Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c₃ from various Desulfovibrio.Abbreviation ELISA enzyme-linked immunoadsorption assay     

Saccharin induces morphological changes and enhances prolactin production in GH4C1 cells

Summary The artificial sweetener saccharin inhibits binding of epidermal growth factor (EGF) to cultured rat pituitary tumor cells (GH₄C₁ cells). Saccharin also causes morphological alterations in these cells, resulting in pronounced elongation, stretching, and firmer attachment of cells to the culture dishes. These alterations in cell shape are similar to those observed after treatment of GH₄C₁ cells with EGF and with thyrotropin-releasing hormone (TRH), both of which enhance prolactin (PRL) production in these cells. After assaying for PRL in saccharin-treated cultures, it was observed that this sweetener is also capable of stimulating PRL production two-to sixfold in a dose-dependent manner. Enhancement of PRL production can be observed at 0.5 mM saccharin, yet this is 10 times less than the saccharin concentration required to alter cell shape. These effects of saccharin on cell morphology and on PRL production are reversible in GH₄C₁ cell cultures. When added to cultures along with maximal concentrations of EGF or TRH, the effects of saccharin on PRL production are additive, suggesting that the actions of saccharin are mediated by a somewhat different pathway from that of the peptide hormones. Pulse labeling studies indicate that the enhancement of PRL production is highly specific inasmuch as saccharin was found to decrease the overall rate of protein synthesis in these cells. Saccharin also causes a decrease in the rate of DNA synthesis under these treatment conditions. Mitomycin C, which similarly inhibited DNA synthesis, had no effect on cell morphology or PRL production. This investigation was supported by a Faculty Research Grant from Wheaton College     

α-Ketoglutarate and Malate Uptake and Metabolism by Synaptosomes: Further Evidence for an Astrocyte-to-Neuron Metabolic Shuttle

This study was undertaken to provide further evidence relevant to the hypothesis that astrocytes supply one or more citric acid cycle intermediates to synaptic terminals, thereby serving an anaplerotic function necessitated by the synthesis and release of amino acid neurotransmitters. In our experiments, two populations of synaptosomes obtained from the brain of rats were separated from myelin and mitochondria by using Percoll to generate continuous density gradients. Both synaptosomal populations readily accumulated 14C-labelled alpha-ketoglutarate and L-malate by high-affinity transport systems. Hofstee plots of uptake velocity as a function of substrate concentration were highly nonlinear, indicating that uptake was mediated by two or more carriers, or was subject to negative cooperativity. At least one carrier was selective for alpha-ketoglutarate and another for malate, whereas a third carrier appeared to be present which transported both substrates. At low concentrations (approximately 1 microM), alpha-ketoglutarate transport was almost totally Na+-dependent, whereas malate uptake exhibited little Na+-dependency. The transport of alpha-ketoglutarate was associated with a net influx, and therefore was not due to a homoexchange process. alpha-Ketoglutarate and malate were metabolized rapidly to glutamate and aspartate, respectively, by both synaptosomal preparations; however, in all cases, label accumulated in gamma-aminobutyric acid rather slowly. The incorporation of label into glutamine from alpha-ketoglutarate was much greater in the high-density synaptosomes that in low-density synaptosomes, an indication that the former contained a higher proportion of astrogliasomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further studies on the involvement of the circadian system in photoperiodic control of antifreeze protein production in the beetle Dendroides canadensis

The role of circadian rhythmicity in the photoperiodic time measuring processes regulating antifreeze protein production in the beetle Dendroides canadensis was further investigated. Using “T” experiments larvae were exposed to environmental light cycle periods close to the period length of the endogenous circadian oscillator. The following light cycles were employed: light/dark 8/13, 8/14, 8/16, 8/18 and 8/19 corresponding to period lengths of 21, 22, 24, 26 and 27 h. Larvae maintained in cycles equal to or less than 24 h displayed a characteristic short-day response, showing significantly (P < 0.01) greater antifreeze protein activity than did those measured on the day of collection in late summer. In contrast, a long-day response was observed in larvae maintained under a 26- or 27-h light cycle in that antifreeze protein activity did not differ from that measured on the initial collection date.

The role of photoperiod and temperature in influencing the photoperiodic timing processes were examined with a series of resonance experiments. The first group consisted of a 24, 36, 48, 60 or 72-h light cycle, each with an 8-h photophase at temperatures of 20 or 17°C. Rhythmic increases in antifreeze protein levels at intervals of 24 h occurred under both temperatures. However, the lower temperature displaced the resonance curve in the vertical direction (i.e. increasing % population response) and reduced the difference between peaks and troughs on the resonance curve. Resonance experiments incorporating a 14-h photophase resulted in low antifreeze protein activity under all conditions except a 36-h light cycle in which a 67% induction was observed.

