Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains

Caveolae are small, plasma membrane invaginations that have been implicated in cell signaling. In A431 cells, approximately half of the total cellular phosphatidylinositol 4,5-bisphosphate (PtdIns 4, 5-P2) was found to be localized in low density, Triton-insoluble membrane domains enriched in caveolin. Treatment of cells with either epidermal growth factor or bradykinin for 5 min at 37 degrees C resulted in approximately a 50% decrease in this caveolar PtdIns 4,5-P2 with no change in the levels of plasma membrane PtdIns 4,5-P2. These data suggest that the PtdIns 4,5-P2 present in cells is largely compartmentalized and that the caveolar PtdIns 4,5-P2 is subject to hydrolysis by hormone-stimulated phospholipase C. As growth factor receptors, seven transmembrane domain receptors, heterotrimeric G proteins, and the inositol trisphosphate receptor have all been shown to be enriched in caveolae, these findings suggest that both the generation and response to inositol trisphosphate is highly compartmentalized within the cell.     

Increased resistance to streptolysin O in mammalian cells treated with oxygenated derivatives of cholesterol

L-cell fibroblast cultures were treated with certain oxygenated derivatives of cholesterol which are known to inhibit cholesterol biosynthesis in mammalian cells. After incubation in the presence of 20-alpha-hydroxycholesterol or 25-hydroxycholesterol for 18 h, the cells became increasingly resistant to streptolysin O. Maximum resistance to toxin was obtained by incubation for 48 h in 0.5 microgram of 20-alpha-hydroxycholesterol or 0.25 microgram of 25-hydroxycholesterol per ml; under these conditions, the cells were 10 to 50 times more resistant than were untreated controls. The ability of the hydroxycholesterol compounds to render the cells resistant was related to the age of the cultures. Maximum protection was found when more sparsely populated cultures were treated with 25-hydroxycholesterol. Older, heavily populated cultures could not be protected even with the high concentrations of 25-hydroxycholesterol. In contrast to control cultures, most of the toxin activity remained in the medium after being incubated with hydroxycholesterol-treated cultures. The results indicate that less toxin binds to the resistant cells and suggest that a reduction in membrane cholesterol content may account for resistance to streptolysin O.     

Serum calcium, phosphate and alkaline phosphate levels in epileptic children treated with phenobarbitone

A longitudinal study to estimate the serum calcium, phosphate and alkaline phosphatase levels of 89 ambulatory epileptic children, aged between 3 years and 12 years, and having generalised tonic-clonic seizures, was carried out. None was on any form of medication for the treatment of seizures prior to presentation. Each patient received only phenobarbitone during the period of study. Serum levels of the biochemical parameters were determined at presentation, 6 months and 12 months, while serum phenobarbitone levels were estimated at 6 months and 12 months. Mean serum calcium, phosphate and alkaline phosphatase of the patients remained within the normal range. Using the paired 't' test, the differences in the levels of the parameters at the three measurements were not statistically significant (P > 0.05). Serum phenobarbitone levels remained within the therapeutic range during the period of study. Our results show that over a 12-month period, serum levels of calcium, phosphate, and alkaline phosphatase, remain normal in ambulant epileptic children treated with phenobarbitone.     

Control of serum phosphate without any phosphate binders in patients treated with nocturnal hemodialysis

