Suppressor analysis of the Saccharomyces cerevisiae gene REC104 reveals a genetic interaction with REC102

REC104 is a gene required for the initiation of meiotic recombination in Saccharomyces cerevisiae. To better understand the role of REC104 in meiosis, we used an in vitro mutagenesis technique to create a set of temperature-conditional mutations in REC104 and used one ts allele (rec104-8) in a screen for high-copy suppressors. An increased dosage of the early exchange gene REC102 was found to suppress the conditional recombinational reduction in rec104-8 as well as in several other conditional rec104 alleles. However, no suppression was observed for a null allele of REC104, indicating that the suppression by REC102 is not "bypass" suppression. Overexpression of the early meiotic genes REC114, RAD50, HOP1, and RED1 fails to suppress any of the rec104 conditional alleles, indicating that the suppression might be specific to REC102.     

Biochemical characterization of a dihydromethanopterin reductase involved in tetrahydromethanopterin biosynthesis in Methylobacterium extorquens AM1

During growth on one-carbon (C1) compounds, the aerobic alpha-proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H4MPT) derivative dephospho-H4MPT as a C1 carrier in addition to tetrahydrofolate. The enzymes involved in dephospho-H4MPT biosynthesis have not been identified in bacteria. In archaea, the final step in the proposed pathway of H4MPT biosynthesis is the reduction of dihydromethanopterin (H2MPT) to H4MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase. A gene encoding a dihydrofolate reductase homolog has previously been reported for M. extorquens and assigned as the putative H2MPT reductase gene (dmrA). In the present work, we describe the biochemical characterization of H2MPT reductase (DmrA), which is encoded by dmrA. The gene was expressed with a six-histidine tag in Escherichia coli, and the recombinant protein was purified by nickel affinity chromatography and gel filtration. Purified DmrA catalyzed the NAD(P)H-dependent reduction of H2MPT with a specific activity of 2.8 micromol of NADPH oxidized per min per mg of protein at 30 degrees C and pH 5.3. Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8. While the existence of an H2MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea. Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase. This may be a consequence of different electron donors, NAD(P)H versus reduced F420, used, respectively, in bacteria and methanogenic archaea.     

Adaptation level effects in discrimination of flicker frequency

Pigeons accustomed to food reinforcement for responding in the presence of a 25-Hz flickering light were exposed to several sets of flicker-frequency stimuli arranged as increasing and decreasing series. In the first experiment, food was occasionally delivered for key pecks during 30-s periods of 25-Hz flicker appearing at the beginning, midway, and at the end of an ascending and descending series of nine frequencies, ranging from 13 to 37 Hz. These stimuli appeared for 15-s periods with no food available (extinction). Gradients of responding to flicker values in the ascending series differed from those in the descending series, showing displacements in peak responding toward the lower and higher frequency values, respectively. The same effects occurred when the sequence was changed so that a descending series was followed by an ascending series of frequencies. These effects are consonant with an adaptation level (AL) interpretation and were replicated in a second experiment in which durations of the extinction presentations were increased to 30s. In a final condition, only a descending series was presented and displacement of peak responding from 25 Hz to a higher frequency stimulus, 28 Hz, was observed.     

Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173-Asp174, Ser180-Asp181, Ser217-Asp218, and Gln221-Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.     

The effects of 8 weeks of creatine monohydrate and glutamine supplementation on body composition and performance measures

Twenty-nine (17 men, 12 women) collegiate track and field athletes were randomly divided into a creatine monohydrate (CM, n = 10) group, creatine monohydrate and glutamine (CG, n = 10) group, or placebo (P, n = 9) group. The CM group received 0.3 g creatine.kg body mass per day for 1 week, followed by 0.03 g creatine.kg body mass per day for 7 weeks. The CG group received the same creatine dosage scheme as the CM group plus 4 g glutamine.day(-1). All 3 treatment groups participated in an identical periodized strength and conditioning program during preseason training. Body composition, vertical jump, and cycle performances were tested before (T1) and after (T2) the 8-week supplementation period. Body mass and lean body mass (LBM) increased at a greater rate for the CM and CG groups, compared with the P treatment. Additionally, the CM and CG groups exhibited significantly greater improvement in initial rate of power production, compared with the placebo treatment. These results suggest CM and CG significantly increase body mass, LBM, and initial rate of power production during multiple cycle ergometer bouts.     

