Human epidermal growth factor-on molecular forms present in urine and blood.

A sensitive enzyme-linked immunosorbent assay (ELISA) for quantitation of human epidermal growth factor (EGF) was employed to study EGF in urine and blood. The EGF/creatinine ratio in urine was significantly higher for women (range and (median); 0.20-0.83 (0.50) nmol EGF/mmol creatinine) than for men (0.17-0.63 (0.30) nmol EGF/mmol creatinine). We were not able to demonstrate EGF in plasma (median plasma EGF < 0.01 nmol/l) whereas serum contained a range and (median) of 0.02-0.31 (0.12) nmol EGF/l. The amount of EGF in serum showed a weak correlation to the platelet count (r = 0.327). EGF was partly purified by affinity chromatography from urine (urine EGF) and from activated platelets in platelet rich plasma (blood EGF). Both blood and urine contained a high molecular weight form of EGF (HMW EGF) as well as 6 kDa EGF. HMW EGF from blood was similar to HMW EGF from urine concerning behaviour upon gel filtration, pI and apparent affinity constant for binding to the EGF receptor. However, HMW EGF constituted approx. 40% of blood EGF but only 10% of urinary EGF. The 6 kDa EGF from both blood and urine contained two isopeptides with pI around 4.40 and 4.15 but in various proportions. The apparent affinity constant for binding to the EGF receptor for blood 6 kDa EGF was 1.8 x 10(10) l/mol compared to 1.0 x 10(10) l/mol for urinary 6 kDa EGF and 0.8 x 10(10) l/mol for HMW EGF from both blood and urine. The present study suggests that the processing of the EGF precursor differs in the blood and in the kidneys and that 6 kDa EGF from blood and urine binds to the EGF receptor with a higher apparent affinity constant than does HMW EGF.     

Effects of gastric inhibitory polypeptide, somatostatin and epidermal growth factor on lipogenesis in ovine adipose explants

Feeding raises the plasma concentrations of a number of gut-related hormones that may, in turn, influence the metabolism of peripheral tissues. This study investigated the effects of gut-related hormones on lipogenesis in explants from three differing adipose depots in lambs (aged 4-9 months). Incorporation of [14C]-acetate into lipid was measured over a 2-h period, following 24 h pre-incubation in the presence of hormone combinations. In perirenal fat explants, gastric inhibitory polypeptide (GIP) in the concentration range 0.01-10 nM stimulated lipogenesis. Maximal effects were seen at 1 nM (an average increase of 64% over basal values). In contrast, in the presence of insulin (0.1 nM), a dose-dependent decrease in lipogenesis was seen with increasing GIP concentration (P < 0.001 for the insulin x GIP interaction). Epidermal growth factor (EGF) and somatostatin in the same concentration range each inhibited lipogenesis. both in the presence and the absence of insulin (P < 0.001 in each case). Subcutaneous (back) fat and intermuscular (popliteal) fat responded similarly to each other, but significantly differently from the perirenal depot (P < 0.001). Here GIP, somatostatin or EGF (each at 1 nM) all separately stimulated lipogenesis.     

Treatment of thrombocytosis in myeloproliferative disorders with interferon alpha-2a

In an open prospective pilot trial, we tested the effect of recombinant interferon alpha-2 a (rIFN alpha-2 a) on thrombocytosis in myeloproliferative disorders (MPD). Since October 1986, 13 patients with MPD (4 with chronic granulocytic leukemia, 4 with polycythemia vera, 3 with essential thrombocythemia and 2 with myeloid metaplasia) were treated with rIFN alpha-2 a. Platelet counts decreased in all treated patients within 2 to 10 weeks from a median value of 1,050 x 10(9)/l (range 610-1,940 x 10(9)/l) to 340 x 10(9)/l (range 230-495 x 10(9)/l). The response was dose-dependent. In 11 patients we observed a simultaneous reduction of the white blood cell count. Six patients still continue the IFN alpha-2 a therapy. In 7 treatment was discontinued, because of chronic side effects in 3, and because of noncompliance in one. In these patients, thrombocytosis recurred after discontinuation of the therapy. These results show that rIFN alpha-2 a is effective in controlling thrombocytosis in MPD. However, the long-term benefit of interferon in these disorders remains to be established.     

