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.     

Differential role of insulin receptor autophosphorylation sites 1162 and 1163 in the long-term insulin stimulation of glucose transport, glycogenesis, and protein synthesis.

The long-term regulatory effect of insulin on glucose transport activity and glucose transporter expression was examined in Chinese hamster ovary (CHO) transfectants that overexpress either human insulin receptors of the wild type (CHO-R cells) or human insulin receptors mutated at two major autophosphorylation sites, Tyr1162 and Tyr1163 (CHO-Y2 cells). Previous studies showed that, when acutely stimulated by insulin, CHO-Y2 cells exhibit decreased receptor kinase activity along with decreased signaling of several pathways, including that for glucose transport, as compared with CHO-R cells. We now report the following. (i) When treated for 24 h with insulin (10(-10) to 10(-6) M), CHO-R and CHO-Y2 cells displayed closely similar concentration-dependent increases in 2-deoxyglucose uptake. In both transfectants, the maximal insulin-induced increase (approximately 3.5-fold) in uptake was cycloheximide-sensitive and was paralleled by equivalent increases in the levels of GLUT-1 immunoreactive protein and mRNA. (ii) By contrast, under similar conditions, CHO-Y2 cells exhibited a marked decrease in their response to insulin for [U-14C]glucose incorporation into glycogen (decreased sensitivity and maximal responsiveness) and for [U-14C]leucine incorporation into protein (decreased sensitivity) as compared with CHO-R cells. (iii) After a 24-h treatment with 10(-7) M insulin, CHO-R (but not CHO-Y2) cells showed a decreased ability to respond to a subsequent acute insulin stimulation of either receptor exogenous kinase activity or 2-deoxyglucose uptake as compared with respective untreated controls. These results indicate that (i) insulin receptors mutated at Tyr1162 and Tyr1163 retain normal signaling of the long-term stimulatory effect of insulin on glucose transport activity and GLUT-1 expression, but not on glycogenesis and overall protein synthesis; (ii) these three insulin signaling pathways may be triggered by distinct domains of the insulin receptor beta-subunit; and (iii) wild-type (but not twin-tyrosine mutant) receptors undergo negative regulation by chronic insulin treatment for subsequent signaling of acute biological actions of insulin.     

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.     

Comparative electron paramagnetic resonance study of radical intermediates in turnip peroxidase isozymes

The occurrence of isozymes in plant peroxidases is poorly understood. Turnip roots contain seven season-dependent isoperoxidases with distinct physicochemical properties. In the work presented here, multifrequency electron paramagnetic resonance spectroscopy has been used to characterize the Compound I intermediate obtained by the reaction of turnip isoperoxidases 1, 3, and 7 with hydrogen peroxide. The broad (2500 G) Compound I EPR spectrum of all three peroxidases was consistent with the formation of an exchange-coupled oxoferryl-porphyrinyl radical species. A dramatic pH dependence of the exchange interaction of the [Fe(IV)=O por(*+)] intermediate was observed for all three isoperoxidases and for a pH range of 4.5-7.7. This result provides substantial experimental evidence for previous proposals concerning the protein effect on the ferro- or antiferromagnetic character of the exchange coupling of Compound I based on model complexes. Turnip isoperoxidase 7 exhibited an unexpected pH effect related to the nature of the Compound I radical. At basic pH, a narrow radical species ( approximately 50 G) was formed together with the porphyrinyl radical. The g anisotropy of the narrow radical Delta(g) = 0.0046, obtained from the high-field (190 and 285 GHz) EPR spectrum, was that expected for tyrosyl radicals. The broad g(x) edge of the Tyr* spectrum centered at a low g(x) value (2.00660) strongly argues for a hydrogen-bonded tyrosyl radical in a heterogeneous microenvironment. The relationship between tyrosyl radical formation and the higher redox potential of turnip isozyme 7, as compared to that of isozyme 1, is discussed.     

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.