Cryoultramicrotomy: electrostatic transfer of dry ultrathin frozen sections on grids applied to the central nervous system.

Due to the extreme fragility of ultrathin frozen sections of brain tissue, the cryoultramicrotomy of non-embedded tissue has not been sufficiently used for immunocytochemical studies of the central nervous system. Sections are easily disrupted by the liquid surface tension when a droplet of sucrose is used (method by Tokuyasu, 1973) for their transfer on the grids. Use of silicotungstic acid (Tsuji, 1986) in place of sucrose improved the preservation of the ultrastructure but still could not resolve the difficulty. This report describes a new procedure for transferring dry ultrathin frozen sections by means of electrostatic attraction induced on the membrane covering the grids. Once attached electrostatically to the membrane, the sections were retained by van der Waals' forces. The dry ultrathin frozen sections obtained from both fresh and fixed brains displayed good preservation of their ultrastructures over a large surface. This new method which electrostatically transfers dry ultrathin frozen sections, avoiding the use of any liquid, is expected to serve for the immunocytochemical identification of neuronal cell bodies and terminals as well as their neurotransmitters and enzymes in both fresh and fixed brains.


Structure of lymphatics in rat cecum with special reference to submucosal collecting lymphatics endowed with smooth muscle cells and valves. I. A scanning electron microscopic study.

The three-dimensional structure of lymphatic vessels in the rat cecum was studied by KOH-collagenase digestion/scanning electron microscopy (SEM), and corrosion casting/SEM. Abluminal surfaces of the lymphatic vessels show flat elliptical nuclear regions and flat cytoplasmic processes interdigitated with adjacent ones. The lymphatic capillaries closed by interdigitations of flat endothelial processes at their initial portion begin at the various levels of the mucosa. They descend and pass through the muscularis mucosa to connect with the lymphatic vessels in the submucosa. They form polygonal meshwork, the distances between intersections being about 0.2-0.5 mm. They also have valves, the distances between adjacent valves being about 0.1-0.6 mm. Most of the submucosal lymphatic vessels are surrounded by either periendothelial cells or typical smooth muscle cells. The polygonal meshworks made up of stellate periendothelial cells with many irregular processes embrace the initial segment of the collecting lymphatics. As they proceed proximally, the periendothelial cells become elongated and branch out by threes or fours, thus presenting the appearance of smooth muscle cells. These branches are connected side by side and run obliquely along the vessels, thus forming polygonal meshworks around the vessels. The more proximal collecting lymphatic vessels are surrounded by circularly oriented smooth muscle cells. Our results indicate that most of the lymphatic vessels in the submucosa are collecting ones and possess smooth muscle cells as well as valves. This suggests that the lymphatic vessels in the submucosa actively contract and propel the lymph towards the mesenteric lymphatic vessels.


Structural and endocytotic differences of fibroblasts and macrophages in the tail fin of amphibian larvae during metamorphosis.

Different features in the fibroblasts and the macrophages, which are prominent cell types in the dermis of the dorsal tail fin of the larval axolotl, Ambystoma mexicanum, and the tadpole, Rana japonica, were examined by light and electron microscopy. At the non-metamorphic stages, the cytoplasm of the macrophage, loaded with numerous lysosomes, is generally located in the cell periphery. Outstanding was the presence of many ruffles or microvillous projections of different shapes and sizes in the plasma membrane. In contrast, the fibroblasts are spindle-shaped and possess less numerous microvillous projections compared with the macrophages, and extracellular spaces neighboring the fibroblasts are loaded with collagen fibers. The fibroblasts are located superficially and sometimes contact each other by the desmosomes. The macrophages are situated relatively deep in the dermis. At the metamorphic stages, both fibroblasts and macrophages contain many phagolysosomes in common, but the desmosomes still remain between the fibroblasts, and therefore the fibroblasts are distinguished from the macrophages. The macrophages are characterized by the their phagolysosomes which contain degenerating cells or cell debris of fibroblasts, myelinated nerve fibers and neutrophils. The macrophages also exhibit a higher endocytotic activity than the fibroblasts for the injected foreign particles (FITC-dextran or latex beads). On the other hand, the phagolysosomes (or autophagolysosomes) of the fibroblasts are characterized by intracellular collagen fibers. These different phagolysosomes between the macrophages and the fibroblasts mean that they may play different roles in the degeneration of the cellular or the extracellular components, respectively, of the tail fins during metamorphosis.


Scanning electron microscopical study of skeletal muscle fiber ends in normal and dystrophic mice.

Muscle fiber ends at myotendinous junctions were examined by scanning electron microscopy after removal of tendon connective tissue components by HCl hydrolysis in the extensor digitorum longus muscle of 30-, 60- and 120-day-old normal and dystrophic (dy) mice. A remarkable morphological difference between normal and dystrophic mice was observed. In normal 30-day-old mice, muscle fiber ends had already assumed a complicated three-dimensional morphology with many thin cytoplasmic processes and lateral longitudinal clefts. On the other hand, in dystrophic animals at the 60th day, muscle fiber ends were characterized by a simple conical form with a rather smooth surface in which existed a number of pit-like sarcolemmal invaginations, and short and narrow longitudinal slits, possibly an indication of developmental immaturity. Thereafter, the slits increased in number and in length, although the linear elongation of the slits seems to be caused by the fusion of adjacent pits to one another and to existing slits. From these findings, the fiber ends in the adult (120-day-old) dystrophic mouse are suggested to retain a preceding state of their unique structural differentiation.


Alumina ceramic prostheses for bone tumor surgery.

Between 1979 and 1990 reconstruction using a ceramic prosthesis with a polycrystal alumina segment and a monocrystal alumina stem was carried out in 65 patients after the resection of malignant or benign aggressive bone tumors. Resection of 18 osteosarcomas, 5 chondrosarcomas, 9 other sarcomas, 10 giant cell tumors, 20 metastatic bone tumors, and 3 other bone tumors was followed by replacement of 17 proximal femurs, 12 distal femurs, 12 proximal tibia, 11 proximal humeri, 3 distal radii, 5 midshafts of the long bone, 2 pelvises, and 3 other parts. Results were rated excellent in 4 cases, good in 43, fair in 13, and poor in 4. In the cases with benignly aggressive or low-grade malignant tumors and those with tumors of the proximal femur, proximal tibia, or midshaft, satisfactory results can be obtained. Four skin ulcers, three dislocations, three loosenings, two infections, and two breaks were noted. Close interfacing between the ceramic prosthesis and the bone was observed radiologically in all cases with cementless fixation except in cases with high-grade malignancies in the knee joint. These results demonstrate that the ceramic prosthesis can be beneficial for the management of patients with benignly aggressive or low-grade malignant bone tumors who have retained adequate muscle strength around the joint even after tumor resection.


Bone-cement removal with the excimer laser in revision arthroplasty.

The excimer laser was thought to be an appropriate tool for the removal of bone cement without damaging the bone. However, due to its low ablation rate, its clinical use in total hip revision arthroplasty proved to be impossible. This experimental study was designed to evaluate the maximal ablation rate by adjusting the laser's parameters. Energy density, frequency, pulse duration, radiation area, quantity of pulses, and environmental conditions were varied in the experimental setup. Even with the best set of parameters the excimer laser was about ten times slower than, e.g., the carbon dioxide laser. The removal of 10 g bone cement takes about 1 h. Thus, complete cement removal by means of the excimer laser alone is not possible. However, selective application of the excimer laser in combination with other techniques could be discussed.


Moderate heat treatment of bone allografts. Experimental results of osteointegration.

The use of bone allografts is often essential in orthopedic surgery. Strict donor screening, including HIV testing 3 months postoperatively, is mandatory before a transplant may be used. Yet these measures do not definitely rule out the possibility of HIV transmission, as there is a window period before infection is revealed by blood testing. Accordingly, there is a need for virus inactivation methods that can be used on bone allografts. As radiation treatment and chemical methods have a number of disadvantages, we chose a moderate heat treatment of 65 degrees C for a series of animal experiments. In 12 rabbit femoral condyles, moderate-heat-treated bone allografts were implanted into 6-mm drill holes. Twelve normal allografts and 12 empty drill holes served as controls. Radiologic and histological evaluation up to 12 weeks postoperatively revealed slow spontaneous bone remodeling from the rim to the center of the empty cavities. Normal deep frozen allografts were quickly intergrated after a short period of osteoclast reaction around the transplant, with occasional bone bridges between host and allograft. The examination of heat-treated allografts showed no differences to the controls, including morphologic aspects and the time course of osteointegration. Five zones of bone repair and osteointegration were distinguished. We conclude that thermal treatment of bone allografts has adverse effects on osteointegration in the rabbit femoral condyle. Thus, it may contribute to improving safety in human bone transplantation.


Osteochondrosis dissecans of the talus. Comparison of results of surgical treatment in adolescents and adults.

Clinical results in two groups of patients treated for osteochondrosis dissecans tali were examined in a follow-up examination: adolescents up to age 16 years versus adults. Thirteen patients were reexamined in each group. Clinical comparison revealed distinct differences between the groups. In adolescents there were eight excellent and three good results, only one fair, and one poor result. In contrast, adults demonstrated excellent results in only two and good results in five patients; five patients showed fair results and one a poor outcome. No signs of osteoarthrosis were detected in adolescents whereas in four adult patients slight radiological signs of osteoarthritis were visible. In both groups clinical and radiological results were better when the cartilage layer at the talar dome was found to be intact at the time of surgical intervention. Detectable cartilage damage at the time of operation was accompanied by a worse result at the time of follow-up examination. In conclusion, adolescents without any signs of joint locking or loose body formation should first be treated conservatively. The older the patient the shorter the period of nonsurgical treatment should be. In cases of increasing stages, joint locking, or formation of loose bodies surgical intervention is recommended.


A small effect of weight bearing in promoting fracture healing.

We treated 66 consecutive type IV comminuted femoral shaft fractures with static Grosse-Kempf interlocking nails and followed them up for at least 1 year (median 27 months). Although the effect of weight bearing was completely lost, 60 fractures healed primarily without dynamization. We conclude that weight bearing contributes only an auxiliary role in promoting fracture healing, and that the most decisive role is adequate mechanical stability with sufficient nutrition.


Ciprofloxacin-impregnated poly-L-lactic acid drug carrier. New aspects of a resorbable drug delivery system in local antimicrobial treatment of bone infections.

