Characteristics of the purified uteroglobin-like protein from rabbit lung.

A uteroglobin-like protein was prepared from lung extracts of female rabbits by absorption to immobilized anti-uteroglobin immunoglobulin and purified to homogeneity by gel filtration on Sephacryl S-200. The final preparation is indistinguishable from uteroglobin according to its behaviour in Ouchterlony double-diffusion, polyacrylamide gel electrophoresis under denaturing and non-denaturing conditions, ultraviolet spectrum, tryptic peptide analysis, and progesterone-binding properties. Progesterone binding to the lung protein exhibits an affinity similar to that observed with authentic uteroglobin and is equally enhanced by reduction of the protein with dithiothreitol. Competition experiments with non-radioactive steroids demonstrate a similar steroid-specificity for both proteins. Progesterone binding causes a perturbation in the ultraviolet absorbance of tyrosine residues of the lung protein similar to that observed with uteroglobin. These data suggest that the proteins prepared from both sources are biochemically identical.     

Evidence of a uteroglobin-like protein in epithelial cells of reproductive and non-reproductive tissues of the rabbit.

An antiserum raised in a goat to a uteroglobin-like protein isolated from uterine fluid of oestrous rabbits was used in an immune fluorescence test to localize an antigen present in the reproductive tract of oestrous and pseudopregnant rabbits and mammary gland tissue. The antigen was also present in the vas deferens and seminal vesicle, but not in testis. Non-reproductive tissues, such as lung, small intestine, bladder and thyroid showed specific fluorescent staining which was eliminated or significantly reduced by absorption of the antiserum with a purified uteroglobin preparation.     

Detection and synthesis of a progestagen-dependent protein in human endometrium

Immunologiocal and biochemical methods were employed to demonstrate the presence of progestagen-dependent proteins in human endometrium. Cytosols were prepared from proliferative and secretory phase endometria of cycling women, from decidua and decidua-rich tissues of women in early pregnancy and from decidua of tubal pregnancy. Antisera were raised in rabbits against the antigens of decidua of tubal pregnancy and decidua-rich tissues. Immunoelectrophoresis, Ouchterlony's immunodiffusion test and polyacrylamide gel electrophoresis using native gels revealed 2 antigenic proteins, designated antigens A and B, in secretory endometria, decidua-rich tissues, decidua, and in decidua of tubal pregnancy. However, only 1 antigenic protein was detected by SDS-gel electrophoresis: antigens A and B may therefore be two different proteins or two forms of a single protein. The antigens could not be detected in non-pregnancy sera or in term placentae. Double isotopic labelling (incubation of tissues with [3H]- and [14C]leucine) followed by protein fractionation methods were used to compare the in-vitro synthesis rates of antigens in poroliferative tissues with those in decidua or secretory endometria. The rate of synthesis of antigens A and B was markedly higher in the decidua and secretory endometria than in the proliferative endometria. We conclude, therefore, that during progestagen-dependent transformation of proliferative phase endometria into secretory endometria and decidua in women, there is a selective stimulation of at least one species of pregnancy-associated protein.     

Metabolism of testosterone in human,rat and rabbit fetal lungs and in rabbit neonatal lung in vitro

• 1.1. Metabolism of 4-¹⁴C-testosterone was investigated in human, rat and rabbit fetal lung subcellular fractions and also in rabbit neonatal lungs. Androst-4-ene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and both 5α- and 5β-androstane-3α,17β-diols were identified as metabolites of testosterone.
• 2.2. The microsomal fraction produced mainly 5α-reduced epimers while the cytosol incubations resulted in 5β-reduced metabolites.
• 3.3. No conjugation was found.
• 4.4. The amounts of polar metabolites in the microsomal incubations and the amounts of dihydroxy-lated metabolites in the soluble fraction incubations were statistically significantly greater in the neonatal rabbit lung incubations compared with those of fetal lungs.

