Calcium accumulation and release by the sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular cardiomyocytes.

We have developed a model for characterizing calcium handling by the intact cardiac sarcoplasmic reticulum (SR) that yields data consistent with both mathematical simulations of in situ SR Ca2+ uptake and deduced behavior of the Ca2(+)-induced Ca2+ efflux channels in mechanically skinned single cardiac cells. In Na(+)-based media (37 degrees C, pH 7.2, 50 mM Pi, 10 mM MgATP, pMg 3.3, 10 mM phosphocreatine), SR 45Ca2+ uptake by digitonin-lysed rat myocytes as a function of free [Ca2+] peaked at pCa 6.2, declined until pCa 5.6 and increased again at lower pCa. When Ca2(+)-induced Ca2+ efflux was inhibited with 30 microM ruthenium red and 10 mM procaine, uptake was saturable with a Vmax of 160 +/- 5 nmol.min-1.mg-1, K0.5 of 500 nM free [Ca2+] and slope factor of 1.6. In K(+)-based media, maximum Pi- and oxalate-supported uptake increased to 220 and 260 nmol.min-1.mg-1, respectively. Without phosphocreatine, 45Ca2+ uptake declined under all conditions; this was correlated with a decrease in ATP/ADP. Vmax for 45Ca2+ uptake was increased 20% in hyperthyroid myocytes but depressed 30% in myocytes from heart failure-prone rats. In canine myocytes, Vmax was the same as in normal rat cells, but K0.5 was 830 nM. Without efflux inhibitors, ryanodine caused a concentration-dependent decline in net Pi-supported 45Ca2+ uptake at pCa 6.3 (K0.5 = 1 microM), while 10 microM ryanodine depressed uptake at all pCa between 7.2 and 5.6. Ruthenium red/procaine fully reversed this effect.     

Functional consequences of human immunodeficiency virus escape from an HLA-B*13-restricted CD8+ T-cell epitope in p1 Gag protein

The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI_429-437 (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness.     

Energy-dependence exchange of K+ in heart mitochondria. K+ efflux.

The efflux ⁴²K⁺ from isolated beef heart mitochondria under conditions of near steadystate K⁺ is increased by repiration and is sensitive to uncouplers and to exogenous Mg² The respiration-dependent efflux is strongly activated by inorganic phosphate in the presence of external K⁺, but not Na⁺, and is inhibited by oxidative phosphorylation. Low concentrations of mersalyl also activate respiration-dependent efflux of ⁴²K⁺ in the absence of net alteration in matrix K⁺. Acetate in the presence of mersalyl brings about net accumulation of K⁺ with retention of internal ⁴²K⁺. The results are consistent with a model in which nearly constant matrix K⁺ is maintained by the regulated interplay between a K⁺ uniport (which is responsive to membrane potential and which is the pathway for K⁺ influx) and a $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchanger (which responds to the transmembrane pH differential and which is the pathway for net K⁺ efflux).     

Expression of Cathepsin D and pS2 in imprint smears of breast carcinoma

athanassiadou p.p., athanassiades p. h., daffaris p., petrakakou e. i., fflerffa ch. i. and kffirkou k. a. (1998) Cytopathology 9, 240–247
Expression of Cathepsin D and pS2 in imprint smears of breast carcinoma
The aim of this study was to add to existing information on the effects of certain tumour markers expressed by breast cancers on tumour malignancy as evidenced by size of primary and occurrence of lymph node invasion. One hundred freshly resected breast cancers were examined by immunocytochemical staining of imprint smears for Cathepsin D and pS2. Oestrogen receptor (ER) and progesterone receptor (PR) were tested for by dextrose-coated charcoal (DCC) assay and the results correlated with tumour size, histology and presence or absence of lymph node metastases at the time of surgery using χ² analysis. A significant positive correlation was demonstrated between Cathepsin D positivity and ER, PR and pS2 positivity. In tumours < 2 cm in diameter at surgery a positive correlation was observed between Cathepsin D positivity and the presence of lymph node metastases. The findings support the hypothesis that Cathepsin D may promote early metastasis, possibly by its proteolytic activity.     

Widespread fluorescence in terrestrial and marine samples investigated from India

The discovery of green fluorescent protein (GFP) from jellyfish Aequorea victoria has provoked numerous studies to unveil the myriads of biomedical applications. Consequently, several studies also revealed the prevalence of fluorescence in different marine and terrestrial organisms. However, since GFP's discovery or the Nobel prize award on GFP, the fluorescence has not been explored so far from India. The current study presents the widespread fluorescent organisms resources available in India for biomedical and toxicological applications. Fluorescence emission from different plant and animal components were examined by direct observations using UV torchlight. Investigation revealed that blue light excited fluorescence in several organisms. For the first time, this study observed GFP like fluorescence in many terretrial and marine organisms of India. Observations are indicating that fluorescent proteins have essential ecological functions that are yet to be determined. The examined plant and animal components were not bioluminescent. In comparison, the potential untouched research areas on fluorescence aspects are detailed. A thorough review of fluorescent organisms reported hitherto is also provided to spread the current knowledge on fluorescence.     

Modulation of human microvascular endothelial cell bioenergetic status and glutathione levels during proliferative and differentiated growth

During angiogenesis, formerly differentiated human microvascular endothelial cells (HMECs) return to a proliferative growth state. Many fundamental questions regarding HMEC function, such as how HMECs adapt to changes in bioenergetic requirements upon return to proliferative growth, remained unanswered. In this study, we evaluated whether modifications in HMEC bioenergetic profiles and glutathione (GSH) levels accompanied the cellular transition between differentiated and proliferative growth. To provide insight into the continuum of cellular adaptations that occur during this transition, we used a method recently developed in our laboratory that induces a state of morphological and functional predifferentiation in HMECs. Cellular morphology, in conjunction with flow cytometric DNA analyses and HMEC functional assays (the directed migration and intercellular association involved in microtubule formation) were employed to validate the HMEC culture state of growth. Analysis of the HPLC nucleotide profiles disclosed several findings common to all culture growth states. These uniform findings, e.g., cellular energy charges > 0.90, and highly reduced redox states, revealed that cultured HMECs maintain high rates of oxidative metabolism. However, there were also significant, culture growth state related differences in the nucleotide profiles. Proliferative HMECs were shown to possess significantly higher (relative to both large vessel endothelial cells, and differentiated HMECs) levels of GSH and specific nucleotides which were related with a return to the active cell cycle-ATP, GTP, UTP, and CTP, and NADPH. Further, the nucleotide profiles and GSH levels of the predifferentiated HMECs were determined to be intermediate between levels obtained for the proliferative and differentiated HMECs. The results of this study demonstrate that the capacity to modulate their cellular bioenergetic status during growth state transitions is one of the adaptations that enable HMECs to retain a growth state reciprocity. In addition, our findings also show that HMECs, especially during the proliferative growth state, are biochemically distinct from endothelial cells harvested from large vessels, and therefore suggest that HMECs are the cells of choice to employ when studying diseases that affect the human microvasculature.     

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.