Probabilities of negative estimates of genetic variances

Summary The probability of negative analysis of variance estimates of genetic variance components due to sampling error (Ps) was investigated. The objectives were to evaluate the magnitude of Ps, to compare Ps for estimates of _A ² and _D ² , and to compare Ps for genetic variance component estimates from the nested and factorial mating designs. Ps was defined in terms of ratios of mean squares and the F distribution was used to calculate probabilities of the negative estimates. The results indicated that Ps is often greater than 0.20 for _D ² . It is generally lower for _A ² than for _D ² , and lower for the factorial mating design than the nested mating design.Technical Contribution No. 2589 from the South Carolina Agricultural Experiment Station, Clemson University     

Mapping of four light chain-associated idiotopes of a human monoclonal rheumatoid factor

In an effort to analyze both IgM rheumatoid factor (RF) repertoire and regulation of RF production in humans, we developed a panel of four mouse monoclonal antibodies (mAb) defining distinct K light chain-associated idiotopes (id) of a human monoclonal IgM RF (Alt). These mAb (A75, AM1, AM2, AM3) had equivalent reactivities with the immunizing RF during classic inhibition of antigen-binding assays. These anti-id reagents were reacting to neither other tested monoclonal IgM RF nor normal polyclonal IgM. It was possible to distinguish the id defined by the mAb from the results of four sets of experiments: dissociation of Alt RF heavy (H) and light (L) chains showed that A75, AM1, and AM2 reacted to id located on the L chain, whereas AM3 defined a conformational RF id; recombination experiments of H and L chains showed that A75 and AM2 reacted well with both homologous (Alt H + Alt L) and heterologous (Alt L + unrelated H) recombinants, whereas AM1 reacted better with the homologous recombinant than with the heterologous one; the relative affinities of the mAb were drawn from their ability to shift already bound labeled Alt RF from solid phase IgG; and radiolabeling of two mAb (A75 and AM3) and experiments of inhibition of id binding with cold anti-id and cold anti-CK showed that A75 recognized a proximal id (close to the K constant region), whereas AM3 defined a more distal id, AM2 and AM1 being located between A75- and AM3-defined sites. This topographic mapping of K light chain-associated id of a human RF with anti-id of known relative affinities could help in studying idiotypic regulation in humans.     

Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.     

Analysis of CD3-antibody-mediated inhibition of T-cell activation

In this study the influence of a non-mitogenic anti-CD3 antibody on accessory cell-dependent antigen and mitogen-induced T-lymphocyte proliferation has been investigated. The antibody was found to completely inhibit PHA, Con A, PWM, and tetanus toxoid stimulation, with no effect on the proliferation induced by the calcium ionophore A23187. VIT3 completely abrogated the production of IL-2 by lectin-stimulated T cells. It had no effect, however, on the IL-2-dependent proliferation of preactivated T-cell blasts. In addition, the antibody was able to elevate free cytoplasmic Ca2+ levels within minutes after the addition to T cells. Detailed time kinetic analyses revealed that the time interval critical for inhibition was significantly dependent on the interaction between T cells and accessory cells. Under standard conditions, in the presence of 10% non-T cells as accessory cells 50% inhibition was still achieved when VIT3 was added to PHA-stimulated T cells as late as 8 hr after the onset of culture. Delayed addition or a decrease in the number of added accessory cells significantly prolonged this time period. Lectin-stimulated T cells can thus obviously be inhibited via CD3 as long as they have not received all signals including those delivered by accessory cells. Although the underlying mechanisms are not clear so far, the observation that VIT3 at the same time triggers an early cytoplasmic Ca2+ response might indicate that it thereby actively interferes with antigen and lectin-initiated activation processes.     

Biochemistry of radioiodinated free fatty acids

Summary Radioiodinated free fatty acids have been developed to study myocardial metabolism non-invasively in man. In the present study the distribution of radiolabeled lipids in the myocardium and in arterial and coronary sinus blood was evaluated following injection of three commonly used iodinated fatty acids in fasted (n = 5) and lactate loaded (n = 3) dogs. Five minutes after simultaneous i.v. injection of radioiodinated 17-I-heptadecanoic acid (IHDA),15-(p-I-phenyl) pentadecanoic acid (IPPA) and 15-(p-I-phenyl)-3,3-dimethylpentadecanoic acid (DMIPPA) a biopsy specimen and samples of arterial and coronary sinus blood were taken. After extraction and TLC the relative distribution of radioactivity in the aqueous phase (containing the oxidation products), pellet and organic phase was calculated. The organic phase was further divided into phospholipids, diglycerides, free fatty acids, triglycerides and cholesterolesters. Seventy two percent of IHDA was oxidized, 36% of IPPA and 7% of DMIPPA. The organic phase consisted primarily of triglycerides and phospholipids. The ratios of triglycerides to phospholipids were about the same for IHDA, IPPA and DMIPPA (0.58, 0.65 and 0.50, respectively). Free IHDA in tissue samples was low (4%) and elevated for IPPA and DMIPPA, (17% and 37%). During lactate loading triglycerides were higher for all three fatty acids. For IHDA and IPPA this increase was paralleled by a decrease in the aqueous phase, in case of DMIPPA the aqueous phase remained the same. Five minutes after injection most of the organic phase of both arterial and coronary sinus blood consisted of the injected fatty acids, the aqueous phase contained oxidation products. There were only minor differences during lactate loading. During the evaluation of scintigraphic patterns of the radioiodinated fatty acids under normal conditions (eg at rest) and during elevated lactate levels (eg during exercise) the differences in distribution must therefore be considered.     

