Stimulus coding in crayfish antennal mechanoreceptors estimated by noise analysis

Two different kinds of mechanoreceptive hairs (smooth and feathered) on the second antennae of the freshwater crayfish, Orconectes virilis, have been investigated for their stimulus coding propertics. These mechanoreceptors show a great deal of non-linear behaviour both in threshold and in directionality. An effective appraoch for the investigation of such systems is noise analysis in the frequency domain. This method has been used here to calculate zero-, first- and second-order kernels. Sensory cells reveal different first- and second-order kernels, depending on which type of hair is being stimulated. The first-order kernel has a pronounced peak in the frequency response at 110 Hz if a feathered hair is stimulated and at 60 Hz if a smooth hair is stimulated. The second-order kernel shows a number of pronounced peaks in the frequency response between 40 and 110 Hz, but only if a feathered hair is stimulated. Smooth hair stimulation results in less sharp peaks but in higher gain for the same range of stimulus frequencies.     

Alterations in protein kinase activity following exposure of cultured human lymphocytes to modulated microwave fields

Cultures of human tonsil lymphocytes were exposed in a Crawford cell to a 450-MHz field (peak envelope intensity 1.0 mW/cm2), sinusoidally amplitude modulated (depth 80%) at frequencies between 3 and 100 Hz for periods up to 60 min. The Crawford cell was housed in a temperature-controlled chamber (35 degrees C) and control cultures were placed in the same chamber. Activity of cAMP-dependent protein kinase relative to controls remained unaltered by fields modulated at 16 or 60 Hz with exposures of 15, 30, and 60 min. By contrast, total non-cAMP-dependent kinase activity fell to less than 50% of unexposed control levels after 15 and 30 min exposures, but, despite continuing field exposure, returned to control or preexposure levels by 45 and 60 min. A smaller reduction (20-25%) also occurred with 60-Hz modulation and was also restricted to exposure durations of 15 and 30 min. CW 450-MHz fields were without effect. Reduced enzyme activity occurred with 16-, 40-, and 60-Hz modulation frequencies, but not with 3-, 6-, 80-, or 100-Hz modulation. The specific identity of this kinase is unknown. This rapid but transient reduction in lymphocyte protein kinase activity restricted to modulation frequencies between 16 and 60 Hz and to less than 30 min exposure is consistent with "windowing" with respect to modulation frequency and exposure duration.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

Comparative use of fructose and glucose in human liver and fibroblastic cell cultures

Summary The effect of fructose as a substitute for glucose in cell culture media was investigated in human skin fibroblast and liver cell cultures. Cells were grown for between 2 and 10 days in identical flasks in four different media, containing 5.5, mmol·1⁻¹ and 27.5 mmol·I⁻¹ glucose and fructose, respectively. In the presence of fructose, cell growth was stimulated, but less in liver cells than fibroblasts. At Day 6, increases were observed in [³H]thymidine incorporation, protein levels, and amino acid consumption, and a reduction was noted in ATP levels. In media containing 5.5, mmol·1⁻¹ glucose or fructose, consumption of fructose was four times lower than that of glucose at Day 3 and did not rise until Day 6. In fructose media, the lactate production was very low (four to five times less than that of glucose) and the pH values were always higher. Some findings were different for the fibroblasts and liver cells, owing to the specific characteristics of these two cell types in culture; this applied especially to the effects of glucose and fructose concentrations of 27.5 mmol·1⁻¹. Several possible explanation for the stimulation of cell growth in fructose medium were discussed. This work was supported by grants for the Institut National de la Santé et de la Recherche Médicale (ATP 82-79-114) and the Unité d'Enseignement et de Recherche, Le Kremlin-Bicêtre, Université Paris-Sud (C. R. 848).     

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture

Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm²) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to platic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium. Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared in Clin. Res. 30: 93A; 1982.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

Growth characteristics,morphology, and phospholipid composition of human type II pulmonary alveolar cells grown in a collagen-free microenvironment

Summary Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro. This work was supported in part by grants from EPA, R 806638-01 and 131-640-1599A1     

Cysteine: a potential source of error in amino acid analysis of mercaptoethane sulfonic or hydrochloric acid hydrolysates of proteins and peptides

Hydrolysis of proteins and peptides with mercaptoethane sulfonic acid is liable to produce overestimation of the proline content owing to the production of ninhydrin-positive material (probably cysteine) which coelutes with proline on many ion-exchange analytical systems. A similar error occurs with HCl hydrolysis (especially in the presence of mercaptoethanol or thioglycollic acid) if care is not taken to oxidize cysteine during reconstitution of the hydrolysate before amino acid analysis.     

Determination of choline in erythrocytes using high-resolution proton nuclear magnetic resonance spectroscopy: Comparison with a choline oxidase method

Detailed operating conditions are reported for the determination of choline in human erythrocytes using proton nuclear magnetic resonance spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence. The results of the NMR method were in excellent agreement with those obtained using an enzymatic (choline oxidase) assay; however, they were approximately three times higher than those reported using gas chromatography/mass spectrometry techniques. The differences may be partly due to the method of preparing or sampling cells since there is a distribution of choline in cells of different ages. However, choline levels were not affected by the methods used in the present study for storing or preparing cells.     

Family resemblance for neuromuscular performance in a Kansas Mennonite community

Familial phenotypic resemblance for six quantitative neuromuscular performance traits is analyzed by path analysis using data from the Mennonite community of Goessel, Kansas. Of the six traits only one, dominant hand strength, shows no evidence of parent-offspring transmission (t2 = 0.001) and only one, trunk flexibility, shows evidence of a high degree of transmissibility (t2 = 0.662). The four remaining traits display low to moderate levels of transmissibility (t2 = 0.073 to t2 = 0.245). A substantial residual sibling resemblance due to shared environmental effects is seen for all six traits. It is suggested that the high heritabilities found for many of these traits by other methods result from the inability of these methods to account for the shared nongenetic effects.