Open access clinic providing HIV-I antibody results on day of testing: the first twelve months.

OBJECTIVES--To determine the sociodemographic profile, risk category, and prevalence of HIV-I infection among people attending a clinic providing counselling, medical advice, and results of HIV-I antibody testing on the day of consultation; to determine the stage of infection and peripheral blood CD4 cell count among attenders with detectable HIV-I antibodies. DESIGN--Analysis of prospectively collected data for the 12 months from March 1989. SETTING--Same day testing clinic run by the HIV/AIDS team at an urban teaching hospital. PATIENTS--561 consecutive people choosing to attend and proceeding to HIV-I testing. RESULTS--The demand for the service caused it to run to capacity within six months. The median age of those attending was 28 years and 65% (364 patients) were male. The overall prevalence of HIV-I infection was 3.9% (22 patients). The greatest prevalence was in men reporting their primary risk as homosexual contact (11.9%, 13/109). The median CD4 cell count in the 22 patients who had detectable HIV-I antibodies was 0.31 x 10(9) cells/l (normal range 0.5 x 10(9)/l to 1.2 x 10(9)/l). Twenty of these patients were asymptomatic (Centers for Disease Control stages II or III), 14 had CD4 cell counts below 0.5 x 10(9)/l. CONCLUSIONS--There is a recognisable demand for a service providing rapid results of HIV-I antibody testing in this setting. The overall seroprevalence of 3.9% is comparable with the 5.8% reported from freestanding clinics in the United States. Most patients with HIV-I antibodies detected in this way are asymptomatic but could benefit from early medical intervention because of low CD4 cell counts.     

Three classes of epidermal growth factor receptors on HeLa cells

The kinetics of 125I-labeled epidermal growth factor (EGF) binding to receptors on HeLa cells were investigated. Scatchard analysis revealed the presence of 22,000 high affinity receptors (Kd = 0.12 nM) and 25,000 low affinity receptors per cell (Kd = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2E9, which prevents specifically EGF binding to low affinity receptors. The study of EGF binding to only low affinity receptors was performed with cells pretreated with the phorbol ester phorbol 12-myristate 13-acetate, which induces a conversion of high affinity receptors to low affinity receptors. This kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors. High affinity receptors were found to consist of one receptor type (type I) with a kinetic association constant (kass) of 6.2 x 10(5) M-1.s-1 and a kinetic dissociation constant (kdis) of 3.5 x 10(-4) s-1. The low affinity receptors were found to consist of two kinetic distinguishable sites: type II or fast sites with kass = 3.3 x 10(6) M-1.s-1 and kdis = 8.1 x 10(-3) s-1 and the type III or slow sites with kass = 3.2 x 10(4) M-1.s-1 and kdis = 1.6 x 10(-4) s-1. The regulatory mechanism which may determine the EGF binding characteristics is discussed.     

