Studies of the silencing of baculovirus DNA binding protein

Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P10 and GP64 was unaffected. In the absence of DBP, viral DNA replication sites were formed, indicating replication of viral DNA. Electron microscopy studies, however, revealed a loss of formation of polyhedra and virus envelopment, suggesting that the primary role of DBP is viral formation rather than viral DNA replication.     

Innervation pattern of suboesophageal ventral unpaired median neurones in the honeybee brain

In honeybees (Apis mellifera), the biogenic amine octopamine has been shown to play a role in associative and non-associative learning and in the division of labour in the hive. Immunohistochemical studies indicate that the ventral unpaired median (VUM) neurones in the suboesophageal ganglion (SOG) are putatively octopaminergic and therefore might be involved in the octopaminergic modulation of behaviour. In contrast to our knowledge about the behavioural effects of octopamine, only one neurone (VUMmx1) has been related to a behavioural effect (the reward function during olfactory learning). In this study, we have investigated suboesophageal VUM neurones with fluorescent dye-tracing techniques and intracellular recordings combined with intracellular staining. Ten different VUM neurones have been found including six VUM neurones innervating neuropile regions of the brain and the SOG exclusively (central VUM neurones) and four VUM neurones with axons in peripheral nerves (peripheral VUM neurones). The central VUM neurones innervate the antennal lobes, the protocerebral lobes (including the lateral horn) and the mushroom body calyces. Of these, a novel mandibular VUM neurone, VUMmd1, exhibits the same branching pattern in the brain as VUMmx1 and responds to sucrose and odours in a similar way. The peripheral VUM neurones innervate the antennal and the mandibular nerves. In addition, we describe one labial unpaired median neurone with a dorsal cell body, DUMlb1. The possible homology between the honeybee VUM neurones and the unpaired median neurones in other insects is discussed. This work was supported by the DFG ME 365/24-2.     

Tropheryma whipplei in the Environment: Survey of Sewage Plant Influxes and Sewage Plant Workers

We studied the prevalence of Tropheryma whipplei in influxes to 46 sewage treatment plants and in stool, mouthwash fluids, and dental plaques of 64 healthy workers in those facilities and 146 disease control patients. T. whipplei was found in sewage water, in stool of healthy individuals, and significantly more often in stool of workers exposed to sewage water.     

Y chromosome polymorphism in various breeds of cattle (<Emphasis Type="Italic">Bos taurus</Emphasis>) in Switzerland

The evolutionary development of mammals involves mutations and fixations of chromosomal types. The Y chromosome polymorphism in cattle is important for the breeding strategy, since chromosomal incompatibilities in crossings result in fertility problems. In bulls of various breeds in Switzerland, data on chromosome status have been collected for over 20 years. Data from 7 years were analysed in this study through chromosome measurements and their normalization. Some highly significant differences were found between the 7 groups of breeds, especially between Holsteins and the original Swiss breeds Braunvieh and Simmental. Fleckvieh (purebred or crossbred) did not differ significantly from Black or Red Holsteins. The results were discussed with respect to fertility problems. The observed Y chromosome polymorphism should be taken into account in breeding, and research in this field should be continued.     

Functional interactions between Prp8, Prp18, Slu7, and U5 snRNA during the second step of pre-mRNA splicing

After the second transesterification step of pre-mRNA splicing, the Prp22 helicase catalyzes release of spliced mRNA by disrupting contacts in the spliceosome that likely involve Prp8. Mutations at Arg1753 in Prp8, which suppress helicase-defective prp22 mutants, elicit temperature-sensitive growth phenotypes, indicating that interactions in the spliceosome involving Prp8-R1753 might be broken prematurely at 37 degrees C. Here we report that mutations in loop I of the U5 snRNA or in Prp18 can suppress the temperature-sensitive prp8-R1753 mutants. The same gain-of-function PRP18 alleles can also alleviate the growth phenotypes of multiple slu7-ts mutants, indicating a functional link between Prp8 and the second step splicing factors Prp18 and Slu7. These findings, together with the demonstration that changes at Arg1753 in Prp8 impair step 2 of pre-mRNA splicing in vitro, are consistent with a model in which (1) Arg1753 plays a role in stabilizing U5/exon interactions prior to exon joining and (2) these contacts persist until they are broken by the helicase Prp22.     

Erythropoietin in Cerebrospinal Fluid: Age-related Reference Values and Relevance in Neurological Disease

We aimed to establish age-related reference values for Erythropoietin (EPO) in cerebrospinal fluid (CSF) and to evaluate concentrations in neurological diseases. CSF and serum EPO was measured in controls with tension-type headache (CTTH), in patients with ALS, dementia and depression using ELISA technique. Stability experiments showed CSF EPO to be stable for two and a half months and over two thaw/freeze cycles. A positive correlation of CSF EPO with age was found (P < 0.01). We found a CSF/serum EPO concentration ratio of 0.126, pointing towards an intrathecal synthesis of EPO. The ALS group showed significantly lowered CSF EPO compared to age-matched CTTH (P < 0.012), whereas the dementia and depression group showed no significant differences compared to CTTH. The establishment of age-related reference values in a large cohort of controls will improve the interpretation of future CSF EPO evaluations in neurological diseases. The authors have not reported any conflicts of interest.     

Immunogold labeling of cryosections from high-pressure frozen cells

Immunogold labeling of cryosections according to Tokuyasu (Tokuyasu KT. A technique for ultracyotomy of cell suspensions and tissues. J Cell Biol 1973;57:551–565), is an important and widely used method for immunoelectron microscopy. These sections are cut from material that is chemically fixed at room temperature (room temparature fixation, RTF). Lately in many morphological studies fast freezing followed by cryosubstitution fixation (CSF) is used instead of RTF. We have explored some new methods for applying immunogold labeling on cryosections from high‐pressure frozen cells (HepG2 cells, primary chondrocytes) and tissues (cartilage and exocrine pancreas). As immunolabeling has to be carried out on thawed and stable sections, we explored two ways to achieve this: (1) The section fixation method, as briefly reported before (Liou W et al. Histochem Cell Biol 1996;106:41–58 and Möbius W et al. J Histochem Cytochem 2002;50:43–55.) in which cryosections from freshly frozen cells were stabilized in mixtures of sucrose and methyl cellulose and varying concentrations of glutaraldehyde, formaldehyde and uranyl acetate (UA). Only occasionally does this method reveal section areas with excellent cell preservation and negatively stained membranes like Tokuyasu sections of RTF material. (Liou et al.) (2) The rehydration method, a novel approach, in which CSF with glutaraldehyde and/or osmium tetroxide (OsO₄) was followed by rehydration and cryosectioning as in the Tokuyasu method. Especially, the addition of UA and low concentrations of water to the CSF medium favored superb membrane contrast. Immunogold labeling was as efficient as with the Tokuyasu method.