We compared the efficacy and the long-term effects of nocturnal hemodialysis (NHD) versus conventional hemodialysis (CHD) in controlling serum phosphate levels in patients with end-stage renal disease (ESRD). Patients underwent thrice weekly CHD and were subsequently switched to NHD six nights weekly. In the "acute" study serum and dialysate phosphate were measured during and after dialysis, and the total dialysate was collected to calculate mass solute removal. Although pre-dialysis (1.7 +/- 0.6 vs. 1.5 +/- 0.8 mM) serum phosphate levels were similar in CHD and NHD, respectively, post-dialysis levels were slightly lower with CHD (0.7 +/- 0.2 vs. 0.8 +/- 0.2 mM, P < 0.05). The measured phosphate removed per session of CHD or NHD was comparable, 25.3 +/- 7.5 versus 26.9 +/- 9.8 mumol/session, respectively. On the other hand, the cumulative weekly phosphate removal was significantly higher with NHD as compared to CHD, 75.8 +/- 22.5 versus 161.6 +/- 59.0 mumol/week (P < 0.01). In the "chronic" study serum phosphate levels were measured monthly for five months on CHD and for five months after the patients were switched to NHD. Dietary phosphate intake and the dosage of phosphate binders were tabulated. Serum phosphate levels fell during NHD: 2.1 +/- 0.5 mM at the beginning of the study and 1.3 +/- 0.2 mM five months after being switched to NHD (P < 0.001). At the same time dietary phosphate intake increased by 50%. By the fourth month of NHD therapy none of the patients was taking any phosphate binders. In conclusion, NHD is more effective in controlling serum phosphate levels than CHD, allowing patients to discontinue their phosphate binders completely and to ingest a more liberal diet.     

Biochemical effects of retinoic acid on GTP-binding Protein/Transglutaminases in HeLa cells. Stimulation of GTP-binding and transglutaminase activity, membrane association, and phosphatidylinositol lipid turnover

Treatment of HeLa cells with retinoic acid (RA) gives rise to a marked stimulation in the incorporation of [alpha-32P]GTP into an approximately 87-kDa cytosolic protein that cross-reacts with a monoclonal antibody raised against tissue transglutaminases. In the absence of RA treatment, the transglutaminase immunoreactivity elutes from a gel filtration column with an apparent size of approximately 600 kDa (designated TGa), whereas following RA treatment, a second peak of transglutaminase immunoreactivity (designated TGb) is detected with an apparent size of approximately 150 kDa. The TGa fractions show little or no GTP-binding or GTP hydrolytic activity and very little transglutaminase activity. However, the TGb fractions show all three activities. Retinoic acid treatment also promotes the association of the GTP-binding protein/transglutaminase with membrane fractions, as detected by Western blotting and photoaffinity cross-linking with [alpha-32P]GTP. In addition, the TGb fraction shows a markedly enhanced ability (relative to TGa) to associate with membranes from control (non-RA-treated) cells. The ability of the GTP-binding protein/transglutaminase to bind to membranes is correlated with the stimulation of a membrane-associated phospholipase C activity. Thus, these findings indicate that RA treatment results in a number of changes in the biochemical properties of a GTP-binding protein/transglutaminase which strongly enhance its ability to bind GTP, associate with plasma membranes, and stimulate phosphoinositide lipid turnover.     

Increased immunogenicity of TA3-Ha cells treated with the antitumor antibiotic macromomycin (B)

Suspension cultures of TA3-Ha mouse mammary tumor cells were treated in vitro with a single dose of macromomycin(B) (MCR) at 1 mug/ml for 24 hours. This dose, which is cytostatic but not lethal, increased the immunogenicity of the cells. Adoptive transfer of spleen cells sensitized to MCR/TA3-Ha cells evoked an immune response against MCR-treated TA3-Ha cells but not against normal TA3-Ha cells. The therapeutic effect of MCR treatment (in this case) was, to a very small extent, due to the cytostatic action and more profoundly to the increased immunogenicity of the cells so treated.     

Humoral antibody and cell-mediated cytotoxicity responses of mice to rat skin xenografts

Aspects of both the humoral and cell-mediated immune responses of mice to rat skin xenografts were studied sequentially and quantitated. Antirat lymphocytotoxic and hemagglutinating antibodies were first detectable at 7 and 6 days, respectively, after primary grafting and their appearance in serum corresponded to the time of graft rejection. Lymphocytotoxic titers were low after primary grafting but increased greater than 4-fold after secondary grafting. Cytotoxicity of mouse spleen and axillary lymph node cells for 51Cr-labeled rat lymphocyte target cells was first detectable 5 days after primary grafting but was quite low showing a peak of only 7% specific 51Cr release 6 days after grafting. In contrast, the cytotoxicity of mouse spleen and lymph node cells for allogeneic target cells after primary skin allografting was significantly greater. It is suggested that the magnitude of the cell-mediated immune response to skin xenografts is less than the response to allografts and that the quicker and more vigorous rejection of skin xenografts is due to a larger participation of humoral antibody in the rejection process.     

Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity

The transit of immature to mature sIgM+ B cells, the life span, maturation kinetics and response to polyclonal activators have been analyzed with the help of a new mAb (493), that distinguishes immature, 493+ from mature, 493 B cells in a variety of mouse strains tested. Analysis of the turnover of immature 493+ B cells by bromodeoxyuridine (BrdU) labeling kinetics indicate that only 10-20 % of the cells reach the spleen as immature 493+ cells. The life span of 493+ B cells in bone marrow and spleen is around 4 days. BrdU chase experiments show that most of the immature cells in spleen enter the pool of mature, 493+ B cells where they gain a longer life span of 15-20 weeks. Immature and mature B cells respond equally well to LPS stimulation; anti-CD40, however, stimulates mature B cells better than immature B cells. IgM cross-linking of mature B cells results in proliferation, while it induces apoptosis in immature B cells. This apoptosis of immature cells can be inhibited by costimulation with anti-CD40 or by overexpression of bcl-2. We speculate that Ig receptor ligand-mediated apoptosis (negative selection) plays a major role in the transit of immature B cells from bone marrow to spleen, but only a minor role in the transit from immature B cells to mature B cells in the spleen.     

Changes in radioiodine turnover in patients with autonomous thyroid adenoma treated with percutaneous ethanol injection

In 24 patients with autonomous thyroid adenoma, we studied the hormonal pattern (free thyroxine, free triiodothyronine and thyroid stimulating hormone) and markers of radioiodine turnover before and after nodule ablation with percutaneous ethanol injection. METHODS: The hormonal pattern was studied before treatment and at various intervals after nodule ablation. Changes in radioiodine turnover were studied measuring 131I protein-bound iodine and the biologic half-life of radioiodine in the thyroid (calculated from thyroid uptake at 24 and 48 hr) before and after ethanol treatment. RESULTS: The hormonal pattern was normalized by treatment in all patients and remained normal for the follow-up period. Before treatment, protein-bound 131I was elevated in all patients but 4; after treatment, it normalized in 15 patients with the disappearance of the adenoma on scintigraphy. In the remaining 9 patients with only partial nodule destruction on scintigraphy, protein-bound 131I remained elevated although markedly reduced. Biologic half-life was shortened in 18 of 24 patients before treatment; after treatment, it was normal in 18 of 24 patients (13 of 15 with complete nodule ablation and 5 of 9 with partial ablation). CONCLUSION: Ethanol treatment normalized the hormonal pattern in all patients. Measures of radioiodine turnover were better markers of residual disease in that they normalized in almost all patients with complete nodule ablation, whereas they remained abnormal in a high proportion of patients with incomplete ablation. Thyroid hormones remained normal over a follow-up period of 3-7 yr in all patients.     

The estimation of the number of binding sites for antibody on antigen molecules with reference to factor VIII and its antibody

The theoretical basis for determining the number of antibody sites on antigen molecules is examined. The theoretical considerations are applied to factor VIII molecules. Examples based on data available at the Oxford Haemophilia Centre are calculated to illustrate the approach. It is concluded that there are few sites on each factor VIII molecule for human antibody. The three antibodies for which reasonable data were available suggest 1-3 sites for human antibody. The data for rabbit antibody suggest 5-6 sites per factor VIII molecule.     

Isolated lichen planus of the lip successfully treated with chloroquine phosphate

Solitary lesions of lichen planus (LP) are often confused with other inflammatory or malignant epithelial lesions. We describe a 51-year-old woman who had had an LP of the lower lip for 11 years. She had undergone several oral and topical therapies with little improvement. The patient had an excellent response to chloroquine phosphate within 3 months. This report represents the third case of LP exclusively localized on the lower lip.     

