Seasonal reproductive cycles in three commercially exploited fishes from the slope waters off New Zealand

Reproductive cycles were investigated in orange roughy, Hoplostethus atlanticus , smooth oreo, Pseudocyttus maculatus , and black oreo dory, Allocyttus sp., from mid-slope waters (600–1300 m) around New Zealand from 1982 to 1985. Orange roughly displayed a mid-winter spawning period in July and August, whereas both dory species spawned in November and December. In all three species, the timing of spawning was consistent from year to year. Ovarian development in orange roughy and black oreo dory was found to be synchronous, with a single clutch of oocytes being matured for each spawning season. In males, testes of a given macroscopic stage were dominated by a single gamete stage, supporting the existence of a brief rather than prolonged spawning period. The possible relationship of spawning period to seasonal changes in the productivity of the surface water is discussed.     

Blood's critical Taylor number and its flow behavior at supercritical Taylor numbers

When the inner cylinder of a fluid-filled Couette viscometer is rotated rapidly, a vortical flow pattern develops when a dimensionless value referred to as the critical Taylor number (Tc) is reached. We have determined its magnitude in our viscometer for three Newtonian fluids and for blood at 37 degrees C, using the inflection point of torque/RPM vs. RPM (sudden rise in apparent viscosity). Its position was identified by least squares line fitting. Because blood was studied, the viscosity used in Tc calculation was the apparent bob shear stress/shear rate ratio at the inflection marking vortical flow onset. For glycerol-water mixtures Tc was 41.8 +/- 0.3 (N = 11), for propylene glycol 42.0 +/- 0.2 (N = 14), for silicone oil 41.8 +/- 0.2 (N = 11). For healthy blood Tc was 40.7 +/- 0.9 (N = 140). This evidence against blood's increased resistance to flow instability was accompanied by a slower rate of rise in torque both above and below Tc compared to the three Newtonian fluids. Newtonian fluids and blood both developed wavy vortical flow at a rotation rate moderately higher than Tc. Blood resisted this unstable flow behavior more than the Newtonian fluids but it also experienced a slower rate of rise in torque with increasing rotation rate above the critical Taylor number. Shear-thinning is the simplest explanation for blood's mildly altered Taylor vortex behavior; blood's resistance to flow instability is otherwise not found to be sufficient to affect its flow stability in man.     

The association of H-2Ld with human beta-2 microglobulin induces localized conformational changes in the alpha-1 and- 2 superdomain

We have analyzed changes in the antigenicity of major histocompatibility complex class I molecules resulting from the association of human beta-2 micro-globulin (B2m) with the mouse class I heavy chain. In particular, the H-2L^d molecule exhibited enhanced crossreactivity for the 34-1-2 monoclonal antibody. In order to assess the nature of this structural alteration induced by human B2m, we utilized H-2 class I hybrid molecules in the mapping of the 34-1-2 determinant to the helical region of the alpha-1 domain. H-2L^d class I hybrid molecules were then used to establish the importance of the alpha-2 and- 3 domains in the observed increase of 34-1-2 cross-reactivity following exchange with human B2m. The H-2L^d hybrids suggest that alterations in interdomain contact are responsible for enhanced 34-1-2 cross-reactivity on the H-2L^d molecule. It is likely that this alteration arises through changes in class I conformation at regions of the molecule distant from points of contact between B2m and the class I molecule. This suggests that perturbations induced by association of human B2m with H-2L^d can affect the conformation of the alpha-1 and- 2 superdomain. That class I antigenic determinants are altered by the association of human B2m with mouse class I further suggests that the class I molecule is structurally flexible and may reflect the ability of the class I molecule to bind and present a vast array of disparate peptides to the T-cell receptor.     

Metabolism of unsaturated fatty acids by RBL-1 5-lipoxygenase: influence of substrate solubility and product inactivation

Several alternative fatty acid substrates have been employed to characterise the kinetics of rat basophilic leukaemia cell (RBL-1) 5-lipoxygenase. Using arachidonic acid (AA) as substrate, enzymes rates declined at high substrate concentrations (greater than 25 microM) and were associated with pronounced lag phases. The concentrations of AA at which apparent substrate inhibition and lag phases were observed were comparable with those at which AA induced emulsion formation in aqueous media. No evidence for substrate inhibition or lag phases was observed using eicosapentaenoic acid (EPA), a more soluble substrate which did not induce emulsion formation at concentrations up to 100 microM. Reactions catalysed by RBL-1 5-lipoxygenase terminated before exhaustion of substrate. AA and EPA induced time-dependent enzyme inactivation at concentrations 100-fold lower than their apparent Km values for the enzyme. The ability of several fatty acids to induce time-dependent inactivation was directly proportional to their substrate potency. We conclude that apparent substrate inhibition is a consequence of a change from monomeric to micellar substrate which has a lower affinity for the enzyme and that premature termination of the enzyme reactions is a consequence of product-induced enzyme inactivation.     

