Gender-dependent differences in biological rhythms of mice

The advantage of a variable's rhythm resides in its optimal time-phasing. This implies that, for a given function, members of a species will strive to exhibit identical time-phasing namely, their inter-individual genetic differences will be masked. To examine the generality of this assumption we explored if inbred mice exhibit gender dependent differences in rhythm parameters of biochemical variables. Male and female mice, entrained by exposure to 12:12 light:dark illumination were sacrificed, every 3 hours over a 27 hours period. Activities of creatine-phosphokinase (CK) and alkaline- phosphatase (AP), white blood cell (WBC) counts and urea nitrogen (UN) concentration were determined at each time point. For each significant rhythm four parameters were computed: period, acrophase, mesor and amplitude. In addition two derived parameters were also calculated: relative-amplitude (RA) and the rate of change in RA (CRA) which provide information about the slope and width of the peak. Patterns of most variables exhibited a compound rhythm containing two significant periodicities. Gender dependent differences were documented in the parameters of most rhythms indicating that the genetic and physiological differences limit to a certain extent the phasing ability of the entraining signals and point to an independent control of each of the rhythm parameters.     

Origin and spread of the 1278insTATC mutation causing Tay-Sachs disease in Ashkenazi Jews: genetic drift as a robust and parsimonious hypothesis

The 1278insTATC is the most prevalent -hexosaminidase A (HEXA) gene mutation causing Tay-Sachs disease (TSD), one of the four lysosomal storage diseases (LSDs) occurring at elevated frequencies among Ashkenazi Jews (AJs). To investigate the genetic history of this mutation in the AJ population, a conserved haplotype (D15S981:175–D15S131:240–D15S1050:284–D15S197:144–D15S188:418) was identified in 1278insTATC chromosomes from 55 unrelated AJ individuals (15 homozygotes and 40 heterozygotes for the TSD mutation), suggesting the occurrence of a common founder. When two methods were used for analysis of linkage disequilibrium (LD) between flanking polymorphic markers and the disease locus and for the study of the decay of LD over time, the estimated age of the insertion was found to be 40±12 generations (95% confidence interval: 30–50 generations), so that the most recent common ancestor of the mutation-bearing chromosomes would date to the 8th–9th century. This corresponds with the demographic expansion of AJs in central Europe, following the founding of the Ashkenaz settlement in the early Middle Ages. The results are consistent with the geographic distribution of the main TSD mutation, 1278insTATC being more common in central Europe, and with the coalescent times of mutations causing two other LSDs, Gaucher disease and mucolipidosis type IV. Evidence for the absence of a determinant positive selection (heterozygote advantage) over the mutation is provided by a comparison between the estimated age of 1278insTATC and the probability of the current AJ frequency of the mutant allele as a function of its age, calculated by use of a branching-process model. Therefore, the founder effect in a rapidly expanding population arising from a bottleneck provides a robust parsimonious hypothesis explaining the spread of 1278insTATC-linked TSD in AJ individuals.Electronic database information: URLs for the data in this article are as follows: , ,     

Differences in rhythms of enzymatic activity of maternal and fetal blood

Blood specimens were obtained at different daily times from the umbilical cord and brachial vein from 53 women within 10 minutes after delivery. Enzyme activities were measured in the white blood cells (WBCs) and serum of each sample. For each enzyme, the results were grouped according to sampling (delivery) times and arrayed to form a 24h time series. Separate time series were generated for maternal and fetal enzymes. Analysis of variance (ANOVA) and cosine best-fit analyses were applied to elucidate significant differences between enzyme activity patterns of mothers and fetuses with regard to time dependency, number of peaks, and acrophases. These and previously documented results indicate that not all mothers and fetuses have rhythms that are concordant. For some enzymes of fetuses, the activity rhythms differ in phase and shape of the time series pattern from those of the mothers; for other enzymes, the activity rhythms develop after birth. (Chronobiology International, 17(2), 221-228, 2000)     

