Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by differential scanning calorimetry

The cyclic AMP receptor protein (CRP) regulates the expression of many genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. In this study, we have used differential scanning calorimetry (DSC) and circular dichroism (CD) to measure the enthalpy change and melting temperature of the apo-CRP and CRP complexes with cAMP or DNA sequences lac, gal, and palindromic ICAP. DSC and CD measurements showed irreversible thermal denaturation process of CRP. Enthalpy of dissociation of the protein–DNA complex, as measured by DSC, depends on the DNA sequence. The thermal transition of the protein in CRP-DNA complexes, measured by CD, indicates that the protein stability in the complex is also DNA sequence-dependent.     

A new look at the cellular scaffold by embedment-free electron microscopy method

The basic scaffold of most cells is afforded by the cytoskeleton (comprising microfilaments, intermediate filaments and the microtubules). The conventional methods of electron microscopy fail to visualize filamentous cell structure. They can show only these filaments lying at the section surface. Heavy metal staining (I), and the optical properties of the resins used for embedding are similar to those of proteins hence most proteinaceous structures remain unresolved and the cytoplasm seems to be quite homogenous (II). Aldehyde fixation could cross-link proteins and lead to the emergence of artificial structures (III). These limitations may be overcome by the use of the embedment-free electron microscopy (EF-EM). This technique present cellular scaffold as a purified, isolated, three-dimensional network with various thickness of filaments. Our study on the dynamic aspect of cellular scaffold indicate that the thickness and arrangement of filaments depend on cell type and both physiological or pathological environments. Thank also to the adaptation of immunocytochemistry to EF-EM it was possible to understand the nuclear matrix and cytomatrix structure in relation to function. Thus, combination these methods revealed findings suggesting the nuclear homing of proapoptotic proteins and their association with intermediate filaments.     

Morphological diversity of androgenic carrot plants

The aim of this study was to evaluate phenotypic variation of R0 androgenic plants obtained from four seed sources and donor plants by anther culture. Several morphological traits (leaf size, petiole length, leaf division, cortex colour) and the range of diversity were evaluated. There was large variation in all traits among the donor varieties. Especially leaf division and cortex colour differed significantly among the androgenic plants that came from different seed sources. The plants regenerated from four donor plants of variety 62 were significantly different in most traits except for leaf width and cortex colour. Evaluation of R1 plants will demonstrate whether the R0 variation observed is due to genetic variation or physiological differences from tissue culture.     

The toxin-antitoxin system of the streptococcal plasmid pSM19035

pSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-number plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-positive bacteria. We show here that the omega-epsilon-zeta operon of this plasmid constitutes a novel proteic plasmid addiction system in which the epsilon and zeta genes encode an antitoxin and toxin, respectively, while omega plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-positive Bacillus subtilis and bacteriostatic for the gram-negative Escherichia coli. The toxic effects of zeta gene expression in both bacterial species are counteracted by proper expression of epsilon. The epsilon-zeta toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.     

Tonic component of myocardial contraction

Calcium transients and contractions of cardiac myocytes consist of phasic component, relaxing spontaneously independently of membrane voltage and of the tonic component (TC) relaxing only upon repolarization. Experimental data reviewed in this article suggest that most Ca(2+) activating TC is released from sarcoplasmic reticulum (SR) via the ryanodine receptors (RyRs). Most likely these RyRs are activated by sustained Ca(2+) influx. However, its route may differ depending on species and state of the cells. It seems that in rat RyRs responsible for TC are activated by the sustained Ca(2+) current. In guinea-pig the blockers of Ca(2+) current or reverse mode Na(+)/Ca(2+) exchange do not inhibit TC, so these routes seem unlikely. In myocytes of the failing human hearts TC is activated mostly via the reverse mode Na(+)/Ca(2+) exchange and contribution of SR is negligible. The mechanism of TC in the normal human cardiomyocytes has not been investigated. Thus, despite investigation of TC for half a century many problems concerning the mechanism of its activation and maintenance as well as its physiological meaning remain unsolved.     