Effects of high pressure on the viability,morphology, lysis,and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris

Viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris MG1363 and SK11 were determined after exposure to pressure. Both strains were completely inactivated at pressures of 400 to 800 MPa but unaffected at 100 and 200 MPa. At 300 MPa, the MG1363 and SK11 populations decreased by 7.3 and 2.5 log cycles, respectively. Transmission electron microscopy indicated that pressure caused intracellular and cell envelope damage. Pressure-treated MG1363 cell suspensions lysed more rapidly over time than did non-pressure-treated controls. Twenty-four hours after pressure treatment, the percent lysis ranged from 13.0 (0.1 MPa) to 43.3 (300 MPa). Analysis of the MG1363 supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed pressure-induced lysis. Pressure did not induce lysis or membrane permeability of SK11. Renaturing SDS-PAGE (zymogram analysis) revealed two hydrolytic bands from MG1363 cell extracts treated at all pressures (0.1 to 800 MPa). Measuring the reducing sugars released during enzymatic cell wall breakdown provided a quantitative, nondenaturing assay of cell wall hydrolase activity. Cells treated at 100 MPa released significantly more reducing sugar than other samples, including the non-pressure-treated control, indicating that pressure can activate cell wall hydrolase activity or increase cell wall accessibility to the enzyme. The cell suspensions treated at 200 and 300 MPa did not differ significantly from the control, whereas cells treated at pressures greater than 400 MPa displayed reduced cell wall hydrolase activity. These data suggest that high pressure can cause inactivation, physical damage, and lysis in L. lactis. Pressure-induced lysis is strain dependent and not solely dependent upon cell wall hydrolase activity.     

Relationship between leaf area index and yield in double-crop and full-season soybean systems

Previous research indicates a correlation between leaf area index (LAI) and yield of full-season soybean [Glycine max (L.) Merrill], which is a single crop planted early in the season. Leaf area index values of at least 3.5-4.0 in the reproductive stages are required for maximum potential yield. It is unknown how yields of double-crop soybean, which is planted late into harvested small grain fields, respond to changes in leaf area index. We hypothesized that double-crop soybean would be more sensitive to defoliation than full-season soybean. This study used linear and linear plateau models to describe the yield response of full-season and double-crop soybean to reductions in leaf area index through manual defoliation, and evaluated the yield response of double-crop soybean to reductions in leaf area index through natural insect defoliation. From 15 manual defoliation experiments over 3 yr, significant linear decreases in yield occurred in both full-season and double-crop soybean when leaf area index values were below 3.5-4.0 by developmental stages R4 to R5, whereas yields usually reached a plateau at higher leaf area index levels. Average yield loss was 769 +/- 319 kg ha(-1) for each unit decrease in leaf area index below the plateau; average maximum yield was 3,484 +/- 735 kg ha(-1). From eight field experiments over 2 yr, insect defoliators had no effect on double-crop soybean yield; leaf area index levels were above 4.0 by the developmental stage when leaf area index estimates were taken (R3 to R6). Therefore, double-crop soybean that maintains leaf area index values above the 3.5-4.0 critical level by mid-reproductive developmental stages can tolerate defoliating pests.     

The effects of turbulent jet flows on plant cell suspension cultures

Cell suspensions of Morinda citrifolia were subjected to turbulent flow conditions in a submerged jet apparatus, to investigate their hydrodynamic shear susceptibility. The suspensions were exposed to repeated, pressure-driven passages through a submerged jet. Two nozzles, of 1 mm and 2 mm diameter, were employed. Average energy dissipation rates were in the range 10(3)-10(5) W/kg and cumulative energy dissipation in the range 10(5)-10(7) J/m3. System response to the imposed conditions was evaluated in terms of suspension viability (determined using a dye exclusion technique) and variations in both chain length distribution and maximum chain length. Viability loss was well-described by a first-order model, and a linear relationship was identified between the specific death rate constant and the average energy dissipation rate. This relationship was consistent with results obtained using the same suspension cultures in a turbulent capillary flow device. Morphological measurements indicated that exposure to the hydrodynamic environment generated in the jet resulted in a significant reduction in both the average and maximum chain lengths, and the reduction in the maximum chain length was identified as an appropriate measure of sustained damage. Analysis of both viability and chain length in terms of cumulative energy dissipated revealed good agreement with results reported by other authors for morphologically different plant cell systems. Copyright 1998 John Wiley & Sons, Inc.     

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.