Transforming growth factor a (TGFa) shares with epidermal growth factor (EGF) structural homology (35%), a common cell-surface membrane receptor (TGFa/EGF receptor), and a nearly identical spectrum of biological activity, including inhibition of gastric acid secretion. Herein, we report expression of TGFa mRNA in normal gastric mucosa of the adult guinea pig, rat, and dog. TGFa mRNA was also detected in matched surgically resected gastric mucosa and adjacent gastric carcinoma from 10 patients, and in gastric mucosa adjacent to a benign ulcer from an additional patient. TGFa protein was quantitated by radioimmunoassay and was present in tumor and adjacent mucosa. TGFa/EGF receptor mRNA was also detected in gastric mucosa from all species studied. Localization of TGFa and TGFa/EGF receptor mRNA expression was examined in samples of unfractionated guinea pig gastric mucosa and from chief cell-enriched and parietal cell-enriched fractions. All samples exhibited TGFa and TGFa/EGF receptor expression. The TGFa signal was greatest in the parietal cell fraction (5.8-fold increase), but was also enhanced in the chief cell fraction (1.9-fold increase) relative to the unfractionated gastric mucosa. Like TGFa expression, TGFa/EGF receptor mRNA expression was most intense in the parietal cellenriched fraction (7.8-fold increase), but was also increased in the chief cell-enriched fraction (2.7-fold increase) relative to the unfractionated guinea pig gastric mucosa. We conclude that TGFa and TGFa/EGF receptor genes are expressed in normal adult mammalian gastric mucosa. These findings, when interpreted in light of described actions of TGFa and EGF, provide evidence that local production of TGFa could play an important role in the regulation of acid secretion and mucosal renewal in the stomach.

Thermostable and thermoactive ␤-mannanase (1,4-␤-D-mannan mannanohydrolase [EC 3.2.1.78]), ␤-mannosidase (␤-D-mannopyranoside hydrolase [EC 3.2.1.25]), and ␣-galactosidase (␣-D-galactoside galactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media. The ␤-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92؇C, with half-lives (t 1/2) of 34 h at 85؇C, 13 h at 90؇C, and 35 min at 100؇C. The ␤-mannosidase and ␣-galactosidase were found primarily in cell extracts. The ␤-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87؇C, with t 1/2 of 18 h at 85؇C, 42 min at 90؇C, and 2 min at 98؇C. The ␣-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103؇C and t 1/2 of 9 h at 85؇C, 2 h at 90؇C, and 3 min at 100؇C. These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T. neapolitana for nutritional purposes. The significance of such substrates in geothermal environments remains to be seen.

An α‐galactosidase and a β‐mannanase produced by the hyperthermophilic bacterium, Thermotoga neapolitana 5068 (TN5068), separately and together, were evaluated for their ability to hydrolyze guar gum in relation to viscosity reduction of guar‐based hydraulic fracturing fluids used in oil and gas well stimulation. In such applications, premature guar gum hydrolysis at lower temperatures before the fracturing process is completed is undesirable, whereas thermostability and thermoactivity are advantageous. Hyperthermophilic enzymes presumably possess both characteristics. The purified α‐galactosidase was found to have a temperature optimum of 100–105°C with a half‐life of 130 minutes at 90°C and 3 min at 100°C, while the purified β‐mannanase was found to have a temperature optimum of 91°C and a half‐life of 13h at this temperature and 35 min at 100°C. These represent the most thermostable versions of these enzymes yet reported. At 25°C, TN5068 culture supernatants, containing the two enzyme activities, reduced viscosity of a 0.7% (wt) guar gum solution by a factor of 1.4 after a 1.5‐h incubation period and by a factor of 2.4 after 5 h. This is in contrast to a viscosity reduction of 100‐fold after 1.5 h and 375‐fold after 5 h for a commercial preparation of these enzymes from Aspergillus niger. In contrast, at 85°C, the TN5068 enzymes reduced viscosity by 30‐fold after 1.5 h and 100‐fold after 5 h compared to a 2.5‐fold reduction after 5 h for the control. The A. niger enzymes were less effective at 85°C (1.6‐fold reduction after 1.5 h and a 4.2‐fold reduction after 5 h), presumably due to their thermal lability at this temperature. Furthermore, it was determined that the purified β‐mannanase alone can substantially reduce viscosity of guar solutions, while the α‐galactosidase alone had limited viscosity reduction activity. However, the α‐galactosidase appeared to minimize residual particulate matter when used in conjunction with the β‐mannanase. This could be the result of extensive hydrolysis of the α‐1,6 linkages between mannose and galactose units in guar, allowing more extensive hydrolysis of the mannan chain by the β‐mannanase. The use of thermostable enzymatic breakers from hyperthermophiles in hydraulic fracturing could be used to improve well stimulation and oil and gas recovery. © 1996 John Wiley & Sons, Inc.

Transforming growth factor a and 01 (TGFa and TGF 01)

The binding of nerve growth factor (NGF) to specific cell surface receptors initiates a variety of effects that lead to the morphological and biochemical differentiation of clonal pheochromocytoma, PC12, cells. The lectin wheat germ agglutinin (WGA) alters the characteristics of NGF-receptor interaction. We have found that treatment of PC12 cells with WGA dramatically and reversibly inhibits the ability of NGF to elicit three distinct biological effects characteristic of NGF action. Two of these events, the rapid ruffling of cell-surface membranes and the stimulation of the phosphorylation of a 250-kD cytoskeletal protein in situ, occur rapidly and are an immediate consequence of receptor occupancy. Both of these effects are blocked by pretreatment of the cells with WGA. WGA was also found to inhibit the NGFstimulated regeneration of neurites that occurs over 1-2 d. Both the WGA inhibition of neurite outgrowth and the phosphorylation of the 250-kD cytoskeletal protein were reversed upon addition of the specific sugar N-acetylglucosamine. These data demonstrate that the WGAinduced changes in the NGF-receptor interaction reflect important alterations in the ability of the receptor to transmit biological signals, resulting in the abrogation of the biological effects of NGF on these cells.