Influence of the human blood serum on contractility and β-adrenoreactivity of the isolated human myocardium

Influence of adrenaline (10⁻⁹ to 10⁻⁴ g/ml) on the contraction amplitude caused by electrostimuli (1Hz, 5 ms, 25–30 V) and inotropic and adrenomodulation activities of blood serum of nonpregnant women (at dilutions of 1 : 10 000, 1 : 1000, 1 : 500, 1 : 100, 1 : 50, 1 : 10, and 1 : 5) have been studied. The study has been carried out on isolated myocardium strips of the right atrial auricle that were taken from 43 patients with ischemic illness of the heart and 9 patients with valvular heart diseases of various etiologies upon venous cannula insertion during an aortocoronary bypass. Direct dependence of the contraction amplitude on the cardiac output according to Teicholz has been found. This meant that strips of the right atrial auricle reflected the contractility of the left ventricle myocardium. Adrenaline has been shown to dose-dependently increase the amplitude of evoked contractions in the concentration interval from 10⁻⁷ to 10⁻⁶ g/ml and had no influence from 10⁻⁹ to 10⁻⁸ g/ml (dissociation constant, 2 × 10⁻⁷ g/ml), which proved a decrease in the β-adrenoreceptor’s (β-AR) activation. Blood serum in a dilution range from 1 : 10 000 to 1 : 50 had no effect on the contraction amplitude, but an enhanced effect has been found in a dilution range from 1 : 10 and 1 : 5. The presence of the endogenous activator of myocytes contractility (EAMC) has explained this enhanced effect. The β-adrenomodulation activity of blood serum has been explained by the presence of the endogenous sensitizer of β-AR (ESBAR) and the endogenous blocker of β-AR (EBBAR). The ESBAR activity of blood serum (dilutions: 1 : 1000, 1 : 500, 1 : 100, and 1 : 50) has been found in experiments with a subthreshold adrenaline concentration (10⁻⁸ g/ml). ESBAR (dilutions: 1 : 50 and 1 : 10) and EBBAR (dilution 1 : 500) activities of blood serum have been found in experiments with the maximum effective concentration of adrenaline (10⁻⁶ g/ml). Therefore, blood serum endogenous modulators of β-adrenergic reactivity, ESBAR and EBBAR, can modulate the activation of β-AR of human cardiomyocytes. These prove the prospects of the ESBAR analogue application in cardiology.     

Ukhtomski? and the nature of the human "ego"]

A. Ukhtomski? is one of those Russian naturalists of the beginning of the 20th century (K. Tsiolkovski?, V. Vernadski?, K. Timiriazev, I. Pavlov), whose scientific work is characterized by a combination of the valuable contribution to a specific field of science and deep philosophical conceptualizations. Ukhtomski? enriched modern neurophysiology by the ideas on dominant--a stable focus of enhanced excitability, determining the organism reactions to the environmental stimuli. Unlike artificial experimental models, natural dominant--vector of the goal-directed behaviour--represents the needs of immediate satisfaction, dominating at the present moment. Formation of needs hierarchy, characteristic of the given personality, occurs during the process of people communication. Namely, another man serves to the subject a mirror, looking into which he realizes himself as a human being (K. Marx). By his teaching on the dominant, Ukhtomski? has revealed the dual nature of the personality's self-consciousness in the process of its perception of the surrounding people. He showed that involuntary projection of its dominating vital and social needs (motives, intentions) to the image of another man makes the latter only the "double" of the observer, which does not promote the development and enrichment of personality. Actually productive is only the perception which is motivated by the ideal need of learning another personality, sincere interest in him, what makes another person a "well-deserved colocutor", a source of new knowledge about people and himself. The ideas of Ukhtomski? on the nature of human "self" are close to ideological searchings of L. Tolsto? and F. Dostoevski?.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of the human γ-secretase activating protein

