Further characterization of four lipocortins from human peripheral blood mononuclear cells

Four calcium and phospholipid binding proteins purified from mononuclear cells were characterized for PKC and EGF phosphorylation, actin binding capacity, and partial tissue distribution. Those named 35K, 32K, and 73K are equivalent, respectively, to lipocortin III, endonexin II and the 67 kDa calelectrin; 36K is a fragment of 73K. After purification, 35K and 73K were phosphorylated by protein kinase C in vitro but 36K nor 32K were not. None were phosphorylated by the epidermal growth factor receptor kinase in vitro; 73K bound F-actin in a calcium-dependent manner, whereas 35K, 36K, and 32K did not. Using Western blotting analysis, 32K and 73K were detected in high amounts in human lymphocytes, monocytes, liver, and placenta and in rat adrenal medulla; but 32K was not detected in polymorphonuclear cells, and 36K and 35K were detected in high amounts only, respectively, in human blood lymphocytes and polymorphonuclear cells. Thus, 32K and 73K appear to have a wide tissue distribution, whereas 35K has a much more restricted distribution.     

Analysis of molecular deletions with cDNA probes in patients with Duchenne and Becker muscular dystrophies

In the course of a systematic survey of DMD and BMD patients with intronic probes and with cDNA probes covering three-fourths of the coding sequence, 45 molecular deletions within the DMD gene were investigated. Forty-two percent of the breakpoints were located in the intronic sequence containing probe P20, whereas the other deletions were widespread around the more proximal part of the gene. Most of the BMD deletions were in the P20 region. Pulsed field gel electrophoresis was used to determine the size of some deletions and allowed us to estimate the physical distance between the intronic probes JBir and P20. The reading frame was checked in 11 cases with proximal deletions and found to be disrupted in 6 of 7 DMD patients, in 1 intermediate case, and, unexpectedly, in 3 BMD patients.     

Analysis of mammalian pigmentation at the molecular level

There has been great interest lately in the cloning of pigment-related genes; several laboratories have succeeded in isolating melanocyte-specific genes which have many of the characteristics expected for tyrosinase. In this paper, we review the selection criteria, the physical properties, and the functional characteristics of several of these gene products. Two of the clones map to the brown (b) and albino (c) loci, genes that are involved in the regulation of the quantity and quality of melanin production. The functional characteristics of these gene products are not easily reconciled with existing schemes of melanogenesis, and a reevaluation of our concepts of melanogenic regulation may be necessary. The altered expression of these gene products in normal and in transformed melanocytes, and the alternative mRNA processing that occurs in those cells, makes this system an appropriate and interesting one for studies of normal metabolic regulation of gene expression, as well as altered gene expression by neoplastic cells.     

Differential expression of the receptors for epidermal growth factor and insulin in the developing human placenta

The detailed cellular distribution of epidermal growth factor (EGF) receptors and insulin receptors during the development of the human placenta was examined. We show that EGF receptors are expressed by villous cytotrophoblast cells in first trimester human placentae. However, where these cells proliferate to form extravillous cytotrophoblast cell columns, there is a dramatic decrease in EGF receptor expression. There is no such differential expression of insulin receptors on this cell population. In contrast, both EGF-and insulin-receptors are present throughout gestation on the microvillous membrane of the terminally differentiated and non-proliferative syncytiotrophoblast although, at term, EGF-but not insulin-receptors are also found on the basolateral membrane of this epithelium. We further show that EGF receptors isolated from first trimester and term human placentae have functional tyrosine kinase activities but differ in their extent of glycosylation. These results suggest that EGF receptors probably play several distinct functional roles in these epithelial cells depending on their proliferative capacity and differentiation status.     

Zinc acexamate reduces gastric damage induced by platelet-activating factor

We have tested the ability of zinc acexamate (ZAC) to prevent platelet-activating-factor (Paf) induced gastric damage in rats. Lesions were characterized by a vascular congestion affecting the entire mucosa, oedema, haemorrhage and frequent necrosis of the more superficial areas. The gastric damage appearing after Paf was accompanied by degranulation of gastric mast cells. Leukocytes were often seen at the submucosal level. Oral pretreatment with ZAC reduced in a dose-dependent manner both gastric damage and mast cell degranulation observed after Paf. ZAC administered orally at a dose of 100 mg kg-1 statistically inhibited (p less than 0.01) gastric damage and mast cell degranulation. ZAC did not affect the hypotension induced by Paf confirming that gastric damage and hypotension appearing in rats after Paf administration are two independent phenomena. The present findings indicate that the inhibitory effect of ZAC upon gastric lesions induced by Paf may be related to the different protective actions exhibited by this zinc compound in a wide variety of experimental models of gastric ulcer.     