     

The glycosaminoglycans of human tracheobronchial cartilage

1. The glycosaminoglycans of human tracheobronchial cartilages from subjects of various ages were liberated by proteolysis of the tissue and purified by ion-exchange chromatography. Purified glycosaminoglycans were fractionated on Dowex 1 resin and cetylpyridinium chloride was used to separate chondroitin sulphates and keratan sulphates occurring in the same fraction. 2. The total chondroitin sulphate content of the cartilages decreased linearly with increasing age. Age-dependent changes in the chemical heterogeneity of chondroitin sulphate were observed, a low-sulphated compound making up 25% of the total glycosaminoglycan at birth but rapidly diminishing in content during the first 6 months of life. Of the total chondroitin sulphate the 6-isomer became rather more prominent than the 4-isomer with increasing age. 3. The total keratan sulphate content of the cartilages increased from trace amounts only at birth to a plateau value by the beginning of the fifth decade. Of the total keratan sulphate approx. 70% was due to a high-molecular-weight compound with a sulphate/hexosamine ratio of 1.5-1.8: 1.0. The degree of sulphation varied between compounds isolated from different individuals. The remaining 30% of the keratan sulphate appeared to be intimately associated with chondroitin 6-sulphate and could only be separated from it after treatment with 0.45m-potassium hydroxide. The hybrid glycosaminoglycans were of lower molecular weight and had a lower sulphate/hexosamine ratio than the major keratan sulphate compound.     

Identification of a rabbit liver cytosolic binding protein for human growth hormone.

A specific growth hormone (GH) binding protein of Mr approx. 100000 has been demonstrated in the cytosolic fraction (200000g supernatant) of pregnant-rabbit liver by gel filtration techniques. This binding species was detectable by a standard charcoal separation procedure but not by the widely used poly(ethylene glycol) precipitation method. The GH binding protein had similar binding characteristics to those of classical membrane-bound GH receptors. The kinetics of association and dissociation, binding affinity (2.56 X 10(9)1/mol) and hormonal specificity have been established. There appears to be equal or greater amounts of GH binding protein in the cytosol than in the membrane fraction. The presence of the GH binding protein in rabbit liver cytosol was substantiated by its selective purification on a GH-Affigel 15 affinity column. This technique has resulted in a 200-300-fold purification with no substantial change in binding affinity. The ability of a concanavalin A-Sepharose affinity column to also bind the cytosolic binding protein indicates that, like the membrane-bound GH receptor, it is a glycoprotein. This is the first report of a cytosolic binding protein for GH and raises important questions regarding its potential physiological role in the mechanism of action of GH.     

Action of aldosterone on protein expression in cultured collecting duct cells from neonatal rabbit kidney

To investigate whether 'aldosterone-induced proteins' could be detected in mammalian species, cultured renal collecting duct epithelia from neonatal rabbit kidneys were labelled under aldosterone administration with radioactive methionine and subsequently fractionated into cytosolic and coarse membrane protein fractions. Newly synthesized proteins were then analyzed by SDS-PAGE, isoelectric focussing and two-dimensional electrophoresis. Quantitative estimates of individual newly synthesized proteins were performed utilizing gel slicing, scintillation counting and autoradiography. The labelling experiments demonstrated that, in comparison to controls, aldosterone (1 X 10(-6) M) generally increased the amount of radioactive protein. No qualitative changes in the pattern of newly synthesized proteins and, therefore, no classical aldosterone-induced proteins were observed. The increase of radioactive protein was already seen after 1, 6, and 18 h of hormone treatment. The effect could be blocked partially by spironolactone (1.5 X 10(-4) M), and totally by amiloride (1 X 10(-6) M), g-strophantin (5 X 10(-4) M), and cycloheximide (1 X 10(-6) M. Thus, the interference of aldosterone action at the receptor level, the Na+ channels and the Na+/K(+)-ATPase pump demonstrate that the expression of proteins in cultured renal collecting duct cells is a sensitive system and seems to be controlled by aldosterone at the receptor level, but also counter-controlled by specific plasma membrane sites.     