Alzheimer's disease is a fatal neurological disorder that is a leading cause of death, with its prevalence increasing as the average life expectancy increases worldwide. There is an urgent need to develop new therapeutics for this disease. A newly described protein, the γ-secretase activating protein (GSAP), has been proposed to promote elevated levels of amyloid-β production, an activity that seems to be inhibited using the well-establish cancer drug, imatinib (Gleevec). Despite much interest in this protein, there has been little biochemical characterization of GSAP. Here we report protocols for the recombinant bacterial expression and purification of this potentially important protein. GSAP is expressed in inclusion bodies, which can be solubilized using harsh detergents or urea; however, traditional methods of refolding were not successful in generating soluble forms of the protein that contained well-ordered and homogeneous tertiary structure. However, GSAP could be solubilized in detergent micelle solutions, where it was seen to be largely α-helical but to adopt only heterogeneous tertiary structure. Under these same conditions, GSAP did not associate with either imatinib or the 99-residue transmembrane C-terminal domain of the amyloid precursor protein. These results highlight the challenges that will be faced in attempts to manipulate and characterize this protein.     

Origin and fate of the nucleolar channel system of normal human endometrium

Human normal endometrium was examined in ultrathinsections.Nucleolar channel system(NCS)appeared in theendometrial epithelial cells during the early and mid secre-tory phase of menstrual cycle.The NCS was a hollow balllike structure of different sizes and was composed of 2 to 5rows of tubules embedded in an amorphous matrix.On itssurface there were numerous electron dense particles resem-bling ribosomes.It was usually located within or associatedwith the nucleolus.Sometimes,it was close to the nuclearenvelope or protruding out from the nucleus.On occasion,NCS with simplified structure was found in the perinuclearcytoplasm.Concepts concerning the genesis,involution andfunction(s) of the NCS were discussed.     

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (R_P/S_P). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (k_on = 174 M^?1 s^?1) was ca. 8-fold more potent than the d-isomer (k_on = 21.5 M^?1 s^?1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be S_P. The S_P-isomers inhibited human GGT (k_on = 21.5–174 M^?1 s^?1), while the R_P-isomers were inactive even at concentrations of 0.1 mM.     

Structure and function of the N‐terminal domain of the human mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti‐/pro‐apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N‐terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake‐1 and mitochondrial calcium uptake‐2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca²⁺ uptake in a stable MCU knockdown HeLa cell line and exerted dominant‐negative effects in the wild‐type MCU‐expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.     

Do age and extended culture affect the architecture of the zona pellucida of human oocytes and embryos?

Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, > 38 years; younger, < or = 38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean +/- SD: 24.1 +/- 2.5 microm vs 23.1 +/- 3.3 microm; p = 0.01) but ZP density was equal (2.8 +/- 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 +/- 2.2 microm vs 21.7 +/- 1.6 microm; p = 0.28) and density (2.9 +/- 0.7 nm vs 2.8 +/- 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 +/- 2.7 microm vs 19.1 +/- 5.0 microm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 +/- 0.5 nm vs 3.3 +/- 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 +/- 0.5 nm vs 2.9 +/- 0.7 nm, p < 0.001; younger: 3.3 +/- 1.0 nm vs 2.8 +/- 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.     

Physicochemical measurement of the base composition of mRNA-related sequences of the human α and β globin genes

Hybrids formed between human and globin cDNA and total human cellular DNA have been studied by thermal denaturation and cesium chloride density gradient centrifugation. From these studies, the weight average G+C content of human globin cDNA has been determined to be 62%±2% and that of human globin cDNA 51%±2%. These values correlate well with the results of G+C content of the human and globin cDNAs as determined by direct nucleotide sequence analysis of the cDNAs. Thermal denaturation and cesium chloride density gradient centrifugation of DNA-cDNA hybrids can therefore provide accurate information on the base composition of mRNA related sequences of any single copy gene for which a relatively pure cDNA can be obtained, without the necessity for direct nucleotide sequence analysis.     

