On the interaction of tritiated 2-ketobutyrate with 2-keto-3-deoxygluconate-6P aldolase ofPseudomonas putida. Evidence suggesting enzyme-mediated,uncatalyzed tritium exchange

2-Keto-3-deoxygluconate-6P aldolase ofPseudomonas putida mediates exchange between hydrogen isotope at the methylene carbon of 2-ketobutyrate and water. This occurs with aK _m of 20 mM, 100 times the corresponding value for pyruvate, and a V_max approximating 1/710 that of KDPG cleavage. Ketobutyrate is competitive with both pyruvate and 2-keto-3-deoxygluconate-6P for the enzyme. In addition, there is no evidence for C-C synthesis between ketobutyrate andd-glyceraldehyde-3P. A comparison of relativeV/K values for hydrogen exchange shows pyruvate to be 17,600 times better as a substrate than ketobutyrate. The detritiation of [3-³H]ketobutyrate is stereochemically random. In addition, the reaction proceeds with ak _H/k _T isotope effect of 15.3, consistent with C-H bond turnover being rate-determining. The E-ketobutyrate complex is reductively trapped, inactivating the enzyme. Reductive inactivation kinetics of E-ketobutyrate compared to E-pyruvate suggests more of the complex may be partitioned to ketimine in the ketobutyrate case than in the pyruvate case. A mechanism is considered in which ketobutyrate is bound as a ketimine in an orientation such that the active site acid/basic group cannot mediate catalytic ketimine/eneamine interconversion. Thus, exchange would result from hydrogen ionization at C-3′ of the ketimine, a slow spontaneous step compared to overall complex turnover. This noncatalyzed deprotonation would explain dissymmetry in exchange, the poorV/K compared to pyruvate, and a large tritium isotope effect.     

The inhibitory effect of ethanol on ethanol production by Zymomonas mobilis

Summary In an effort to establish the reasons for the limitations in the final ethanol concentration of Zymomonas mobilis fermentation, the effects of CO₂ and ethanol on the fermentation were investigated using continuous and fed-batch cultivation systems. The nucleation and stripping out of CO₂ from the fermenter using diatomaceous earth or nitrogen gas or both exhibited a profound effect on the glucose uptake rate during the early stages of fed-batch fermentation, but did not improve final ethanol yields. The addition of ethanol together with above mentioned experiments confirmed conclusively that ethanol inhibition is responsible for the final ethanol concentration obtainable during Zymomonas mobilis fermentation. The final concentration lies between 90 and 110 gl⁻¹ or approximately 12–15% (v/v) ethanol.     

Reduced lipogenesis in cafeteria-fed rats exhibiting diet-induced thermogenesis

Fatty-acid synthesis has been measured in vivo with³H₂O in cafeteria-fed rats exhibiting diet-induced thermogenesis. Synthesis was decreased in brown adipose tissue, the liver, white adipose tissue, and the carcass of the cafeteria-fed animals compared to rats fed the normal stock diet. Whole-body synthesis was also decreased in the cafeteria-fed group. Diet-induced thermogenesis, in contrast to cold-induced non-shivering thermogenesis does not lead to increased fatty-acid synthesis and this is presumably due to the inhibitory effects on lipogenesis of the high dietary fat intake characteristic of cafeteria diets. The results also indicate that the energy cost of body fat deposition in cafeteria-fed rats is lower than in animals fed a low-fat/high-carbohydrate stock diet.     

Wind drift,compensation, and the use of landmarks by nocturnal bird migrants

In this paper we describe fall nocturnal migration at three localities in eastern New York, one adjacent to the Hudson River, the other two 30 km to the west in a topographically more uniform area. Migrants at both study areas moved southwest in winds not out of the west and were, therefore, seemingly unaffected by the river. In west winds, however, birds away from the river moved south-southeast whereas those in the vicinity of the river flew a track west of south paralleling the river. In addition, a relative increase in the number of migrants along the river compared to away was observed in west winds as birds presumably became concentrated near the river. We conclude that on most autumn nights migrants passing through this area have a preferred track direction toward the southwest and in strong winds from the west and northwest they are drifted. Upon reaching the vicinity of the Hudson River, some birds alter their headings yielding a track direction that closely parallels the river resulting in at least a partial compensation for wind drift. No alternative hypothesis is consistent with all the data.     

Postembryonic nongonadal cell lineages of the nematode Panagrellus redivivus: Description and comparison with those of Caenorhabditis elegans

The postembryonic nongonadal cell lineages of the nematode Panagrellus redivivus are described and compared with those of Caenorhabditis elegans. The newly hatched larvae of P. redivivus females and males and C. elegans hermaphrodites and males are very similar. An almost identical set of blast cells divides postembryonically in P. redivivus and C. elegans to produce similar changes in the neuronal, muscular, hypodermal, and digestive systems. Most of these cell lineages are invariant; however, there is substantial variability in the number of cell divisions in the relatively extensive lineages of the lateral hypodermis of P. redivivus. Typically, in P. redivivus females, 55 blast cells generate 635 surviving progeny and 29 cell deaths; in P. redivivus males, 59 blast cells generate 758 surviving progeny and 35 cell deaths. The lineages generating the cells of the male tails of P. redivivus and C. elegans are almost identical; thus, the grossly different characteristics of these structures must reflect differences in the morphogenesis of cells equivalent in lineage history. Laser ablation experiments demonstrate that the gonad induces vulva development and that cell-cell interactions are important in specifying the fates of hypodermal precursor cells. The lateral hypodermal lineages provide striking examples of the apparent construction of complex lineages from modular sublineages; one simple pattern of cell divisions and cell fates occurs 70 times in the P. redivivus female. The differences in cell lineage between P. redivivus and C. elegans are relatively minor, and many appear to have involved two types of evolutionary change: the replacement of sublineages, and the modification of sublineages by the four classes of lineage transformations previously proposed based on a comparison of P. redivivus and C. elegans gonadal cell lineages (Sternberg and Horvitz, 1981). These types of differences suggest that the genetic programming of cell lineage includes instructions specifying where and when a particular sublineage is utilized, and other instructions specifying the nature of that sublineage.     

