concentrations (0.1–0.3%), most of vegetative cells increased in volume and became oval or spherical after cessation of cell division in the culture. Epifluorescence microscopy with nucleic acid-specific fluorochromes (DAPI and acridine orange) revealed the presence of multiple (six to nine) nucleoids in these cells. The nucleoids were located at the cell periphery in close contact with the cytoplasmic membrane. These nucleoids became the centers (poles) for forespore formation. Thus, in the early stationary phase transversion from bipolar to multipolar cells occurred. Cessation of cell division combined with continuing replication of the nucleoids resulted in formation on multinuclear cells. The multiplicity of nucleoides and multipolarity of these cells were prerequisites determining endogenous polysporogenesis, occurring as synchronous formation of three to seven twin spores in many of the oval and spherical cells.     

Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency,the PEX5 mRNA and protein levels and induction of peroxisomal deficiency

The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine^® 2000, FuGENE 6, HiPerFect^®, INTERFERin?, RiboJuice?) as well as microporation using the Neon? Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0–70 %), to the reduction of PEX5 mRNA (by 90 vs. 0–50 %) and PEX5 protein levels (by 70 vs. 0–50 %). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3′ fragment (15 %) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded to transiently knockdown PEX5 mRNA and its protein level leading to functional consequences similar as observed in peroxisome deficiencies.     

Length polymorphism of the B2-VNTR minisatellite repeat of the bradykinin B2 receptor gene in healthy Russians and patients with coronary heart disease

Bradykinin B2 receptor is involved in many processes, including the regulation of blood pressure and smooth muscle contraction, vasodilation, inflammation, edema, cell proliferation, and pain. This receptor attracts special attention as one of the factors that have cardioprotective and infarct-limiting effects. Certain genetic variants of the coding and noncoding regions of the bradykinin B2 receptor gene (BDKRB2) may play a role in modulating its expression. The 3′-untranslated region of BDKRB2 exon 3 harbors a minisatellite repeat (B2-VNTR), which affects the mRNA stability. Hence, it is of interest to study a possible association of B2-VNRT alleles with various forms of coronary heart disease (CHD). In our work the allele and genotype frequency distributions of B2-VNTR were compared between healthy individuals and patients with CHD (angina pectoris or myocardial infarction (MI)) of the Russian ethnic group. Based on its length polymorphism, B2-VNTR was classed with low-polymorphic non-hypervariable minisatellites. Three B2-VNTR alleles, which consisted of 43, 38, and 33 repeats, were observed in all investigated cohorts. The alleles with 43 and 33 repeats were the most prevalent. The allele and genotype frequencies of B2-VNTR did not significantly differ between males and females in control group, and also between healthy males and males with angina pectoris or MI. Thus, B2-VNTR length polymorphism was not associated with these clinical forms of CHD in males. However, we do not exclude the possibility of an association of the short B2-VNTR alleles (38 and 33 repeats) with a cardioprotective effect in females with CHD. This hypothesis requires further investigation.     

Transplantation and immunoincompatibility reactions among reef-building corals

Intracolony or syngeneic transplants of pieces of living coral, either occurring naturally on reefs or produced experimentally, were consistently compatible. In contrast, intercolony allografts performed extensively inAcropora formosa and inPorites andrewsii were incompatible, as were allografts occurring naturally in additional species observed in reef communities. Diverse interspecific combinations of coral xenografts generally displayed greater incompatibilities. Four levels of immuno-reactivity, with manifestations ranging from mild to severe, were distinguishable as follows: a) contact avoidance reactions, b) allogeneic contact incompatibility, c) chronic xenogeneic incompatibility, and d) acute interspecific aggression.Allogeneic incompatibility, most studied inA. formosa, was characterized by soft tissue contact avoidance wherever possible; absence of tissue death despite extensive enforced contact (18–20 days); and late interfacial cementation terminating allogeneic soft tissue contact. The properties ofchronic xenogeneic incompatibility as found in diverse species combinations, are slow onset (>7 days), bidirectional or unidirectional killing in contact zones, and short-range or localized effectiveness.Acute interspecific aggression initiated byFungia fungites as a dominant “aggressor ” is distinguished by early occurrence (2–7 days) and unidirectional contact killing. The presence or absence of these acute xenogeneic reactions sharply discriminated between species within each of the generaAcropora, Pocillopora, andPorites. No hierarchy of aggressive interactions was evident at the generic level.     

Quantitative description of morphological changes during growth of a pleomorphic budding bacterium

Temporal variations in the different morphological forms observed during growth of the pleomorphicHyphomicrobium T37 have been quantified by the use of a “Population Index” (P _i). This index relates the “Cell Number” (CN) in each “Colony-Forming Unit” (CFU) to the total number of CFU's at any particular time. Such measurements have proved of value in explaining the nature of an observed pseudo-log phase in swarmer-inoculated cultures and of a discontinuity in the log phase of growth.     

