Acceleration of cheese ripening

The characteristic aroma, flavour and texture of cheese develop during ripening of the cheese curd through the action of numerous enzymes derived from the cheese milk, the coagulant, starter and non-starter bacteria. Ripening is a slow and consequently an expensive process that is not fully predictable or controllable. Consequently, there are economic and possibly technological incentives to accelerate ripening. The principal methods by which this may be achieved are: an elevated ripening temperature, modified starters, exogenous enzymes and cheese slurries. The advantages, limitations, technical feasibility and commercial potential of these methods are discussed and compared.     

Multiple peptide synthesis

The synthesis of large numbers of peptides can be very labor intensive and, if a conventional peptide synthesizer is used, only small numbers of peptides can be produced within a reasonable time. The techniques described below can make large numbers of different peptides simultaneously with varying degrees of mechanization, ranging from the wholly manual methods, to those involving complete mechanization of the whole synthesis process. Most of the multiple synthesis methods are primarily intended for small scale production ranging from microgram amounts up to a few tens of milligrams. All of the systems are economical in use of solvents and reagents, enabling cost-effective synthesis. The techniques described can also be used to prepare peptide libraries, containing several millions of peptide sequences, to enable the rapid screening of all possible permutations of amino acids within short peptides. However, it is considered that multiple synthesis methods are not particularly suited where extreme high purity or very long peptides are required.     

Effect of tumor necrosis factor-α and interferon-γ on the growth of a human salivary gland cell line

Interferon-γ (IFN-γ) is a product of activated T-lymphocytes, and tumor necrosis factor-α (TNF-α) is a product of both lymphocytes and macrophages. These cell types are often present at sites of tissue damage secondary to chronic infection or autoimmune disease. The purpose of this study was to characterize the effects of TNF-α and IFN-γ on a human submandibular gland epithelial cell line (HSG). IFN-γ caused a concentration-dependent decrease in HSG cell growth (～70% in 6 days). Conversely, TNF-α alone had little effect on the growth of these cells. When these cytokines were added in combination (20 units/ml TNF-α and 1,000 units/ml of IFN-γ), there was a synergistic antiproliferative effect; no apparent cell growth was observed. The cytokine-induced antiproliferative effect was reversible. After the apparent cessation of cell growth for 3–6 days, removal of the cytokines permitted complete growth recovery. Further, cells that recovered and exhibited growth patterns that were similar to control cells remained susceptible to the antiproliferative effects of the cytokines. Flow cytometry revealed that the percentage of cells in G0/G1 with the combination of cytokines was significantly increased by 24 h. The antiproliferative effect of IFN-γ alone and that of IFN-γ and TNF-α in combination were blocked completely using an antibody to the IFN-γ receptor. A hypothesized mechanism of tissue damage in autoimmune inflammatory disorders is via up-regulation of cell surface markers such as intercellular adhesion molecule type I (ICAM-1) and histocompatibility antigen HLA-DR which can exacerbate the inflammatory process. Treatment of HSG cells with IFN-γ, with or without TNF-α, resulted in increased levels of ICAM-1 and the acquisition of HLA-DR expression. These aggregate data suggest that IFN-γ alone can regulate the expression of cell surface markers involved in the inflammatory process as well as cause a potent yet reversible inhibition of HSG cell growth that is modulated by the presence of TNF-α. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Host-associated fitness variation in a seed beetle (Coleoptera: Bruchidae): evidence for local adaptation to a poor quality host

The geographic distributions of many generalist herbivores differ from those of their host plants, such that they experience coarse-grained spatial variation in natural selection on characters influencing adaptation to host plants. Thus, populations differing in host use are expected to differ in their ability to survive and grow on these host plants. We examine host-associated variation in larval performance (survivorship, development time, and adult body weight) and oviposition preference, within and between two populations ofStator limbatus (Coleoptera: Bruchidae) that differ in the hosts available to them in nature. In one population,Acacia greggii (Fabaceae: Mimosoideae) andCercidium microphyllum (Fabaceae: Caesalpininoideae) are each abundant, while in the second population onlyC. floridum andC. microphyllum are present. In both populations, egg-to-adult survivorship was less than 50% onC. floridum, while survivorship was greater than 90% onA. greggii. Most of the mortality onC. floridum occurred as larvae were burrowing through the seed coat; very low mortality occurred during penetration of the seed coat ofA. greggii. Significant variation was present between populations, and among families (within populations), in survivorship and egg-to-adult development time onC. floridum; beetles restricted toCercidium in nature, without access toC. floridum, survived better and developed faster onC. floridum than beetles that had access toA. greggii. Large host effects on body size were detected for female offspring: females reared onA. greggii were larger than those reared onC. floridum, whereas male offspring wee approximately the same size regardless of rearing host. Trade-offs between performance onC. floridum andC. floridum were not detected in this experiment. Instead, our data indicate that development time and survivorship onC. floridum may be largely independent of development time and survivorship onA. greggii. Patterns of oviposition preference corresponded to the observed patterns of host suitability: in laboratory preference tests, beetles with access toA. greggii in nature tended to prefer this host more than beetles without access to this host in nature.     

