Phosphorus eutrophication in the SW Frisian lake district 2. Phosphorus balances and simulation of reduction scenarios

The lakes and interconnecting canals in the S.W. Frisian lake district are highly eutrophic. In the mid-1980's a project on eutrophication and lake restoration research was started. This project was aimed at modelling water transport and phosphorus (P) dynamics and at simulating management scenarios. A simple dynamic P-balance model was used to calculate total phosphorus (TP) balances and to simulate three TP reduction scenarios in each of three lakes: Tjeukemeer, Groote Brekken and Slotermeer. The model covered three periods in 1985, 1986 and 1987. The external loads to Tjeukemeer were highest, moderate to Groote Brekken, and lowest to Slotermeer. The major P sources in the area were discharges from the surrounding polders, used mainly for agriculture, and from IJsselmeer.Despite a 75% TP-reduction in water from the surrounding polders the 0.07 mg l^–1 target level could be reached only occasionally in Tjeukemeer, while in the other two lakes this level was not even approached. The effect of a 75% reduction in water from IJsselmeer was greatest in Groote Brekken (but again approached the target only occasionally), moderate in Tjeukemeer and least in Slotermeer. The simulations showed that only a 75% reduction in both external loads (from polders and from IJsselmeer) will lead to achieving the target level in Tjeukemeer and Groote Brekken during the summer periods. In Slotermeer, a relatively isolated lake, other measures are necessary to reach the target level. The results are confirmed by an approximate theoretical analysis of the effects of load reduction.     

Putting the heat on sex determination

Sex determination and differentiation are inherently fascinating to both layperson and geneticist. Major advances have accelerated interest in the molecular genetic events mediating these processes in nematodes, flies, mice and humans. Far less attention has been paid to those organisms, particularly reptiles, where sex is determined by environmental cues. However, recent experimental evidence suggests that the two modes of sex determination may not only share common genetic elements, but may also be regulated by similar mechanisms. We argue that the ability to manipulate sex by temperature provides a particularly suitable model for exploring the molecular basis of this fundamental biological process.     

Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to- pole motion

During prometaphase and metaphase of mitosis, tubulin subunit incorporation into kinetochore microtubules occurs proximal to the kinetochore, at the plus-ends of kinetochore microtubules. During anaphase, subunit loss from kinetochore fiber microtubules is also thought to occur mainly from microtubule plus-ends, proximal to the kinetochore. Thus, the kinetochore can mediate both subunit addition and loss while maintaining an attachment to kinetochore microtubules. To examine the relationship between chromosome motion and tubulin subunit assembly in anaphase, we have injected anaphase cells with biotin-labeled tubulin subunits. The pattern of biotin-tubulin incorporation was revealed using immunoelectron and confocal fluorescence microscopy of cells fixed after injection; chromosome motion was analyzed using video records of living injected cells. When anaphase cells are examined approximately 30 s after injection with biotin-tubulin, bright "tufts" of fluorescence are detected proximal to the kinetochores. Electron microscopic immunocytochemistry further reveals that these tufts of biotin-tubulin-containing microtubules are continuous with unlabeled kinetochore fiber microtubules. Biotin-tubulin incorporation proximal to the kinetochore in anaphase cells is detected after injection of 3-30 mg/ml biotin-tubulin, but not in cells injected with 0.3 mg/ml biotin-tubulin. At intermediate concentrations of biotin-tubulin (3-5 mg/ml), incorporation at the kinetochore can be detected within 15 s after injection; by approximately 1 min after injection discrete tufts of fluorescence are no longer detected, although some incorporation throughout the kinetochore fiber and into nonkinetochore microtubules is observed. At higher concentrations of injected biotin-tubulin (13 mg/ml), incorporation at the kinetochore is more extensive and occurs for longer periods of time than at intermediate concentrations. Incorporation of biotin-tubulin proximal to the kinetochore can be detected in cells injected during anaphase A, but not during anaphase B. Analysis of video records of microinjection experiments reveals that kinetochore proximal incorporation of biotin-tubulin is accompanied by a transient reversal of chromosome-to-pole motion. Chromosome motion is not altered after injection of 0.3 mg/ml biotin-tubulin or 5 mg/ml BSA. These results demonstrate that kinetochore microtubules in anaphase cells can elongate in response to the elevation of the tubulin concentration and that kinetochores retain the ability to mediate plus-end-dependent assembly of KMTs and plus-end-directed chromosome motion after anaphase onset.     

A case of endometriosis in the macaque diagnosed by nuclear magnetic resonance imaging.

A case of spontaneous endometriosis was diagnosed in the pigtailed macaque (Macaca nemestrina nemestrina) with the aid of high-field (2.35 T), T2-weighted (TE50), C1H2-suppressed, oblique nuclear magnetic resonance imaging (MRI). Postmortem histology was obtained. A variety of endometriotic lesions was seen with MRI, including extrauterine hyperintense apparently cystic regions, extrauterine hypointense regions apparently associated with intracellular paramagnetic iron proteins, and an enlarged myometrium exhibiting adenomyosis foci.     

Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR far-red light - R red light - WL white light - WL + FR white light supplemented with FR - HIR high-irradiance response - PAR photosynthetically active radiation - Pr, Pfr R- and FR-absorbing forms of phytochrome - Ptot total phytochrome - phyA (PhyA) gene (encoded protein) for phytochrome - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line.     

