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.     

Constraints on flowering phenology in a phryganic (East Mediterranean shrub) community

The phenological pattern of flowering at the community level was studied in a Greek phryganic ecosystem near Athens for 4 consecutive years. Flowering is strongly seasonal: 80% of the insect-pollinated flora, which consists of 133 species, blooms between February and June. There is a variably expressed secondary flowering period in autumn. The pollinating fauna follows a strongly correlated pattern of abundance. Two types of plants were distinguished: pauciflorous species bearing <10 flowers that are large compared to the plant body, and multiflorous species with many small flowers. Pauciflorous species flower in the winter half of the year, while multiflorous species flower mainly in the summer half. The mean flower life spans are 9 and 3 days, respectively. The duration of flowering (DF) for each species is 55 days on average, which is long compared to other communities. The DF shows year-to-year variations, concomitant with the vicissitudes of the climate. The start of flowering of a species is statistically correlated with the temperature in the previous month, not with rainfall; its end date of flowering only partly compensates for the time gained or lost. DF is maximal in winter. The average flower life span of species flowering at any given date varies strongly and independently of the average DF. We tested the hypothesis that flowering phenology is set by phylogenetic and life form constraints. This could not be corroborated for phylogeny, evidently because of the overriding influence of the mediterranean climate, and probably also for biogeographical reasons. In contrast, life forms and multiflorous and pauciflorous species show strong differences. Many (51) of the species are therophytes; we tested the hypothesis that because of their annual habit they would be more dependent on pollination than perennials. Thus we anticipated that therophytic species would be differentiated from perennials in their flowering phenologies. This is not corroborated. We therefore conclude that the seed bank plays a role that is analogous to that of a perennial plant body.     

<Emphasis Type="Italic">CER1</Emphasis>gene variations associated with bone mineral density,bone markers,and early menopause in postmenopausal women

Background

Osteoporosis has a multifactorial pathogenesis characterized by a combination of low bone mass and increased fragility. In our study, we focused on the effects of polymorphisms in CER1 and DKK1 genes, recently reported as important susceptibility genes for osteoporosis, on bone mineral density (BMD) and bone markers in osteoporotic women. Our objective was to evaluate the effect of CER1 and DKK1 variations in 607 postmenopausal women. The entire DKK1 gene sequence and five selected CER1 SNPs were amplified and resequenced to assess whether there is a correlation between these genes and BMD, early menopause, and bone turnover markers in osteoporotic patients.

Results

Osteoporotic women seem to suffer menopause 2 years earlier than the control group. The entire DKK1 gene sequence analysis revealed six variations. There was no correlation between the six DKK1 variations and osteoporosis, in contrast to the five common CER1 variations that were significantly associated with BMD. Additionally, osteoporotic patients with rs3747532 and rs7022304 CER1 variations had significantly higher serum levels of parathyroid hormone and calcitonin and lower serum levels of osteocalcin and IGF-1.

Conclusions

No significant association between the studied DKK1 variations and osteoporosis was found, while CER1 variations seem to play a significant role in the determination of osteoporosis and a potential predictive role, combined with bone markers, in postmenopausal osteoporotic women.

     

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.