Raman spectroscopic studies of hen egg-white lysozyme at high temperatures and pressures

In situ high-temperature, high-pressure Raman experiments on 3 mM (pH 5) aqueous solutions of hen egg-white (HEW) lysozyme show a decrease in the relative height of the 505 cm^–1 band associated with S-S stretching vibrations at 72°C (1 bar). The peak height changes are accompanied by significant band broadening, and the integrated band intensity does not change within experimental error. The effect of increased pressure at 72°C was to hinder broadening of the 505 cm^–1 band. HEW lysozyme (2.4 mM,pH 5) was also heated at 76°C, 80°C, and 95°C for different periods of time, and aliquots were quenched to room temperature for Raman and enzymatic activity measurements. After 9 hr at 76°C, the protein exhibits enzyme activity less than 50% of the initial value, and approximately 50% reduction in activity is achieved after 3 hr at 80°C or 1 hr at 95°C. The Raman results suggest that different irreversibly denatured conformations are attained during prolonged exposures at these different temperatures. It is apparent from these studies that the S-S stretch intensity is decreased irreversibly.     

Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures: Comparison of different substrata and tissue culture media formulations

Summary Long-term monolayer cultures of adult rat hepatocytes were tested for their ability to glucuronize phenol red and to maintain initial levels of cell proteins, glucose consumption, and lactic acid production. Lactate dehydrogenase leakage served as an index of culture status because a high value indicates cell death. Three tissue culture (TC) media formulations were the main variables introduced to determine ideal conditions for cell survival in vitro. Investigations of long-term cultures were preceded by studies of hepatocyte attachment to polystyrene surfaces. This attachment was influenced by the amount of substrate deposited and the number of cells seeded, but not by the uniformity of the substrate coating. A statistical analysis of our data revealed that in the absence of fetal bovine serum (FBS), air dried collagen (ADC) and Biomatrix (BMX) were superior to saline precipitated collagen and fibronectin as attachment substrates. In the presence of 10% FBS, all of the substrates performed equally. Chee's Medium (CEM) proved to be the best for preserving cell proteins over a time course of 28 d and Williams' E medium also performed adequately up to 14 d. The glucuronization of phenol red was at 50% of initial values at Day 7 in CEM-ADC hepatocytes in contrast to 30% for cells in Williams' E medium and 5% for cells grown in Waymouth's. At 14 d glucuronization was still present at 40% of original values in CEM-ADC cells but had ceased in the other two media. When BMX was used, none of the TC media supported glucuronization levels comparable to ADC cells. This research was supported in part by grant 1R01-AM-26520 from the National Institute of Arthritis, Diabetes and Digestive Kidney Diseases, NIH, Bethesda, Maryland.     

The expression of human glycerol-3-phosphate dehydrogenase in human/rodent somatic-cell hybrids

Our previous studies using rodent/human somatic-cell hybrids suggested that the expression of human mitochondrial glycerol-3-phosphate dehydrogenase (GPDM) is dependent on the presence of human mitochondria. This has now been tested directly by analysis of GPDM activity in a series of nine hybrid-cell lines, four segregating human chromosomes and five losing rodent chromosomes (reverse segregants). The chromosome composition of the hybrids was deduced from analysis of biochemical markers and examination of G- and G11-banded metaphase spreads and the mitochondrial content was determined by Southern blot analysis, using cloned mouse and human mtDNA sequences as probes. We found that the mtDNA species present in these hybrids correlated exactly with the pattern of chromosome segregation such that the conventional hybrids contained rodent mtDNA and the reverse segregants human mtDNA. However, the pattern of GPDM expression was not directly correlated with the species of chromosomes or mitochondria present: all the hybrids showed strong rodem GPDM activity and two from each class of hybrid also showed human GPDM activity but the other hybrids were negative for human GPDM. We conclude that rodent GPDM readily integrates into human mitochondria, that the expression of rodent GPDM is not dependent on the presence of rodent mitochondria, and that GPDM is not coded by mtDNA. Human GPDM either is not capable of being inserted into the rodent mitochondrial membrane or is regulated in some way in the hybrid cells by an unidentified rodent factor.     

Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interface

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus exhibits half-of-the-sites reactivity and negative cooperativity in binding of tyrosine. Protein engineering has been applied to the enzyme to determine whether it can be reversibly dissociated into monomers and if the monomers are active. The target for mutation is the residue Phe-164. The side chain of Phe-164 in one subunit interacts with its symmetry-related partner in the other. Mutation of Phe-164----Asp-164 gives a mutant [TyrTS(Asp-164)] that undergoes dissociation at high pH when the aspartate residues are ionized. The monomer is inactive and does not bind tyrosine. Dissociation is enhanced at low concentrations of enzyme by a mass action effect. Kinetic and binding measurements on TyrTS(Asp-164) with tyrosine and tyrosyl adenylate show that the monomer has very weak affinity for these ligands. Accordingly, dimerization is favored by high concentrations of tyrosine and ATP since the dimeric form has a high affinity for the ligands. The presence of tRNA does not encourage dimer formation, and so it must bind to the monomer. TyrTS(Asp-164) is fully active at pH 6 where dimerization is favored but has low activity at pH 7.8 where dissociation is favored. It should now prove possible to engineer heterodimers that may be used to investigate the subunit interactions further.     

Human glyceraldehyde-3-phosphate dehydrogenase: mRNA levels and enzyme activity in developing muscle.

Analysis of human glyceraldehyde-3-phosphate dehydrogenase mRNA revealed that levels in adult skeletal muscle are 12-fold greater per microgram of polyadenylated RNA than in fetal skeletal muscle, whereas in cardiac muscle RNA levels were about equal in fetal and adult tissue. The mRNA levels correlate well with glyceraldehyde 3-phosphate dehydrogenase enzyme activities. There was no evidence for fetus- or tissue-specific forms.