Dynamics of beta2-adrenergic receptor-ligand complexes on living cells

The agonist-induced dynamic regulation of the beta(2)-adrenergic receptor (beta(2)-AR) on living cells was examined by means of fluorescence correlation spectroscopy (FCS) using a fluorescence-labeled arterenol derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II cell line A549. Alexa-NA specifically bound to the beta(2)-AR of neurons with a K(D) value of 1.29 +/- 0.31 nM and of A549 cells with a K(D) of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and 19.9 +/- 2.0 microm(-2) in A549 cells (rho(A549)). Kinetic experiments revealed comparable on-rate constants in both cell types (k(on) = 0.49 +/- 0.03 s(-1) nM(-1) in neurons and k(on) = 0.12 +/- 0.02 s(-1) nM(-1) in A549 cells). In addition to the free ligand diffusing with a D(free) of (2.11 +/- 0.04) x 10(-6) cm(2)/s, in both cell types receptor-ligand complexes with two distinct diffusion coefficients, D(bound1) (fast lateral mobility) and D(bound2) (hindered mobility), were observed [D(bound1) = (5.23 +/- 0.64) x 10(-8) cm(2)/s and D(bound2) = (6.05 +/- 0.23) x 10(-10) cm(2)/s for neurons, and D(bound1) = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D(bound2) = (1.01 +/- 0.46) x 10(-9) cm(2)/s for A549 cells]. Fast lateral mobility of the receptor-ligand complex was detected immediately after addition of the ligand, whereas hindered mobility (D(bound2)) was observed after a delay of 5 min in neurons (up to 38% of total binding) and of 15-20 min in A549 cells (up to 40% of total binding). Thus, the receptor-ligand complexes with low mobility were formed during receptor regulation. Consistently, stimulation of receptor internalization using the adenylate cyclase activator forskolin shifted the ratio of receptor-ligand complexes toward D(bound2). Intracellular FCS measurements and immunocytochemical studies confirmed the appearance of endocytosed receptor-ligand complexes in the cytoplasm subjacent to the plasma membrane after stimulation with the agonist terbutaline (1 microM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin.     

125I-labelled hCG binding characteristics in theca interna and other tissues from Romney ewes and from Booroola x Romney ewes with and without a major gene influencing their ovulation rate

Specific receptors for 125I-labelled hCG in ovarian follicle wall were located in the theca interna. No specific binding of 125I-labelled hCG was found in theca externa and/or stromal tissue. The kinetics of 125I-labelled hCG binding to theca interna followed second order kinetics with calculated association rate constants (ka +/- s.d.) of 1.57 +/- 0.16 X 10(6) and 0.57 +/- 0.02 X 10(6) litres mol-1 sec-1 at 37 degrees C and 22 degrees C respectively. Dissociation of specifically bound 125I-labelled hCG from theca interna was minimal at 37 degrees C and 22 degrees C. The binding of 125I-labelled hCG to theca interna could be displaced with PMSG, FSH-P and sheep LH but other sheep pituitary hormones and LH-releasing hormone showed little or no cross-reaction. The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hCG binding to theca interna did not differ between Romney ewes and Booroola x Romney ewes with and without the fecundity (F) gene on Day 10 of the oestrous cycle, during anoestrus or at 36 h after an injection of cloprostenol on Day 10 of the oestrous cycle. When the data for Day 10 and anoestrus were pooled, the median (range) Bmax and Kd values in non-atretic follicles (greater than or equal to 3 mm diameter) were 12.0 (5.1-23.5) fmol/mg protein and 0.10 (0.05-0.16) nM respectively. At 36 h after cloprostenol injection the respective median (range) Bmax and Kd values in non-atretic follicles (greater than or equal to 3 mm diam.) increased to 46.9 (28.4-70.3) fmol/mg protein and 0.23 (0.13-0.65) nM respectively. In corpora lutea the hCG binding characteristics were similar in all the above breeds/genotypes. On Day 10 of the cycle, the mean Bmax but not the mean Kd value was significantly higher (P less than 0.01) than the corresponding value at 36 h after cloprostenol injection. In granulosa cells, from follicles of greater than or equal to 5 mm diameter of Romney and Booroola x Romney (++) ewes and from follicles of greater than or equal to 3 mm diameter of Booroola x Romney (F+) ewes, the hCG binding characteristics were similar. In granulosa cells from smaller sized follicles from the above breeds/genotypes, no specific hCG binding was noted.     

Injury in nonischemic lung after unilateral pulmonary ischemia with reperfusion.