Adipose conversion of Ob17 preadipocytes : Relationships between cell division and fat cell cluster formation

Adipose conversion of Ob17 preadipocyte cells proceeds after confluence with the formation of fat cell clusters, due to the coexistence of cells susceptible or not to adipose conversion. In order to determine whether commitment to differentiation occurs after quiescence or during exponential growth, the spatial arrangement of Obl7 cells was destroyed at different times before and after confluence by trypsinization followed by cell reinoculation. The resulting distribution of lipid-filled cells, as compared to non-replated control cells, indicates that both insusceptible and susceptible cells are present during the growth phase in the absence of insulin. It is shown that the formation of fat cell clusters of large size is due to mitoses of susceptible cells during a limited period of time after confluence. Blockade of post-confluent mitoses by selective elimination of cells in the S phase abolishes the formation of clusters of large size, but single differentiated cells and clusters containing a few cells remain present. Therefore post-confluent mitoses are not necessary for the differentiation to occur, but rather they serve to amplify the proportion of adipose cells relative to non-adipose cells.     

Replication and amplification of recombinant plasmid molecules as extra chromosomal elements in transformed mammalian cells

Mouse thymidine kinase negative (TK^?) L cells, F4-12B2TK^? Friend erythroleukemic cells and hamster BHKTK^? cells were transformed in the absence of carrier DNA with closed circular molecules of herpes simplex virus 1 TK DNA cloned in plasmid pAT153 (pTK-1). The physical status of donor DNA in the transformed cells was studied by Southern blot hybridization and spot hybridization techniques. Up to 65 copies of pTK-1 DNA molecules/cell were present in transformed cells grown under selective conditions. Whereas a steady increase in the number of pTK-1 copies/cell was found during the first few weeks of growth in selective medium, 2–8 months later copy numbers varied within the same cell line and their numbers rarely exceeded fifty copies/cell. Donor DNA sequences were found both in the Hirt precipitate and in the supernatant showing that some of the pTK-1 molecules existed in circular form. Many of the cell lines gave a Southern blot hybridization pattern indicative of pTK-1 sequences integrated into high molecular weight DNA as well as present in a circular configuration.     

The IL-4 receptor: biochemical characterization of IL-4-binding molecules in a T cell line expressing large numbers of receptors

Cross-linking of 125I-IL-4 to the surface of cells expressing IL-4R yields as the major IL-4-binding molecules, polypeptide chains with inferred m.w. of approximately 70,000 (p70) and approximately 120,000 to 140,000 (p120-p140). The demonstration that the functional product of the IL-4R cDNA clone has m.w. of approximately 140,000 and that no p70 product is detected in transfected COS-7 cells has led to an uncertainty regarding the nature of p70. To study this issue, we examined the relationship of the IL-4-binding molecules p120 and p70 and, in parallel, attempted to immunoprecipitate p70 from surface and internally labeled cells using IL-4 and two anti-IL-4R antibodies (M1 and M2), bound to Affigel 10, as ligands. Cross-linked complexes containing 125I-IL-4 and p70 or p120 were isolated and digested with chymotrypsin or with V8 protease. Three distinct IL-4-binding peptides could be compared; these were indistinguishable for cross-linked p70 and p120, strongly implying that p70 and p120 were structurally related. Furthermore, immunoprecipitates made with IL-4 or anti-IL-4R-Affigel did not contain p70. This led us to conclude that p70 is a breakdown product of p120. A second IL-4-binding molecule of 40,000 Da (p40) expressing the M1 and M2 epitopes of the IL-4R was detected and appears to be the product of an mRNA coding for the soluble form of the receptor. mRNA for p40 was detected in both the T cell line CT.4R and the mast cell line CFTL.12 using polymerase chain reaction primers unique to this species of message. Pulse-chase studies of IL-4R in [35S] methionine-labeled cells indicates that p40 is derived from a 42,000-Da precursor that is detectable at the end of the pulse period, and thus, further argue that p40 is an independently translated molecule and not a degradation product of p120. Although p40 has been previously shown to be a soluble, truncated form of the receptor, we failed to observe secretion of p40 into the medium by internally labeled CT.4R cells.     

A review of the salt sensitivity of the Australian freshwater biota

In Victoria, Australia, both dryland salinity and salinity in irrigation regions are serious agricultural problems. One option to control the latter is to pump groundwater to maintain it below the surface. However, this leaves a saline wastewater for disposal, probably into local streams or wetlands. This review of the salt sensitivity of the biota of Australian streams and wetlands gives information of interest to those responsible for developing controls on these discharges. The review addresses the lethal and sub-lethal effects of salinity on microbes (mainly bacteria), macrophytes and micro-algae, riparian vegetation, invertebrates, fish, amphibians, reptiles, mammals, and birds. Data suggest that direct adverse biological effects are likely to occur in Australian river, stream and wetland ecosystems if salinity is increased to around 1 000 mg L⁻¹. The review highlights a general lack of data on the sensitivity of freshwater plants and animals to salinity increases.