Detection of adenocarcinoma in peritoneal washings by staining with monoclonal antibody B72.3

Peritoneal washings are routinely performed in the staging evaluation of carcinomas of the ovary and in "second-look" explorations; major problems in the evaluation of these specimens continue to be the distinction between atypical mesothelium and adenocarcinoma and the identification in an otherwise inflammatory specimen of rare cells of adenocarcinoma, which may be undetected by the most trained individual. Monoclonal antibody (MAb) B72.3, reactive with an oncofetal, tumor-associated glycoprotein (termed TAG-72; MW greater than 1000 kd) expressed in a variety of epithelial malignancies but not generally expressed in benign or malignant mesothelium, was reacted with sections from the paraffin-embedded cell blocks of 185 peritoneal washings from 180 patients with extant cancer or a prior history of malignancy. One hundred four of the washings were initially interpreted as atypical mesothelium, with no evidence of malignancy; when reacted with MAb B72.3, 6 of these specimens demonstrated groups of metastatic adenocarcinoma cells not appreciated by the usual cytologic criteria. Of the 81 washings interpreted as showing cells of adenocarcinoma, 73 demonstrated expression of TAG-72 from both gynecologic and nongynecologic malignancies. In the remaining 49 cases without an associated malignant process, MAb B72.3 did not stain atypical mesothelium, but did react, however, with benign endometrial cells and müllerian inclusions in two cases. MAb B72.3 may be used as a diagnostic adjunct to the routine cytologic evaluation of malignancy in peritoneal washings; reactivity with MAb B72.3 may indicate the need for further evaluation to define the presence of a malignancy or an advanced cancer.     

Immunolocalization of a Na-K-2Cl cotransporter in human tracheobronchial smooth muscle.

Inhibition of the Na-K-2Cl (NKCC) cotransporter by loop diuretics is associated with airway relaxation, but there has been no direct evidence for the expression of this protein in airway smooth muscle. Thus we hypothesized that a NKCC cotransporter is present and functional in airway smooth muscle cells. Monoclonal and polyclonal antibodies were used first to demonstrate the presence of a NKCC cotransporter protein in isolated human fetal trachea and normal human bronchial smooth muscle cells (BSMC) by Western blotting. The cotransporter protein was then localized by immunohistochemical staining to airway smooth muscle cells in culture and in situ. The localization was confirmed by indirect immunofluorescence and laser confocal microscopy in the BSMC. Cotransporter function in BSMC was also confirmed in vitro by bumetanide-mediated inhibition of rubidium uptake. Our present findings thus document the presence of a functional NKCC cotransporter in human airway smooth muscle, providing a basis for defining the role of this ion cotransporter in airway smooth muscle function.     

Structural analysis of purified human tracheobronchial mucins

Light scattering has been used to investigate the structure of human tracheobronchial mucin glycoproteins (HTBM) from the sputum of cystic fibrosis patients. The specimen was extracted using 6M guanidinium hydrochloride solution and fractionated by gel exclusion chromatography on Sephacryl S-1000. The fractionated HTBM was purified by density gradient ultracentrifugation. Purity of the resulting material was confirmed by SDS polyacrylamide gel electrophoresis and uv spectroscopy. Light scattering measurements on the fractionated mucins yield weight-average molecular weights M_w, and z-average radii of gyration R_{g, z}. The native cystic fibrosis HTBM consisted of a high molecular weight fraction with M_w = 9.3 × 10⁶ daltons and a lower molecular weight fraction contanining partly degraded mucins. After reduction and carboxymethylation of the high molecular weight native fraction, the resulting material was separated into three pools with M_w values of 5.1 × 10⁶, 1.6 × 10⁶, and 400,000. The derived molecular weights for the protein cores M_p,w, and the experimental radii of gyration are found to be consistent with the M_p,w – R_g relation established previously for submaxillary, cervical, and gastric mucins. These results imply that HTBM has the same extended-coil conformation reported for other mucins and has a molecular structure consisting of subunits, linked into linear chains via covalent (disulfide) bonds.     

Regulation by human chorionic gonadotropin of protein kinases in rabbit endometrium

The effect of human chorionic gonadotropin (CG) on protein kinase levels has been studied in rabbit endometrium. A good correlation was observed between alterations of total protein kinase activity and DNA content in the tissue. The level of type I cyclic AMP-dependent protein kinase had slightly increased by 6 h after human CG treatment, and then decreased later. In contrast, the type II enzyme gradually increased after 3 days. The increase of the activity at 7 days was mainly due to that of type II enzyme, and the ratio of type II to type I enzyme increased. The activity pattern at the later stage closely resembled that of treatment with progesterone.     