Comparison of Representative Strains of Infectious Hematopoietic Necrosis Virus by Serological Neutralization and Cross-Protection Assays

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of young salmon and trout. Viral epizootics among these fish in private and public rearing facilities have been a problem in the northwestern United States from California to Alaska, and an IHNV vaccine has been sought by the aquaculture experts. Since an IHNV vaccine must be designed to immunize against all viral serotypes, an analysis of IHNV serotypes was made. A large number of viruses from widely separated geographic locations and different fish species had already been placed in one of five electropherotypes by the migration of the virion proteins in sodium dodecyl sulfate-polyacrylamide gels. Also, there was evidence that some of these virus isolates had differences in virulence for chinook salmon, rainbow trout, or kokanee salmon. Previous serological studies with polyclonal rabbit antisera and three IHNV isolates indicated that there was only one serotype (B. B. McCain, J. L. Fryer, and K. S. Pilcher, Proc. Soc. Exp. Biol. Med. 137:1042-1046, 1971). A substantial number of new IHNV isolations have been made since that study, and thus a more extensive comparison was made of 10 different IHNV isolates representing the five electropherotypes. This report shows that the glycoprotein from a single isolate of IHNV can induce a protective immune response in vivo to the five IHNV electropherotypes. Plaque reduction neutralization assays indicated that there was only one serotype. Thus, despite the differences observed in the migration of the structural proteins for IHNV isolated from separate geographic locations and different fish species, only one neutralizing virus type was identified.     

A new structural model for the thyrotropin (TSH) receptor, as determined by covalent cross-linking of TSH to the recombinant receptor in intact cells: evidence for a single polypeptide chain.

The most widely held model for the human TSH receptor is of holoreceptor of 80 kDa with two subunits of approximately 50 and 30 kDa linked by disulfide bridges, with the former subunit containing the major hormone-binding site. We reexamined this model by covalently cross-linking radiolabeled TSH to the recombinant human TSH receptor stably expressed in Chinese hamster ovary (CHO) cells. When cross-linking was performed after the preparation of CHO membranes, analysis of hormone-receptor complexes under reducing and nonreducing conditions provided results supporting the two-subunit TSH receptor model. In contrast, however, cross-linking of TSH to the TSH receptor in intact CHO cells before membrane preparation revealed, even under reducing conditions, an approximately 100-kDa receptor as well as an approximately 54-kDa hormone-binding subunit. The approximately 100-kDa holoreceptor size is consistent with the size of the TSH receptor, as predicted from its derived amino acid sequence. The proportions of the approximately 100-kDa TSH receptor and the 54-kDa fragment varied in different experiments, suggesting the occurrence of proteolytic cleavage. Cross-linking of radiolabeled TSH to intact cells expressing a mutant TSH receptor (TSHR-D1) lacking amino acids 317-366 localized the proteolytic cleavage site to just up-stream of amino acid residue 317. In summary, the present data obtained by cross-linking TSH to recombinant human TSH receptors in intact cells provides evidence that the receptor exists in vivo as an approximately 100-kDa glycoprotein with a single polypeptide chain with intramolecular disulfide bridges.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of the human thyrotropin receptor: role of asparagine-linked oligosaccharides in the expression of a functional receptor

We studied the role of glycosylation in the expression of a functional human TSH receptor. Oligonucleotide-directed mutagenesis was used to replace, separately or together, the Asn codons with Gln in each of the six potential glycosylation sites in the receptor. Recombinant wild-type and mutated TSH receptors were stably expressed in Chinese hamster ovary cells. High affinity TSH binding and the cAMP response to TSH stimulation were abolished in the receptor mutated at Asn77 as well as in the receptor mutated at all six potential glycosylation sites. In the receptor mutated at Asn113, the affinity of TSH binding was markedly decreased (Kd, 2.6 x 10(-8) 3.3 x 10(-10) M in the wild-type receptor). This affinity was too low to permit the transduction of a signal, as measured by an increase in intracellular cAMP generation. Substitution of Asn at positions 99, 177, 198, and 302 did not appreciably affect the affinity of the TSH receptor for TSH binding or its ability to mediate an increase in intracellular cAMP levels. Therefore, either these four potential glycosylation sites are not glycolysated, or alternatively, oligosaccharide chains at these positions do not play a major role in the folding, intracellular trafficking, stability, or expression of a functional receptor on the cell surface. Conversely, our data suggest that N-linked glycosylation of Asn77 and Asn113 does play a role in the expression of a biologically active TSH receptor on the cell surface.     

Extracellular domain chimeras of the TSH and LH/CG receptors reveal the mid-region (amino acids 171-260) to play a vital role in high affinity TSH binding

We constructed a series of TSH-LH/CG receptor chimeras by homologous substitution of relatively small regions of the TSH receptor extracellular domain for the corresponding region of the extracellular domain of the LH/CG receptor. Constructs were stably expressed in Chinese hamster ovary cells. Of the five chimeric receptors, only TSH-LHR-14, which contains mid-region domain C (amino acid residues 171-260) of the extracellular component of the TSH receptor, exhibited TSH binding of relatively high affinity. Consistent with this TSH binding, chimera TSH-LHR-14 was the only one that demonstrated a functional response to TSH stimulation in terms of intracellular cAMP generation. These data indicate that domain C plays a vital role in TSH receptor function.