Resorbable poly-L-lactic acid (PLLA) cylinders (3.5 mm diameter, 5 mm in length) carrying 6% of weight ciprofloxacin (Ciprobay, Bayer AG, Leverkusen, FRG) were investigated in vitro to explore their properties as a slow-release antibiotic deposit. Forty bioactive cylinders stored in test tubes were covered with phosphate buffer (pH 7.4 at 37 degrees C) and 40 with fresh human blood plasma and tested under various conditions. For comparison a gentamicin-polymethylmethacrylate (PMMA) chain (Septopal, E. Merck, Darmstadt, FRG) was exposed to similar test conditions. The quantities of ciprofloxacin and gentamicin released were analysed by a microbiological method (bioassay). The concentrations of ciprofloxacin released were analysed by a microbiological method (bioassay). The concentrations of ciprofloxacin released from 40 cylinder were initially very high (up to 180 mg/l) but they decreased rapidly within the first 5 days (4.2-22.5 mg/l). Early release of gentamicin reached up to 227.5 mg/l but dropped to of 22 mg/l on the 14th day. Complete degradation of the PLLA-cylinders was not seen in the observed period of 92 days. The mean loss of mass was 8.4%. The recovery of incorporated ciprofloxacin was 6.5% on average.


Osteoblastoma of the coccyx. A report of two cases.

Osteoblastoma, defined as a benign bone tumor by Jaffe and Lichtenstein, together with osteoid osteoma and multifocal osteoblastoma, belongs to the osteoblastic tumors. Due to the variable features of this tumor, differential diagnosis comprises fibrous dysplasia, giant cell tumor, aneurysmal bone cyst and osteoid osteoma; histological analysis is the only way to obtain a definite answer about the tumor type. Occasionally differential diagnosis from aggressive osteoblastoma and osteosarcoma may be difficult. The history of symptoms is usually long and diagnosis may be difficult both clinically and histologically.


Stress fracture of the radius following non-union of an isolated fracture of the ulna.

Non-union of the ulnar diaphysis after isolated ulnar shaft fracture is uncommon. Complications due to non-union of the ulna must therefore be very rare. A case of a stress fracture of the radius in a postmenopausal woman following an isolated ulnar shaft fracture and non-union is presented. The mechanism of the stress fracture and propositions for preventing non-union of the ulnar shaft after an isolated fracture in the osteoporotic patient are discussed.


Bilateral rupture of the patellar tendon without predisposing systemic disease.

Bilateral rupture of the patellar ligament in a 49-year-old woman is reported. She sustained a relatively minor trauma and no underlying systemic disease could be found. This is the 14th case of simultaneous spontaneous rupture of the patellar tendons reported in the literature. The diagnostic features were diffuse swelling around the knees, visible and palpable infrapatellar defects, and an inability to extent either leg.


Traumatic rupture of the quadriceps tendon in a 16-year-old girl. A case report.

Traumatic rupture of the quadriceps tendon is well known to occur in middle-aged men in patients on hemodialysis for renal failure and in patients with diabetes, but only very rarely in young and healthy women. We report a case of traumatic rupture of the quadriceps tendon of a girl without any of the predisposing factors listed.


Patellar tendon rupture. Description of a simplified operative method for a current therapeutic problem.

A simple operative method for the treatment of patellar tendon rupture is introduced. The operative procedure is described and we present the short-term results of the first seven patellar tendon ruptures that have been treated accordingly at our clinic. The technique is easily reproducible and utilizes a minimum of foreign body implant. A secondary procedure for removal of suture material is rarely necessary.


MRI of the knee joint with a 3-D gradient echo sequence. Equivalent to diagnostic arthroscopy?

Three-dimensional (3-D) gradient echo sequences offer the advantage of volume imaging within a few minutes scanning time and creating thin contiguous slices. An optimized sequence (FISP 3D 40) was evaluated for its potential to detect lesions of the menisci, cruciate ligaments, and articular cartilage in 80 patients compared to arthroscopy as the standard. Magnetic resonance imaging showed a sensitivity of 97.9%, a specificity of 97.3%, and an accuracy of 97.5% for meniscal lesions, and a sensitivity of 100%, a specificity of 97.1%, and an accuracy of 97.5% for lesions of the cruciate ligaments. Based upon morphological criteria normal cartilage was identified in 91.5%, but fibrillation of different depths (grades 1-3) was poorly recognized. All full-thickness defects (grade 4) were recognized. Measurement of mean signal intensity (95% confidence intervals) was more useful to discriminate normal from abnormal cartilage (grades 1-3). This method promises the capability to detect early stages of oestoarthritis in future. The present results show MRI of the knee joint with a 3-D fast imaging technique to be a noninvasive alternative to diagnostic arthroscopy.


Practical approaches to pediatric dermatology.

Pediatric dermatology has evolved as a subspecialty of dermatology. The ability to recognize and adequately treat the most common pediatric dermatoses represents an important skill for all dermatology nurses.


Staff nurse knowledge and perceptions about prevention of pressure sores.

A survey of 245 staff nurses from multiple health care settings revealed that nurses had a good knowledge of risk factors for skin breakdown. However, a variety of environmental and clinical factors were cited as impediments to their ability to maintain patient skin integrity. Development of nursing interventions that are effective and also practically feasible in a particular care setting are needed.


Skin care in the space environment.

Envisioned in the 21st century is the United States Space Program's establishment of a permanent human presence in a space station and a moon colony. Mid-21st century humans will take part in an exploratory trip to Mars from the moon (Collins, 1988; Ride, 1987). Fundamental to achieving these space goals will be human health care management. Skin, the largest human organ, will require care. The purpose of this article is to provide an understanding of skin care in the space environment.


Power at meetings: strategies to move people.

Opportunities exist during meetings to become visible, make impressions, gain control, and be an initiator of decisions. Selective strategies and group dynamic skills enhance the nurse's ability to generate influence and power at meetings.


Oxyquinoline-containing ointment vs. standard therapy for stage I and stage II skin lesions.

This study compared DermaMend and A & D for treating Stage I and Stage II skin lesions in nursing home residents. DermaMend led to more healing (p < .05) and more rapid healing (p < .03 Stage II lesions). Nurses preferred DermaMend over A & D 79.5% to 4% (16.5% no opinion) (p < 0.01).


Methylprednisolone treatment of patients with steroid-resistant nephrotic syndrome.

Treatment with a combination of pulse methylprednisolone (MP) and an alkylating agent has been reported to induce long-term remission of proteinuria in patients with steroid-resistant nephrotic syndrome (SRNS). We have treated 13 patients with SRNS with a course of pulse MP. There were 8 black patients and 5 white; 10 had a biopsy diagnosis of focal segmental glomerulosclerosis (FSGS) and 3 nil lesion. Initially 5 patients responded and 2 partially responded. Of the responding patients, 5 relapsed while treated with alternate-week MP therapy. Of these relapsing patients, 3 received a second course of MP plus chlorambucil; 2 responded. The patients were observed for a mean of 47 months (range 4-64 months). When last seen only the 3 patients with a biopsy diagnosis of nil lesion were protein free. There were no complications of steroid therapy. Six patients currently have end-stage renal disease and 2 have renal insufficiency. All of the 6 patients with no response to treatment were black. These data suggest that a course of pulse MP therapy alone induces short-term remission of the nephrotic syndrome in some white patients with FSGS, but in almost no blacks. Patients who relapse may respond to retreatment, but addition of an alkylating agent does not appear to induce long-term remission in patients with FSGS.


Creatinine for estimation of glomerular filtration rate.

The aim of this study was to evaluate the plasma creatinine concentration (PCr) and creatinine clearance (CCr) for estimation of glomerular filtration rate (GFR). Inulin clearance (Cin) was used as the reference standard for GFR. Thirty-nine concurrent Cin and CCr studies provided data for comparing Cin with the measured CCr and with the calculated CCr (calc-CCr). (Calc-CCr = k.L/PCr, where L = height in centimeters and k is the proportionality constant.) Thirty-one children 5.3-20.8 years of age, with Cin ranging from 2.8 to 138.8 ml/min per 1.73 m2, participated in these studies at The Children's Mercy Hospital. The measured CCr was 16.7 +/- 10.3 ml/min per 1.73 m2 (P < 0.001) greater than the Cin, and the calc-CCr overestimated Cin by a mean of 31.6 +/- 20.8 ml/min per 1.73 m2 (P < 0.001). Although there is good correlation between Cin and CCr (r = 0.96), and Cin and calc-CCr (r = 0.90), the 95% confidence intervals are quite broad. Hence, the CCr and the calc-CCr, derived using Schwartz values for k, consistently overestimate GFR. However, if the k value in the equation GFR = k.L/PCr is derived from k = Cin/L, rather than from k = CCr.PCr/L, a more accurate estimate of GFR may be obtained.


Renal cortical and renal medullary necrosis in the first 3 months of life.

Renal cortical necrosis, renal medullary necrosis, and combined renal cortical-medullary necrosis result from renal ischemia without vascular occlusion. Renal hypoperfusion and ischemic injury in infants have been ascribed to massive blood loss, hemolytic disease, septicemia, and severe hypoxemia. In a postmortem study we identified 82 cases among 1,638 autopsies during the 20 years between 1970 and 1989 in infants 3 months old or less at the time of death. The frequency of renal necrosis in autopsy cases increased significantly during the last 6 years of the study. The distribution of the renal lesion was cortical in 28, medullary in 23, and combined in 31. Forty infants carried diagnoses of congenital heart disease, 17 of asphyxial shock, 9 of sepsis, 3 of infectious myocarditis, 9 of major malformations, 4 of anemic shock, 1 of vascular malformation, and 1 of gastroenteritis and dehydration. A significantly higher proportion of babies with congenital heart disease had cortical involvement. Comparison of clinical characteristics revealed a significantly higher frequency of prematurity, respiratory distress syndrome, bleeding diathesis, and possibly sepsis in the children with congenital heart disease, suggesting that these factors are important in the pathogenesis of the renal lesion. Fourteen infants underwent cardiac catheterization; there was no demonstrable association between the renal lesions and the use of radiographic contrast medium. We conclude that severe congenital heart disease itself is a risk factor for life-threatening renal cortical and medullary necrosis.


Plasma atrial natriuretic peptide and endothelin levels in acute poststreptococcal glomerulonephritis.