Evidence of homology between the β-chain of human haptoglobin and the chymotrypsin family of serine proteases

Amino acid sequences from the β-chain of human haptoglobin are compared with those sequences known for the serine proteases of the chymotrypsin family. In a comparison of some 171 residues of the haptoglobin β-chain (approximately 60% of the protein molecule), approximately 30% of these are identical to residues occurring in sequences of either bovine trypsin, bovine chymotrypsin A, bovine chymotrypsin B, porcine elastase, or bovine thrombin B-chain, and an additional 10% are chemically similar. A combined comparison of the haptoglobin β-chain with the above five serine proteases gave an identity of 56% and a chemical similarity of 11%. Similarity of primary structure is also striking around two of the five half-cystinyl residues so far characterized in long lengths of sequence. These data provide substantial evidence that the β-chain of haptoglobin is homologous to the chymotrypsin family of serine proteases. Proposals are also presented to explain the occurrence of internal homology in the N-terminal region of the β-chain.     

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ɛ (Polɛ) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polɛ is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polɛ possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polɛ heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polɛ in vitro. However, similar studies of the human Polɛ heterotetramer (hPolɛ) have been limited by the difficulty of obtaining hPolɛ in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolɛ from insect host cells has allowed for isolation of greater amounts of active hPolɛ, thus enabling a more detailed kinetic comparison between hPolɛ and an active N-terminal fragment of the hPolɛ catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolɛ. We observe that the small subunits increase DNA binding by hPolɛ relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolɛ is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolɛ and sway hPolɛ toward DNA synthesis rather than proofreading.     

“Neutral theory” and the dynamics of the evolution of “Modern” human morphology

There is a widespread assumption, even among those who reject the Synthetic Theory of Evolution, that the form of “modern”Homo sapiens is somehow superior to that of archaicHomo sapiens (Tattersall 2000). Those who accept the general outlook of evolutionary biology also tend to assume that “modern” form emerged because it was selected for, which also implies that it was better than that which preceded it. However, after years of using craniofacial measurements to compare human populations, I finally came to realize that, with only a few exceptions, the dimensions measured have no relation to differences in adaptation (Brace 1989, 1996, 2000; Brace et al., 1993). Elsewhere the conclusion has been supported that what is shown by craniometrics is selectively neutral on the average (Relethford 2002). With the documentation that approximately 95% of the genome is not functional, molecular genetics has proved to be useful in documenting the length of time of separation of related human populations by investigating the differences that have accumulated in the neutral parts of the genome. Not surprisingly, the picture revealed by the study of genetic differences is very similar to the one revealed by the study of craniometric differences (Brace et al., 2001). For this reason, the logic behind the “neutral theory” in molecular genetics is very similar to that applied to what happens to morphological characteristics when selection ceases (Brace 1963; Kimura 1968). The difference is that random changes in the neutral part of the genome have no other consequences. However, random changes in the genes that produce specific aspects of morphology will be visible even when selection is no longer controlling the particular trait in question. From an assessment of what random changes in genes controlling morphological traits are likely to do, it follows that the most likely change will probably be a reduction in the trait in question, i.e. the Probable Mutation Effect will produce structural reduction. When survival in the temperate zone during the last glaciation dependend on “obligatory cooking”, one of the unintended consequences was a reduction in the selective pressures maintaining a Middle Pleistocene-sized dentition. The result was a gradual reduction in tooth size and a conversion, of a Neanderthal-sized face into one of “modern” dimensions. The manufacture and use of string for snares and nets similarly reduced the selective pressures maintaining post-cranial levels of robustness and muscularity. The reduction in the latter resulted in the emergence of moderm post-cranial robustness out of what had been a Neanderthal level,in situ wherever the technology can be documented and without any need for invasions and replacements.     

Identification, localization and characterization of the human γ-synuclein gene

We have identified and characterized a new member of the human synuclein gene family, γ-synuclein (SNCG). This gene is composed of five exons, which encode a 127 amino acid protein that is highly homologous to α-synuclein, which is mutated in some Parkinson’s disease families, and to β-synuclein. The γ-synuclein gene is localized to chromosome 10q23 and is principally expressed in the brain, particularly in the substantia nigra. We have determined its genomic sequence, and established conditions for sequence analysis of each of the exons. The γ-synuclein gene, also known as BCSG1, was recently found to be overexpressed in advanced infiltrating carcinoma of the breast. Our survey of the EST database indicated that it might also be overexpressed in an ovarian tumor. Received: 6 February 1998 / Accepted: 8 April 1998