Effect of Repetitive Lysine-Tryptophan Motifs on the Eukaryotic Membrane

In a previous study, we synthesized a series of peptides containing simple sequence repeats, (KW)_n–NH₂ (n = 2,3,4 and 5) and determined their antimicrobial and hemolytic activities, as well as their mechanism of antimicrobial action. However, (KW)₅ showed undesirable cytotoxicity against RBC cells. In order to identify the mechanisms behind the hemolytic and cytotoxic activities of (KW)₅, we measured the ability of these peptides to induce aggregation of liposomes. In addition, their binding and permeation activities were assessed by Trp fluorescence, calcein leakage and circular dichrorism using artificial phospholipids that mimic eukaryotic liposomes, including phosphatidylcholine (PC), PC/sphingomyelin (SM) (2:1, w/w) and PC/cholesterol (CH) (2:1, w/w). Experiments confirmed that only (KW)₅ induced aggregation of all liposomes; it formed much larger aggregates with PC:CH (2:1, w/w) than with PC or PC:SM (2:1, w/w). Longer peptide (KW)₅, but not (KW)₃ or (KW)₄, strongly bound and partially inserted into PC:CH compared to PC or PC:SM (2:1, w/w). Calcein release experiments showed that (KW)₅ induced calcein leakage from the eukaryotic membrane. Greater calcein leakage was induced by (KW)₅ from PC:CH than from PC:SM (2:1, w/w) or PC, whereas (KW)₄ did not induce calcein leakage from any of the liposomes. Circular dichroism measurements indicated that (KW)₅ showed higher conformational transition compared to (KW)₄ due to peptide-liposome interactions. Taken together, our results suggest that (KW)₅ reasonably mediates the aggregation and permeabilization of eukaryotic membranes, which could in turn explain why (KW)₅ displays efficient hemolytic activity.     

Differential effect of steady versus oscillating flow on bone cells

Loading induced fluid flow has recently been proposed as an important biophysical signal in bone mechanotransduction. Fluid flow resulting from activities which load the skeleton such as standing, locomotion, or postural muscle activity are predicted to be dynamic in nature and include a relatively small static component. However, in vitro fluid flow experiments with bone cells to date have been conducted using steady or pulsing flow profiles only. In this study we exposed osteoblast-like hFOB 1.19 cells (immortalized human fetal osteoblasts) to precisely controlled dynamic fluid flow profiles of saline supplemented with 2% fetal bovine serum while monitoring intracellular calcium concentration with the fluorescent dye fura-2. Applied flows included steady flow resulting in a wall shear stress of 2 N m(-2), oscillating flow (+/-2 Nm(-2)), and pulsing flow (0 to 2 N m(-2)). The dynamic flows were applied with sinusoidal profiles of 0.5, 1.0, and 2.0 Hz. We found that oscillating flow was a much less potent stimulator of bone cells than either steady or pulsing flow. Furthermore, a decrease in responsiveness with increasing frequency was observed for the dynamic flows. In both cases a reduction in responsiveness coincides with a reduction in the net fluid transport of the flow profile. Thus. these findings support the hypothesis that the response of bone cells to fluid flow is dependent on chemotransport effects.     

Long-term outcome after marrow transplantation for severe aplastic anemia

We reviewed the records and reevaluated 212 patients with aplastic anemia transplanted at the Fred Hutchinson Cancer Research Center (FHCRC) between 1970 and 1993 who survived >/=2 years and who have been followed for up to 26 years. Parameters analyzed included hematopoietic function, chronic graft-versus-host disease (GVHD), skin disease, cataracts, lung disease, skeletal problems, posttransplant malignancy, depression, pregnancy/fatherhood, and the return to work or school, as well as patient self-assessment of physical and psychosocial health, social interactions, memory and concentration, and overall severity of symptoms. Survival probabilities at 20 years were 89% for patients without (n = 125) and 69% for patients with chronic GVHD (n = 86) (the status was uncertain in 1 surviving patient). All patients had normal hematopoietic parameters. Skin problems occurred in 14%, cataracts in 12%, lung disease in 24%, and bone and joint problems in 18% of patients. Eleven patients (12%) developed a solid tumor malignancy and 19% of patients experienced depression. Chronic GVHD was the dominant risk factor for late complications. Seventeen patients died at 2.5 to 20.4 years posttransplant; 13 of these had chronic GVHD and related complications. At 2 years, 83% of patients had returned to school or work; the proportion increased to 90% by 20 years. At least half of the patients preserved or regained the ability to become pregnant or father children. Patients rated their quality of life as excellent and symptoms as minimal or mild. In conclusion, marrow transplantation in patients with aplastic anemia established long-term normal hematopoiesis. No new hematologic disorders occurred. The major cause of morbidity and mortality was chronic GVHD. However, the majority of patients who survived beyond 2 years returned to a fully functional life.     