Plasma levels of atrial natriuretic peptide (ANP) and of endothelin (ET) were significantly elevated (87.7 +/- 13.9 pg/ml and 79.7 +/- 10.8pg/ml, respectively) during the acute phase of acute poststreptococcal glomerulonephritis (APSGN). Plasma renin levels were normal, fractional excretion of sodium (FENa) was 0.5 +/- 0.1% and creatinine clearance (CCr) averaged 82.2 +/- 18.3 ml/min per 1.73 m2. In the recovery phase of the disease (n = 12), levels of ANP (23.6 +/- 6.7 pg/ml) and ET (43.1 +/- 2.4 pg/ml) fell and were not significantly different from those measured in 11 control subjects. FENa increased to 1.3 +/- 0.1% and CCr to 113.5 +/- 12.1 ml/min per 1.73 m2 (all values mean +/- standard error). ANP did not correlate with PRA, blood pressure, CCr or FENa. There was an inverse relationship between the ET level and FENa in the acute phase of the disease (r = 0.489, P < 0.05), but no significant correlation between ET and blood pressure, PRA, CCr or ANP was found. We suggest that, despite the sodium retention, the increased ANP level in APSGN indicates unresponsiveness of the kidneys to ANP; the increased ET levels may contribute to this.


Plasma renin activity as a marker for growth failure due to sodium deficiency in young rats.

We evaluated the effect of feeding diets of varying sodium content on growth and plasma renin activity (PRA) in young rats. In the first study, four groups of rats were offered 10 g/100 g body weight per day of diets containing either 0.005%, 0.015%, 0.03%, or 0.3% sodium; weight gain per day of each rat was followed for 10-14 days and PRA was then measured. A control group was fed a sodium-replete tryptophan-deficient diet which caused protein calorie malnutrition and inhibited growth. Weight gain (g/day) among the rats on the sodium-deficient diets varied directly (r = 0.81, P < 0.001) and PRA inversely (r = -0.82, P < 0.001) with dietary sodium content. PRA varied inversely with weight gain (r = -0.84, P < 0.001). Insulin-like growth factor-1 (IGF-1), which is depressed in calorie-deficient growth failure, was depressed in all the rats on the low-sodium intakes relative to ad libitum-fed controls, but did not vary in relation to dietary sodium or weight gain within those groups. In rats fed the tryptophan-deficient diet, both IGF-1 and weight gain were severely depressed; PRA was normal. In the second study, rats in each of two groups were pair fed, the diet containing either 0.03% or 0.3% sodium matched to rats fed the 0.005% sodium diet; weight gain was followed for 28 days. Both length and weight gain were retarded; PRA again varied inversely with dietary sodium content and with weight gain.(ABSTRACT TRUNCATED AT 250 WORDS)


Increased insulin-like growth factor-II tissue concentrations during compensatory kidney growth in infantile rats.

Insulin-like growth factor (IGF)-I and IGF-II serum and kidney tissue concentrations were measured in compensatory kidney growth in infantile and adult rats. We hypothesized that the known switch from IGF-II in fetal life to IGF-I in adult life may be responsible for the different modes of compensatory kidney growth, which are mainly characterized by hyperplasia in infantile rats and hypertrophy in adult rats. While IGF-I serum concentrations increased with age in infantile rats, kidney tissue concentrations of IGF-I showed a similar increase in both age groups after uninephrectomy. In adult rats, serum and kidney tissue concentrations of IGF-II were unchanged by uninephrectomy. In infantile rats, however, a significant increase in both serum and kidney concentrations of IGF-II was observed with a maximum at day 5 after uninephrectomy. To investigate if compensatory kidney growth is dependent on hyperperfusion of the remnant kidney, the left renal artery was clipped in infantile rats. The clipped kidney showed growth retardation despite normal kidney tissue concentrations of IGF-I and IGF-II. The contralateral kidney was enlarged and IGF-II kidney concentrations were elevated. However, animals with one clipped kidney and nephrectomy of the contralateral kidney showed compensatory kidney growth of the clipped kidney combined with increased IGF-II kidney tissue concentrations. We conclude that IGF-II mainly promotes compensatory kidney growth in infantile rats by hyperplasia. Hyperperfusion of the remnant kidney seems to be unnecessary for initiation of compensatory kidney growth.


Developmental differences in renal sulfate reabsorption: transport kinetics in brush border membrane vesicles.

Renal tubular reabsorption of inorganic sulfate is greater in younger than older guinea pigs. To determine the mechanism of this difference, we studied the transport of inorganic sulfate in renal brush border membrane vesicles (BBMV) obtained from young (< 25 days) and adult guinea pigs (> 60 days). BBMV were obtained by mechanical and osmotic disruption of dissected renal cortices followed by magnesium precipitation and differential centrifugation. After the membranes were incubated for 10 s in solutions containing inorganic sulfate at several concentrations (0.1-10 mM) and trace amounts of 35sulfate, intravesicular uptake was measured. Based on 35sulfur uptake, reabsorption transport kinetics (Vmax and Km) were estimated. BBMV obtained from young guinea pigs demonstrated higher sodium-sulfate cotransport, Vmax (51.79 +/- 4.34 pmol/mg protein per s) than those obtained from adult animals (Vmax = 34.28 +/- 9.17 pmol/mg per s), P < 0.05. Vmax values are represented as means plus or minus standard deviation. No differences in Km were observed. Our results indicate that age-related differences in renal inorganic sulfate reabsorption are due to a higher Vmax for sodium-sulfate cotransport in the younger animals, suggesting a higher density of sodium-sulfate cotransporters or an increased cotransport turnover rate in this age group.


The effect of erythropoietin on the cellular defence mechanism of red blood cells in children with chronic renal failure.

The glutathione redox system, haemoglobin (Hb) oxidation, the activity of antioxidant enzymes and the lipid peroxidation product malonyl dialdehyde (MDA) were studied in red blood cells (RBCs) during administration of recombinant human erythropoietin (rhEPO) over 12 weeks in ten children maintained on haemodialysis. A rapid increase in the reticulocyte count was accompanied by a slower rise in total Hb concentration. The mean level of oxidized glutathione (GSSG) increased from 13.2 +/- 5.3 nmol/g Hb to 56.7 +/- 15.8 nmol/g Hb 4 weeks after the start of rhEPO (P < 0.001), followed by a fall to the basal value. Reduced glutathione (GSH) levels showed a smaller though constant elevation during rhEPO therapy (P < 0.001). Before rhEPO treatment, incubation of RBCs for 1 h with acetylphenylhydrazine induced a decrease in GSH concentration compared with controls (P < 0.001), which became more pronounced in the first few weeks of rhEPO therapy (P < 0.001). In addition, the percentage of Hb derivatives (metHb and haemichrome) increased in the first 4 weeks of rhEPO therapy (P < 0.001). Although there was no significant difference between the values obtained preEPO and during EPO treatment, MDA levels were continuously higher and superoxide dismutase, catalase and glutathione peroxidase concentrations were lower than in the controls (P < 0.001). These results are compatible with oxidative damage to the RBCs in the early period of rhEPO therapy in children with end-stage renal failure. The GSH-GSSG system, as an important cellular defence mechanism of the RBCs, appears to be severely affected.


Peritoneal morphology in children treated by continuous ambulatory peritoneal dialysis.

Fifty peritoneal biopsies (PB) from 35 patients with end-stage renal disease, treated by continuous ambulatory peritoneal dialysis (CAPD) and aged 2 months to 18 years, were examined by light microscopy (n = 50) and/or scanning electron microscopy. PB were performed during surgical procedures immediately before the start of, during, or after the cessation of CAPD treatment. PB from 15 children without renal disease undergoing laparatomy were examined similarly. Before the start of CAPD, a scarcity and shortening of the mesothelial microvilli was observed by scanning electron microscopy. During and after CAPD, variable alterations of mesothelium, interstitium and capillaries were found. The mesothelial layer was absent in all 5 PB obtained during episodes of active peritonitis. In patients treated by CAPD for longer than 6 months, mesothelial denudation was observed more frequently (6/11) than in children treated for shorter periods (1/7) (P < 0.08). Fibrosis of the peritoneal membrane was present in about 50% of patients during or after the cessation of CAPD without impairment of peritoneal function. No correlation was found between the presence of fibrosis and the frequency of peritonitis or the duration of CAPD treatment.


Acute tubulointerstitial nephritis with uveitis syndrome presenting as multiple tubular dysfunction including Fanconi's syndrome.

We describe an 11-year-old male patient with acute tubulointerstitial nephritis with uveitis (TINU) syndrome. He presented with easy fatigability, pallor, nocturia and weight loss. Laboratory examination disclosed anaemia, polyclonal hypergammaglobulinaemia, low molecular weight proteinuria, glycosuria, aminoaciduria, proximal and distal renal tubular acidosis, a urine concentration defect and decreased creatinine clearance. The multiple renal tubular dysfunction and slight glomerular dysfunction subsided spontaneously. Bilateral anterior uveitis was manifested 7 months after the onset of the disease. This is the first reported case of TINU syndrome with multiple proximal and distal tubular dysfunction including a complete type of Fanconi's syndrome.


Cerebral infarct with recurrence of hemolytic-uremic syndrome in a child following renal transplantation.

A white girl with a history of atypical hemolytic-uremic syndrome (HUS) and persistent microangiopathic anemia, and thrombocytopenia for 2 months after the initial presentation at age 7 months, received her first cadaveric renal transplant at age 3 years. During the first 2.5 days post transplant, she developed progressive thrombocytopenia and anemia followed by tonic-clonic seizures and loss of consciousness, secondary to a diffuse cerebral infarction of the left hemisphere. Renal histology showed evidence of glomerular microthrombi and microangiopathy. A large cerebral infarct, previously described in patients during their initial presentation with HUS, presented in our patient as part of the recurrence of the disease post renal transplantation.


Treatment of steroid-resistant post-transplant nephrotic syndrome with cyclophosphamide in a child with congenital nephrotic syndrome.

A child with congenital nephrotic syndrome underwent renal transplantation, was treated for acute rejection, and then developed nephrotic syndrome and renal failure. He was felt to have minimal change disease on allograft biopsy, but failed to respond to therapy with corticosteroids. Cyclophosphamide was substituted for cyclosporine and rapidly induced a complete remission of his nephrotic syndrome. We feel that this case not only represents an important example of a useful therapeutic approach to the child with congenital nephrotic syndrome who develops nephrotic syndrome post transplantation, and also raises questions concerning the pathogenesis of congenital nephrotic syndrome.


Hypertension associated with diffuse small artery calcification: a case report.

Renal ultrasound examination, performed following a urinary tract infection in a 4.5-year-old girl with triple X syndrome, showed multiple echogenic foci at the corticomedullary junction in both kidneys. She was asymptomatic but had hypertension with echocardiographic evidence of left ventricular hypertrophy. Computerised tomographic scan revealed foci of calcification in the kidneys, spleen and pancreas. On biopsy calcification was found in the internal and external elastic laminae of the superficial temporal artery and in the internal elastic lamina of a renal arcuate artery. Intimal fibrosis was mild and focal. No other arterial calcification was demonstrated radiographically or by ultrasound. Biochemical and hormonal profiles revealed no abnormality except hypercalciuria. The aetiology and prognosis of this child's condition are unknown. Although similar ultrasound and histological appearances have been described in pseudoxanthoma elasticum and in idiopathic arterial calcification of infancy, there is no evidence that the child has either of these conditions.


