The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

The water frogs of Greece: Bioacoustic evidence for a new species

The structure of the mating call of lake frogs (referred to as R. ridibunda) from 16 populations in Greece was analyzed for local variation using multivariate statistics. The populations of Thrace and of the island of Samothraki form a group giving the same type of mating call, whereas the mating call of the other populations differs in the degree of temperature dependence of four parameters, and specifically in the number of pulses/pulse group and pulse groups/call. Discriminant functions distinguish even single call series with a probability of 97%, intermediate mating calls are absent, and there is a significant, but slight differentiation of external morphological characters. These results have strong taxonomic implications. We conclude that the lake frogs of Greece comprise two species. The mating call of the lake frogs from Thrace resembles in all parameters that of the Rana ridibunda in the terra typica restricta (Guryev, CIS). Accordingly, the lake frogs of eastern Greece belong to R. ridibunda. The mating call of these lake frogs consists of 20 pulses/pulse group and of 7 pulse groups/call on the average. Most of Greece is inhabited by the second taxon, Rana balcanica sp. n. Its mating call is characterized by 27 pulses/pulse group and 4 pulse groups/call on the average. The two species in Greece do not differ with respect to coloration and size, but several standardized indices vary significantly: body length/digitus primus length; body length/callus internus length; body length/snout-eye distance; body length/tympanum diameter; tibia length/callus internus length; maximal head width/snout-eye distance.     

Multiple stressors on biotic interactions: how climate change and alien species interact to affect pollination

Global change may substantially affect biodiversity and ecosystem functioning but little is known about its effects on essential biotic interactions. Since different environmental drivers rarely act in isolation it is important to consider interactive effects. Here, we focus on how two key drivers of anthropogenic environmental change, climate change and the introduction of alien species, affect plant–pollinator interactions. Based on a literature survey we identify climatically sensitive aspects of species interactions, assess potential effects of climate change on these mechanisms, and derive hypotheses that may form the basis of future research. We find that both climate change and alien species will ultimately lead to the creation of novel communities. In these communities certain interactions may no longer occur while there will also be potential for the emergence of new relationships. Alien species can both partly compensate for the often negative effects of climate change but also amplify them in some cases. Since potential positive effects are often restricted to generalist interactions among species, climate change and alien species in combination can result in significant threats to more specialist interactions involving native species.     

Monitoring and manipulation of the pH of single cells using infrared spectromicroscopy and a molecular switch

Background

The pH of a biological system is a crucial determinant of the structures and reactivity of its components and cellular homeostasis of H⁺ is critical for cell viability. Control and monitoring of cellular acidity are highly desirable for the purpose of studying biochemical processes in vivo.

Methods

The effect of photolysis of a caged strong acid, the ester 1-(2-nitrophenyl)-ethylhexadecyl sulfonate (HDNS) is used to cause a controlled drop in pH in single cells. An isolated cell is selected under the IR microscope, irradiated with near-UV light and monitored by FTIR.

Results

We demonstrate the use of FTIR spectromicroscopy to monitor light-induced acidification of the cellular medium by measuring the increased concentration of CO₂ and corresponding decrease of HCO₃⁻ in the cell and in the surrounding medium.

Conclusions

We have demonstrated a method to control and accurately monitor the changes in pH of a cellular system by coupling a caged proton-releasing agent with FTIR spectromicroscopy detection. The overall implementation of photolysis and spectroscopic detection in a microscope optical configuration ensures single cell selectivity in both acidification and monitoring. We show the viability of monitoring of pH changes by FTIR spectromicroscopy with sensitivity comparable to that of glass electrodes, better than the existing methods for determining cell pH.

General significance

Reporting the effect of small variations of cellular acidity provides a major improvement in the understanding of the interplay between molecular properties as assessed in vitro and cell physiology.     

Bone morphogenetic proteins (BMPs) expression in the femoral heads of patients with avascular necrosis

Avascular necrosis (AVN) is a disorder of the bone repair process which usually results in femoral head (FH) destruction. Bone morphogenetic proteins (BMPs) are the key proteins regulating bone remodelling and healing. BMPs gene expression levels were analyzed in the normal and necrotic sites of osteonecrotic FHs. Quantitative RT-PCR for BMP-2, -4, -6, -7 genes was performed in bone tissue samples from 47 osteonecrotic FHs. Protein levels of BMP-2, -4, -6 were estimated by Western Blot. Statistical analysis was performed using the Wilcoxon signed rank test. BMP-2 and BMP-6 mRNA levels were higher in the normal than the necrotic site (normal/necrotic: 16.8/6.8 and 1.75/1.64, respectively). On the contrary, BMP-4 mRNA levels were higher in the necrotic (0.75) than the normal (0.62), while BMP-7 mRNA levels were extremely low. At the protein level, BMP-2 continued to have a higher expression in the normal region (normal/necrotic: 0.67/0.64). BMP-4 and -6 were detected at higher levels in the necrotic site (normal/necrotic: 0.51/0.61 for BMP-4, 0.51/0.56 for BMP-6), while BMP-7 was not detectable. Different BMP levels between the normal and necrotic site, as well as discrepancies between the gene and protein expression pattern suggest a different regulation mechanism for BMPs between the two regions of FHs. The understanding of the expression pattern and the correlation of BMPs could lead to a more successful use in the prevention and treatment of AVN.     

The Soluble Interleukin-6 Receptor Is a Mediator of Hematopoietic and Skeletal Actions of Parathyroid Hormone

Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6^+/+ and IL-6^−/− mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6^−/− compared with IL-6^+/+ bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6^−/− earlier than IL-6^+/+ marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6^−/− marrow. In the skeletal system, although intermittent PTH administration to IL-6^+/+ and IL-6^−/− mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-β1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.