Rupture recurrence after surgical repair of postinfarction ventricular septal rupture. Influence of early thrombolysis

OBJECTIVES: The aim of this study was to identify factors causing rupture recurrence after surgical repair of postinfarction ventricular septal rupture and to evaluate the indication for reoperation. PATIENTS: Recurrence of rupture was analysed in 25 out of a series of 109 patients who underwent surgical repair for postinfarction ventricular septal rupture between 1980 and 1992 in our institution. RESULTS: The mean interval between initial operation and recurrence was 3.6 days with a median of 2 days. Multivariate logistic regression analysis identified early thrombolysis after infarction (P = 0.0085) as a risk factor for recurrence of the rupture. Rupture recurrence occurred more in the anterior then in the posterior infarction site, although non-significant. Reoperation was indicated in 15 patients, in 13 for postrecurrent cardiac failure. The main determinant of cardiac failure was a large postrecurrent shunt (P = 0.05). The mean interval between initial operation and reoperation was 136 days with a median of 101 days. In 6 patients a combined apical ventricular septal rupture recurrence and anterior ventricular aneurysm was found, in 9 patients the recurrent rupture was proximally located, without concomitant aneurysm formation. Of 15 patients who were reoperated, one died in hospital and three after the in-hospital period. Of 10 patients treated conservatively, one died in hospital and two after the in-hospital period. One residual ventricular septal rupture closed spontaneously. CONCLUSIONS: Rupture recurrence is mainly determined by early thrombolysis. Postrecurrent cardiac failure, as the main indication for reoperation, is dependent on postrecurrent shunt size.     

Results of irradiation treatment in patients with non-resectable oesophageal cancer

Forty-eight patients with non-resectable cancer of the oesophagus and oesophagogastric junction (Group A: Stage I/II, 32; Group B: Stage III/IV, 16) underwent intraluminal Iridium-192 high dose-rate afterloading therapy (5-7 Gy/session, total dose: 5-21 Gy, mean: 12.4 Gy) and external beam irradiation (Karnofsky > or = 80% 50-60 Gy/2 Gy per day; Karnofsky 60-79%: 30 Gy/3 Gy per day). Durable satisfactory palliation (intake of at least semi-solid food) was demonstrated in 96% of patients. The mean survival for group A was 19.1 months and that for group B, 6.9 months, with a 12-month survival rate of 66% (group A) and 0% (group B) (P < 0.001). Local tumour response and complication rate were significantly dose-related with a predicted response rate of 70.5%, and a complication rate of 50% at ERD 129.3 Gy.     

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes

The inhibitors of the Na+/H+-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Nai) and calcium (Cai) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na+ and Ca2+ (pHi, Nai and Cai) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2, 7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pHi 7.2 to pHi 6.8) and an increase in Cai from about 50 nmol/l to about 1 micromol/l. The decrease in pHi began before the cells underwent hypoxic (rigor) contracture, whereas Cai only began to rise after rigor shortening had taken place. After reoxygenation, pHi returned to its control value and Cai oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 micromol/l Hoe 642 was present from the beginning of the experiment, pHi and Cai were not significantly different from control experiments. At reoxygenation, pHi did not recover, but Cai oscillated and returned to its resting level. To monitor Nai, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 micromol/l ouabain, Nai increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 micromol/l) did not prevent the increase in Nai. In contrast, the blocker of the persistent Na+ current R56865 (10 micromol/l) attenuated the CN--induced rise in Nai. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell's autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Cai overload, nor CN--induced Nai and Cai overload. Hoe 642 prevented the recovery of pHi from anoxic acidification. This low pHi maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca2+ overload in mitochondria, cannot be ruled out. The increase in Nai during anoxia is possibly due to an influx of Na+ via persistent Na+ channels.     

Unresolved issues in the pathogenesis of Bartter's syndrome and its variants

Recent physiologic information concerning the renal response to potassium deprivation has been used to reevaluate potassium wasting in Bartter's syndrome. Experimental patient data support the notion that failure of potassium conservation is due to an imbalance between tubular secretory and reabsorptive processes. Suggestions are presented for the further evaluation of potassium reabsorptive pathways in the distal tubule.     