IgA nephritis in a patient with Alagille syndrome and a transplanted liver.

Alagille syndrome (arteriohepatic dysplasia) is a major cause of intrahepatic cholestasis in infancy. The present report describes a patient with Alagille syndrome who presented with hematuria and IgA nephritis 7 years after an orthotopic liver transplantation and immunosuppression. This patient suggests that glomerular lipidosis is not an inherent feature of the Alagille syndrome, and that IgA nephritis may develop in spite of ongoing immunosuppressive treatment.


Arteriovenous fistula after biopsy of renal transplant kidney: diagnosis and treatment.

An 11-year-old renal transplant recipient was noted to have a bruit over her transplant graft 26 months post transplant and 17 months following percutaneous renal biopsy during an episode of rejection. Diagnosis of an arteriovenous (AV) fistula was made by ultrasound examination with Doppler flow and was confirmed with arteriography. The AV fistula was occluded by transcatheter embolotherapy with placement of a steel coil into the fistula from the renal vein approach. This procedure allowed nonsurgical closure of the AV shunt without significant change in renal function.


Tubulointerstitial nephritis.

Tubulointerstitial nephritis (TIN) describes a range of pathological processes that are at least partly responsible for the progression of renal disease of nearly all aetiologies. TIN is frequently the most important pathological manifestation of progressive glomerulonephritis, obstructive uropathy, reflux nephropathy and cystic diseases, although it may also present as a primary disease process associated with infection, drug use or other immunologically mediated disease. Recent clinical and laboratory research has increased our knowledge of tubulointerstitial structure, physiological function and tubulointerstitial response to injury. This review presents a classification of TIN in which acute and chronic tubulointerstitial diseases are recognized as forming a continuum. Primary TIN and TIN associated with glomerulonephritis, obstructive nephropathy and chronic progressive renal disease are discussed from both clinical and pathogenic aspects. It is argued that chronic TIN is a disease process in which inflammation is accompanied by a destructive tubulopathy and fibrogenesis. In acute TIN there is a cessation and reversal of this process. It is suggested that most forms of TIN have an immunological basis because of the presence of immune cell infiltrates, the occurrence of TIN in several immune diseases and immunological animal models of TIN. However, to date TIN has not been convincingly modified in patients by immune manipulation. Experimental evidence suggesting an important pathogenic role for proteinuria and antigenuria, and the renal tubule cell acting as an antigen-presenting cell is discussed.


Erythropoiesis in multiple myeloma: defective red cell production due to inappropriate erythropoietin production.

We investigated the pathophysiology of erythropoiesis in 62 patients with multiple myeloma and examined whether it would establish a rational basis for the treatment of their anaemia with recombinant human erythropoietin. Erythropoietin (Epo) production was evaluated by serum levels and erythropoiesis was quantitated by serum transferrin receptor (TfR) levels, both assessed relative to the degree of anaemia. Instead of the expected stimulation of erythropoiesis in response to anaemia, haematocrit correlated positively with marrow erythropoietic activity, indicating that the mechanism of anaemia was primarily defective red cell production. Erythropoiesis decreased and anaemia worsened significantly with advancing clinical stage. 25% of the patients had inadequate Epo production and this proportion increased to 50% in stage 3. Inappropriate Epo production was seen in 60% of patients with renal impairment but was also observed in a number of patients with normal renal function. Erythropoiesis correlated strongly with the adequacy of Epo production, particularly in advanced disease. We conclude that most myeloma patients have defective red cell production even in the absence of massive marrow infiltration and that inappropriate Epo production contributes to their anaemia.


Polyethylene glycol (PEG) modification of granulocyte-macrophage colony stimulating factor (GM-CSF) enhances neutrophil priming activity but not colony stimulating activity.

PEG-modified proteins have numerous advantages over their unmodified counterparts (increased half life, reduced antigenicity, improved solubility), but almost without exception, they show a modest to marked reduction in biological or enzymatic activity. However, while investigating a new protocol for the preparation of PEG-proteins, we compared PEG-modified and unmodified GM-CSF with respect to their polymorphonuclear neutrophil granulocyte (PMN) priming activities. PEG-GM-CSF was unexpectedly more active than GM-CSF in its ability to prime neutrophils to respond to the synthetic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) with an oxidative burst (assessed both by nitroblue tetrazolium reduction and ferricytochrome c reduction). These results were in contrast to the findings for colony stimulating activity and with GM-CSF induced thymidine uptake, where the biological activity was unchanged or reduced. The enhanced neutrophil priming activity of PEG-GM-CSF was confirmed using FPLC fractionated PEG-modified GM-CSF. This showed changes in the bioactivity profile consistent with both the shift in protein elution profile and enhanced activity of the PEG-modified material (reflected in the increased area under the bioactivity curve). We also excluded a neutrophil priming action for PEG-modified fetal calf serum proteins, carrier proteins and 'irrelevant' cytokine, erythropoietin. The dissociation of the two bioactivities was confirmed using individual FPLC fractions. These results suggest the presence of differences in either binding, receptor/ligand processing or signal transduction for neutrophils versus progenitors, that are differentially affected by PEG-modification of GM-CSF. The demonstration that PEG-modification can partially dissociate two biological activities suggests the feasibility of using PEG-modification to produce proteins with subtly altered spectra of biological activity and hence new ranges of clinical applications.


Long-term treatment results for acute megakaryoblastic leukaemia patients: a multicentre study.

The prognosis and long-term results of a group of 57 acute megakaryoblastic leukaemia (M7-AML) patients was analysed from a multicentre perspective. Ages ranged from 4 to 83 years, median 49 years; 30 were males and 27 were females. The median follow-up time was 7 months, range 1-24 months. Early exits occurred in 12 cases, their median age being 71 years. Forty-five patients were treated with combined aggressive chemotherapy (CT) (n = 26) or low-dose cytarabine (LD-AraC) (n = 19). The following results were obtained with combined CT or AraC, respectively. Complete remission (CR) rates were 73% and 84%, 12-month survival (SV) were 37% and 26%, 24-month SV were 12% and 11%, median SV 10 and 4 months, and relapse rates (RR) were 68% and 94%. These differences were not statistically significant. Irrespective of the treatment modality, the results were better for children (n = 10) than for adults (n = 35): RR rates were 90% and 74%, median SV: 7 and 5 months, 12-month SV: 40% and 22%, 24-month SV; 30% and 9%, and RR: 78% and 81%, respectively; these differences also were not statistically significant. In addition, a literature review of 42 patients from 18 previous reports is presented, including seven cases treated with allogeneic bone marrow transplantation (BMT). The best results were obtained with BMT: 12 and 24 month SV was 86% and the RR was 0%. On the above-mentioned basis, we feel that children and young adults with M7-AML should be offered BMT. In patients over 60 years old or not eligible for aggressive chemotherapy or BMT, an interesting possibility would be the use of LD-AraC which allows a high CR rate, followed by a classical consolidation regimen in order to prevent early relapses.


Multiple independent immunoglobulin class-switch recombinations occurring within the same clone in myeloma.

Multiple myeloma (MM) is characterized by the expansion of terminally differentiated plasma cells. It is still uncertain whether the clonogenic fraction is confined to the plasma cell or pre-plasma cell compartment. We examined the immunoglobulin (Ig) rearrangement of myeloma heavily infiltrated bone marrow cells with a probe from the heavy chain J region (JH) and the BamHI, EcoRI and HindIII restriction enzymes which are appropriate for the detection of clonal VDJ recombination. In 23/39 MMs, clonal Ig gene rearrangement was detected with BamHI, EcoRI and HindIII enzymes. Unexpectedly, in 14/39 patients both BamHI and EcoRI failed to detect Ig rearrangement, whereas HindIII consistently demonstrated VDJ recombination. The 5' sites of BamHI, EcoRI and HindIII restriction fragments are precisely defined by the VDJ rearrangement. Since the 3' ends of BamHI and EcoRI restriction fragments are downstream from the switch mu region and change in size during switch recombination, the absence of rearranged bands is determined by several autonomous recombinations affecting the switch region. By contrast, the 3' ends of HindIII restriction fragments are upstream, their size does not vary during isotype switch allowing the constant detection of clonality. Accordingly, in 35% of patients the clonogenic fraction seems to originate from a pre-switch B cell. This B cell will differentiate to a mature plasma cell developing multiple independent switch recombinations, as the variable mechanism of switch recombination suggests.


Argyrophilic nucleolar organizer region counts and prognosis in multiple myeloma.

The prognostic significance of argyrophilic nucleolar organizer regions (AgNORs) has been evaluated in bone marrow trephine biopsies from 64 patients with multiple myeloma (MM) prior to therapy. The univariate Kaplan-Meyer survival analysis showed a significant correlation between survivals and AgNOR counts (median of survival 51.3 months for cases with < or = 4.62 AgNORs per plasma cell (PC) versus 16 months for cases with > 4.62 AgNORs per PC; P = 0.0000) or AgNOR distribution in PC nucleus (AgNOR configuration) (median of survival 71.67 months for cases with tightly grouped AgNORs, 16.26 for partially grouped and 11.74 for dispersed AgNORs; P = 0.001). Significant prognostic correlations were also found for monoclonal immunoglobulin type (P = 0.008), platelet counts (P = 0.0078), serum creatinine level (P = 0.0001), Durie's clinical stage (P = 0.02), percentage of plasma cells in bone marrow biopsies (BMPC%) (P = 0.005), pattern of medullary involvement (P = 0.003) and PC atypia (P = 0.009). Borderline result was detected for the percentage of PCs in aspirates (P = 0.06). No significant correlation was found between prognosis and patients age, sex, haemoglobin level, serum albumin or calcium level, marrow cellularity and excess of haemosiderin. Multivariate survival analysis showed that only two variables were significantly correlated with prognosis: AgNOR counts (P = 0.003) and AgNOR configuration (P < 0.001). In addition, the analysis of variance showed significant association between AgNOR number and platelet counts, haemoglobin level, calcaemia, creatininaemia, clinical stage, percentage of PCs in aspirates, BMPC%, pattern of medullary involvement, PC atypia, marrow cellularity and configuration of AgNORs. Our results indicate that AgNOR counts and configuration have prognostic and diagnostic value and therefore they are useful independent parameters to assess the pretherapeutic aggressiveness of multiple myeloma.


Follicular dendritic cells in non-Hodgkin-lymphoma express adhesion molecules complementary to ligands on neoplastic B-cells.

