Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40

The Ag CDw40 (p50, Bp50) is a phosphoprotein expressed on the surface of both B lymphocytes and on certain malignant cell types of nonhemopoietic origin. Antibodies to this Ag have been shown to act as a potent co-mitogen for B cells. In order to elucidate the function of this Ag, we have now investigated some of its biochemical characteristics as well as the relationship of B cell derived CDw40 to that derived from urinary bladder carcinoma (transitional cell carcinoma, TCC) cells. CDw40 from normal B cells or from the Burkitt lymphoma line Raji showed a characteristic pattern of three bands when analyzed by SDS-PAGE and Western blotting: a main band of 47 kDa, a degradation product of 43 kDa, and a dimer of 85 kDa. The dimer was disrupted by reduction with 2-ME but was reformed spontaneously from the purified monomers under nonreducing conditions. CDw40 from two bladder cancer cell lines gave a similar pattern but formed little or no dimer. Thirty amino acids of the amino terminal end of CDw40 from Raji and 22 amino acids of that from TCC cells (HU549) were sequenced. The sequences were unusually rich in cysteines and differed only in that the cysteine in position 6 in Raji CDw40 had been replaced by glutamine in HU549. In addition there were two conservative changes in positions 15 and 19. Taken together these results show that CDw40 derived from B cells or from TCC cells are the same or closely related molecules. Comparisons of the amino acid sequence and biochemical characteristics of CDw40 with proteins having receptor functions indicated a close structural resemblance of CDw40 to the nerve growth factor-receptor.     

Quantitative ultrastructural immunocytochemistry using a computerized image analysis system

Secretory granules in human pituitary adenoma cells have been examined indirectly for hormone epitopes by immunogold labelling of resin-embedded ultrathin sections. The specific binding of different immunoglobulin-gold complexes to the antigrowth hormone antibodies over the secretory granules was measured using a computerized image analysis system. This facilitated the assessment of the preferential binding to the target granules of gold particles with three different average particle diameters (Au7, Au11, Au17). The time of pretreatment of sections with H2O2 or a buffer was found to influence the staining considerably. The scanning electron microscopic findings of protruded secretory granules with a mountain-like surface might be relevant to the uneven distribution of immunolabels seen over the secretory granules in the adenohypophysis.     

Diet and urinary estrogen profile in premenopausal omnivorous and vegetarian women and in premenopausal women with breast cancer

The urinary estrogen profile was studied in the midfollicular phase twice, and diet four times during 1 yr in 10 premenopausal breast cancer (BC) patients consuming an omnivorous normal Finnish diet and in two control groups, one consuming an omnivorous (n = 12) and the other a lactovegetarian (n = 11) diet. Total fat intake in relation to caloric intake was almost identical in all three groups. Only with regard to grain fiber intake did the BC patients differ significantly from both other groups. No differences were found between the groups with regard to urinary excretion of 13 individual estrogens and total estrogens, with the exception of 4-hydroxyestrone (4-OH-E1), which was significantly lower (P less than 0.05) in the BC group than in the vegetarians. A high carbohydrate to protein ratio in the diet had a negative correlation with the excretion of 2-hydroxyestrogens and 2-hydroxyesterone (2-OH-E1) to 4-OH-E1 ratio. The BC group had significantly higher urinary 2-OH-E1 to E1 ratio (P less than 0.05) compared to the vegetarians. The 2-OH-E1 to 4-OH-E1 ratio was highest in the BC group (= 7.1) and differed significantly from that of the omnivores (= 4.3; P less than 0.02) and vegetarians (= 3.6; P less than 0.005). This ratio showed a negative correlation with intake of carbohydrates, starch, total and grain fiber. Urinary excretion of 4-OH-E1 correlated positively with total and grain fiber intake and plasma SHBG. Protein intake correlated positively with urinary 2-methoxy-E1 excretion, and retinol intake positively with catechol estrogen, E1 and E2 excretion. It is concluded that estrogen production and urinary estrogen profile in premenopausal breast cancer patients is normal with the exception of a low 4-OH-E1 excretion and high urinary 2-OH-E1 to 4-OH-E1 ratio. This ratio, which seems to depend on diet, is the only urinary estrogen parameter separating premenopausal BC patients from the control omnivorous and lactovegetarian women.     

Purification of thymocyte growth peptide (TGP) from sheep thymus. Relationship to FTS/thymulin

Thymocyte growth peptide (TGP) initiates DNA synthesis in immature thymocytes and has previously been characterized as an acidic peptide isolated from calf thymus. We now report the isolation of TGP from sheep thymus and show it to be a nonapeptide with a large N-terminal blocking moiety characterized by high UV absorbance. The amino acid composition is identical to FTS, consisting of 2 Gly, 2 Ser, 2 Glx, 1 Ala, 1 Lys, 1 Asx. In contrast to FTS, TGP is acidic with an apparent isoelectric point of 4.2 and a high UV absorbance at 270–280 nm. Reverse phase chromatography of TGP at an acidic pH results in a change of the molecule and the appearance of two new compounds TGP-A and TGP-B, both with less than 50% of the original TGP activity. Full activity could be restored by the addition of ZnCl₂ to TGP-A. Both TGP-A and B have some amino acid composition and high UV absorbance as native TGP. We propose that TGP consists of a non-peptide moiety bound to the N-terminal of the nonapeptide Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn and that the active molecule is stabilized by Zn²⁺.     