We developed an in vivo intact canine model to study pulmonary ischemia-reperfusion (IR) injury. The surgical approach simulates that of unilateral lung transplantation but is free of technical difficulties and other factors related to lung preservation. Serial measurements of regional pulmonary blood flow (rPBF), extravascular density (EVD), and transcapillary protein flux were made with the quantitative imaging technique of positron emission tomography. Eleven experimental and six control animals were studied. After 2 h of warm ischemia followed by reperfusion, no significant change occurred in rPBF despite significantly increased EVD, which was greater on the ischemic than on the nonischemic side. Protein flux, measured as a rate constant, was also greater on the ischemic than on the nonischemic side (median 181 x 10(-4)/min, range 104-619, vs. median 90, range 33-132) immediately after reperfusion. Both sides were also significantly different from control values (median 37, range 21-57). On both sides, protein flux decreased over time and at 5 h after reperfusion was not different from that of controls. Data from the control animals showed that these findings in the experimental animals were not due to surgical technique, deterioration in the surgical preparation, or hyperperfusion of the nonischemic lung. Thus IR injury of one lung can lead to similar, but less severe, injury in the contralateral lung. Because injury in the nonischemic lung develops only after reperfusion of the ischemic lung, injury to the nonischemic lung is probably humorally mediated. The model is a useful and relevant method for studying the physiological consequences of pulmonary IR injury.     

Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells

Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 x 10(4) sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 x 10(6) sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1 x 10(-3) s-1 (1.0-1.3 x 10(6) sites per cell) and a slow-dissociating site, with a k-1 of 3.5 x 10(-5) s-1 (0.6-0.7 x 10(6) sites per cell). The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that approximately 5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 x 10(-3) s-1) and a slow-dissociating component (k-1 = 9.1 x 10(-5) s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4 degrees C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4 degrees C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.     

EGFR expression and EGF stimulation of proliferation in human liver carcinoma cells

Epidermal growth factor receptor (EGFR) gene expression and growth stimulation of EGF on human hepatoma cells of cell lines BEL-7404 and SMMC-7721 were studied. 125I-EGF binding assay was used to measure the binding characteristics and the amounts of EGFR on these cells. The binding time course and the binding competition assay showed that the binding of 125I-EGF to 7404 cells was saturable and specific. Scatchard analysis of EGF binding curve indicated that 7404 and 7721 cells expressed approximately 1.1 x 10(5) and 0.7 x 10(5) EGFRs per cell with binding affinity (Kd) 2.1 nM and 1.8 nM respectively. Northern hybridization and immunoblotting analysis showed the EGFR gene expression products in 7404 and 7721 cells were 5.6 Kb mRNA and 170 Kilo-dalton glycoprotein. Anchorage-dependent growth of 7404 and 7721 cells was stimulated in the presence of nanogram quantities of EGF in medium containing 10% calf serum or 0.5% calf serum. The factors in serum appeared to act synergitically in stimulating of cell proliferation. EGF also stimulated the anchorage-independent growth of 7404 and 7721 cells in soft agar. The results suggest that EGFR is actively expressed in human hepatoma 7404 and 7721 cells and EGF may be one of the mitogens needed for the growth of hepatoma cells.     

Exocrine and endocrine secretion of renin and epidermal growth factor from the mouse submandibular glands

Renin and epidermal growth factor (EGF) are synthesized in large amounts by the male mouse submandibular glands. We report the peptides to be secreted mainly in an exocrine manner. The highest values in saliva are obtained upon stimulation with the alpha-adrenergic agonist phenylephrine. The median value for renin is 54 700 nmol/l and the median value for EGF is 211 800 nmol/l. Aggressive behaviour and beta-adrenergic stimulation also increase salivary output of both peptides, while vasoactive intestinal polypeptide (VIP) plus pilocarpine selectively stimulate the secretion of renin. The pattern of increase in plasma is comparable to that in saliva though the substance concentration is lower by a factor of 2 to 70 for renin and a factor of 280 to 12 000 for EGF.     

