Expression of rat renal gamma-glutamyltransferase cDNA in Escherichia coli

To obtain the expression of rat kidney gamma-glutamyltransferase (GGT) cDNA in E. coli, plasmids containing the cDNA sequences coding for various parts of GGT were constructed. Transformation of E. coli cells by these hybrid vectors results in a production of unglycosylated recombinant proteins, immunologically recognized by specific antirat kidney GGT antibodies. Plasmid, expressing the complete coding sequence of GGT cDNA, allows the production of enzymatically active proteins localized in the periplasmic space, while the same sequence without the N-terminal hydrophobic region results in a production of cytoplasmic proteins. These recombinant proteins present a very basic isoelectric point (pI greater than 9). These results suggest that the presence of the N-terminal region seems to be necessary to direct the expressed proteins enzymatically active in the periplasmic space.     

Predictors of success in hypertensives treated with biofeedback-assisted relaxation

This paper describes differences in response in seventeen patients with essential hypertension who participated in a treatment program consisting of electromyograph biofeedback assisted relaxation training. Responders were found to have higher treatment values of urinary and plasma cortisol, Trait Anxiety and forehead muscle tension compared to treatment failures. Responders also sustained greater decreases in plasma, and urinary cortisol after treatment. These data are discussed in light of the ability to predict which hypertensive patients may be most benefitted by a relaxation based treatment.We would like to thank Dr. Charles Spielberger for his permission to use the State-Trait Anxiety Inventory. We thank Michael Robinson for assistance with statistical analysis.     

The effect of biofeedback-assisted relaxation training on blood pressure and selected biochemical parameters in patients with essential hypertension

The effect of EMG biofeedback-assisted relaxation on blood pressure and selected biochemical parameters was evaluated in 38 patients with essential hypertension. Training consisted of 8 weeks of biofeedback and home practice of relaxation exercises. Mean blood pressure decreased in the experimental group from 144/90 to 133/84 mm Hg while the control group remained unchanged. Statistically significant decreases in the experimental group also occurred in muscle tension levels, in plasma aldosterone, and in urinary cortisol. Both aldosterone and cortisol are secreted by the adrenal cortex. It was concluded that the technique taught to the experimental group produced a reduction in skeletal muscle tension and a decrease in stress responding mediated by the adrenal cortex.This work was supported by the Northwestern Ohio Heart Association Grant No. 93298. It was presented as a citation paper at the meetings of the Biofeedback Society in 1979 and 1980.     

Sustained effects of biofeedback-assisted relaxation therapy in essential hypertension

The usefulness of biofeedback-assisted relaxation as an adjunct or substitute for pharmacotherapy in essential hypertension can be enhanced if the effects are shown to persist after formal treatment has ended. Patients with essential hypertension successfully treated with biofeedback-assisted relaxation were recalled for follow-up yearly after the termination of treatment. Twenty-six of 40 patients met the BP criterion for success. At one-, two-, and three-year follow-up, 31%, 38%, and 27% of the successful completers continued to meet the criterion for success. The pretreatment-posttreatment decreases in BP were accompanied by decreases in forehead muscle tension and urinary cortisol. Forehead muscle tension, urinary cortisol, and anxiety levels were significantly lower than pretreatment one year after the end of treatment. Self-report data were used to assess continued relaxation practice. No relationship was found between practice and any other dependent measure. It appears that some patients trained in biofeedback-assisted relaxation can maintain lowered blood pressure, muscle tension, anxiety, and cortisol levels over the long term; however, the role of relaxation practice in maintaining these lowered levels remains unclear.     

Neuraminidase-associated plasminogen recruitment enables systemic spread of natural avian Influenza viruses H3N1

Repeated outbreaks due to H3N1 low pathogenicity avian influenza viruses (LPAIV) in Belgium were associated with unusually high mortality in chicken in 2019. Those events caused considerable economic losses and prompted restriction measures normally implemented for eradicating high pathogenicity avian influenza viruses (HPAIV). Initial pathology investigations and infection studies suggested this virus to be able to replicate systemically, being very atypical for H3 LPAIV. Here, we investigate the pathogenesis of this H3N1 virus and propose a mechanism explaining its unusual systemic replication capability. By intravenous and intracerebral inoculation in chicken, we demonstrate systemic spread of this virus, extending to the central nervous system. Endoproteolytic viral hemagglutinin (HA) protein activation by either tissue-restricted serine peptidases or ubiquitous subtilisin-like proteases is the functional hallmark distinguishing (H5 or H7) LPAIV from HPAIV. However, luciferase reporter assays show that HA cleavage in case of the H3N1 strain in contrast to the HPAIV is not processed by intracellular proteases. Yet the H3N1 virus replicates efficiently in cell culture without trypsin, unlike LPAIVs. Moreover, this trypsin-independent virus replication is inhibited by 6-aminohexanoic acid, a plasmin inhibitor. Correspondingly, in silico analysis indicates that plasminogen is recruitable by the viral neuraminidase for proteolytic activation due to the loss of a strongly conserved N-glycosylation site at position 130. This mutation was shown responsible for plasminogen recruitment and neurovirulence of the mouse brain-passaged laboratory strain A/WSN/33 (H1N1). In conclusion, our findings provide good evidence in natural chicken strains for N1 neuraminidase-operated recruitment of plasminogen, enabling systemic replication leading to an unusual high pathogenicity phenotype. Such a gain of function in naturally occurring AIVs representing an established human influenza HA-subtype raises concerns over potential zoonotic threats.     