Microbial selective plugging and enhanced oil recovery

Summary The ability of indigenous populations of microorganisms in Berea sandstone to improve the volumetric sweep efficiency and increase oil recovery by in situ growth and metabolism following the injection of nutrients was studied. Cores of differing permeabilities connected in parallel without crossflow and slabs of sandstone with differing permeabilities in capillary contact to allow crossflow were used. The addition of a sucrosenitrate mineral salts medium stimulated the growth and metabolism of microorganisms in the sandstone systems. This resulted in a preferential decrease in permeability in the core or slab with the higher initial permeability, diverted flow into the lower-permeability core or slab and improved the volumetric sweep efficiency. Injectivity into the slab with the lower initial permeability in the crossflow system increased during subsequent nutrient injections. Thus, microbial selective plugging does occur in laboratory systems that have the complex flow patterns observed in petroleum reservoirs without losing the ability to inject fluids into the formation. In situ microbial growth and metabolism increased oil recovery 10 to 38% of the original oil in place. Biogenic gas production accompanied oil production, and much of the gas was entrained within the produced oil suggesting that gas production was an important factor leading to increased oil recovery. Quantitation of the amount of phospholipid in the core confirmed that microbial growth preferentially occurred throughout the core with the higher initial permeability. These data showed that in situ microbial growth in the high-permeability regions improved not only the volumetric sweep efficiency but also the microscopic oil displacement efficiency.     

Stability of the heart rate power spectrum over time in the conscious dog

The purpose of this experiment was to test the stability of the heart rate (HR) power spectrum over time in conscious dogs. HR was recorded for 1 h for each of six animals on 2 days. A Fast Fourier transform was used to derive the HR power spectrum for the 12 contiguous 5-min epochs comprising the 1-h recordings. Changes in frequency and amplitude of the various spectral peaks were quantitatively examined. We confirm the presence of two major concentrations of power centered around 0.02 (low frequency peak) and 0.32 Hz (high frequency peak). However, we observed variations in these spectral peaks, especially their amplitudes, both within each hour and from day 1 to day 2. The amplitudes of these two spectral peaks tended to vary reciprocally. HR power spectra based on 5 min of recorded data were also derived from an additional eight animals in both the lying and standing positions; the power spectra from these short recordings were sufficiently sensitive to detect redistributions in power due to changes in posture in all eight dogs. We conclude that: 1) data should be recorded for relatively long periods (e.g., 1 h) to characterize the HR power spectrum; 2) some variability in frequency and amplitude will persist across spectra even when based on longer data bases; 3) care should be taken to ensure that the subject's behavioral state is stable within the recording period; 4) shorter (e.g., 5 min) data bases are not suitable except for detecting relatively robust changes in the HR power spectrum.     

An inexpensive tag for short-term studies in milkfish (Cbanos chanos Forsskal) and in seabass (Lates calcarifer Bloch)

An opercular tag for marking adult milkfish ( Chanos chanos Forsskal) and seabass ( Lates calcarifer Bloch) is described. High tag retention and relatively low mortality rates were observed in adult fish handled two to ten times during 14-to 60-day tests. The features and advantages of the tag for marking large-sized fish in short-term studies are discussed.

Zusammenfassung

Eine preiswerte Markierung für Kurzzeitstudien des Milchfisches (Chanos chanos Forsskal) und der Centropomidae (Lates calcarifer Bloch)
Eine Kiemendeckel-Markierung für adulte Milchfische (Chanos chanos Forsskal) und Centropomidae (Lates calcacifer Bloch) wird beschrieben. Sie zeichnet sich durch gute Haltbarkeit aus und verursacht relativ geringe Mortalität bei adulten Fischen, die in Versuchen von 14 bis 60 Tage Dauer 2-bis 10mal untersucht wurden. Die Eigenschaften und Vorteile dieser Markierung für große Fische in Kurzzeitstudien werden diskutiert.

Résumé

Un marquage économique pour des études de courte durée du chanidé (Chanos chanos Forsskal) et du centropomidé (Lates calcarifer Bloch)
Un marquage d'opercule pour les chanidés (Chanos chanos Forsskal) et les centropomidés (Lates calcarifer Bloch) adultes est décrit. Une bonne conservation et une mortalité relativement basse ont été observées chez des poissons adultes examinés 2 à 10 fois pendant des expériences d'une durée de 14 à 60 jours. Les caractéristiques et les avantages du marquage de poissons de grande taille pendant des expériences d'une courte durée sont discutés.     

Quantitative autoradiography of [3H]CTOP binding to mu opioid receptors in rat brain

[3H]H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ([3H]CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with [3H]CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited [3H]CTOP binding with high affinity (IC50 values of 0.2-2.4 nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69, 593 were very weak inhibitors of [3H]CTOP binding (IC50 values of 234-3631 nM). Light microscopic autoradiography of [3H]CTOP binding sites revealed regions of high density (nucleus of the solitary tract, clusters in the caudate-putamen, interpeduncular nucleus, superior and inferior colliculus, subiculum, substantia nigra zona reticulata, medial geniculate, locus coeruleus and dorsal motor nucleus of the vagus) and regions of moderate labeling (areas outside of clusters in the caudate-putamen, cingulate cortex, claustrum and nucleus accumbens). The cerebral cortex (parietal) showed a low density of [3H]CTOP binding.