Structural and functional properties of adult rat heart myocytes lysed with digitonin

Low concentrations of digitonin disrupt the sarcolemma of adult rat heart myocytes selectively and completely. When the digitonin lysis is carried out in the presence of 10 mM Mg-ATP, the permeabilized cells retain the rod-cell morphology typical of heart cells in situ and show spontaneous phasic contractions. The rate of contraction is a function of the free Ca2+ concentration from a pCa of 7.2 to 5.2. Higher levels of free Ca2+ result in hypercontracture of the myocytes into round cells with characteristically distorted morphology. The sarcoplasmic reticulum of digitonin-lysed myocytes takes up Ca2+ in an ATP-dependent reaction that is inhibited and reversed by caffeine and strongly enhanced by procaine or ruthenium red. The Ca2+ accumulation has a Km of 0.6 microM Ca2+, depends on Pi (Km of 13 mM), and is strongly inhibited by bicarbonate ion. The hypercontracture of digitonin-lysed myocytes is a function of both the pCa and the Mg-ATP concentration of the suspending medium. Hypercontracture requires ATP. Hypercontracture due to Ca2+ overload occurs at lower Ca2+ concentrations when Mg-ATP is decreased from 10 to 1 mM. However, at low concentrations of Mg-ATP (in the range from 1 to 10 microM), hypercontracture also occurs and is essentially Ca2+-independent. Since hypercontracture of heart myocytes appears analogous to the formation of contraction bands in situ, these observations may be relevant to the phenomena of oxygen paradox and of Ca2+ paradox in intact myocardial tissue.     

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.     

Influence de l'âge des grand-parents sur l'apparition des mâles chez le Rotifère Notommata copeus Ehr.

When parental females (♀♀P = mothers) of the rotifer Notommata copeus are placed in light conditions inducing the appearance of mictic females in their offspring (F₁), the age of the parents of these parental females significantly affects the ratio of mictic females in the F₁ generation.

The preparental females (♀♀PP = grandmothers), the parental females and the F₁ females are isolated in a medium changed at each generation and, in the second experimental series, changed daily: the preparental age effect implicates the transmission of substances on two generations.

This influence of the preparental age is rhythmic: the ratio of mictic females in F₁ related to this age varies in a sinusoidal manner. This influence is endogenous: it persists, always in a sinusoidal form, when the medium in which each grandmother is placed is changed daily.

Furthermore, the net reproduction ratio, Ro, does not vary significantly with the preparental age during these experiments.     

A benefit-finding intervention for family caregivers of persons with Alzheimer disease: study protocol of a randomized controlled trial

Background

Patients with ST-elevation myocardial infarction (STEMI) not treated with primary or rescue percutaneous coronary intervention (PCI) are at risk for recurrent ischemia, especially when viability in the infarct-area is present. Therefore, an invasive strategy with PCI of the infarct-related coronary artery in patients with viability would reduce the occurrence of a composite end point of death, reinfarction, or unstable angina (UA).

Methods

Patients admitted with an (sub)acute myocardial infarction, who were not treated by primary or rescue PCI, and who were stable during the first 48 hours after the acute event, were screened for the study. Eventually, we randomly assigned 216 patients with viability (demonstrated with low-dose dobutamine echocardiography) to an invasive or a conservative strategy. In the invasive strategy stenting of the infarct-related coronary artery was intended with abciximab as adjunct treatment. Seventy-five (75) patients without viability served as registry group. The primary endpoint was the composite of death from any cause, recurrent myocardial infarction (MI) and unstable angina at one year. As secondary endpoint the need for (repeat) revascularization procedures and anginal status were recorded.

Results

The primary combined endpoint of death, recurrent MI and unstable angina was 7.5% (8/106) in the invasive group and 17.3% (19/110) in the conservative group (Hazard ratio 0.42; 95% confidence interval [CI] 0.18-0.96; p = 0.032). During follow up revascularization-procedures were performed in 6.6% (7/106) in the invasive group and 31.8% (35/110) in the conservative group (Hazard ratio 0.18; 95% CI 0.13-0.43; p < 0.0001). A low rate of recurrent ischemia was found in the non-viable group (5.4%) in comparison to the viable-conservative group (14.5%). (Hazard-ratio 0.35; 95% CI 0.17-1.00; p = 0.051).

Conclusion

We demonstrated that after acute MI (treated with thrombolysis or without reperfusion therapy) patients with viability in the infarct-area benefit from a strategy of early in-hospital stenting of the infarct-related coronary artery. This treatment results in a long-term uneventful clinical course. The study confirmed the low risk of recurrent ischemia in patients without viability.

Trial registration

ClinicalTrials.gov: NCT00149591.     

Cultured AIDS-related kaposi's sarcoma (AIDS-KS) cells demonstrate impaired bioenergetic adaptation to oxidant challenge: Implication for oxidant stress in AIDS-KS pathogenesis

Despite its recognition as the most prevalent HIV associated cancer, speculation still abounds regarding the pathogenesis of AIDS-related Kaposi's sarcoma (AIDS-KS). However, it has been established that both cytokines, e.g. IL-6, and HIV-associated products, e.g., Tat, are integral in AIDS-KS cellular proliferation. Further, both experimental and clinical evidence is accumulating to link reactive oxygen intermediates (ROI) with both cytokine induction (primarily via nuclear factor-κB [NF-κB] dependent routes) as well as the subsequent cytokine, tumor necrosis factor α (TNFα) stimulation of HIV replication. Features of AIDS-KS patients, such as retention of phagocytes, presence of sustained immunostimulation, and a frequent history of KS lesions arising at traumatized sites, make oxidant stress a viable clinical factor in AIDS-KS development. Time course nucleotide profile analyses show that AIDS-KS cells have an inherent, statistically significant, biochemical deficit, even prior to oxidant stress, due to (1) a more glycolytic bioenergetic profile, resulting in lower levels of high energy phosphates (impairing capacity for glutathione [GSH] synthesis and DNA repair); (2) lower levels of NADPH (compromising the activities of GSSG reductase and peroxidase function of catalase); and (3) reduced levels of GSH (impeding both GSH peroxidase and GSH-S-transferases). Following exposure to physiologically relevant levels of H₂O₂ only the human microvascular endothelial cells (a putative AIDS-KS progenitor cell) responded with bioenergetic adaptations that reflected co-ordination of energy generating and cytoprotective pathways, e.g., retention of the cellular energy charge, increased NAD⁺, and an accentuation of the ATP, NADPH, and total adenine nucleotide differences relative to AIDS-KS cells. Also, some of the AIDS-KS strains retained intracellular GSSG subsequent to oxidant challenge, inviting the formation of deleterious protein mixed disulfides. While the results of our study address some AIDS-KS issues, they also raise an etiological question, i.e., Does the inability to tolerate oxidant stress arise in conjunction with AIDS-KS neoplastic development, or is it pre-existing in the population at risk? Regardless, use of antioxidant therapy (low risk/potentially high benefit) in both the “at risk” population as well as in those individuals with active disease may prove a useful preventative and/or treatment modality. © 1995 Wiley-Liss, Inc.