Detection of Epstein-Barr virus in salivas and throat washings in healthy adults and children

It is well known that Epstein-Barr virus (EBV) is excreted from oral regions in the patients with infectious mononucleosis. We analyzed the prevalence of EBV in saliva and throat washings from healthy people in Japan by the polymerase chain reaction assay. EBV DNA was detected in 43 (90%) of the 48 throat washings from healthy adults (21 to 57 years old) and in 35 (38%) of the 93 salivas from healthy children (0 to 6 years old). The percentages of the EBV DNA-positive ratio in salivas increased in proportion relative to the increase of the children's ages. EBV type 1 was predominant and was detected in 86 and 94% of adults and children, respectively. Umbilical cord lymphocytes were transformed by some throat washings from EBV seropositive donors. EBV DNA was detected in throat washings from two healthy adults whose EBV antibody was not detected. In both cases, higher amounts of EBV DNA were detected in their peripheral blood mononuclear cells than in those of other, EBV antibody-positive donors. These results demonstrated the incidence of EBV excretion in oral regions of healthy individuals in Japan and defined a novel type of EBV infection in healthy adults.     

Viscoelastic properties of human tracheobronchial mucin in aqueous solution

Human tracheobronchial mucin isolated from cystic fibrosis patients (CF HTBM) was purified using a combination of gel filtration and density gradient centrifugation. The resulting mucin was fractionated to reduce polydispersity and to facilitate studies of the molecular weight dependence of mucin viscoelasticity in concentrated solution. The viscoelastic properties of CF HTBM were examined in distilled water, 0.1M salt solutions and chaotropic solvents. In controlled strain experiments (strain ≥ 5%) with increasing mucin concentration, a crossover from sol to gel behavior is observed. The gel strength, as measured by the magnitude of the storage modulus at comparable mucin concentrations, is greatest for distilled water, intermediate for 0.1M NaCl, and lowest far 6M GdnHCl. In distilled water, high molecular weight mucin undergoes a sol-gel transition at ～ 12 mg/mL, and shows evidence of a plateau modulus at higher concentrations. The storage and loss moduli of concentrated high molecular weight fractions in 6M GdnHCl exhibit a power law dependence on frequency typical of weak gels near the sol–gel transition at 20 mg/mL. Similar rheology is observed in 0.1M NaCl and 0.091M NaCl/3 mM CaCl₂, but with evidence for additional weak associations at low frequency. The power law exponent in these systems is 0.70 ± 0.02, in good agreement with prediction for networks formed by a percolation mechanism. Low molecular weight fractions in these solvents exhibit a fluid-like viscoelastic response. However, low molecular weight mucin in distilled water shows a strain-dependent increase in elasticity at low frequency indicative of weak intermolecular associations. Comparison of the rheological behavior of CF HTBM with our earlier studies of ovine submaxillary mucin lends support to the idea that carbohydrate side-chain interactions are important in the gelation mechanism of mucins. © 1995 John Wiley & Sons, Inc.     

Frequency potentiation in the neonatal rabbit hippocampus

The formation of properties of frequency potentiation in the entorhinal afferent pathway of the hippocampus was studied in unanesthetized rabbits aged from 1 to 15 days. In areas CA₁ and CA₃ of the dorsal hippocampus in newborn rabbits repetitive (1–20 Hz) electrical stimulation of the perforant path led to an increase in amplitude of the slow wave of the field potential by 20–100% compared with the control and to an increase in the probability of response discharges from the neurons from 0–0.5 in the control to 0.8–1.0 during tetanization. In rabbits aged 2–3 days potentiation was more marked at a frequency of 4–6 Hz, whereas depression of the responses developed rapidly to a higher frequency of stimulation. The frequency optimum of 4–15 Hz was established on the 5th day. Potentiation of the first component of the field potential was observed starting from the 8th–10th day of life. The experimental results show that the property of frequency potentiation in the cortical afferent connections of the hippocampus is found in rabbits actually at birth, and it acquires the adult form at the beginning of the second week of life.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 533–539, November–December, 1979.