In non-Hodgkin-lymphoma (NHL) with nodular growth patterns, follicular dendritic cells (FDC) form a spherical network which contains neoplastic B-cells. In order to dissect the basis of this close FDC/B cell association, the antigenic profile of adhesion molecules expressed by individual FDC and NHL-B-lymphocytes was evaluated. FDC isolated from NHL were found to express C3bi receptors (CD11b), the very late antigen (VLA) alpha-5- and alpha-6-chain (CD49e, CD49f), and the intercellular adhesion molecule-1 (ICAM-1; CD54). Only a percentage of the FDC population was positive for the VLA beta-1- and alpha-3-chain (CD29, CD49c), the vitronectin receptor (CD51) and the vascular cell adhesion molecule-1 (VCAM-1). B-cells obtained from the lymph nodes of patients with centroblastic-centrocytic lymphoma expressed several ligands complementary to the adhesion molecules detected on FDC. These include LFA-1 alpha- and beta-chains (CD11a, CD18), and ICAM-1 (CD54). Surprisingly, monoclonal lymphocytes in the peripheral blood of patients with a leukaemic course of this lymphoma entity were devoid of these antigens. It seems likely then that neoplastic B-cells without CD11a, CD18 and CD54 surface molecules are unable to associate with FDC and now invade other compartments. Thus, the adhesive interactions which do occur between FDC and NHL-B-cells may directly influence the peculiar growth pattern and spread of centroblastic-centrocytic lymphoma.


Quantitative determination of the hybrid Bcr-Abl RNA in patients with chronic myelogenous leukaemia under interferon therapy.

In vitro amplification of the Bcr-Abl cDNA has been widely used to assess for the presence of minimal residual disease in patients with chronic myelogenous leukaemia (CML) presenting with complete clinical and cytogenetic remission. However, the level of sensitivity achieved in different laboratories remains largely unknown. Moreover, the results are usually expressed as positive or negative, making a precise follow-up of the patients difficult. In an attempt to overcome these limitations, we devised a quantitative method to measure the amount of Bcr-Abl mRNA in clinical samples. This methodology involves a single reverse transcription step, followed by separate amplifications of Bcr-Abl and total Abl mRNA. These two amplifications are performed in the presence of different dilutions of a same internal standard. This standard consists of Bcr-Abl sequences with an eight bases deletion in exon 2 of Abl. One of the primers used in each separate reaction is labelled with fluorescein. Following amplification, PCR products derived from cellular RNA and those from the internal standard are separated and their relative fluorescence is determined using a laser fluorescent DNA sequencer (ALF, Pharmacia). The number of Bcr-Abl and total Abl mRNA molecules initially present in each sample is then calculated. The accuracy and reproducibility of this method was assessed by studying serial dilutions of K562 RNA into normal RNA. Blood samples from 10 patients in cytogenetic remission under interferon therapy were studied. Only one sample was found negative while the others contained between 0.05 and 17 hybrid Bcr-Abl mRNA molecules per 1000 molecules of Abl mRNA. These results suggest that a variable number of malignant cells are present in the majority of CML patients in cytogenetic remission following interferon therapy.


Detection of minimal residual disease by polymerase chain reaction of bcr/abl transcripts in chronic myelogenous leukaemia following allogeneic bone marrow transplantation.

The prognostic significance of detecting minimal residual disease by polymerase chain reaction (PCR) amplification of bcr/abl mRNA transcripts was investigated in 27 bone marrow samples from 20 patients with Philadelphia chromosome (Ph1) positive chronic myelogenous leukaemia (CML) in complete cytogenetic remission following allogeneic bone marrow transplantation. Sixteen were transplanted in first chronic phase, two were in second chronic phase, one was in accelerated phase and one was in blast crisis. All 20 achieved complete cytogenetic remission post transplant and 15 patients had detectable bcr/abl mRNA by PCR from 2 to 22 months following the procedure. One of these patients had graft failure and one died from graft-versus-host-disease at 7 months. Of the remaining 13 PCR-positive patients, only one (8%) relapsed after 23 months; the other 12 were alive and still in remission after a median follow-up of 16+ months (ranging 5+ to 29+ months). Five patients were PCR negative; all are alive in complete clinical and cytogenetic remission at 10+, 11+, 19+, 25+ and 25+ months post transplant. In this study, detection of subclinical Ph1-positive cells by PCR was not associated with imminent clinical or cytogenetic relapse. Since late recurrence may potentially occur, long-term follow-up is required to definitely determine the prognostic value of the PCR assay.


The influence of therapeutic blocking of Gp IIb/IIIa on platelet alpha-granular fibrinogen.

Recent evidence suggests that platelet alpha-granule fibrinogen (fg) is derived from the plasma pool. Since platelets from patients with Type I Glanzmann's thrombasthenia (GT) are deficient in intracellular fibrinogen (fg) it was hypothesized that Gp IIb/IIIa could mediate the uptake of fg. To study the potential role of Gp IIb/IIIa in intracellular fg trafficking, the influence of therapeutic blocking of Gp IIb/IIIa on platelet fg was studied in 12 patients with stable ischaemic heart disease. Patients were either given a single intravenous dose of the monoclonal antibody 7E3 Fab (n = 4) or a combination of bolus and continuous infusion up to 24 (n = 3), 36 (n = 3) or 96 h (n = 2). All patients showed grossly prolonged bleeding times with a significant reduction of ex-vivo ADP induced aggregation. Although, surface Gp IIb/IIIa binding sites were consistently reduced in all patients, there was a variable but delayed decrease in platelet fg relative to vWf:Ag in only six out of the 12 patients studied. The reduction in fg appeared dependent upon both dosage and duration of Gp IIb/IIIa blockade. The study provides further evidence for the novel role of Gp IIb/IIIa in the intracellular trafficking of fg to platelet and megakaryocytic alpha-granules.


Adjusted dose continuous infusion of factor VIII in patients with haemophilia A.

Surgical interventions in patients suffering from haemophilia A require infusions of large doses of factor VIII (F VIII) concentrates. These are expensive and may pose a burden on the immune system, which is already compromised in many haemophiliacs. F VIII is usually given as bolus injections, although there are reports on fixed dose continuous infusion. We have developed a continuous infusion programme with dosage adjusted according to daily calculation of the clearance of F VIII. Twenty-four haemophiliacs received a total of 205 d of continuous infusion with F VIII (168 d in hospital, 37 d home therapy). Eighteen patients underwent surgeries (11 elective, seven emergency) and six were treated for serious haemorrhages. We observed a significant, progressive decrease of the clearance of F VIII over the first 5 d from 3.2 (2.8-3.5) to 1.7 (1.3-1.9) ml/kg/h (median and interquartile range). The decrease of the clearance together with the fact that a continuous infusion requires less drug than bolus injections to keep the same minimal concentration, contributed to much lower doses of F VIII than reported in the literature or than required by historical controls, matched for the type of operation. Other advantages of our method are improved safety with stable activities of F VIII, lack of hazardous troughs below the haemostatic range, and the convenience, which permits ambulant therapy even when high doses of F VIII are required.


Molecular characterization of a novel 10.3 kb deletion causing beta-thalassaemia with unusually high Hb A2.

A family of Asian-Indian descent has a variant form of beta-thalassaemia characterized by unusually high levels of Hb A2 in the heterozygous state. The propositus who is homozygous for the mutation has thalassaemia intermedia. Restriction endonuclease mapping suggested the presence of a 10.3 kilobase (kb) deletion removing the whole of the beta-globin gene. Subsequently, molecular analysis was performed by directly sequencing a specifically amplified region of genomic DNA. A 10329 basepair deletion was precisely defined which results in the loss of the 5' beta promoter region and the entire beta-globin gene. The deletion extends from 3011 bp 5' to the mRNA cap site to an L1 repeat element downstream of the beta-globin gene and is very similar to the 12.6 kb deletion of Dutch beta zero-thalassaemia. In common with four other mutations, both these deletions remove the 5' promoter region of the beta gene and all are associated with unusually elevated levels of Hb A2 in the heterozygous state.


Incompletely processed N-glycans of serum glycoproteins in congenital dyserythropoietic anaemia type II (HEMPAS).

Congenital dyserythropoietic anaemia type II, or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test) is a genetic disease caused by membrane disorganization of erythroid cells. The primary defect of this disease lies in the gene encoding enzyme(s) which is responsible for the biosynthesis of Asn-linked oligosaccharides chains of glycoproteins (Fukuda et al, 1990). In order to know whether this gene defect affects the glycosylation in the cells other than the erythroid cells, the carbohydrate structures of the transferrin isolated from the sera of HEMPAS patients were analysed. Fast atom bombardment mass spectrometry analysis showed the presence of high mannose type and hybrid type oligosaccharides in the HEMPAS transferrin which is in contrast to the complex-type oligosaccharides found in the normal transferrin. The results strongly suggest that biosynthesis of Asn-linked oligosaccharide chains in HEMPAS hepatocytes is disturbed. As a result, the serum glycoproteins with incompletely processed carbohydrates are circulating in the plasma in HEMPAS patients, but they must have been absorbed by the cells in the liver and the reticuloendothelial cells. Upon intravenous infusion into rats, as much as 30% of the HEMPAS transferrin was cleared from the plasma circulation. The majority of the HEMPAS transferrins was taken up by the liver, and transferrin was distributed both in the hepatocytes and the Kupffer cells. The presence of enormous amounts of aberrantly glycosylated serum glycoproteins may lead to the liver cirrhosis and secondary tissue siderosis seen in HEMPAS patients.


Red cell autoantibody production by colonic mononuclear cells from a patient with ulcerative colitis and autoimmune haemolytic anaemia.

We have studied the ability of mononuclear cells extracted from the colon (CMC), draining lymph nodes (LNMC), peripheral blood (PBMC) and spleen (SMC) of a patient with ulcerative colitis and severe autoimmune haemolytic anemia to produce red cell autoantibodies. The LNMC, PBMC and SMC secreted immunoglobulin in vitro in pokeweed mitogen culture but not spontaneously. They also produced immunoglobulin when transferred to severe combined immunodeficient (SCID) mice but no anti-red cell activity was demonstrable. In contrast, in vitro cultures of CMC produced immunoglobulin spontaneously, showing the presence of activated B-cells and plasma cells, but anti-red cell activity could not be demonstrated. However, CMC transferred to SCID mice were able to produce IgG with anti-red cell activity. These results concur with clinical observations suggesting that the colon is the source of red cell autoantibodies in these patients.


Rheological properties of rosettes formed by red blood cells parasitized by Plasmodium falciparum.