The activity of alpha1,6-fucosyltransferase during human megakaryocytic differentiation

alpha1,6-Fucosyltransferase (6FucT, E.C. 2.4.1.68) is one of the enzymes involved in the synthesis of N-linked glycans of the GpIIb/IIIa complex (CD41a) which is present on megakaryocytes (MKs) and platelets. In this study, we examined 6FucT activity in ex vivo cultures of immunoselected cord blood CD34(+) cells grown in a medium promoting megakaryocytopoiesis. Our results show that the activity of 6FucT increased ahead of, and thereafter concomitantly with, cells expressing the CD41a antigen. When the CD41a(+) subpopulation of cells was immunoselected (using anti-CD61 i.e. anti-GpIIIa antibodies), its 6FucT activity increased proportionally to the yield of CD61(+)(+)(+) cells. Taking into account the heavy load of 6FucT in platelets and megakaryocytes, we regard this enzyme as a candidate for the earliest marker of MK-commitment in cultured hematopoietic stem cells. Such a marker should allow an earlier detection and earlier transplantation of patients' own, ex vivo expanded, Mk progenitors.     

The impact of neuraminidase on apoptosis in cultures of blood lymphocytes isolated from rats bearing morris hepatoma

Lymphocytes were obtained by heart-punction from rats bearing Morris hepatoma. In the short term, 18-hour cultures of these lymphocytes exhibited a significantly higher amount of apoptotic cells than lymphocyte cultures from the healthy, control animals. Neuraminidase, injected into the caudal vein of the rats with Morris hepatoma, caused a marked lowering in the amount of apoptotic blood-lymphocytes and an elevation of the amount of viable cells. The possible mechanism of neuraminidase preventing the apoptosis of blood-circulating lymphocytes in tumour hosts is discussed herein.     

Functioning of the TA cassette of streptococcal plasmid pSM19035 in various Gram-positive bacteria

Toxin-antitoxin (TA) systems are common in microorganisms and are frequently found in the chromosomes and low-copy number plasmids of bacterial pathogens. One such system is carried by the low copy number plasmid pSM19035 of the pathogenic bacterium Streptococcus pyogenes. This plasmid encodes an omega-epsilon-zeta cassette that ensures its stable maintenance by post-segregational killing of plasmid-free cells. In this study, the activity of the ω-ε-ζ cassette was examined in various Gram-positive bacteria with a low G/C content in their DNA. The broad host range of pSM19035 was confirmed and the copy number of a truncated derivative in transformed strains was determined by real-time qPCR.     

MTHFR 677T is a strong determinant of the degree of hearing loss among Polish males with postlingual sensorineural hearing impairment

Hearing impairment (HI) is the most common sensory handicap. Congenital HI often has a genetic basis, whereas the etiology of nonsyndromic postlingual HI (npHI) usually remains unidentified. Our purpose was to test whether the MTHFR C677T (rs1801133) polymorphism affecting folate metabolism is associated with the occurrence or severity of npHI. We studied rs1801133 genotypes in 647 npHI patients (age <40, sudden sensorineural loss excluded, HI characterized as mean of better ear hearing thresholds for 0.5-8 kHz) and 3273 adult controls from the background population. Genotype distribution among patients and controls was similar, but among male cases (n = 302) we found a dose-dependent correlation of MTHFR 677T with the degree of HI (mean thresholds in dB: 38.8, 44.9, and 53.3, for CC, CT, and TT genotypes, respectively; p = 0.0013, p(cor.) = 0.017). Among male patients rs1801133 TT significantly increased the risk of severe/profound HI (odds ratio = 4.88, p = 0.001). Among controls the known effect of MTHFR 677T on plasma total homocysteine was more pronounced in men than in women (p<0.00004 for genotype-sex interaction) suggesting that in Poland folate deficiency is more prevalent in males. In conclusion, we report a novel strong effect of MTHFR 677T among males with npHI. The functional significance of rs1801133 suggests that these patients may benefit from folate supplementation-an intervention which is simple, cheap, and devoid of side effects.