Which patients with antiphospholipid antibody should be treated and how?

The tests for antiphospholipid antibody are relatively crude but usable. Asymptomatic persons with incidentally discovered antiphospholipid antibody do not need treatment. Those with symptoms are best treated with anticoagulation, but data specifically supporting aspirin, heparin, or warfarin or combinations thereof remain to be generated. High-dose corticosteroid therapy has at best equivocal efficacy and much toxicity and should be used only for lupus activity and not for phenomena of the antiphospholipid antibody syndrome. The roles of low-dose corticosteroid therapy, immunosuppressive agents, and other treatments are unknown.     

Subcellular distribution and turnover of presenilins in transfected cells

The mechanisms by which mutations in presenilin-1 (PS1) and presenilin-2 (PS2) result in the Alzheimer's disease phenotype are unclear. Full-length PS1 and PS2 are each processed into stable proteolytic fragments after their biosynthesis in transfected cells. PS1 and PS2 have been localized by immunocytochemistry to the endoplasmic reticulum (ER) and Golgi compartments, but previous studies could not differentiate between the full-length presenilin proteins and their fragments. We carried out subcellular fractionation of cells stably transfected with PS1 or PS2 to determine the localization of full-length presenilins and their fragments. Full-length PS1 and PS2 were principally distributed in ER fractions, whereas the N- and C-terminal fragments were localized predominantly to the Golgi fractions. In cells expressing the PS1 mutant lacking exon 9 (DeltaE9), we observed only full-length molecules that were present in the ER and Golgi fractions. The turnover rate was considerably slower for the DeltaE9 holoprotein, apparently due to decreased degradation within the ER. Our results suggest that that full-length presenilin proteins are primarily ER resident molecules and undergo endoproteolysis within the ER. The fragments are subsequently transported to the Golgi compartment, where their turnover rate is much slower than that of the full-length presenilin in the ER.     

Telomere loss in cells treated with cisplatin

Telomeres play an important role in the immortalization of proliferating cells. The long tandem repeats of 5'-TTAGGG-3' sequences in human telomeres are potential targets for the anticancer drug cisplatin, which forms mainly intrastrand d(GpG) and d(ApG) cross-links on DNA. The present study reveals that telomeres in cisplatin-treated HeLa cells are markedly shortened and degraded. A dose that killed 61% of the cells but allowed one round of cell division resulted in shortened telomeres before the induction of apoptosis. Higher doses of cisplatin halted cell cycle progression during the first S phase and triggered apoptosis followed by degradation of telomere repeats. A model in which both cell division with incomplete replication and induction of apoptosis by cisplatin could occur was devised to explain the drug-induced telomere loss.     

Studies on gonococcus infection. VIII. 125Iodine labeling of gonococci and studies on their in vitro interactions with eukaryotic cells

Intact gonococci (GC) have been labeled with 125iodine by the lactoperoxidase plus hydrogen peroxide procedure. The specific activities of types 2, 4, and 4 GC have been determined and are found to show small differences as follows: T4 greater than T2 greater than T4. 125I-labeled GC have been studied for their associations with both leukocytes and tissue culture cells. 125I-labeled GCshow the following relative order of association with the leukocytes: T2 equals T4* greater than T4. This contrasts with the relative degree of interaction between the GC and tissue culture cells, which follows the relative order: T2 greater than T4 equals T4*. Trypsin pretreatment of GC markedly reduces the association of all three types (T2, T4 AND T4*) with leukocytes but does not alter the level of attachment of any of the gonococcal types with tissue culture cells.     

Spontaneous hemobilia in a hemophiliac treated by factor VIII replacement and nasobiliary drainage

We report a case of spontaneous hemobilia in a hemophilia patient presenting with classical Sandblom's triad of symptoms. Endoscopic retrograde cholangiography was helpful in establishing the diagnosis. Hemobilia subsided with factor VIII replacement and endoscopic nasobiliary drainage. Endoscopic nasobiliary drainage resulted in lysis of the clots in the biliary tree and relief of obstructive jaundice.