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Organization and potential function of the mrjp3 locus in four honeybee species.

Royal jelly is a nutritious secretion produced by nurse honeybees to provision queens and growing larvae. Major proteins of royal jelly are mutually similar, and they all belong to the MRJP/yellow protein family (pfam03022). The mrjp3 loci in four traditional honeybee species (Apis mellifera, Apis cerana,Apis dorsata, and Apis florea) were sequenced and found to share high sequence and structural similarities. PCR analyses confirmed the presence of an extensive repetitive region, which showed size and sequence polymorphisms in all species. The evolutionary history of mrjp genes and their repetitive regions was reconstructed from their nucleotide sequences. The analyses proved that the repeat region appeared early in the evolution of the mrjp gene family and that the extreme elongation of the repeat is mrjp3 specific. In the MRJPs was documented a correlation between nitrogen content and repeat length. Therefore, it is argued that the repeat occurred due to a selection for an increase in nitrogen storage for a more efficient nutrition of queens and larvae.

Amino Acid Sequence↗

Effect of intrauterine growth retardation on postnatal weight change in preterm infants.

To investigate the cause or causes of early postnatal weight change, we measured total body water and fluid and energy balances in 14 preterm infants who were appropriate in size for gestational age (AGA) and in 5 weight-matched, preterm, small-for-gestational-age (SGA) infants. On the first day of life, AGA and SGA infants had the same weight and total body water content. At 6 +/- 2 days (mean +/- SD), AGA infants had had significant weight loss (94 +/- 45 gm) and body water loss (67 +/- 80 ml), whereas weight and total body water content in the SGA infants at the same age (5 +/- 1 days) did not differ from the values at birth. Loss of weight and total body water in AGA infants was accompanied by a greater diuresis than in SGA infants at the same amount of fluid intake. At the end of week 1, AGA and SGA infants had the same total energy expenditure (184 +/- 33 vs 171 +/- 17 kJ.kg-1 x day-1); energy intake, which had exceeded total energy expenditure from the third day of life and beyond, already provided 188 +/- 46 (AGA) or 209 +/- 109 kJ.kg-1 x day-1 (SGA), respectively, for energy storage. Nitrogen balance was positive. Subsequent weight gain occurred at the same rate in AGA and SGA infants; both total body water and solids increased. Energy intake, total energy expenditure, and the amount of energy stored (measured during stable weight gain on a regimen of full enteral feedings) had significantly increased compared with week 1, but both groups maintained similar energy storage.(ABSTRACT TRUNCATED AT 250 WORDS)

Body Composition↗

Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis.

BACKGROUND: Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine. RESULTS: High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2) of Ort1p/Arg11p was able to suppress the growth deficiency of arg11. RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers. Promoter-GUS fusions showed preferential expression in flowers, i.e. pollen, in the vasculature of siliques and in aborted seeds. Variable expression was observed in leaf vasculature. Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH4NO3, arginine or ornithine as sole nitrogen sources. CONCLUSION: AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis. The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds.

Arabidopsis↗

Nitrogen deprivation strongly affects photosystem II but not phycoerythrin level in the divinyl-chlorophyll b-containing cyanobacterium Prochlorococcus marinus.

Effects of nitrogen limitation on Photosystem II (PSII) activities and on phycoerythrin were studied in batch cultures of the marine oxyphotobacterium Prochlorococcus marinus. Dramatic decreases in photochemical quantum yields (F(V)/F(M)), the amplitude of thermoluminescence (TL) B-band, and the rate of Q(A) reoxidation were observed within 12 h of growth in nitrogen-limited conditions. The decline in F(V)/F(M) paralleled changes in the TL B-band amplitude, indicative of losses in PSII activities and formation of non-functional PSII centers. These changes were accompanied by a continuous reduction in D1 protein content. In contrast, nitrogen deprivation did not cause any significant reduction in phycoerythrin content. Our results refute phycoerythrin as a nitrogen storage complex in Prochlorococcus. Regulation of phycoerythrin gene expression in Prochlorococcus is different from that in typical phycobilisome-containing cyanobacteria and eukaryotic algae investigated so far.

Chlorophyll↗

Amino acid kinetics during the anhepatic phase of liver transplantation.

Alanine and glutamine are interorgan nitrogen/carbon carriers for ureagenesis and gluconeogenesis, which are mainly but not necessarily only hepatic. The liver is central to alanine and glutamine metabolism, but most organs can produce and use them. We studied amino acid kinetics after liver removal to depict initial events of liver failure and to provide a model to study extrahepatic gluconeogenesis and nitrogen disposal in humans. We measured amino acid kinetics with [5,5,5-(2)H(3)]leucine and [3-(13)C]alanine or [1,2-(13)C(2)]glutamine tracers in 21 subjects during and after the anhepatic phase of liver transplantation: 12 were at 7 months posttransplantation, and 7 were healthy control subjects. Anhepatic leucine kinetics, including proteolysis, was unchanged. Alanine plasma and whole-body contents increased 3x and 2x, with a halved metabolic clearance and a doubled production, 2% greater than disposal. Free whole-body glutamine decreased 25% but increased 50% in plasma. Glutamine clearance was halved, and the production decreased by 25%, still 2% greater than disposal. Liver replacement decreased alanine and glutamine concentrations, leaving leucine unchanged. Liver removal caused doubled alanine fluxes, minor changes in glutamine, and no changes in leucine. The initial events after liver removal are an accumulation of three-carbon compounds, an acceleration of alanine turnover, and limited nitrogen storage in alanine and glutamine.

Alanine↗

Ion chromatographic analysis of selected free amino acids and cations to investigate the change of nitrogen metabolism by herbicide stress in soybean (glycine max).