Human monocytes convert leukotriene B4 to two dihydro-leukotriene B4-metabolites

Human monocytes metabolize LTB4 by an additional pathway different from omega-oxidation. Reverse-phase high performance liquid chromatography showed four metabolites: 20-COOH-LTB4, 20-OH-LTB4 and two metabolites less polar than LTB4 with an UV maximum at 232 nm. Gas-chromatography mass-spectrometry showed nearly identical mass spectra for both metabolites. The main mass fragments of the two metabolites were increased by two mass units compared to LTB4. Our findings suggest that LTB4 had been reduced to a known and a new dihydro-metabolite of LTB4. Both metabolites together amounted to 85% of total metabolites. The remaining 15% were omega-oxidation products. Thus, the major pathway of LTB4 metabolism by human monocytes is reduction to dihydro-LTB4.     

Incidence of peptic ulcer disease in Gothenburg, 1985.

OBJECTIVE--To determine the incidence and age distribution of peptic ulcer disease in adults in Gothenburg. DESIGN--Retrospective study of patients with symptoms over one year. SETTING--All gastroenterology and x ray departments. PATIENTS--Any patient found to have an active ulcer crater during 1985. MAIN OUTCOME MEASURES--Sex, age, past history of gastrointestinal ulcers, and smoking habit. RESULTS--In 1985, 1402 peptic ulcers were diagnosed in 1137 adults. Over half (403; 54%) of the ulcers in men and 393 (60%) ulcers in women were in patients aged over 60. All types of ulcer showed increasing incidence with age. The sex ratio of patients aged 40-50 with peptic ulcers was 1:1. Nearly half (109; 48%) of ulcers diagnosed for the first time in men and 129 (57%) of such ulcers in women were in patients aged over 60. Elderly men and women were also more likely to develop haemorrhage. CONCLUSIONS--In Gothenburg there is a surprisingly high incidence of peptic ulcer disease, which increases considerably with age, possibly explained by the availability of modern diagnostic techniques as 1121 (80%) ulcers had been diagnosed by gastroscopy. Compared with earlier studies there was no difference in the incidence between men and women aged 40-50.     

Untreated asymptomatic bacteriuria in girls: II--Effect of phenoxymethylpenicillin and erythromycin given for intercurrent infections.

OBJECTIVE--To investigate the effects of phenoxymethylpenicillin and erythromycin on urinary isolates from patients with untreated asymptomatic bacteriuria. DESIGN--Retrospective study of subgroup of patients from cohort followed up till the end of 1986. SETTING--Outpatient clinic for children with urinary tract infections. PATIENTS--51 Girls aged under 15 with untreated asymptomatic bacteriuria. INTERVENTIONS--Before 1982 intercurrent infections (mostly tonsillitis or otitis) were treated with phenoxymethylpenicillin; after 1982 erythromycin treatment was preferred. END POINTS--Change of bacterial strain in urinary tract and symptomatic recurrences of disease. MEASUREMENTS AND MAIN RESULTS--Bacteria identified by serotype and electrophoretic type and compared before and after antibiotic treatment. Bacteriuria eradicated and replaced by new strains in most patients treated with phenoxymethylpenicillin, leading to symptomatic recurrences in about 15%. Conversely, patients given erythromycin rarely showed change in bacteriuria and none suffered symptomatic recurrence. CONCLUSIONS--In girls with untreated asymptomatic bacteriuria the use of phenoxymethylpenicillin for intercurrent infections may lead to a change of urinary bacteria and leave them at substantial risk of acute pyelonephritis. With erythromycin this risk is small (2/20 courses in this series).     

Untreated asymptomatic bacteriuria in girls: I--Stability of urinary isolates.

OBJECTIVE--To assess the frequency of spontaneous changes of bacterial strains in patients with untreated asymptomatic bacteriuria. DESIGN--Retrospective analysis of samples from all patients with renal scarring and random sample of patients with normal kidneys. SETTING--Outpatient clinic for children with urinary tract infections. PATIENTS--54 Girls aged 3.3-15.5 years with untreated asymptomatic bacteriuria caused by Escherichia coli. INTERVENTION--None. END POINT--Change in bacterial strain. MEASUREMENTS AND RESULTS--Serotyping and electrophoretic analysis of sequential bacterial isolates, representing 151 patient years of untreated asymptomatic bacteriuria. A total of 24 changes of strain were identified. Eleven were related to medical interference such as treatment of other infections with antibiotics. CONCLUSIONS--Spontaneous changes of strain were uncommon, one change in 11.6 patient years, and thus are not a characteristic feature of the course of asymptomatic bacteriuria.     

Evidence for probenecid-sensitive organic anion transporters on polarized thyroid cells in culture

Epithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half-maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen-coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.