Detection of drug-induced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells

A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs. This work was supported by Hungarian National Research Foundation (OTKA I/352 and OTKA II/2622).     

Synaptic connections linking cones and horizontal cells in the retina of the pikeperch (Stizostedion vitreum)

Cones and horizontal cells of the pikeperch retina were studied with morphological and physiological techniques. Gap junctions were observed between cone pedicles and basal processes emitted by neighboring cones. Intracellular recordings showed that the light-evoked hyperpolarizing cone response was enhanced by light falling upon neighboring receptors within a radius of 50 microns. We suggest that the network of gap junctions between cones mediates the summative lateral interaction described. Three sub-classes of horizontal cells (H1, H2, H3) send dendrites to cones; H1 and H2 cells appear to contact twin cones, exclusively or preferentially, whereas H3 cells appear to synapse only with single cones. Horizontal cells of the same sub-class are joined by gap junctions between dendrites or at the lateral faces of perikarya. These unions extend over several micron 2 and as seen in transmission electron microscopy consist of patches of close apposition alternating with areas of membrane separation, folding and occasional zonulae adherents. Freeze-fracture profiles of horizontal gap junctions show localized areas of dense particle aggregation on the P-face and pits on the E-face flanked by regions of unspecialized membrane. These morphological findings provide support for the known spatial and color-coding properties of pikeperch horizontal cells.     

Steady-state kinetics of imipramine in patients

Steady-state plasma level kinetics were studied in 76 patients given imipramine (IP) 150 to 225 mg/day for 2–5 weeks. IP was given in three divided doses at 8.00 a.m., 1.00 p.m. and 5.00 p.m. Plasma concentrations of IP and its active metabolite desipramine (DMI) were determined by quantitative in situ thin-layer chromatography. The plasma levels of IP and DMI showed pronounced flucutations throughout the day with a ratio of about 2 between highest and lowest level. Patients with steady-state levels of IP and/or DMI below 50 g/l reached this within 1 week of treatment. Patients with higher steady-state levels reached steady-state concentrations within 2–3 weeks. There were some intraindividual fluctuations in plasma levels from week to week after steady state had been reached (coefficient of variation: 10–20%). Interindividually, the steady-state levels corrected to a dose of 3.5 mg/kg per day varied considerably: IP: 6–356 g/l, DMI: 24–659 g/l and IP+DMI: 58–809 g/l. The steady-state plasma levels showed a skew distribution that became normal by logarithmic transformation. The IP/DMI ratio ranged from 0.07 to 5.5 with a median value of 0.47. Compared to data from amitriptyline treated patients the IP/DMI ratios had significantly lower median value and larger variation than the corresponding plasma level ratios of amitriptyline/nortriptyline. Several statistically significant differences in steady-state levels between age groups were found. For IP: Women aged 30–39 had lower levels than women aged 20–29, 40–49, and 50–59, and men aged 50–59 and 60–65; men aged 30–39 had lower levels than men aged 60–65. For DMI: Women aged 30–39 had lower levels than women aged 50–59.     

Do adaptive changes in metabolic rate favor weight regain in weight-reduced individuals? An examination of the set-point theory

BACKGROUND: Obese persons generally regain lost weight, suggesting that adaptive metabolic changes favor return to a preset weight. OBJECTIVE: Our objective was to determine whether adaptive changes in resting metabolic rate (RMR) and thyroid hormones occur in weight-reduced persons, predisposing them to long-term weight gain. DESIGN: Twenty-four overweight, postmenopausal women were studied at a clinical research center in four 10-d study phases: the overweight state (phase 1, energy balance; phase 2, 3350 kJ/d) and after reduction to a normal-weight state (phase 3, 3350 kJ/d; phase 4, energy balance). Weight-reduced women were matched with 24 never-overweight control subjects. After each study phase, assessments included RMR (by indirect calorimetry), body composition (by hydrostatic weighing), serum triiodothyronine (T(3)), and reverse T(3) (rT(3)). Body weight was measured 4 y later, without intervention. RESULTS: Body composition-adjusted RMR and T(3):rT(3) fell during acute (phase 2) and chronic (phase 3) energy restriction (P: < 0.01), but returned to baseline in the normal-weight, energy-balanced state (phase 4; mean weight loss: 12.9 +/- 2.0 kg). RMR among weight-reduced women (4771 +/- 414 kJ/d) was not significantly different from that in control subjects (4955 +/- 414 kJ/d; P: = 0.14), and lower RMR did not predict greater 4-y weight regain (r = 0.27, NS). CONCLUSIONS: Energy restriction produces a transient hypothyroid-hypometabolic state that normalizes on return to energy-balanced conditions. Failure to establish energy balance after weight loss gives the misleading impression that weight-reduced persons are energy conservative and predisposed to weight regain. Our findings do not provide evidence in support of adaptive metabolic changes as an explanation for the tendency of weight-reduced persons to regain weight.     

