Labile dissolved organic carbon and water temperature as regulators of heterotrophic bacterial activity and production in the lakes of Sub-Antarctic Marion Island

Summary The objectives of the 3 year study were to determine the relationship between bacterial numbers and phytoplankton standing crops (chlorophyll a) in sub-antarctic Marion Island lakes (33) and to determine the relative importance of labile dissolved organic carbon and water temperature as regulators of heterotrophic bacterial activity and production. Bacterial activity (the incorporation and respiration rates of ¹⁴C-labelled substrates) and production (the rate of [methyl-³H]thymidine incorporation into DNA) were measured in oligotrophic Lava Lake and Gentoo Lake, an elephant seal wallow. Samples were incubated under ambient conditions as well as at increased temperature and with additions of labile dissolved organic carbon (DOC). Bacterial numbers ranged from 2.13 × 10⁵ cell ml^–1 to 15.17 × 10⁶ cells ml^–1 in the lake survey. The chlorophyll range was 0.18 to >75 g 1^–1. Bacterial numbers were not correlated to chlorophyll concentration in waters where the chlorophyll content was 5 g 1^–1 but were correlated in waters with larger algal contents. Heterotrophic bacterial activity and production, which were similar to rates recorded for equivalent lower latitude systems, were higher in Gentoo Lake than in Lava Lake. As a result of qualitative and quantitative differences in the DOC pools, DOC was the stronger regulator of bacterial activity and production in Lava Lake, while temperature was the stronger factor in Gentoo Lake.     

Impact of acute temperature transition on enzyme activity levels,oxygen consumption,and exogenous fuel utilization in sea raven (Hemitripterus americanus) hearts

Summary The impact of an acute temperature transition between 5 °C and 15 °C on energy metabolism and performance of sea raven (Hemitripterus americanus) heart was assessed. Maximal in vitro activity of hexokinase was 1.2 and 3.7 mol · min^-1 · g^-1 at 5 °C and 15 °C, respectively. Carnitine palmitoyl transferase and carnitine palmitoleoyl transferase activities were 0.07 mol · min^-1 · g^-1 at 15 °C and declined substantially at 5 °C. Oxygen consumption and power output of perfused isolated hearts offered glucose alone as a metabolic fuel decreased significantly between 15 °C and 5 °C. When palmitoleate was included in the perfusion medium, oxygen consumption and power development remained constant between 15 °C and 5 °C, suggesting that glucose alone was not an adequate metabolic fuel at low temperature. However, maximal in vitro activity of HK implied that the catalytic potential at this locus was quite capable of meeting demands of carbon flow, while the maximal in vitro activity of the carnitine acyl CoA transferases implied that fatty acid metabolism should be greatly compromised at low temperatures. In an effort to resolve the contradiction, hearts were perfused with medium containing ¹⁴C-glucose or ¹⁴C-palmitate. Rates of ¹⁴CO₂ production from labelled metabolic fuels could account for only about 2% of the oxygen consumption rates. Most of the label from ¹⁴C-glucose was incorporated into the glycogen and lipid fractions and label from ¹⁴C-palmitate was incorporated into the lipid fraction. The net incorporation rates of label into intracellular pools were temperature insensitive over the range 5–15 °C. The incorporation of exogenous glucose into the lipid fraction suggests that activity of the entire glycolytic pathway was maintained over the temperature range. Thus, the relatively low rate of oxygen consumption of hearts perfused with glucose alone as an exogenous substrate cannot be attributed to a limitation of glucose catabolism. The alternative explanation is that the presence of fatty acids induces an increase in oxygen consumption, especially at 5 °C. It is speculated that this is due to alterations in Ca²⁺ balance.Abbreviations ATPase adenosine triphosphatase - BSA bovine serum albumin - CoA coenzyme A - C palmitoyl T carnitine palmitoyl transferase - CS citrate synthase - HK hexokinase - MO oxygen consumption - PFK phosphofructokinase - PO ₂ oxygen partial pressure     

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).     