A proportion of red blood cells parasitized by Plasmodium falciparum form rosettes with non-parasitized red cells. Although these rosettes are thought to impair microcirculatory flow, their rheological characteristics have not been fully described. Using dual-micropipette manipulation to pull apart individual rosettes, we found that the forces binding rosettes together were strong (average force for removal of a cell was 4.4 x 10(-10) N, approximately 5 times that required to detach a parasitized cell adhered to cultured endothelium). If disrupted rosettes were re-formed, cells rosetted immediately on contact, but the strength of attachment increased over minutes, and did not apparently reach its maximal level for hours. All non-parasitized cells tested could adhere to rosette-forming parasitized cells. Rosettes could withstand arterial flow stresses (1.4-1.6 Pa) for minutes without disintegration. To test the effects of rosetting on flow resistance, the time required for entry into a 4.3 microns pipette was measured. Entry times depended strongly on the number of cells in the rosette, and averaged 35 times longer than for non-parasitized cells. Our studies show that the cell-cell attachments within rosettes are strong, and suggest that rosettes might survive both the arterial circulation and passage through microvessels and could contribute to the ischaemic complications of falciparum malaria.


Detection of HIV in haemopoietic progenitors.

Peripheral blood cytopenias present a major problem in the management of patients with HIV infection. Their pathophysiology is likely to be multifactorial, although there is controversy as to whether haemopoietic progenitors are a target for HIV. In order to investigate the haemopoietic defect in HIV infection, we looked at bone marrow culture characteristics of marrow from eight HIV+ patients compared to normal controls. We performed long-term liquid culture (LTC) and colony forming assays for granulocyte-macrophage (CFU-GM) and granulocyte, erythroid, megakaryocyte, macrophage (CFU-GEMM). In LTC we found normal stromal appearance and haemopoietic focus formation. There was no difference in colony assays of CFU-GM and CFU-GEMM between HIV+ and normal controls. Colonies taken from CFU-GM and CFU-GEMM were analysed for HIV DNA sequences, and we were able to detect HIV DNA in colonies from all HIV+ patients. Our results indicate that despite infection of haemopoietic progenitor cells by HIV, bone marrow function is preserved. This suggests that HIV-related cytopenias may be due to alternative mechanisms not present in our in vitro system.


Species and tissue specificity of mammalian GTP cyclohydrolase I messenger RNA.

Northern blot analysis of rat RNA from cell lines and isolated organs with a specific rat cDNA probe detected two GTP cyclohydrolase I mRNA species of approx. 1.4 and 3.6 kb. The ratio between these two species varies between 0.6 and 2.4 in different rat organs. Using primers derived from highly conserved regions in the rat and Escherichia coli cDNA sequences a human GTP cyclohydrolase I probe was obtained by means of reverse transcription and PCR (polymerase chain reaction). The human PCR product consisting of 555 bp was cloned and sequenced. It shows a 92% identity with the published sequence of the rat gene. The analysis of various human cell lines with this specific probe shows only one species of GTP cyclohydrolase I mRNA with an approximate size of 3.6 kb.


Sequences of the swine 21-hydroxylase gene (CYP21) and a portion of the opposite-strand overlapping gene of unknown function previously described in human.

We sequenced a 4.8 kb BamHI swine genomic fragment comprising the entire 21-hydroxylase gene (CYP21) and its 5' and 3' flanking segments. The CYP21 coding sequence spanned 3050 bp and as in other species, comprised 10 exons separated by the corresponding introns. The deduced protein corresponded to 492 amino acid residues, 8 of which differed from a previously sequenced swine CYP21 enzyme. The 5' flanking region displayed several putative cis-acting elements which may be involved in either constitutive or cyclic adenosine 3',5'-monophosphate (cAMP) dependent transcriptional expression. We also characterized within the 5' region a 139 bp repetitive element of the short interspersed nucleotide element (SINE) family located on the opposite strand. In addition, we characterized the last five exons of a human-like opposite strand gene (OSG/X) located in the swine at the 3' end of CYP21. The sequenced part of this OSG/X displayed a very strong homology with its human counterpart.


Purification of a telomere-binding protein from Physarum polycephalum.

We have purified a telomere-binding protein (PPT) from the acellular slime mold Physarum polycephalum. As shown previously (Coren, J.S., Epstein, E.M. and Vogt, V.M. (1991) Mol. Cell. Biol. 11, 2282-2290), in vitro this protein binds specifically to the double stranded (TTAGGG)n repeats that are found at the telomeres of extrachromosomal ribosomal DNA from this organism, and also at telomeres of mammalian chromosomes. PPT was purified from Physarum nuclear extracts by heat treatment at 90 degrees C followed by heparin-agarose fractionation and gel filtration chromatography. The most purified fraction contained two major protein bands of 10 and 7 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In gel filtration chromatography PPT migrated with a Stokes radius of 1.6 nm. Along with the previously determined sedimentation coefficient of 1.2 S, this value implies a molecular weight of about 8000, making PPT the smallest known telomere-binding protein.


Expression and regulation of cytochrome P-450I genes (CYP1A1 and CYP1A2) in the rat alimentary tract.

Cytochrome P-450 (P450) enzymes in the mucosa of the alimentary tract may be involved in the activation and/or inactivation of ingested xenobiotics and procarcinogens. Since the multiple P450 enzymes have overlapping substrate specificities and, in some cases, similar antigenic determinants, definitive identification of P450 genes that are expressed in various tissues requires molecular analysis. In this study, a sensitive and specific polymerase chain reaction assay along with hybridization analysis was used to examine the expression of the CYP1A gene family in the rat alimentary tract. CYP1A1 mRNA was expressed throughout the alimentary tract in untreated rats, as determined by the polymerase chain reaction. However, Northern blot analysis detected CYP1A1 mRNA and enzymatic activity only in small intestine and liver, with greater amounts in intestine. After treatment with beta-naphthoflavone, CYP1A1 mRNA and enzymatic activity was markedly induced in each alimentary tissue including esophagus, fore-stomach, glandular stomach, small intestine and colon. A single dose of inducer resulted in a rapid rise in CYP1A1 mRNA which was shown by nuclear run-on assays to be primarily due to an increase in transcription of the CYP1A1 gene. CYP1A2 mRNA was detected in significant amounts only in glandular stomach following induction although the polymerase chain reaction assay identified low levels of CYP1A2 mRNA in several other tissues. The definitive identification of the CYP1A genes that are expressed in alimentary tissue will allow the design of experiments to investigate the importance of these enzymes in the metabolism of carcinogens, and ultimately carcinogenesis, in the gastrointestinal tract. In addition, these data suggest that the aromatic hydrocarbon receptor, which mediates transcriptional induction of multiple genes by xenobiotics, is expressed through the alimentary tract.


Purification of ribonucleoproteins by a novel approach: isolation of the SSB1 ribonucleoprotein from yeast and demonstration that it has no role in mRNA splicing.

A novel approach is described to purify potential ribonucleoproteins (RNP) of yeast. The method assays a yeast RNP complex, assembled in vitro on actin pre-mRNA, by low-ionic strength acrylamide gel electrophoresis. The minimal protein components of this RNP complex were three proteins, one of 30 kDa and two at 42-44 kDa, defined by formation of the complex on biotinylated-RNA, binding of this complex to avidin-agarose, and salt elution of the protein in the biotinylated-RNP complex. Using the assay for RNP complex formation, an RNP protein was purified to homogeneity on the basis of its affinity towards single-stranded DNA and RNA. This RNP protein turned out to be identical to a known RNP protein, the single-stranded binding protein 1 (ssb1) of yeast, on the basis of identical gel electrophoretic migration, antibody cross-reactivity, and identical properties on the gel complex formation assay. In vitro mRNA splicing was normal in extracts made from a yeast strain missing ssb1 (ssb1- strain). Addition of anti-ssb1 antibody to splicing extracts made from a wild type strain did not inhibit or diminish splicing. Instead, mRNA splicing was reproducibly stimulated several fold, indicating competition between ssb1 and splicing factors for binding to single-stranded RNA in the extracts. RNP complexes still formed in the ssb1- strain, demonstrating that it would be possible to purify other RNP proteins from this strain using the gel complex formation assay.


Isolation and characterization of the gene encoding gluconolactonase from Zymomonas mobilis.

The gene encoding the enzyme gluconolactonase (D-glucono-delta-lactone lactonohydrolase, EC 3.1.1.17) has been isolated from a recombinant library of genomic Zymomonas mobilis DNA, by detection of enzyme activity in recombinant clones. The gene encoded a protein of 320 amino acids, which is processed to the mature enzyme of 285 amino acids (31079 Da) by cleavage at an Ala-Ala bond, as determined from N-terminal sequencing of the purified enzyme. A minor sequence commencing at amino acid 6 is suggestive of an alternative start of translation at the ATG codon of amino acid 5; in this case the expressed enzyme would remain cytoplasmic, whereas it is presumed that the main portion is directed to the membrane of periplasm by the leader sequence.


Cloning and sequencing of a cDNA for the delta-subunit of photosynthetic ATP-synthase (EC 3.6.1.34) from pea (Pisum sativum).

lambda gt10 cDNA clones for the nuclear encoded subunit delta of chloroplast ATP-synthase from Pisum sativum have been isolated. The 5' end was completed by PCR. The sequenced cDNA codes for the import precursor. N-Terminal sequencing of the mature protein isolated from chloroplasts revealed that the processing sites of the transit peptide from Pisum sativum and Spinacea oleracea are similar. The overall homology of the deduced amino acid sequences of the mature delta proteins from higher plants is about 40%. The conservation among hydrophilic residues is higher than for hydrophobic ones, indicating that the surface of delta is important for its function within the ATP-synthase.


Sequence analysis of cucumber cotyledon ribulosebisphosphate carboxylase/oxygenase activase cDNA.

The cDNA encoding ribulosebisphosphate carboxylase/oxygenase activase from cotyledons of dark-grown cucumber seedlings has been isolated and sequenced. The 2.0 kilobase pair cDNA encodes a polypeptide with 413 amino acid residues which shows 70% identity with the rubisco activase from green leaves of spinach. The full-length cDNA was used to prepare rubisco activase by in vitro transcription. Subsequent in vitro translation led to the formation of a 46 kDa protein which is the putative precursor of a 42 kDa plastid enzyme.


Sequence characterization of a sheep cDNA for antithrombin III.