An in vitro bioassay for quantitation of human interferons by measurements of antiproliferative activity on a continuous human lymphoma cell line

Interferons have, in addition to their antiviral effects, been shown to possess several non-antiviral activities. In this study, an in vitro bioassay for interferon alpha (IFN-alpha) preparations based on their antiproliferative effect in cultured Daudi cells has been developed. Briefly, about 10(5) cells per ml treated with different concentrations of IFN were incubated under standard culture conditions for 3 days. Two different end points, i.e. incorporation of [3H]thymidine and final cell density, were used and responses were evaluated according to established pharmacopoeial principles for quantification of biomolecules. Both methods gave similar results. However, measurement of final cell density yielded the most precise results. The proposed assay, with an effective assay range of 1-10 IU/ml (approximately 0.2-2 x 10(-12)M, had a high sensitivity and precision as well as a good reproducibility. Compared with antiviral assays, it is less resource demanding. In conclusion, the in vitro bioassay described is well suited for potency determinations of IFN-alpha and probably also IFN-beta preparations.     

Effect of nitrogen concentration on the activity of manganese peroxidase ofPhanerochœte chrysosporium

Manganese peroxidase as an extracellular enzyme is produced by the white rot fungusPhanerochœte chrysosporium under nutrient nitrogen or carbon limitation. The effect of nitrogen concentration on the activity of manganese peroxidase was studied using ammonium nitrate andl-asparagine as nitrogen sources. The highest activity of the enzyme was observed in cultures grown in a medium containing 75 mg/L ammonium nitrate and 0.15 g/Ll-asparagine. Manganese peroxidase was not detectable in cultures grown in the presence 0.5 g/L ammonium nitrate and 1 g/Ll-asparagine.     

Insulin-like growth factor I enhances the formation of type I collagen in hydrocortisone-treated human osteoblasts

We have studied the effect of insulin-like growth factor I (IGF-I) on the formation of osteocalcin and type I collagen in isolated human osteoblasts. IGF-I at and above 0.1 nM stimulated the formation of type I collagen as measured by the type I procollagen carboxyterminal peptide (PICP), in human osteoblasts, incubated for 72 hrs in serumfree conditions. The secretion of osteocalcin was not affected by IGF-I while 1,25(OH)₂ vitamin D₃ significantly enhanced the formation of osteocalcin. When human osteoblast-like cells were incubated with hydrocortisone (1 M), a significant decrease in the release of both PICP and osteocalcin was seen. Addition of IGF-I to human osteoblasts also treated with hydrocortisone normalized the PICP-formation but did not affect the suppressed osteocalcin-formation. These data indicate that IGF-I reverses selective effects of hydrocortisone on bone.     

Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers.

We have expressed glycine receptor (GlyR) alpha and beta subunit cDNAs in HEK-293 cells to study the functional properties of homo- versus hetero-oligomeric GlyR channels. Dose-response curves of whole-cell currents in cells expressing alpha 1 subunits revealed an average Hill coefficient of h = 4.2. Co-expression with the beta subunit markedly increased glycine-gated whole-cell currents, which now exhibited a mean Hill coefficient of only h = 2.5. For alpha 1, alpha 2 and alpha 3 homo-oligomers, the main-state single-channel conductances were 86, 111 and 105 pS, respectively, recorded at symmetrical Cl- concentrations of 145 mM. The mutant alpha 1 G221A gave rise to a main-state of 107 pS. This indicates that the main-state of alpha homo-oligomers depends on residue 221 which is located within transmembrane segment M2. Importantly, the main-state conductances of alpha 1/beta, alpha 2/beta and alpha 3/beta hetero-oligomers were only 44, 54 and 48 pS, respectively. The latter values are similar to those found in spinal neurons, suggesting that native GlyRs are predominantly alpha/beta hetero-oligomers. Co-expression of alpha 1 with mutant beta subunits revealed that residues within and close to segment M2 of the beta subunit determine the conductance differences between homo- and hetero-oligomers.     

High prevalence of asthma in cross country skiers.

OBJECTIVES--To study the prevalence of asthma (asthma symptoms and bronchial hyperresponsiveness) in Swedish cross country skiers compared with non-skiers and monitor changes in symptoms and bronchial hyperresponsiveness during the year. DESIGN--Cross sectional study during the winter ski season and in the summer. SETTING--Six ski clubs for élite skiers (total 47) in two different areas of Sweden. SUBJECTS--42 élite cross country skiers and 29 non-skiing referents. MAIN OUTCOME MEASURES--Bronchial responsiveness, asthma symptoms, and lung function. RESULTS--Bronchial responsiveness was significantly greater and asthma symptoms more prevalent in the skiers than in the referents. There was no difference in bronchial responsiveness within either group between winter and summer. 15 of the 42 skiers used antiasthmatic drugs regularly and 23 had a combination of asthma symptoms and hyperresponsive airways or physician diagnosed asthma, or both. Altogether 33 skiers had symptoms of asthma or bronchial hyperresponsiveness. One of the referents had symptoms of asthma and bronchial hyperresponsiveness, and none used antiasthmatic drugs regularly. CONCLUSIONS--Asthma, asthma-like symptoms, and bronchial hyperresponsiveness are much more common in cross country skiers than in the general population and non-skiers. Strenuous exercise at low temperatures entailing breathing large volumes of cold air is the most probable explanation of persistent asthma in skiers.