Effect of PhoP-PhoQ activation by broad repertoire of antimicrobial peptides on bacterial resistance

Pathogenic bacteria can resist their microenvironment by changing the expression of virulence genes. In Salmonella typhimurium, some of these genes are controlled by the two-component system PhoP-PhoQ. Studies have shown that activation of the system by cationic antimicrobial peptides (AMPs) results, among other changes, in outer membrane remodeling. However, it is not fully clear what characteristics of AMPs are required to activate the PhoP-PhoQ system and whether activation can induce resistance to the various AMPs. For that purpose, we investigated the ability of a broad repertoire of AMPs to traverse the inner membrane, to activate the PhoP-PhoQ system, and to induce bacterial resistance. The AMPs differ in length, composition, and net positive charge, and the tested bacteria include two wild-type (WT) Salmonella strains and their corresponding PhoP-PhoQ knock-out mutants. A lacZ-reporting system was adapted to follow PhoP-PhoQ activation. The data revealed that: (i) a good correlation exists among the extent of the positive charge, hydrophobicity, and amphipathicity of an AMP and its potency to activate PhoP-PhoQ; (ii) a +1 charged peptide containing histidines was highly potent, suggesting the existence of an additional mechanism independent of the peptide charge; (iii) the WT bacteria are more resistant to AMPs that are potent activators of PhoP-PhoQ; (iv) only a subset of AMPs, independent of their potency to activate the system, is more toxic to the mutated bacteria compared with the WT strains; and (v) short term exposure of WT bacteria to these AMPs does not enhance resistance. Overall, this study advances our understanding of the molecular mechanism by which AMPs activate PhoP-PhoQ and induce bacterial resistance. It also reveals that some AMPs can overcome such a resistance mechanism.     

The stroke trial - can we predict clinical outcome of patients with ischemic stroke by measuring soluble cell adhesion molecules (CAM)?

BACKGROUND: Several studies have found that an increased concentration of haemostatic or inflammation markers was associated with worse prognosis in vascular disease. The inflammatory components in ischemic stroke are of current interest, and there is some experimental evidence that they may be linked. HYPOTHESIS: The study was performed to determine the association between the neurological clinical outcome and levels of cell adhesion molecules in the first four days of hospitalization in patients with acute ischemic event. METHODS: This prospective, pilot, case-controlled study examined the association between the clinical outcome and inflammatory markers within the first few days of hospitalization. The neurological evaluation was performed using the NIH score on admission and four days later, and levels of cell adhesion molecules were measured by ELISA methods on admission and four days later. RESULTS: Twenty three patients with an acute cerebral event (mean age 71 +/- 15 y, 12 women and 11 men) were examined neurologically on admission and four days later. Among 19 patients who improved, there was a significant decrease in the NIH neurological scale, from 3.8 +/- 3.2 to 1.3 +/- 1.8 (p = 0.01), which was accompanied by a significant decrease in the cell adhesion molecules that were measured (E-selectin, ICAM-1 and VCAM-1). Of the four patients who did not improve, their mean clinical NIH score was 10 +/- 4.6 and worsened or remained unchanged after four days of follow-up. In this group, we could not demonstrate a significant change in levels of cell adhesion molecules between days one and four. CONCLUSIONS: Patients who improved clinically within the first four days of hospitalization demonstrated a remarkable inhibition of all three cell adhesion molecules that were measured (E-selectin, ICAM-1, and VCAM-1). Patients who did not improve had more severe cerebral infarcts, a higher NIH score on admission (10 +/- 4.6), and no change was observed in levels of cell adhesion molecules during the follow-up period. Measuring cell adhesion molecule levels may predict objectively the clinical outcome in hospitalized patients with acute ischemic stroke.     

Amalgam, an axon guidance Drosophila adhesion protein belonging to the immunoglobulin superfamily: over-expression, purification and biophysical characterization

Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH₂-terminal His₆Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high β-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with R_H = 16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin.