Allium cepa L. Cultivars from Four Continents Compared by Flow Cytometry show Nuclear DNA Constancy

In 1965 Van't Hof estimated the nuclear DNA amount of an unidentifiedAllium cepa L. cultivar as 2C = 33.55 pg (Experimental CellResearch39: 8–58). This value has been adopted by commonusage as the main calibration standard for angiosperm DNA C-valueestimations. However, different cultivars have been used whileassuming species DNA C-value constancy. Surprisingly this assumptionhas never been tested. A. cepa is an outbreeder with telomericheterochromatic segments, so intraspecific variation in C-value,possibly correlated with environmental factors as seen in Zeamays L., might be expected. We used laser flow cytometry tocompare nuclear DNA amounts in roots of six A. cepa cultivarsused as calibration standards or from different environments.Tissues from one cultivar, or similar volumes of tissue fromtwo cultivars, were run and the variance between nuclei in 2Cpeaks compared. Only one shoulderless 2C peak was seen for allpairs of co-chopped cultivars. Thus, no large differences inC-value between cultivars from different environments were found.Moreover, comparing cultivars run singly or as pairs showedno evidence for increased variation in 2C peaks in the latter,and hence of critical differences in DNA amounts between ‘AilsaCraig’ and another cultivar. Such variation was insufficientto make their use as alternative calibration standards, or thepractice of imputing Van't Hof's original C-value estimate tothem, unacceptable for most practical purposes. Given the mechanismsknown which can generate genome size variation, the degree ofconstancy in DNA C-value found seems remarkable. Copyright 2000Annals of Botany Company Allium cepa, onion cultivars, calibration standards, DNA C-value constancy, flow cytometry     

Multicenter evaluation of the performance and clinical utility in longitudinal monitoring of the Bayer Immuno 1 complexed PSA assay.

We conducted a multicenter evaluation of the analytical and clinical performance of the automated Bayer Immuno 1 complexed PSA (cPSA) assay, and compared assay performance to the Bayer Immuno 1 PSA assay. We sought to determine whether measurements of cPSA could be of clinical utility in the management of patients with prostate cancer. Results of the 10-day imprecision across three evaluation sites produced total CV < 2.50% and an analytical sensitivity of 0.02 microgram/L. There was an increased trend in clinical sensitivity for prostate cancer with increasing stage of disease (71-86%). Clinical specificity for patients with benign urogenital disease was 74.8%, and for other nonprostate diseases ranged from 91.1-100%. Retrospective serial monitoring of 155 patients with prostate cancer demonstrated concordance of cPSA measurements to clinical status for 97% of the patients analyzed. Results from the clinical studies using the Bayer Immuno 1 cPSA assay were comparable to results obtained with the Bayer Immuno 1 PSA assay. The Bayer Immuno 1 cPSA assay demonstrates analytical performance and clinical effectiveness in the management of prostate cancer patients during the course of disease and therapy.     

Delayed nucleoid segregation in Escherichia coli

To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry. In pbpB(Ts) and ftsZ strains growing as filaments at 42 degrees C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild-type cells. Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers. From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D' period (time between termination and nucleoid separation). Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28 degrees C) showed a long D' period (42 min versus 18 min in the parent) indicative of an extended segregation time. The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation. We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.     

N-Methyl-D-Aspartate receptors in acquisition of the short-term memory in the honeybeeApis mellifera

Role of NMDA receptors in the process of associative learning has been studied in the honeybeeApis mellifera L. in behavior experiments, using method of conditional reflexes. To determine pharmacological profile of NMDA receptors, effects of Mg²⁺ ions, NMDA, glycine (Gly), antagonist of the glycine site 5,7-dichlorokynurenic acid (DCK), competitive antagonists of NMDA receptors: D,L-2-aminophosphovalerate (APV), L-2-aminophosphobutyrate (APB), and D-glutamyl-aminomethylphosphonic acid (GAMP) as well of antagonists of NMDA receptor ion channels MK-801 and ketamine (Ket) were studied on acquisition of alimentary conditional reflex and its retention in memory. NMDA increased capacity for learning by stimulating shortterm memory. The NMDA receptor co-agonist Gly activated this NMDA effect. DCK eliminated the NMDA and Gly stimulatoty effects. All tested antagonists at millimolar concentrations inhibited associative function. The data obtained confirm our hypothesis about participation of NMDA receptors in processes of formation of short-term memory in the honeybeeApis mellifera and suggest that functional characteristics of the NMDA receptors involved in the process of associative learning in the honeybee resemble those in mammals.     

New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS.

The genetics of resistance to infection by HIV-1 cohort consists of 200 slow and 75 rapid progressors to AIDS corresponding to the extremes of HIV disease outcome of 20,000 Caucasians of European descent. A comprehensive analysis of HLA class I and class II genes in this highly informative cohort has identified HLA alleles associated with fast or slow progression, including several not described previously. A quantitative analysis shows an overall HLA influence independent of and equal in magnitude (for the protective effect) to the effect of the CCR5-Delta32 mutation. Among HLA class I genes, A29 (p = 0.001) and B22 (p < 0.0001) are significantly associated with rapid progression, whereas B14 (p = 0.001) and C8 (p = 0.004) are significantly associated with nonprogression. The class I alleles B27, B57, C14 (protective), and C16, as well as B35 (susceptible), are also influential, but their effects are less robust. Influence of class II alleles was only observed for DR11. These results confirm the influence of the immune system on disease progression and may have implications on peptide-based vaccine development.     

Improved microdialysis technique.

A simple, inexpensive method is described for dialysis of microliter amounts of aqueous samples against large volumes of solution with complete recovery of the fluid dialyzed. An example is given of application of the method to separation of [³H]inulin from a monosaccharide.     

Melanocortin‐1 receptor structure and functional regulation

The melanogenic actions of the melanocortins are mediated by the melanocortin‐1 receptor (MC1R). MC1R is a member of the G‐protein‐coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or α‐melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist‐independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.     

Determining the kinetics of membrane pores from patch clamp data without measuring the open and closed times

We present a new and very time saving way to determine kinetic rate constants from patch clamp data by using the correlation functions utilized in analyzing experiments with photon correlations.