Effects of vanadate on the ATP content,ATPase activity and phosphate absorption capacity of maize roots

Although the sensitivity of the plasma membrane H⁺-ATPase to vanadate is well known, the metabolic response of plant cells to vanadate is less well characterised in vivo and its use as an inhibitor in whole plant experiments has had mixed success. Experiments with maize (Zea mays, L.) roots and with purified plasma membrane fractions from the same tissues showed that exposure to vanadate caused: (i) a reduction in the capacity for phosphate uptake; (ii) a reduction in the extractable ATPase activity from the tissue; and (iii) a significant increase in the ATP level. The measurements on the extractable ATPase activity and the ATP level showed that the effect of vanadate developed slowly, apparently reflecting the slow accumulation of intracellular vanadate. The marked effect of vanadate on the ATP level-exposure to 500 M vanadate for 5 h doubled the ATP content of the roots tips-indicates that there is no stringent control over the ATP level in the roots and that the plasma membrane H⁺-ATPase activity is likely to have a significant role in determining the ATP level under normal conditions.     

Interaction of mithramycin with isolated GC and CG sites

We have studied the interaction of the GC-specific, minor groove-binding ligand, mithramycin, with cloned DNA inserts containing isolated GC and CG sites flanked by regions of (AT)_n and A_n · T_n using DNase I and hydroxyl radical footprinting. We find that mithramycin binds to GC better than CG and that AGCT is a better site than TGCA. Sites flanked by (AT)_n appear to be bound better than those surrounded by A_n · T_n. Although no footprints are produced at T₉GCA₉ and T₁₅CGA₁₅, DNase I cleavage is enhanced with the GC sites suggesting that there is some interaction with the ligand. Mithramycin also alters the DNase I cleavage of (GA)_n · (CT)_n.     

Size and sex allocation in monoecious woody plants

The female size advantage hypothesis predicts that the allocation ratio of female: male reproductive effort should increase with plant size (total reproductive effort). A male height advantage hypothesis has also been proposed, based on the supposed greater advantage of height to male reproductive success in wind-pollinated plants. These ideas were tested with data for wind-pollinated, monoecious trees and shrubs which exhibit a suitably large range of sizes. Number of male inflorescences increased faster with size than did number of female inflorescences in 2 of 9 species; in the remaining 7 species there was no significant difference. The male:female ratio of inflorescence numbers increased with height in 4 of 7 species and did not change significantly in the remaining 3 species, as shown by regression. Height and size are highly correlated and so their effects could not be distinguished. The fact that many conifers place the female cones uppermost in the crown suggests that size and not height favors increased allocation to male function, as does well-established theory connecting the existence of male versus female size advantage to pollen and seed dispersal chacteristics. Regression analysis of the relation between male and female reproductive effort should be done by reduced major axis regression; ordinary least squares regression underestimates slopes; in this study opposite conclusions could be drawn from ordinary least squares and reduced major axis regressions.     

Suppression of a defect in the 5'' untranslated leader of mitochondrial COX3 mRNA by a mutation affecting an mRNA-specific translational activator protein.

Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader. To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader. Both deletions blocked translation without affecting mRNA stability. However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences. Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive. We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader. One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader. The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122.     

Auto and transactivation of FGF expression: Potential mechanism for regulation of myogenic differentiation

Summary Fibroblast growth factors (FGFs) are potent inhibitors of myogenic differentiation. The recent observation that the endogenous expression of acidic and basic FGF by myogenic cells decreases coordinately with differentiation suggests a regulatory role for these growth factors in myogenesis. Inasmuch as other proteins known to influence myogenesis (e.g., MyoD1 and myogenin) activate their own expression as well as the expression of other members of their family, we hypothesized that the FGFs might be capable of similar autoregulation. We examined the effect of exogenously supplied FGF on the abundance of the mRNAs encoding acidic and basic FGF in Sol 8 myoblasts, and demonstrate that either acidic or basic FGF stimulate, through paracrine mechanisms, the accumulation of the mRNAs encoding both of these FGFs. Thus FGFs can auto- and transregulate their own expression in a manner analogous to that observed for the myogenic determination proteins. In addition, similar to that previously observed for MyoD1, both acidic and basic FGF suppress myogenin expression in myoblasts. These results suggest two mechanisms whereby endogenously produced FGFs participate in the maintenance of the undifferentiated state of myogenic cells. These data provide support for paracrine, and suggest potential autocrine, roles for FGFs in the regulation of myogenic differentiation.     

AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5′-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.