Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.     

Pulsed-field gel analysis of human immunoglobulin heavy-chain constant region gene deletions reveals the extent of unmapped regions within the locus

The human immunoglobulin heavy-chain constant region gene locus is organized in three main gene groups, the physical distances of which are unknown. Different types of gene deletions, originated by unequal crossingover, have been found to encompass one or more genes in the locus. We have analyzed some of these deletions by means of pulsed-field gel electrophoresis, which allows resolution of large DNA fragments. By identifying a fragment containing two of the main gene groups and by observing the size reduction of this fragment in subjects with deletions, we were able to estimate the distance between the two groups and better locate the pseudogene in-between.     

Role of tumor necrosis factor-alpha in E1A oncogene-induced susceptibility of neoplastic cells to lysis by natural killer cells and activated macrophages

NIH-3T3 cells transfected with adenovirus E1A oncogene cDNA were found to exhibit cytolytic susceptibility to murine NK cells and activated macrophages associated with a threshold level of oncogene product expression exceeding that required for morphological transformation. A similar correlation was observed between threshold levels of E1A gene product expression and target cell susceptibility to direct cytotoxicity by rTNF. Inhibition of splenic NK cell and peritoneal macrophage cytolysis by antisera specific for murine rTNF confirmed the importance of E1A-induced TNF susceptibility as one determinant of target cell cytolytic susceptibility. Anti-TNF antibody was, however, unable to block killing of E1A-expressing targets by the NK cell line, NKB61A2. These results suggest a direct link between the functions of E1A oncogene products and cellular mechanisms of action of TNF elaborated by host effector cells and indicate that E1A expression also affects target cell susceptibility to TNF-independent cytolytic mechanisms.     

Enumeration of anaerobic oxalate-degrading bacteria in the ruminal contents of sheep

Abstract Concentrations of oxalate-degrading anaerobes in ruminal contents of sheep were determined from counts of colonies producing clear zones on a calcium oxalate medium (D agar with 7 mM CaCl₂). Viable counts of oxalate degraders from a 55-kg sheep fed a diet containing 32% halogeton (4.6% oxalate) averaged 2.6 × 10⁶/ g (dry weight). When the halogeton concentration in the diet was reduced to 16%, counts of oxalate degraders decreased nearly 300-fold. Oxalate-degrading isolates from this sheep were similar to OxB, the type strain of Oxalobacter formigenes . When a 45-kg sheep was fed diets containing 2.2, 1.5, and 0.8% oxalate, viable counts of oxalate degraders (enumerated on D agar with 14 mM CaCl₂ and 20% filter-sterilized ruminal fluid) represented 0.85, 0.52, and 0.06% of the total viable population, respectively; total viable counts were essentially unchanges by these concentrations of dietary oxalate. Similar percentages of oxalate degraders were also observed when a 23-kg sheep was fed diets containing 1.5 or 0.8% oxalate. This report presents the first direct measurements of the concentrations of oxalate-degrading bacteria in the rumen and supports the concept that the availability of oxalate in the diet influences the proportion of oxalate-degrading bacteria in the rumen     

Internal desynchronization of circadian rhythms and tolerance of shift work

Fifteen male subjects including 12 shift workers (oil refinery operators) volunteered to document circadian rhythms in sleep-wake, grip strength of both hands, peak expiratory flow, heart rate, self-rated drowsiness, fatigue and attention. Each of these variables was measured 4 to 6 times/day for 2 to 3 weeks. In addition, both axillary temperature (with a shielded probe) and wrist activity were almost continuously recorded at 15 min intervals during the same time span. Individual time series were analyzed according to several statistical methods (power spectrum, cosinor, chi 2, etc.), in order to estimate the prominent circadian period tau and to evaluate both individual and subgroup differences with regard to tolerance of shift work, age, duration of shift work. The present study confirms for continuously recorded temperature and wrist activity, grip strength of both hands, heart rate and peak expiratory flow that intolerance of shift work is frequently associated with an internal desynchronization. However, this conclusion cannot be extended to circadian rhythms in self-rated drowsiness, fatigue and attention. The internal desynchronization among several circadian rhythms supports the hypothesis that these latter are driven by several oscillators, with presumable differences between right and left hemispheres as suggested by unequal values of tau in rhythms of both hand grip strength. Since an internal desynchronization can be observed in tolerant shift workers having no complaint, it is likely that symptoms of intolerance are related to the subject's sensitivity to internal desynchronization rather than to the desynchronization itself.     

Adrenal glucocorticoids regulate adipsin gene expression in genetically obese mice

Adipsin expression at the protein and mRNA levels is greatly reduced in several distinct syndromes of obesity in the mouse: genetic obesity due to the db/db and ob/ob genes, and a chemically induced model secondary to neonatal exposure to monosodium glutamate. We considered first the possibility that the adipsin gene might be identical to the db or ob locus and the lowered expression of this protein might result from a mutation in this gene. We show here that the adipsin structural gene is located on chromosome 10 and hence is physically distinct from any obesity genes so far identified in the mouse. A major role for the adrenal gland and adrenal glucocorticoids in the aberrant regulation of adipsin in these models of obesity is indicated by several experiments. Adrenalectomy of the ob/ob mouse raises the circulating levels of adipsin protein and the amount of this mRNA in epididymal fat pads (5-fold), although neither is increased to the levels seen in lean controls. Exogenous administration of corticosterone completely blocks the effects of adrenalectomy on adipsin, suggesting that the effect of this endocrine ablation is through reduction of adrenal glucocorticoids. Corticosterone administration also causes suppression in the levels of adipsin mRNA and protein in lean mice, although this decrease is never as severe as that seen in obese mice. The effect of exogenous corticosterone in lean mice occurs within 2 days and hence is not secondary to the obesity which these hormones eventually elicit. These results indicate that glucocorticoids can regulate adipsin expression in vivo and strongly suggest that the hyperglucocorticoid state seen in certain obese models plays a significant role in lowering adipsin mRNA and protein levels. Quantitative analysis of these experiments suggests that other as yet unknown neuroendocrine factors also function to suppress adipsin in obesity.     

A 14-kilodalton selenium-binding protein in mouse liver is fatty acid-binding protein

In a previous study, we purified three selenium-binding proteins (molecular masses 56, 14, and 12 kDa) from mouse liver using column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aim of the present study was to determine the amino acid sequence of the 14-kDa protein thereby establishing any relationship with known proteins. Although the amino terminus of the 14-kDa protein was blocked, separate in situ digestions of the protein with endoproteinases Glu-c and Lys-c gave overlapping peptides that provided a continuous sequence of 93 amino acids. This sequence exhibited a 92.5% sequence homology with rat liver fatty acid-binding protein. In situ enzymatic digestion and partial sequencing of a 12-kDa selenium-binding protein revealed identical homology to the 14-kDa protein. The 14-kDa protein bound specifically to an oleate-affinity column from which the protein and 75Se coeluted. Delipidation or sodium dodecyl sulfate treatment failed to remove 75Se from the protein, indicating that the selenium moiety was tightly bound to the protein. These observations confirm that the mouse liver selenium-binding 14-kDa protein is a fatty acid-binding protein. The nature of the selenium linkage to the protein still needs to be explored.