Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a vermicompost and leachates on <i>Solidago x hybrida</i> and organic carbon mineralization under aerobic incubations

In the floriculture region of Tenancingo in the State of Mexico, the application of stabilized organic matter, such as vermicompost and leachates, contributes to improve the quality of the soil and plant nutrition. However, it is important to know the chemical composition of a vermicompost and the mineralization process. This is because the amount and speed of nutrient release which will be available to the crop will depend on that knowledge. The objective of the study was to evaluate the effect of the application of a vermicompost and leachates on various quantitative variables of Solidago x hybrid, and the mineralization of organic carbon under aerobic incubations. Vermicompost (74 and 36 g/kg soil), leachates (5 and 10 L/kg soil), 0.33 g/kg soil of chemical fertilizer Ca (NO₃)₂, and not treated soil (control) were applied under greenhouse conditions to evaluate their effects on plant growth variables. Mixtures of 100 g of soil with vermicompost and leachates were made in the laboratory which were incubated during 9 weeks to obtain the potentially mineralizable organic carbon (Corg PM) and the rate of mineralization (k) after adjusting an exponential model. In the greenhouse experiment there were no statistical differences after applying vermicompost and leachates on the quantitative variables (number of stems per plant, diameter of the panicle, fresh weight, plant length and stem diameter) with respect to the control (p>0.05). The effect among the applied doses was evident only for variables such as fresh weight, panicle length and stem diameter with respect to the control. In incubated soils, k values ranged between 0.209-0.325 C mg/kg soil/week. Only with the application of leachates in high doses two pools of organic matter were shown: one soluble labile (102.9 mg/kg soil) and the other hydrolysable (819 mg/kg soil). The soluble, labile fraction favored nutrient availability immediately after its application to the soil. However, a single pool of hydrolysable organic C (987-1074 mg/kg soil) was found when vermicompost was applied. It was associated with a release of organic matter during crop development, and to a possible stimulation of microbial activity. High values of electrical conductivity in vermicompost and leachates (8.2-11.7 mS/m) suggest a moderate application of both products.     

Secretory IgA in Urinary Tract Infections

Secretory IgA, measured by radial immunodiffusion, was compared in the urine of children with chronic and recurrent non-obstructive urinary tract infections with that in normal children. IgA, IgG, and IgM were also measured. Absent and low levels of IgA(s) were found in both groups; however, the mean levels of IgA(s) were significantly higher in the infected group compared with normals—3·3 to 0·78 mg./24 hours, respectively. Secretory IgA was found to be locally produced in the bladder. It is suggested that IgA(s) levels reflect an antibody response to infection.     

Effects of caffeine on crayfish muscle fibers. II. Refractoriness and factors influencing recovery (repriming) of contractile responses

When caffeine evokes a contraction, and only then, crayfish muscle fibers become refractory to a second challenge with caffeine for up to 20 min in the standard saline (5 mM K_o). However, the fibers still respond with contraction to an increase in K_o, though with diminished tension. Addition of Mn slows recovery, but the latter is greatly accelerated during exposure of the fiber to high K_o, or after a brief challenge with high K_o. Neither the depolarization induced by the K, nor the repolarization after its removal accounts for the acceleration, which occurs only if the challenge with K had itself activated the contractile system; acceleration is blocked when contractile responses to K are blocked by reducing the Ca in the bath or by adding Mn. Recovery is accelerated by redistribution of intracellular Cl and by trains of intracellularly applied depolarizing pulses, but not by hyperpolarization. The findings indicate that two sources of Ca can be mobilized to activate the contractile system. Caffeine mobilizes principally the Ca store of the SR. Depolarizations that are induced by high K_o, by transient efflux of Cl, or by intracellularly applied currents mobilize another source of Ca which is strongly dependent upon the entry of Ca from the bathing medium. The sequestering mechanism of the SR apparently can utilize this second source of Ca to replenish its own store so as to accelerate recovery of responsiveness to a new challenge with caffeine.     

Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV

The present paper demonstrates the terminal de- and reglycosylation of a rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP IV). Cultured hepatocytes were used in pulse-chase experiments with [3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal carbohydrates, [3H]D-mannose as marker of a core-sugar, and [35S]L-methionine for labeling the protein backbone. Membrane DPP IV was immunoprecipitated with a polyclonal antibody which bound selectively at 4 degrees C to the cell-surface glycoprotein. The times of maximal labeling of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min for [3H]D-mannose, and 25 min for [35S]L-methionine. When antibodies were bound to cell-surface DPP IV at 4 degrees C, the immune complex remained stable for more than 1 h after rewarming to 37 degrees C, despite ongoing metabolic and membrane transport processes. This was shown by pulse labeling with [35S]L-methionine at 37 degrees C, followed by cooling to 4 degrees C, and addition of antibody against plasma membrane DPP IV. During rewarming, the radioactivity in the complex remained constant. In a similar experiment with [3H]L-fucose, the radioactivity in the immune complex declined rapidly, indicating a defucosylation of the plasma membrane glycoprotein. Using the same experimental design with [3H]D-mannose, the radioactivity in the immune complex remained constant, showing that the core-sugar D-mannose is not cleaved from the membrane glycoprotein. Terminal reglycosylation (refucosylation and resialylation) was demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a medium supplemented with tunicamycin in order to block the de novo synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the antibody against DPP IV bound only to cell surface glycoprotein. During the rewarming period at 37 degrees C, radioactivity from [3H]L-fucose and [14C]N-acetyl-D-mannosamine became incorporated into the immune complex. This indicates a fucosylation and sialylation of the glycoprotein originally present at the cell surface. The mechanisms whereby terminal de- and reglycosylation of plasma membrane glycoproteins may occur during membrane recycling are discussed.     

Circadian rhythms in juvenile american shad, Alosa sapidissima

Swimming activity (in cm s⁻¹) of a school (55 individuals) of young-of-the-year ( total length=110 mm) American shad, Alosa sapidissima , was determined under a variety of photoperiod conditions. These included a normal (ambient), a shifted, and constant-light day. Swimming activity was measured over 4-day periods. During normal days swimming speeds followed periods of about 24 h, with fast speeds (up to 45 cm s⁻¹) and schooling occurring during the photoperiod. Under dark conditions speeds were slower (8 cm s⁻¹) with most fish swimming as individuals. During a shifted day swimming speeds and schooling corresponded to the imposed day. Under constant light (equivalent to bright moonlight) no schooling was evident, and a constant, but slow, swimming speed was observed in each 24-h period. These shad demonstrated an exogenous rhythm with respect to the imposed day length. It is hypothesized that an endogenous circadian rhythm would only be of use to a fish required to hunt or chase its prey. Shad, being plankton feeders, do not chase prey and therefore can exhibit an exogenous circadian rhythm with no detrimental feeding results.     

The effects of corticosteroids on immunoregulation in sarcoidosis.

The effects of in vivo hydrocortisone administration on the kinetics and functional capabilities of cells involved in the immune response in sarcoidosis were examined. Untreated sarcoidosis patients have a decrease in the absolute numbers of circulating T lymphocytes (P < 0.05). However, with regard to the proportions of T lymphocyte subpopulations, there is an increase in the relative proportions of IgG Fc receptor positive T cells (T_G) (P < 0.01), which have suppressor capabilities in certain in vitro systems of mitogen-induced antibody production, and a relative decrease in IgM Fc receptor positive T lymphocytes (T^M) which have helper effects in this system (P < 0.05). Additionally, sarcoidosis patients have circulating “suppressor” monocytes capable of suppressing anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) responses by pokeweed mitogen (PWM)-stimulated lymphocytes. The in vitro removal of this cell abrogated this depressed response (P < 0.01). Intravenous administration of hydrocortisone produced a transient absolute T lymphocytopenia (P < 0.01) accompanied by a relative increase in T^G cells (P < 0.01) and a relative decrease in T_M cells (P < 0.02). Four hours after hydrocortisone therapy, at the point of maximal hydrocortisone-induced monocytopenia (P < 0.01), the suppressed ability of sarcoidosis lymphocytes to synthesize and secrete in vitro anti-SRBC antibody after polyclonal activation was corrected (P < 0.01), and PFC responses comparable to those seen in untreated normal subjects were obtained. These studies demonstrate that corticosteroid administration has profound effects on certain in vitro demonstrable immunoregulatory abnormalities in sarcoidosis.