Guidelines for the diagnosis and management of blunt aortic injury: an EAST Practice Management Guidelines Work Group

In summary, BAI is a lethal result of severe blunt trauma. It should be considered in all patients who sustained injury by a deceleration or acceleration mechanism, especially in the face of physical or radiographic findings suggestive of mediastinal injury. Angiography remains the "gold standard" for diagnosis, although CT scanning is taking more of a role, especially for screening. Diagnosis should be followed by prompt surgical repair using some method of distal perfusion to minimize renal and spinal cord ischemia. If prompt repair is not feasible because of other injuries or comorbidities, medical control of blood pressure is warranted in the interim.     

Possible association between maternal recurrent orofacial herpes in pregnancy and a lower rate of preterm birth.

OBJECTIVE: To study the possible association between orofacial herpes during pregnancy and pregnancy complications including preterm birth and low birth weight, since the results of previous studies are inconsistent. METHOD: The population-based large data set of the Hungarian Case-Control Surveillance System of Congenital Abnormalities was used; pregnancies in mothers with and without recurrent orofacial herpes were compared. RESULTS. Of 38,151 newborn infants, 572 (1.5%) had mothers with recurrent orofacial herpes during pregnancy, while 37 577 had mothers with no orofacial herpes. Pregnant women with recurrent orofacial herpes had a higher prevalence of severe nausea and vomiting, threatened preterm delivery, and placental disorders but a lower prevalence of preeclampsia. Mothers with recurrent orofacial herpes during pregnancy also had a somewhat longer (0.4 weeks) gestation (adjusted t = 2.7; p = 0.006) and an obviously lower proportion of preterm births (3.5% vs. 9.3%; adjusted POR with 95% CI = 0.42, 0.27-0.65). However, there was no significant difference in the mean birth weight and rate of low birth weight infants between the two study groups. CONCLUSION: Recurrent orofacial herpes during pregnancy is associated with a smaller proportion of preterm births.     

Voluntary sucrose ingestion, like corticosterone replacement, prevents the metabolic deficits of adrenalectomy

We tested whether corticosterone replacement causes increased sucrose drinking in adrenalectomized (ADX) rats compared to sham-ADX (sham) rats. ADX rats given high doses of corticosterone drank as much sucrose as sham rats, whereas at three lower doses of corticosterone, drinking was similar between groups and was only approximately 40% of that ingested by shams. Compared to sham rats, ADX rats drinking saline, or saline and saccharin, gain weight more slowly, contain less white adipose tissue, and have higher sympathetic outflow as assessed by uncoupling protein content in brown adipose tissue. Allowing sucrose as well as saline to drink restored all of these variables to normal in ADX rats with no- or low-corticosterone. All endpoints from sucrose-drinking ADX rats with no-or low-corticosterone were indistinguishable from those in water-drinking shams. By contrast, sucrose-drinking ADX rats that were given high doses of corticosterone exhibited the usual catabolic effects of corticosterone on body weight gain and, unlike sucrose-drinking shams, were obese. We conclude that (i) high corticosterone stimulates the potability of sucrose and inhibits sympathetic stimulation of uncoupling protein; (ii) sucrose, without corticosterone, normalizes metabolic deficits in ADX rats probably through actions mediated both peripherally and by the central nervous system; and (iii) ADX rats have a distinct sucrose appetite.