We determined the cDNA sequence of the mRNA for antithrombin III (AT III) from sheep liver. It encodes a protein of 465 amino acids, including a signal peptide of 32 amino acids. The amino acid sequence of the mature protein shows a sequence identity of 89.1%, 95.6% and 85.0% to the human, bovine and rabbit equivalents, respectively. Cysteine residues involved in disulfide bonds as well as potential glycosylation sites are conserved between the four species. In contrast, the amino acid sequence of the signal peptide shows a smaller identity, i.e., 68.7% and 56.3% compared to the human and rabbit preprotein, respectively.


AAT1, a gene encoding a mitochondrial aspartate aminotransferase in Saccharomyces cerevisiae.

We have isolated a gene, AAT1, encoding an aspartate aminotransferase (AspAT) from a Saccharomyces cerevisiae genomic library. AAT1 encodes a 451 amino acid protein with a predicted molecular weight of 51,687, which is likely to be the yeast mitochondrial AspAT. Sequence comparison of this yeast AspAT with AspATs from other organisms shows a high degree of homology in regions previously shown to be important for catalysis. However, the yeast mitochondrial AspAT contains four obvious insertions with respect to all other known AspATs, suggesting that the AAT1-encoded protein represents a distinct AspAT.


Nucleotide sequence of a complementary DNA for human ST2.

Human ST2 cDNA, a homologue of murine ST2 that is only expressed in growth-stimulated BALB/c-3T3 cells and a member of the primary response gene family induced by growth factors, was isolated from the cDNA library of an activated human helper T cell line, 5C10. Human ST2 has 67.6% identity in a 327 amino acid overlap to murine ST2. Furthermore, as in the case of murine ST2, human ST2 encodes a protein remarkably similar in sequence to the extracellular portion of human interleukin 1 receptor, both types 1 and 2. The expression of ST2 in human lymphocytes could trigger further investigations into its physiological role in humans.


Molecular characterization of a putative peroxidase gene of Drosophila melanogaster.

We have identified genomic clones and corresponding cDNAs that encode a putative peroxidase of Drosophila melanogaster. The gene (DmPO) appears as a single copy gene located on the third chromosome at position 89 D/E. It is interrupted by seven small introns and one unusually large 5' intron (about 11 kb). Sequence analysis of the cDNA showed an open reading frame of 690 amino acids resulting in a protein of 77 kDa. The deduced amino acid sequence reveals an overall homology to myeloeosinophil and thyroid peroxidase, a human superfamily of peroxidases.


Nucleotide and (derived) amino acid sequence of a novel peptide from a rat (hypercalcemic) Leydig cell tumor.

The nucleotide sequence of a novel peptide from a rat Leydig cell hypercalcemic tumor H-500 was determined. This cDNA encodes a peptide of 93 amino acids and contains a heparin binding domain similar to histone 2-B. Northern blot analysis showed tissue specific expression of this peptide mRNA.


Polarized secretion of plasminogen activators by epithelial cell monolayers.

We have investigated the synthesis and the polarized secretion of plasminogen activators (PAs) in three epithelial cell lines (FRT, derived from rat thyroid; MDCK, from canine kidney, and CaCo-2, from human intestine) grown on filters, in bicameral systems. Confluency and acquisition of functional polarity were assessed by measuring transepithelial resistance and by showing polarized secretion of endogenous proteins. By zymography, before and after immunoprecipitation with specific antibodies, we found that FRT cells synthesized tissue plasminogen activator (tPA) and that tPA activity was mostly confined to the apical cell compartment. MDCK and CaCo-2 cells, instead, synthesized urokinase-type plasminogen activator (uPA). In MDCK cells the uPA activity was found predominantly in the apical cell compartment while in CaCo-2 cells it was mostly basolateral.


Insulin decreases chylomicron production in human fetal small intestine.

The objective of this study was to examine the effect of insulin on lipoprotein synthesis and secretion in human fetal intestine. Jejunal explants were cultured with [14C]oleic acid in Leibovitz medium for 42 h. Although the addition of insulin (30 U) did not alter the incorporation of [14C]oleic acid into triglycerides, phospholipids and cholesteryl esters in the tissue, it significantly decreased (P < 0.05) the level of triglycerides (20%) in the medium. Among the three lipoprotein classes (chylomicron, VLDL, and HDL) isolated by ultracentrifugation, the chylomicron level was found significantly (P < 0.05) diminished in the medium (29%). Neither the lipid chemical composition of CM or that of VLDL, LDL and HDL was affected by the presence of insulin. These results suggest that chylomicron synthesis is modulated by insulin, whereas lipoprotein distribution and lipid composition are not regulated by this hormone.


Regulation of mitochondrial glycerol-phosphate dehydrogenase by Ca2+ within electropermeabilized insulin-secreting cells (INS-1).

(1) A new insulin-secreting cell line (INS-1; Asfari et al. (1992) Endocrinology 130, 167-178) has been used to study the regulation by Ca2+ of mitochondrial FAD-linked glycerol-phosphate dehydrogenase (FAD-GPDH) in situ. (2) Enzyme activity was examined on-line in electropermeabilized cells by a new, sensitive, assay. This involved the reduction of the artificial electron acceptor, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), monitored by the quenching of the fluorescence of rhodamine-18. Using this approach, similar total levels of FAD-GPDH activity (nmol/min per 10(6) cells) were measured in INS-1 cells (1.35 +/- 0.22) and isolated rat islet cells (1.63 +/- 0.02) (3) Ca2+ ions markedly activated the enzyme, lowering the apparent Km-value for added DL-glycerophosphate from 8.8 +/- 1.4 mM to 1.0 +/- 0.1 mM. Ca2+ had no effect on the apparent Vmax. The enzyme displayed cooperative kinetics with respect to DL-glycerophosphate (Hill coefficient of 2.0 +/- 0.2 and 1.6 +/- 0.2 in the absence and presence respectively of Ca2+). Half-maximal effects of Ca2+ were observed in the range 30-130 nM, depending on the concentration of glycerol phosphate. (4) Enzyme activity was weakly (30%) inhibited by diazoxide, but not by the diabetogenic drug, streptozotocin. (5) The data indicate that INS-1 cells represent an excellent model for studying the rôle of FAD-GPDH in the control of insulin secretion.


Glycine, a new regulator of glutamine metabolism in isolated rat-liver cells.

Glycine (0.1-10 mM) caused a dose-dependent increase in the removal of 5 mM [1-14C]glutamine by isolated rat-liver cells; at low concentrations of glycine, an increase in the formation of 14CO2, urea and glucose from glutamine occurred. At 2-10 mM, glycine also caused an accumulation of ammonia, a well-established activator of glutaminase (E.C. 3.5.1.2) and, at concentrations found in the presence of glutamine plus glycine, ammonia stimulated glutamine removal. The inhibition of urea synthesis from glutamine observed with 10 mM glycine was relieved by the addition of ornithine, suggesting that this inhibition occurred by reducing the availability of ornithine for the ornithine transcarbamoylase reaction. The metabolism of glycine as sole substrate led to a small increase in the accumulation of ammonia. Glycine did not alter hepatic glutaminase activity but swelling of rat hepatocytes, a factor considered to stimulate glutamine metabolism, was observed in the presence of glycine (1 mM). It is concluded that stimulation by glycine of hepatic utilization of glutamine is mediated by the accumulation of ammonia arising from both glycine and glutamine metabolism and by hepatocyte osmotic swelling secondary to glycine transport.


Polyamine metabolism in enterocytes isolated from newborn pigs.

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol/10(6) cells, respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM L-arginine or 2 mM L-glutamine was very low in isolated enterocytes (6.4 +/- 3.8 pmol/10(6) cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low L-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from L-arginine remained undetectable despite a higher L-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from L-arginine or L-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.


Ion transport across the epithelium of the rabbit caecum.

The isolated rabbit caecum was studied in vitro. Under our experimental conditions, the rabbit caecum secreted potassium and chloride and absorbed sodium. To characterize the transport properties of the apical and the basolateral barriers, transepithelial electrical and flux (22Na, 36Cl and 86Rb) measurements and their sensitivity to transport inhibitors (furosemide, DIDS, ouabain and barium) are presented together with intracellular measurements with double-barrelled microelectrodes of intracellular electrical potentials and ionic activities. The fluxes of sodium and chloride were insensitive to DIDS and furosemide. The secretion of potassium and the absorption of sodium were both inhibited by ouabain, indicating that they are coupled through the sodium pump. Ouabain induced a slow fall in the chloride net fluxes, suggesting that these fluxes are also driven by the sodium pump, albeit indirectly. The basolateral to apical fluxes of potassium are insensitive to barium added to the apical side, but are accelerated by the replacement of chloride by gluconate on the apical side, suggesting the presence of a K+/Cl- symport in the apical barrier.


Participation of calpain I activation in the ATP release reaction of platelets stimulated with thrombin.

Proteolytic activation of calpain (calcium-dependent neutral protease) I in thrombin-stimulated platelets was determined by following the production of the 76- and 78-kDa forms from the 80-kDa subunit of calpain I as measured by immunoblotting using monospecific antibody to human calpain I, and the correlation between the extents of calpain I activation and ATP release was investigated. When platelets were stimulated with thrombin in the range from 0.01 to 0.5 U/ml, the maximal 60% activation of calpain I was achieved within 15 s after the stimulation, and ATP release began after the maximal activation had been reached. The extent of ATP release decreased in parallel with the decrease in activation ratio of calpain I on treatment of platelets with EGTA or EST, a membrane-permeable inhibitor of calpain. Although pretreatment of platelets with EST did not affect the thrombin-dependent elevation of the cytosolic Ca2+ concentration, both the inhibition of calpain I activation and the reduction of ATP release were observed as a function of EST concentration. These results suggest that calpain I participates in one of the processes leading to the ATP release reaction of platelets stimulated with thrombin.


Three types of Gi alpha protein of the guinea-pig lung: cDNA cloning and analysis of their tissue distribution.

cDNA clones encoding three types of Gi alpha, the alpha subunit of GTP-binding protein (Gi1 alpha, Gi2 alpha, and Gi3 alpha), were isolated from a cDNA library of the guinea-pig lung. Nucleotide sequence analysis revealed a high degree of homology with other mammalian Gi alpha cDNAs. By RNA blot analysis, the expression pattern of Gi1 alpha was more tissue-specific than those of other types of Gi alphas in the guinea-pig tissues examined. While Gi2 alpha and Gi3 alpha mRNAs were ubiquitously expressed in all tissues examined, Gi1 alpha mRNA was mainly expressed in the brain, lung and kidney. These results suggest that each Gi alpha protein may have a different role.


Antiprogestin ZK-98.299 and progesterone display differential binding characteristics in the human myometrial cytosol.