Human biokinetics of strontium. Part I: Intestinal absorption rate and its impact on the dose coefficient of 90Sr after ingestion

Intestinal absorption of strontium (Sr) in thirteen healthy adult German volunteers has been investigated by simultaneous oral and intravenous administration of two stable tracer isotopes, i.e. (84)Sr and (86)Sr. The measured Sr tracer concentration in plasma was analyzed using the convolution integral technique to obtain the intestinal absorption rate. The results showed that the Sr labeled in different foodstuffs was absorbed into the body fluids in a large range of difference. The maximum Sr absorption rates were observed within 60-120 min after administration. The rate of absorption is used to evaluate the intestinal absorption fraction, i.e. the f (1) value for various foodstuffs. The equivalent and effective dose coefficients for ingestion of (90)Sr were calculated using these f (1) values, and they were compared with those recommended by the International Commission on Radiological Protection (ICRP). The geometric and arithmetic means of the f (1) values are 0.38 and 0.45 associated with a geometric standard deviation and a standard deviation of 1.88 and 0.22, respectively. The 90% confidence interval of the f (1) values obtained in the present study ranges from 0.13 to 0.98. Expressed as the ratio of the 95 and 50% percentiles of the estimated probability, the uncertainty for the f (1) value corresponds to a factor of 2.58. The effective dose coefficients of (90)Sr after ingestion are 6.1 x 10(-9) Sv Bq(-1) for an f(1) value of 0.05, 1.0 x 10(-8) Sv Bq(-1) for 0.1, 1.9 x 10(-8) Sv Bq(-1) for 0.2, 2.8 x 10(-8) Sv Bq(-1) for 0.3, 3.6 x 10(-8) Sv Bq(-1) for 0.4, 5.3 x 10(-8) Sv Bq(-1) for 0.6, 7.1 x 10(-8) Sv Bq(-1) for 0.8, and 7.9 x 10(-8) Sv Bq(-1) for 0.9, respectively. Taking the effective dose coefficient of 2.8 x 10(-8) Sv Bq(-1) for an f (1) value of 0.3, which is recommended by the ICRP, as a reference, the effective dose coefficient of (90)Sr after ingestion varies by a factor of 2.8 when the f (1) value changes by a factor of 3, i.e. it decreases from 0.3 to 0.1 or increases from 0.3 to 0.9, respectively.     

Growth inhibition of human nasopharyngeal carcinoma in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies

Monoclonal antibodies (MoAbs) were developed against epidermal growth factor (EGF) receptor on the human epidermoid carcinoma cell line A431. The A431 antigen recognized by the MoAbs has an apparent molecular weight of approximately 170,000, with the same molecular weight as the CNE-2 cell line (poorly differentiated nasopharyngeal carcinoma). Administration of anti-EGF receptor MoAbs inhibited tumor formation, caused by the CNE-2 and A431 cell lines, in athymic mice. When the same MoAbs were used in therapy against Tca8113 (a human tongue carcinoma) and HeLa cells (a human cervical carcinoma), tumor growth was not affected. The number of EGF receptors and the apparent dissociation constants for 125I-EGF on CNE-2 and A431 were 1.3 x 10(5)/cell (Kd 7.7 x 10(-8) M) and 1.4 x 10(6)/cell (Kd 2.4 x 10(-9) M), respectively. Three anti-EGF receptor MoAbs were used in these studies. MoAbs 3 and 176, capable of competing with EGF for receptor binding, showed significant tumor growth inhibition. MoAb 101 was incapable of blocking the binding of EGF to its receptor and was not as effective as MoAbs 3 and 176 in tumor growth inhibition. Our observation is that in vitro, MoAb anti-EGF receptor is cytostatic, rather than cytocidal, against CNE-2 and A431.     