Eight hour resonance experiments were also conducted with D. canadensis collected in early spring to determine whether the circadian system participates in the photoperiodic timing processes influencing the spring termination of antifreeze protein production. Positive resonance results were obtained in that only larvae maintained in cycles of 36 and 60 h displayed significantly (P < 0.01) lower antifreeze activity when compared to animals on the initial collection date.

The combined results emphasize the involvement of the circadian system in the photoperiodic control of antifreeze protein production by D. canadensis during the fall and spring. Furthermore, the induction of antifreeze protein production is a function of light cycle and its waveform (photoperiod). Temperature appears to modify the photoperiodic response in some manner involving the photoperiodic time measuring processes. It is concluded that the photoperiodic response of antifreeze protein production by D. canadensis is dependent upon the entrainment of the circadian system by the light cycle.     

A tandemly reiterated DNA sequence in the long repeat region of herpes simplex virus type 1 found in close proximity to immediate-early mRNA 1.

The 3' end of immediate-early mRNA 1 was mapped precisely within the IRL/TRL genome regions, and the DNA sequences around the 3' end were determined. An AATAAA polyadenylation signal was present 17 base pairs upstream of the 3' end, and eight tandemly repeated copies of a 16-base-pair sequence (GGGGGTGCGTGGGAGT) plus one further closely related copy were located 20 base pairs downstream. Other tandem reiterations present in the herpes simplex virus genome are described and their properties are considered.     

Intracellular Ca and cell injury: A paradoxical role of Ca in complement membrane attack

Disturbances in intracellular Ca2+ are known to be important in cell injury caused by a wide range of toxic factors. The complement system is a major effector of immune damage in vivo, and is known to be involved in the pathogenesis of many immune diseases. We present here evidence that the potentially lethal membrane attack complex of complement causes a rapid increase in intracellular free Ca2+ concentration before any other detectable biochemical changes in the cell. In nucleated cells the increased intracellular free Ca2+ concentration initially stimulates recovery processes, allowing the cell to escape mild complement attack and also activates the production of inflammatory mediators, which may amplify an ongoing inflammatory response. More severe complement membrane attack causes a more rapid rise in intracellular free Ca2+ concentration allowing a threshold to be breached above which recovery processes are overwhelmed, and cell death occurs. The importance of non-lytic effects and recovery processes mediated by Ca2+, and the molecular basis of these effects are discussed, and the hypothesis proposed that the cell-injuring effects of other "pore-forming" toxins are also caused by increases in intracellular free Ca2+.     

Column cellulose hydrolysis reactor: An efficient cellulose hydrolysis reactor with continuous cellulase recycling

Summary The use of a column cellulose hydrolysis reactor with continuous enzyme recycling was demonstrated by incorporating a continuous ultrafiltration apparatus at the effluent end of the column reactor. Using this setup, over 90% (w/v) cellulose hydrolysis was achieved, resulting in an average sugar concentration of 6.8% (w/v) in the effluent stream. The output of the system was 1.98 g of reducing sugar/l/h with a ratio of 87% (w/v) of the reducing sugars being monomeric sugars. Batch hydrolysis reactors were less effective, resulting in 57% (w/v) of the cellulose being hydrolyzed. The output of the batch reactor was 1.33 g of reducing sugar/l/h with similar product concentrations and percentage of monomeric sugars. The ratio of reducing sugar/filter paper unit of cellulase activity for the column method was 69.1 mg/U as compared to only 21.2 mg/U for the batch reactor.     

Structural basis of the polymorphism of human complement components C4A and C4B: gene size, reactivity and antigenicity.

The human complement components C4A and C4B are highly homologous proteins, but they show markedly different, class-specific, chemical reactivities. They also differ serologically in that C4A generally expresses the Rodgers (Rg) blood group antigens while C4B generally expresses the Chido (Ch) blood group antigens. C4A 1 and C4B 5 are exceptional variants which possess their class-specific chemical reactivities, but express essentially the reversed antigenicities. The genes encoding the typical Rg-positive C4A 3a and Ch-positive C4B 3 allotypes and the interesting variants C4A 1 and C4B 5 have been cloned. Characterization of the cloned DNA has revealed that the genes encoding the A 3a, A 1 and B 3 allotypes are 22 kb long, but that encoding B 5 is only 16 kb long. Comparison of derived amino acid sequences of the polymorphic C4d fragment has shown that C4A and C4B can be defined by only four isotypic amino acid differences at position 1101-1106. Over this region C4A has the sequence PCPVLD while C4B has the sequence LSPVIH, and this presumably is the cause of their different chemical reactivities. Moreover, the probable locations of the two Rg and the six Ch antigenic determinants have been deduced. Our structural data on the C4A and C4B polymorphism pattern suggests a gene conversion-like mechanism is operating in mixing the generally discrete serological phenotypes between C4A and C4B.