Conductance and permeability of the residual state of connexin43 gap junction channels

We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell-cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (V(j)) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher V(j)s that are negative on the side of gated hemichannel. The degree of rectification increases when Cl(-) is replaced by Asp(-) and decreases when K(+) is replaced by TEA(+). These data are consistent with an increased anionic selectivity of the residual state. The V(j)-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast V(j)-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.     

Reversal of sexual dimorphism in splenic T lymphocyte responses after trauma-hemorrhage with aging

Previous studies have demonstrated that hemorrhagic shockproduces immunodepression in young male mice, whereas theimmunoresponsivness in young proestrus female mice is enhanced undersuch conditions. This sexually dimorphic immune response to hemorrhageappears to be related to high estrogen and testosterone levels infemales and males, respectively. Nonetheless, it is unknown what impact the age-related decline in the sex steroid levels has on the immune response after hemorrhage. To study this, young (2-3 mo) and aged (18-19 mo) male and female CBA/J NIA mice were subjected tolaparotomy (i.e., soft tissue trauma) and hemorrhage (35 ± 5 mmHg for90 min and fluid resuscitation) or sham operation. Twenty-four hours later, splenocyte responses were assessed in vitro. Splenic T lymphocyte responses [i.e., proliferation, interleukin-2 (IL-2) and interferon- (IFN-) release] were depressed in youngmales and enhanced in young females after trauma-hemorrhage. Incontrast, in the aged male and female groups these parameters ofsplenocyte function were reversed after trauma-hemorrhage (i.e.,increased proliferation and IL-2 release in aged males compared withsuppressed proliferation and IFN- release in aged females).Furthermore, the release of the immunosuppressive cytokine IL-10inversely correlated with the age- and gender-related changes insplenocyte responses after trauma-hemorrhage. Thus the sexuallydimorphic immune response in young males and females totrauma-hemorrhage appears to reverse as sex hormone levels decline with age.

     

ESR Studies of a Series of Phthalocyanines. Mechanism of Phototoxicity. Comparative Quantitation of O2-using ESR Spin-Trapping and Cytochrome c Reduction Techniques

ESR experiments with 2,2,6,6-tetramethyl-4-piperi-done (4-oxo-TEMP) and the spin-trap 5,5-dimethyl pyrroline-N-oxide (DMPO) have been performed on a series of new phthalocyanines: the bis(tri-n-hexyl-siloxy) silicon phthalocyanine ([(nhex)₃SiO]₂SiPc), the hexadecachloro zinc phthalocyanine (ZnPcCl₁₆), the hexadecachloro aluminum phthalocyanine (AlPcCl₁₆), the hexadecachloro aluminum phthalocyanine sulfate (HSO₄A1PcCl₁₆), whose photocytotoxicity has been studied against various leukemic and melanotic cell lines. Type I and Type II pathways occur simultaneously in DMF although the Type II seems to be prevalent. These results are not changed when the bis(tri-n-hexylsiloxy) silicon phthalocyanine is entrapped into liposomes. By contrast, the Type I process is favored in membrane models for all the perchlori-nated phthalocyanines. This modified behavior may be accounted on a possible stacking of phthalocyanines in membranes and a preventing effect of axial ligands against aggregation in the case of the bis(tri-n-hexyl-siloxy) silicon phthalocyanine. The photodynamic action of zinc perchlorinated phthalocyanine is not dependent on singlet oxygen, phototoxicity of this molecule being essentially mediated by oxygen free radicals. Quantitation of the superoxide radical was accomplished, with good agreement, by two techniques: the cytochrome c reduction and the ESR quantitation based on the double integration of the first derivative of the ESR signal. The disproportionation of the superoxide radical or degradation of the spin-trap seem to be avoided in aprotic solvents such as DMF.