Accurate quantification of basal plasma levels of 3-nitrotyrosine and 3-nitrotyrosinoalbumin by gas chromatography-tandem mass spectrometry

Measurement of 3-nitro-L-tyrosine (NO(2)Tyr) and protein-related 3-nitro-L-tyrosine in human plasma is associated with numerous methodological problems which result in highly divergent basal plasma levels often ranging within two orders of magnitude. Recently, we have described an interference-free GC-tandem MS-based method for NO(2)Tyr which yielded the lowest basal plasma NO(2)Tyr levels reported thus far. This method was extended to quantify protein-associated 3-nitrotyrosine and in particular 3-nitrotyrosinated albumin (NO(2)TyrALB) in human plasma. NO(2)TyrALB and albumin (ALB) were extracted from plasma by affinity column extraction and digested enzymatically at neutral pH. 3-Nitro- L-[2H(3)]tyrosine was used as internal standard. In plasma of 18 healthy young volunteers the molar ratio of NO(2)TyrALB to albumin-derived tyrosine (TyrALB), i.e. NO(2)TyrALB/TyrALB, was determined to be 1.55+/-0.54x1:10(6) (mean+/-SD). The plasma concentration of NO(2)TyrALB was estimated as 24+/-4 nM. The NO(2)Tyr plasma levels in these volunteers were determined to be 0.73+/-0.53 nM. In the same volunteers, NO(2)TyrALB/TyrALB, NO(2)TyrALB and NO(2)Tyr were measured 15 days later and the corresponding values were determined to be 1.25+/-0.58x1:10(6), 25+/-6 nM and 0.69+/-0.16 nM. For comparison, NO(2)Tyr and NO(2)TyrALB were measured in six plasma samples from healthy volunteers by GC-MS and GC-tandem MS. Different values were found for NO(2)Tyr, i.e. 5.4+/-2.8 versus 2.7+/-1.5 nM, and comparable values for NO(2)TyrALB/TyrALB, i.e. 0.5+/-0.2x1:10(6) versus 0.4+/-0.1x1:10(6), by these methods. The ratio of the values measured by GC-MS to those measured by GC-tandem MS were 2.9+/-3.1 for NO(2)Tyr and 1.2+/-0.2 for NO(2)TyrALB/TyrALB. The present GC-tandem MS method provides accurate values of NO(2)Tyr and NO(2)TyrALB in human plasma.     

Impaired PBPC collection in patients with myeloma after high-dose melphalan

BACKGROUND: Tandem stem cell transplantation is an important treatment option for patients with myeloma and some additional tumors. In an attempt to reduce the contamination of the stem cell graft with tumor cells, patients with myeloma who entered complete remission after the first transplant underwent a second episode of mobilization to obtain progenitor cells for the second transplant. METHODS: Twenty-two patients with myeloma participated in the study. The first mobilization utilized CY, etoposide and filgrastim. The second mobilization used the same regimen, but seven patients received only filgrastim. The interval between the two collection periods was 6 months (median; range 4-9 months). The preparative regimen for the first transplant consisted of melphalan 200 mg/m(2). RESULTS: The number of total white cells collected during the two collection episodes was similar: 10.8+/-1.6 x 10(8)/kg white cells vs. 11.8+/-1.7 x 10(8)/kg white cells (P=0.63). The collected CD34(+) cell dose was much larger during the first collection: 45.2+/-8.4 x 10(6)/kg vs. 6.9+/-2.7 x 10(6)/kg (P<0.001). Similarly, the collected colony-forming unit (CFU)-GM dose was much larger during the first collection: 295.4+/-59.3 x 10(4)/kg vs. 67.3+/-21.6x10(4)/kg (P<0.001). While the CD34(+) cells collected during the two collection episodes correlated significantly (r=0.55, P<0.01); the first dose was a median of 14.9-fold larger. DISCUSSION: No laboratory parameter was able reliably to predict the results of the second collection. A second mobilization/collection episode as part of a tandem transplant approach carries a considerable risk of failing to obtain sufficient progenitor cells.     