Reducing the bioactivity of Tannerella forsythia lipopolysaccharide by Porphyromonas gingivalis

Tannerella forsythia is considered a pathogen of periodontitis and forms a biofilm with multi-species bacteria in oral cavity. Lipopolysaccharide is a powerful immunostimulator and induces inflammation and shock. The purpose of this study was to investigate the characteristics of T. forsythia LPS in its co-cultivation with Fusobacterium nucleatum or Porphyromonas gingivalis. T. forsythia was co-cultured in the presence and absence of F. nucleatum and P. gingivalis and then T. forsythia LPS was extracted. The extracts were analyzed by SDS-PAGE and NF-κB reporter CHO cell lines. THP-1 cells were treated with the LPS and evaluated induction of cytokine expression by real-time RT-PCR and ELISA. For analysis of the bioactivity of T. forsythia LPS, the binding assay on LPS-binding protein (LBP) and CD14 was processed. The extracts did not contaminate other molecules except LPS and showed TLR4 agonists. Co-cultured T. forsythia LPS with P. gingivalis exhibited a lower level of induction of TNF-α, IL-1β, and IL-6 expression than singleor co-cultured T. forsythia LPS with F. nucleatum in the conditions of human serum. However, the three T. forsythia LPS did not show difference of cytokine induction in the serum free conditions. Co-cultured T. forsythia LPS with P. gingivalis exhibited a lower affinity to LBP and CD14 as binding site of O-antigen and attached at a lower level to THP-1 cells compared to single- or co-cultured T. forsythia LPS with F. nucleatum. The virulence of T. forsythia LPS was decreased by co-culturing with P. gingivalis and their affinity to LBP and CD14 was reduced, which may due to modification of O-antigen chain by P. gingivalis.     

Linkage disequilibrium and haplotypes of the rs1042522, rs1625895, and rs1787862 markers of TP53 in patients with diffuse large B-cell lymphoma

The association of the rs1042522, rs17878362, and rs1625895 polymorphisms of TP53 with risk of non-Hodgkin’s lymphoma (NHL) has not been studied in full detail. NHL has many variants, and each individual polymorphism exerts only a minor effect, requiring the polymorphisms to be studied for particular histological subtypes of lymophomas and as components of haplotype groups. The objective of this work was to analyze the frequencies and linkage disequilibrium for rs1042522, rs1625895, and rs17878362 and their combined haplotype in patients with diffuse large B-cell lymphoma (DLBCL) and control subjects. Differences in linkage disequilibrium structure were observed for rs17878362, rs1042522, and rs1625895 in the Siberian population. The haplotype proved to be more informative than the individual TP53 polymorphisms in a case-control association analysis in DLBCL. Haplotype wArgG was associated with predisposition to DLBCL, while haplotypes wProG and dupProG were found to exert a protective effect. The effect of the haplotype at three TP53 polymorphisms was observed to vary depending on their homozygous or heterozygous state.     

Oxytocin plasma concentrations in children and adolescents with autism spectrum disorder: correlation with autistic symptomatology

Findings from research in animal models and humans have shown a clear role for the neuropeptide oxytocin (OT) on complex social behaviors. This is also true in the context of autism spectrum disorder (ASD). Previous studies on peripheral OT concentrations in children and young adults have reported conflicting results with the initial studies presenting mainly decreased OT plasma levels in ASD compared to healthy controls. Our study therefore aimed to further investigate changes in peripheral OT concentrations as a potential surrogate for the effects observed in the central nervous system (CNS) in ASD. OT plasma concentrations were assessed in 19 male children and adolescents with ASD, all with an IQ > 70 (age 10.7 ± 3.8 years), 17 healthy male children (age 13.6 ± 2.1 years) and 19 young male patients with attention deficit hyperactivity disorder (ADHD) as a clinical control group (age 10.4 ± 1.9 years) using a validated radioimmunoassay. Analysis of covariance revealed significant group differences in OT plasma concentrations (F(2, 48) = 9.574, p < 0.001, η ² = 0.285; plasma concentrations ASD 19.61 ± 7.12 pg/ml, ADHD 8.05 ± 5.49 pg/ml, healthy controls 14.43 ± 9.64 pg/ml). Post hoc analyses showed significantly higher concentrations in children with ASD compared to ADHD (p < 0.001). After Bonferroni correction, there was no significant difference in ASD in comparison with healthy controls (p = 0.132). A significant strong correlation between plasma OT and autistic symptomatology, assessed by the Autism Diagnostic Observation Schedule, was observed in the ASD group (p = 0.013, r = 0.603). Patients with ADHD differed from healthy control children by significantly decreased OT concentrations (p = 0.014). No significant influences of the covariates age, IQ, medication and comorbidity could be seen. Our preliminary results point to a correlation of OT plasma concentrations with autistic symptom load in children with ASD and a modulation of the OT system also in the etiologically and phenotypically overlapping disorder ADHD. Further studies in humans and animal models are warranted to clarify the complex association of the OT system with social impairments as well as stress-related and depressive behavior and whether peripheral findings reflect primary changes of OT synthesis and/or release in relevant areas of the CNS.     