High nucleotide sequence variation in a region of low recombination in Drosophila simulans is consistent with the background selection model

We surveyed nucleotide sequence variation at glucose dehydrogenase (Gld), in a region of low recombination on chromosome 3R, from a population sample of Drosophila simulans. The levels of nucleotide variation were surprisingly high. There was no departure from the expectation of a neutral model for the level of polymorphism, indicating no evidence of a selective sweep in this region. There was a significant deficiency of singleton polymorphisms according to the Fu and Li test, although Tajima and Hudson, Kreitman, and Aguade (HKA) tests do not provide evidence of a significant elevation of variation due to balancing selection. Genetic map data for the D. simulans third chromosome were used to calculate expected values of pi for Gld under a current model of background selection, varying the values for the parameter sh (selection coefficient against deleterious mutations). We show that the recombinational landscape of D. simulans is sufficiently different from that of D. melanogaster that we expect higher variation under the background selection model, even when effective population sizes are assumed to be equal. The data for Gld were tested against the predictions using computer simulations of the distribution of the number of segregating sites conditioned on pi. Background selection alone can explain our observations as long as sh is larger than 0.005 and species-level effective population size is assumed to be several- fold larger than in D. melanogaster. Alternatively, the deleterious mutation rate may be smaller in D. simulans, or balancing selection may be acting nearby, thereby reducing the effect of background selection.      

The aggregation of mutant p53 produces prion-like properties in cancer

The tumor suppressor protein p53 loses its function in more than 50% of human malignant tumors. Recent studies have suggested that mutant p53 can form aggregates that are related to loss-of-function effects, negative dominance and gain-of-function effects and cancers with a worsened prognosis. In recent years, several degenerative diseases have been shown to have prion-like properties similar to mammalian prion proteins (PrPs). However, whereas prion diseases are rare, the incidence of these neurodegenerative pathologies is high. Malignant tumors involving mutated forms of the tumor suppressor p53 protein seem to have similar substrata. The aggregation of the entire p53 protein and three functional domains of p53 into amyloid oligomers and fibrils has been demonstrated. Amyloid aggregates of mutant p53 have been detected in breast cancer and malignant skin tumors. Most p53 mutations related to cancer development are found in the DNA-binding domain (p53C), which has been experimentally shown to form amyloid oligomers and fibrils. Several computation programs have corroborated the predicted propensity of p53C to form aggregates, and some of these programs suggest that p53C is more likely to form aggregates than the globular domain of PrP. Overall, studies imply that mutant p53 exerts a dominant-negative regulatory effect on wild-type (WT) p53 and exerts gain-of-function effects when co-aggregating with other proteins such as p63, p73 and acetyltransferase p300. We review here the prion-like behavior of oncogenic p53 mutants that provides an explanation for their dominant-negative and gain-of-function properties and for the high metastatic potential of cancers bearing p53 mutations. The inhibition of the aggregation of p53 into oligomeric and fibrillar amyloids appears to be a promising target for therapeutic intervention in malignant tumor diseases.     

MAP2 IHC detection: a marker of antigenicity in CNS tissues

Immunohistochemistry (IHC) is used to detect antibody-specific antigens in tissues; the results depend on the ability of the primary antibodies to bind to their antigens. Therefore, results depend on the quality of preservation of the specimen. Many investigators have overcome the deleterious effects of over-fixation on the binding of primary antibodies to specimen antigens using IHC, but if the specimen is under-fixed or fixation is delayed, false negative results could be obtained despite certified laboratory practices. Microtubule-associated protein 2 (MAP2) is an abundant microtubule-associate protein that participates in the outgrowth of neuronal processes and synaptic plasticity; it is localized primarily in cell bodies and dendrites of neurons. MAP2 immunolabeling has been reported to be absent in areas of the entorhinal cortex and hippocampus of Alzheimer’s disease brains that were co-localized with the dense-core type of amyloid plaques. It was hypothesized that the lack of MAP2 immunolabeling in these structures was due to the degradation of the MAP2 antigen by the neuronal proteases that were released as the neurons lysed leading to the formation of these plaques. Because MAP2 is sensitive to proteolysis, we hypothesized that changes in MAP2 immunolabeling may be correlated with the degree of fixation of central nervous system (CNS) tissues. We detected normal MAP2 immunolabeling in fixed rat brain tissues, but MAP2 immunolabeling was decreased or lost in unfixed and delayed-fixed rat brain tissues. By contrast, two ubiquitous CNS-specific markers, myelin basic protein and glial fibrillary acidic protein, were unaffected by the degree of fixation in the same tissues. Our observations suggest that preservation of various CNS-specific antigens differs with the degree of fixation and that the lack of MAP2 immunolabeling in the rat brain may indicate inadequate tissue fixation. We recommend applying MAP2 IHC for all CNS tissues as a pre-screen to assess the quality of the tissue preservation and to avoid potentially false negative IHC results.     