Scatchard analysis of EGF receptor and effects of EGF on growth and TA-4 production of newly established uterine cervical cancer cell line (OMC-1)

Effects of epidermal growth factor (EGF) on growth and tumor antigen-4 (TA-4) production of newly established uterine cervical cancer cell line (OMC-1) are reported. OMC -1 was established from a metastatic lesion of Virchow's lymph node of a large cell non-keratinizing squamous cell carcinoma of the uterine cervix and successively subcultured for about 4 years. Scatchard analysis of EGF binding to OMC-1 cells indicated a single class of binding sites with a dissociation constant (Kd) of 360 pM. The theoretical maximum number of binding sites was 2.4 x 10(4) sites/cell. The growth of OMC-1 cells was stimulated by EGF at 0.01-0.1 nM and inhibited at higher concentrations. The TA-4 production of OMC-1 cells was slightly stimulated by EGF at 0.01-1 nM and significantly stimulated at 10 nM. OMC-1 may serve as one of the available model systems for studies of regulation of proliferation and tumor marker production by EGF, particularly in cervical squamous cell carcinomas.     

Comparison of whole-blood eosinophil counts in extrinsic asthmatics with acute and chronic asthma.

Venous whole-blood eosinophil counts were performed on 50 occasions in 42 patients with varying patterns of asthma. None of the patients studied had received systemic corticosteroids during the previous year. Patients with acute severe asthma, as defined by symptomatic airways obstruction with a tachycardia of at least 120 beats/min, showed eosinopenia (21 x 10(9)/l +/- SD 57 x 10(9)/l). Patients with chronic asthma, as defined by symptomatic airways obstruction with a heart rate of less than 100 beats/min, showed appreciable eosinophilia (1048 x 10(9)/l +/- SD 708 x 10(9)/l). Finally, asymptomatic patients had a variable total eosinophil count but with values lower than those of patients with chronic asthma (345 x 10(9)/l +/- SD 431 x 10(9)/l). Eosinophilia may contain chronic asthma, thereby mediating bronchial damage, whereas absence of eosinophils in acute asthma enables vasoactive mediators to enter the systemic circulation, possibly causing circulatory disturbances.     

The kinetics of ligand binding to plant hemoglobins. Structural implications

The rates of reaction of oxygen, carbon monoxide, and nitric oxide with 14 plant hemoglobins have been determined by relaxation and stopped-flow methods. The combination rates for oxygen lie between 0.12 and 0.26 x 10(9)/M.s, for carbon monoxide between 0.01 and 0.07 x 10(9)/M.s, and for nitric oxide between 0.12 and 0.25 x 10(9)/M.s. The dissociation velocities for oxygen range from 5 to 25/s, and for CO from 0.005 to 0.011 s. The oxygen dissociation constants range only from 36 to 78 nM. Nanosecond relaxation experiments show large differences between the proteins. Five have known primary structures which correlate closely with the nanosecond relaxations and less immediately with the millisecond reactions. The relevant amino acid substitutions are concentrated in the C-E interhelical region.     

Thermodynamic comparison of PNA/DNA and DNA/DNA hybridization reactions at ambient temperature

The thermodynamics of 13 hybridization reactions between 10 base DNA sequences of design 5'-ATGCXYATGC-3' with X, Y = A, C, G, T and their complementary PNA and DNA sequences were determined from isothermal titration calorimetry (ITC) measurements at ambient temperature. For the PNA/DNA hybridization reactions, the binding constants range from 1.8 x 10(6)M(-1)for PNA(TT)/DNA to 4.15 x 10(7)M(-1)for PNA(GA)/DNA and the binding enthalpies range from -194 kJ mol(-1)for PNA(CG)/DNA to -77 kJ mol(-1)for PNA(GT)/DNA. For the corresponding DNA/DNA binding reactions, the binding constants range from 2.9 x 10(5)M(-1)for DNA(GT)/DNA to 1.9 x 10(7)M(-1)for DNA(CC)/DNA and the binding enthalpies range from -223 kJ mol(-1)for DNA(CG)/DNA to -124 kJ mol(-1)for DNA(TT)/DNA. Most of the PNA sequences exhibited tighter binding affinities than their corresponding DNA sequences resulting from smaller entropy changes in the PNA/DNA hybridization reactions. van't Hoff enthalpies and extrapolated Delta G values determined from UV melting studies on the duplexes exhibited closer agreement with the ITC binding enthalpies and Delta G values for the DNA/DNA duplexes than for the PNA/DNA duplexes.