Descriptive and contextual analysis of eye covering behavior in captive rhesus macaques (Macaca mulatta)

An abnormal behavior pattern (covering the eyes with one or both hands) is studied in rhesus macaques (Macaca mulatta). In adults, the eye covering pattern is more frequent and less variable in form than in youngsters; in addition, intermediate forms with play are observed. The frequency of eye covering increases when subjects are placed in an aversive situation (social isolation) and decreases when they perform another behavior (feeding). At the same time, eye covering increases in the evening due to the anticipation of light-dark transition by subjects. The spread of eye covering by way of social transmission is considered.     

Reproductive relationships between annual species ofCalendula (Compositae)

Breeding experiments were carried out inCalendula species. In the annuals, which are selfers, rarely some outcrossing was observed only in the most peripheral flowers. In experimental crosses fruit was produced in all combinations. Fertile F₁ and F₂ hybrids could be grown from crosses between parents with similar chromosome numbers:C. palaestina ×C. pachysperma and crosses of different morphological forms ofC. arvensis. In crosses of species with different chromosome numbers at least partly fertile F₁ hybrids were obtained fromC. tripterocarpa ×C. stellata andC. tripterocarpa ×C. arvensis and crosses of the latter withC. palaestina. Fertile F₂ plants were grown from the combination ofC. arvensis ×C. tripterocarpa. Considering this information and previously obtained data, a scheme is proposed for explaining speciation in the genusCalendula.     

Production of the mosquito-parasitic fungus,Coelomomyces dodgei,through synchronized infection and growth of the intermediate copepod host,Cyclops vernalis

High yields of the copepodCyclops vernalis infected with the mosquito-parasitic fungusCoelomomyces dodgei Couch & Dodge were obtained by infecting nauplii in large synchronously developing populations. Exposure of 2000 48 or 72 h old nauplii to 6×10³ sporangia at the time of meiospore release yielded ca. 1500 infected copepods. Based on yields of infected copepods, susceptibility ofC. vernalis toC. dodgei decreased as copepods developed. Infection rates were 75% for copepods exposed as 48 or 72 h old nauplii but declined to 32 and 9.6%, respectively, for those exposed as copepodids or adults. The relevance of these results for domestication of other species ofCoelomomyces and studies on non-target organisms is discussed, and improved procedures for routine production ofC. dodgei are described.     

Interspecific hybridization in the grain legumes—a review

The current position regarding reports in the literature on interspecific hybridization in the pulses is confused. Two major reasons can he advanced in explanation. The first is that frequently conspecific wild and cultivated forms have received different binomials and crosses between such are frequently regarded as interspecific hybrids. The second arises from mistaken interpretation of results of attempted interspecific hybridization when progeny strongly resembling the maternal parent are produced. It is suggested that these progeny might have been produced by failures in emasculation or rare accidental apomixis. All cultigens of the generaArachis, Cajanus, Cicer, Phaseolus andPisum are able to some extent to produce viable true interspecific hybrids as are the Asiatic forms ofVigna. True interspecific hybrids have not been produced withVicia faba nor withVigna unguiculata the cowpea.     

Streptosporangium subfuscum sp. nov., isolated from the rhizosphere of marigold (Tagetes erecta L.)

A Gram-stain positive, filamentous bacterial strain, designated strain NEAU-TWSJ13^T, was isolated from the rhizosphere of a marigold (Tagetes erecta L.) plant collected in Heilongjiang Province, northeast China, and characterized using a polyphasic approach. The strain was observed to form abundant aerial hyphae differentiated into spherical sporangia. 16S rRNA gene sequence similarity studies showed that strain NEAU-TWSJ13^T belongs to the genus Streptosporangium, being most closely related to Streptosporangium fragile DSM 43847^T (98.6 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that it formed a phyletic line with S. fragile DSM 43847^T, Streptosporangium jomthongense NBRC 110047^T (98.4 % 16S rRNA gene similarity) and Streptosporangium violaceochromogenes DSM 43849^T (97.6 % 16S rRNA gene similarity). A combination of DNA–DNA hybridization results and some phenotypic characteristics indicated that strain NEAU-TWSJ13^T can be distinguished from S. fragile DSM 43847^T and S. jomthongense NBRC 110047^T. Moreover, strain NEAU-TWSJ13^T can also be differentiated from S. violaceochromogenes DSM 43849^T and other Streptosporangium species showing high 16S rRNA gene sequence similarity (>98.0 %) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-TWSJ13^T represents a novel species of the genus Streptosporangium, for which the name Streptosporangium subfuscum sp. nov. is proposed. The type strain is NEAU-TWSJ13^T ( = CGMCC 4.7146^T = DSM = 46724^T).