Selective inactivation of peroxisomal and cytosolic 3-ketothiolase IB by 2-chloro-6-phenylhexanoate in intact hepatocytes

Rat liver mitochondria and cytosol contain two types of 3-ketothiolases, namely 3-ketothiolases IA and IB, which cleave 3-ketoacyl-coenzyme A (CoA) esters containing four or more carbons and 3-ketothiolases IIA and IIB, which cleave 3-ketoacyl-CoA esters containing four carbons, i.e. acetoacetyl-CoA (Aragon, J.J., and Lowenstein, J.M. (1983) J. Biol. Chem. 258, 4725-4733). We now report that rat liver peroxisomes also contain 3-ketothiolases IA and IB and show that incubation of hepatocytes with 2-chloro-6-phenylhexanoate causes the selective inactivation of peroxisomal and cytosolic 3-ketothiolase IB, while mitochondrial 3-ketothiolases are not appreciably affected. The basis of the selectivity of the inhibitor for peroxisomal and cytosolic 3-ketothiolases can be accounted for in terms of the specificities of the enzymes in the different pathways of beta-oxidation. Evidence is presented that 2-chloro-6-phenylhexanoate is metabolized to 2-chloro-3-keto-6-phenylhexanoyl-CoA, which then alkylates 3-ketothiolase and thereby inactivates the enzyme. Evidence is presented which suggests that cytosolic 3-ketothiolases IA and IB are not artifacts of homogenization and organelle preparation.     

Quantitative positron emission tomography of mGluR5 in rat brain with [18F]PSS232 at minimal invasiveness and reduced model complexity

Imaging the density of metabotropic glutamate receptor 5 (mGluR5) in brain by positron emission tomography (PET) is of interest in relation to several brain disorders. We have recently introduced [¹⁸F]PSS232, an F‐18‐labeled analog of the mGluR5‐targeting [¹¹C]ABP688. Quantitative PET requires kinetic modeling with an input function (IF) or an appropriate reference tissue model. We aimed at minimizing invasiveness of IF recording in rat and employing this protocol for mGluR5 quantitative PET with [¹⁸F]PSS232. We further aimed at defining models of low complexity for quantitative PET with [¹⁸F]PSS232. The IF was recorded in an arterio‐venous shunt applied by minimally invasive cannulation. PET data were analyzed with a modified two‐tissue compartment model including a single variable for radiometabolite correction in brain. We further evaluated a simple reference tissue model. Receptor‐dependent accumulation was similar to [¹¹C]ABP688 at lower unspecific accumulation of unchanged [¹⁸F]PSS232, in agreement with its higher plasma protein binding and lower lipophilicity. The minimally invasive protocol revealed similar results as the invasive shunt method and parameters calculated with the modified two‐tissue compartment model were similar to those calculated with the standard model. The simple area under the curve ratios agreed with the Logan reference method. [¹⁸F]PSS232 is a promising radioligand for mGluR5 quantification.

     

Fitness variation in response to artificial selection for reduced cell area,cell number and wing area in natural populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background

Genetically based body size differences are naturally occurring in populations of Drosophila melanogaster, with bigger flies in the cold. Despite the cosmopolitan nature of body size clines in more than one Drosophila species, the actual selective mechanisms controlling the genetic basis of body size variation are not fully understood. In particular, it is not clear what the selective value of cell size and cell area variation exactly is. In the present work we determined variation in viability, developmental time and larval competitive ability in response to crowding at two temperatures after artificial selection for reduced cell area, cell number and wing area in four different natural populations of D. melanogaster.

Results

No correlated effect of selection on viability or developmental time was observed among all selected populations. An increase in competitive ability in one thermal environment (18°C) under high larval crowding was observed as a correlated response to artificial selection for cell size.

Conclusion

Viability and developmental time are not affected by selection for the cellular component of body size, suggesting that these traits only depend on the contingent genetic makeup of a population. The higher larval competitive ability shown by populations selected for reduced cell area seems to confirm the hypothesis that cell area mediated changes have a relationship with fitness, and might be the preferential way to change body size under specific circumstances.