Effects of the combinations propofol/alfentanil and midazolam/fentanyl on blood pressure and contact phase system during coronary surgery

Perioperative haemodynamic changes leading to severe circulatory problems during open-heart surgery still represent dreaded complications. The aim of this study was to examine the relationship between the use of applied anaesthetic agents and alterations of the contact phase of the intrinsic blood-clotting system, as changes within the kallikrein-kinin system can lead to a fall in blood pressure. In a randomized study, parameters of the kallikrein-kinin system, coagulation and fibrinolysis were determined for 36 patients with aortocoronary bypass operations. The patients had been given either midazolam/fentanyl or propofol/alfentanil to maintain anaesthesia. Perioperative blood pressure values were registered at seven fixed points. The measured values of the factor XIIa-like activity and the kallikrein-like activity suggested a higher activation of the contact phase, when propofol/alfentanil was given. From the start of the extracorporeal circulation (ECC) to the end of the operation, the kallikrein-like activities in the propofol/alfentanil group were significantly higher than those of the midazolam/fentanyl group. Also, the results of the kallikrein inhibition capacity and the indicators of fibrinolysis (t-PA and D-dimers) indicate a stronger activation of the contact phase--at least at the beginning of recirculation--and as a result of it, a stronger fibrinolysis within the propofol/alfentanil group. In addition, the hypotensive side-effects differed significantly between the two groups. Patients receiving propofol/alfentanil needed the triple amount of antihypotonicum to maintain the mean arterial blood pressure above 75 mmHg. With the results of this study, a correlation between the application of propofol/alfentanil, contact phase activation, with activation of the kallikrein-kinin-bradykinin system and the observed hypotension, can be presumed.     

The dimensionality of schizophrenia concepts in first-episode psychosis

OBJECTIVE: To evaluate the efficacy of unreamed retrograde intramedullary (IM) nailing of fractures of the femoral shaft in a second series of patients using modifications suggested from our initial study. DESIGN: Prospective. SETTING: Level I trauma center. METHODS: Based on the findings of a previous study, we began a clinical series incorporating changes consisting of (a) inclusion of any patient with a femoral shaft fracture amenable to IM nailing (i.e., closed physes), (b) primary use of a split patellar tendon intercondylar distal femoral entry portal, and (c) the use of a full-length femoral implant having variable size availability and dynamization capability. Over a twelve-month period, thirty-four patients with thirty-five femoral shaft fractures were treated. The protocol called for planned dynamization in statically locked stable fractures and unstable fractures showing minimal healing at six to twelve weeks. Functional outcome was assessed by using the Knee Society clinical rating system. RESULTS: Incorporating the concepts of canal fill and early dynamization, there were only two nonunions (6 percent) in this series as compared with 14 percent in the previously reported series with an overall shorter time to union (12.6 versus 15 weeks). There were no infections or malunions. Postoperative complaints of knee pain were minimal (knee score average: 98 points) and knee function was excellent (knee score average: 97 points). CONCLUSIONS: Although not advocated as a replacement for other techniques, unreamed retrograde nailing is presented as a safe and beneficial fracture fixation method that should be added to the orthopaedic surgeon's treatment armamentarium. The operative technique is quick and simple, and blood loss is minimal. Early nail dynamization and early weight-bearing are important in minimizing the risk of nonunion.     

Mutations in the EXT1 and EXT2 genes in hereditary multiple exostoses

In an in-vitro preparation of gastric mucosae of Rana pipiens, the effect of adding melittin to a concentration of 5x10-6 M in the secretory solution on the transepithelial potential difference (PD), resistance (R) and short-circuit current (Isc) was studied. In 20 min, melittin decreased the PD by 9.3 mV and R by 148 ohm cm2. These changes can be explained by a decrease in the resistance, RP, of the paracellular pathway. To determine whether specific-ion pathways were responsible for the decrease in R, the effect of melittin on the partial conductances of Cl-, K+ and Na+ was also studied using the ion substitution method. Melittin decreased the PD response to changes in nutrient Na+, K+ and Cl- and the PD response to changes in secretory Cl-, but did not affect PD responses to changes in secretory Na+ or K+. Therefore, melittin decreased the nutrient membrane partial conductances of Cl-, K+ and Na+ and secretory membrane partial conductance of Cl-, without affecting the secretory partial conductances of Na+ or K+. Initially, melittin increased Isc in regular and Cl--free but not in Na+-free solutions. There was a delayed decrease in Isc. The results indicate that melittin decreases RP, increases the Na+ conductance of the secretory membrane and inhibits, eventually, the Na+/K+-ATPase pump.