To investigate whether the synthetic progesterone antagonist ZK-98.299 binds to progesterone receptor or also has distinct binding sites, the binding characteristics of ZK-98.299 were compared with those of progesterone in the human myometrial cytosol. [3H]ZK-98.299 and [3H]progesterone showed specific binding in the myometrial cytosol and the binding of each radiolabelled ligand could be displaced with the respective ligand in a dose-response manner. However, while the binding of [3H]progesterone could be completely blocked with progesterone or ZK 98.299, the binding of [3H]ZK-98.299 could not be displaced more than 50%. The non-specific binding of [3H]ZK-98.299 was very high as compared to that of [3H]progesterone. Using [3H]progesterone, the relative binding affinity (RBA) of progesterone was more than that of ZK 98.299, whereas using [3H]ZK-98.299 the RBA of ZK 98.299 exceeded that of progesterone. Treatment of myometrial cytosol with increasing concentrations of -SH-modifying agents (iodoacetamide (IA) 0-10 mM or N-ethylmaleimide (NEM) 0-1000 nM) decreased the binding of progesterone by over 80%, whereas similar treatment did not have appreciable effect on the binding of [3H]ZK-98.299. Although both preformed ligand-receptor complexes were relatively stable in the presence of IA and NEM, the [3H]progesterone-receptor complex was more sensitive as compared to the [3H]ZK-98.299-receptor complex. The addition of 20 mM molybdate in the cytosol had a protective effect against the -SH-modifying agents. [3H]ZK-98.299 and [3H]progesterone-receptor complexes also showed differential stability when incubated at elevated temperatures (25 degrees C and 37 degrees C), [3H]ZK-98.299-binding sites being more thermolabile as compared to [3H]progesterone binding sites. Prior occupation of the receptor by the two ligands gave the complexes the ability to resist an elevated temperature of 25 degrees C. Moreover, molybdate stabilized both the liganded and unoccupied receptors at 25 degrees C. When the ligand-receptor complexes were applied onto a prefocused polyacrylamide gel, the progesterone and ZK-98.299-receptor complexes were resolved and focused at pH 7.2 and 8.4, respectively. The results of this study suggest that although progesterone and ZK-98.299 are mutually competitive for binding to progesterone receptor, ZK-98.299 also has distinct binding sites.


Cooperative effects of ethanol and protein kinase C activators on phospholipase-D-mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 fibroblasts.

In a previous study, ethanol was shown to enhance the stimulatory effect of phorbol 12-myristate 13-acetate (PMA), a prominent activator of protein kinase C (PKC), on phospholipase-D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts (Kiss et al. (1991) Eur. J. Biochem. 197, 785-790). Here, the mechanism and possible significance of ethanol-stimulated PtdEtn hydrolysis was further studied. In [14C]ethanolamine-labeled NIH 3T3 fibroblasts, 10 mM ethanol enhanced PMA-induced hydrolysis of PtdEtn 1.5-2.0-fold during a 2.5-15-min incubation period. Other alcohols, including glycerol, methanol, and 1-propanol, also enhanced PMA-induced PtdEtn hydrolysis. Of the other PLD activators tested, ethanol potentiated the PKC-dependent stimulatory effect of bombesin but failed to alter the apparently PKC-independent stimulatory effect of serum. Pretreatment of [14C]ethanolamine-labeled fibroblasts with 200 mM ethanol for 20 min resulted in increased (approx. 2-fold) hydrolysis of [14C]PtdEtn in isolated membranes. In membranes from ethanol-treated, but not from untreated, cells, PMA further enhanced (approx. 1.5-fold) the production of [14C]ethanolamine. Ethanol exerted none of the above stimulatory effects on phosphatidylcholine hydrolysis. These results suggest that the specific stimulatory action of ethanol on PLD-mediated PtdEtn hydrolysis can occur in vivo and may involve increased binding of a regulatory PKC-isoform to membranes.


Development of fibroblast-type-II cell communications in fetal rabbit lung organ culture.

The development of the fetal lung is regulated by fibroblast-type-II cell communications which involve fibroblast pneumonocyte factor (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming growth-factor beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day 19 of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the fibroblast. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.


Simultaneous use of two retroviral vectors in human gene marking trials: feasibility and potential applications.

Two Moloney murine leukemia virus (Mo-MLV)-based neoR retroviral vectors--LNL6 and G1Na--were used to transduce various human tumor-infiltrating lymphocytes (TIL) populations. These groups included bulk CD(8+)- and CD(4+)-enriched TIL from human renal cell carcinomas and melanomas. Transduction efficiencies averaged 5% for single 4-hr supernatant infections. Integrated provirus could be detected for up to 4 weeks of in vitro culture. LNL6 provirus could be distinguished from G1Na provirus using specific polymerase chain reaction (PCR) primers. A single neomycin phosphotransferase (neoR) gene copy could be detected in 10(5) TIL. Using quantitative PCR, the relative ratio of LNL6 to G1Na copies in the same sample could be determined even at low copy numbers. These preclinical studies demonstrate the feasibility of using two retroviral marking vectors in human gene therapy efforts.


Correction of purine nucleoside phosphorylase deficiency by retroviral-mediated gene transfer in mouse S49 T cell lymphoma: a model for gene therapy of T cell immunodeficiency.

To determine the effectiveness of retroviral-mediated purine nucleoside phosphorylase (PNP) gene transfer and expression for metabolic correction of PNP deficiency, we used as a gene transfer target the NSU-1 subline of murine S49 T lymphoma cells, an in vitro genetic model of PNP deficiency. NSU-1 cells were transduced with recombinant retroviruses that express either the murine or human PNP coding sequences under transcriptional regulation of the Moloney murine leukemia virus (Mo-MLV) long terminal repeat (LTR), resulting in expression of substantial levels of PNP activity. Untransduced or control virus-transduced NSU-1 cells were extremely sensitive to deoxyguanosine, a PNP substrate that is toxic for lymphoid cells. However, PNP-virus transduction of NSU-1 cells metabolically corrected the sensitivity of these cells to deoxyguanosine, resulting in near wild-type levels of growth inhibition. These results demonstrate that retroviral-mediated PNP gene transfer and expression corrects the metabolic defect observed in PNP-deficient murine lymphoid cells, suggesting that PNP gene transfer and expression in human lymphoid cells might similarly correct substrate-mediated toxicity and provide an effective genetic therapy.


Toward gene therapy for Niemann-Pick disease (NPD): separation of retrovirally corrected and noncorrected NPD fibroblasts using a novel fluorescent sphingomyelin.

The neurologic (type A) and nonneurologic (type B) forms of Niemann-Pick disease (NPD) both result from deficiencies of acid sphingomyelinase (ASM) activity leading to the accumulation of sphingomyelin and other related lipids within lysosomes. Recently, the full-length cDNA and genomic sequences encoding ASM have been isolated and the nature of the molecular lesions causing NPD has been investigated. Although these developments have facilitated diagnosis for this debilitating disease, no effective treatment is currently available. Toward this latter goal, our laboratories recently reported the effectiveness of retroviral-mediated gene transfer for the in vitro correction of the cellular pathology in NPD fibroblasts (Suchi et al., 1992). In addition, novel selection procedures were developed to separate retrovirally corrected and noncorrected NPD fibroblasts based on the receptor-mediated delivery of a fluorescently (pyrene)-labeled sphingomyelin (P12-SPM) to the lysosomes of cells using liposomes coated with apolipoprotein E. In this study, we have used a different, fluorescent derivative of sphingomyelin (lissamine-rhodamine dodecanoyl sphingomyelin; LR12-SPM) to extend and improve this selection system. LR12-SPM offers a number of advantages over P12-SPM, including the facts that apolipoprotein E is not required for its efficient uptake and targeting to lysosomes and that the product of LR12-SPM degradation by ASM is efficiently transported out of cells. Thus, when analyzed in a fluorescence-activated cell sorter (FACS), there was complete separation (i.e., no overlap) of retrovirally corrected and noncorrected NPD cells after the administration of LR12-SPM.(ABSTRACT TRUNCATED AT 250 WORDS)


Gene transfer in vivo with DNA-liposome complexes: lack of autoimmunity and gonadal localization.

Direct gene transfer into localized arterial segments can be performed in vivo by transfection with DNA-liposome complexes. This technique holds promise for the treatment of human diseases, including malignancy and cardiovascular disorders. We have previously characterized the potential toxicity of this form of treatment in mice in vivo (Stewart et al., 1992). In this report, we examined two issues relevant to long-term expression of foreign recombinant genes: (i) the potential for autoimmune damage to major organs and (ii) DNA localization in gonadal tissue. Autoimmunity and toxicity of allogeneic major histocompatibility (MHC) gene transfer was assessed in mice after induction of an immune response to a recombinant murine class I MHC gene by direct gene transfer in vivo. Histological examination of brain, heart, lung, liver, kidney, spleen, and skeletal muscle revealed no clinically significant immunopathology or organ damage. The toxicity of gene delivery by DNA liposomes was also analyzed in pigs and rabbits in vivo. No histopathology was observed following the introduction of plasmids encoding several different gene products, and analysis of serum following DNA liposome delivery revealed no abnormalities of serum biochemical parameters. The potential for transfer of recombinant DNA into testes and ovary in animals was evaluated by the polymerase chain reaction. Although evidence of recombinant plasmid was consistently observed in transfected, but not untransfected, arterial sites and occasionally in lung, kidney, spleen, and liver, no plasmid DNA was detected in testes or ovary. These studies suggest that uptake of recombinant DNA following direct gene transfer by liposomal transfection in major organs is not associated with autoimmunity, toxicity, or gonadal localization.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatic gene therapy: persistent expression of human alpha 1-antitrypsin in mice after direct gene delivery in vivo.

The liver represents an excellent target organ for gene therapy. The current strategy for hepatic gene therapy involves the isolation of primary hepatocytes from a resected liver lobe, transduction of therapeutic genes in vitro followed by autologous hepatocellular transplantation. This ex vivo approach is a rather complex procedure in its entirety; thus, a simple method for direct gene delivery into hepatocytes in vivo has been developed. The procedure involves partial hepatectomy followed by the portal vein infusion of recombinant retroviral vectors. Histological analysis of hepatocytes after in vivo delivery of a recombinant retrovirus bearing the E. coli beta-galactosidase gene showed that 1-2% of the parenchymal cells were transduced. Direct hepatic transfer of human alpha 1-antitrypsin cDNA under the transcriptional direction of the albumin promoter-enhancer led to constitutive expression of the human protein in the sera of recipients at concentrations of 30-1,400 ng/ml for at least 6 months. The experimental animals showed no signs of illness and histologic analysis of the liver revealed no evidence of pathologic abnormalities. The results suggest that the in vivo approach is an attractive alternative for hepatic gene therapy.