A simple and reliable method for the determination of NH4+, K+, Na+, aspartic acid, asparagine, glutamine, and alanine by ion chromatography has been developed. It is suitable for monitoring changes of nitrogen metabolism in soybean because it can accurately measure concentrations o asparagine and NH4+, two key substances for nitrogen storage and transport in this plant species Analysis of asparagine distribution in soybean indicated that higher levels (up to 18.4 micromol g(-1) of fresh mass) occur in stems and lower levels in roots (2.0 micromol g(-1) of fresh mass) and leaves (1.6 micromol g(-1) of fresh mass). When the herbicide metsulfuron-methyl (0.5, 5, and 50 ppb) was applied via the nutrient solution to the root system, asparagine concentrations increased 3-6 times in stems roots, and leaves. Metsulfuron-methyl is known to impair the synthesis of branched amino acids and, in consequence, protein synthesis. Thus, nitrogen consumption was limited, leading to ar accumulation of asparagine. The possible use of this physiological response in agricultural practice to identify herbicide stress in soybean and to detect low-level residues of sulfonylurea herbicides ir the soil is discussed.

Alanine↗

Pulmonary cell death in warm ischemic rabbit lung is related to the alveolar oxygen reserve.

BACKGROUND: If lungs could be retrieved for transplantation from non-heart-beating cadavers, the shortage of donors might be significantly alleviated. METHODS: We studied the effect of different postmortem lung conditions on pulmonary cell death. Lungs from 208 New Zealand white rabbits were flushed with trypan blue vital dye solution at intervals after circulatory arrest, fixed, and mounted for histologic examination. Pulmonary cells were judged to be viable on the basis of their ability to exclude trypan blue dye. In the control group, lungs were excised immediately after death and immersed in cold (4 degrees C) saline solution. In the other groups, cadavers were left at room temperature with lungs deflated, ventilated with room air or 100% oxygen or 100% nitrogen, or inflated with room air or 100% oxygen. RESULTS: There was a gradual increase in percentage (mean +/- SEM) of nonviable cells in the control group from 2.5%+/-0.9% (preischemic value) to 18.1%+/-2.8% at 24 hours after death (p < 0.001). In cadavers with lungs deflated, 79.7%+/-2.1% of cells were nonviable at 24 hours after circulatory arrest (p < 0.001 versus control group). In contrast, room air-ventilated cadavers showed only 21.4%+/-2.7% nonviable cells at this interval (p < 0.001 versus deflated group; not significant versus control group). Values in oxygen-ventilated animals were similar. Nitrogen-ventilated cadavers, however, had significantly more nonviable lung cells (73.8%+/-3.2%; p < 0.001 vs room air and oxygen-ventilated group, not significant vs deflated group). Oxygen-inflated lungs showed a parallel decrease in cell viability up to 4 hours after death when compared with room air-inflated cadaveric lungs, but thereafter more cells became nonviable in the latter group (11.1%+/-0.7% vs 19.6%+/-3.2% at 6 hours and 48.7%+/-7.2% vs 75.5%+/-4.6% at 24 hours, respectively; p < 0.01). CONCLUSIONS: Postmortem room air ventilation is as good as oxygen ventilation in delaying pulmonary cell death, and its effect is comparable to cold storage; nitrogen ventilation, however, is ineffective and not different from deflation; oxygen inflation will preserve ischemic cells for longer intervals as opposed to room air inflation. Therefore the alveolar oxygen reserve seems to be the critical factor to protect-the lung parenchyma from warm ischemic damage.

Animals↗

Expression of a vegetative-storage-protein gene from Arabidopsis is regulated by copper, senescence and ozone.

Emerging data suggest that the mechanisms regulating plant copper homeostasis could be implicated in stress and senescence signal transduction pathways. To gain insight into copper-modulated patterns of gene expression, copper-treated Arabidopsis thaliana (L.) Heynh. plants were analysed by mRNA differential display. The experimental conditions were selected using aggregation of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a molecular sensor to monitor copper-induced oxidative stress. Two copper-induced messengers encoding a vegetative storage protein (VSP2) were isolated by this technique. Both clones differed in the length of their 3'-untranslated region according to the presence of two polyadenylation signals in this region. VSP2 expression was further studied under natural senescence and various conditions causing oxidative stress, such as ozone exposure, paraquat and H2O2 treatments. The expression of other messengers related to copper homeostasis and detoxification processes was followed in parallel to that of VSP2. Here, we describe specific gene-expression responses to copper treatment, and present arguments connecting copper homeostasis, senescence and antioxidative responses in plants. Our results are consistent with the role of VSPs as temporary nitrogen-storage proteins which accumulate if nutrients are abundant, either in developing organs or in cotyledons and mature leaves subjected to generalized protein mobilization, such as those conditions created under severe oxidative stress.

Arabidopsis↗

Preservation of Mycoplasma strains by freezing in liquid nitrogen and by lyophilization with sucrose.

Procedures for maintaining Mycoplasma strains are described. Liquid nitrogen storage provided an adequate means for keeping stock cultures stable. Over 74 strains of approximately 26 species have been preserved in this way, some for as long as 9 years. Mycoplasma strains can also be recovered satisfactorily from the freeze-dried state when the procedure described includes the use of 12% (v/v) sucrose as an additive. Fifteen strains representing 12 or more species were subjected to a freeze-drying program with and without sucrose added to concentrated cell suspensions in growth medium. Cell counts indicate improved survival with sucrose.

Bacteriological Techniques↗

Vacuolar transporters and their essential role in plant metabolism.

Following the unequivocal demonstration that plants contain at least two types of vacuoles, scientists studying this organelle have realized that the plant 'vacuome' is far more complex than they expected. Some fully developed cells contain at least two large vacuoles, with different functions. Remarkably, even a single vacuole may be subdivided and fulfil several functions, which are supported in part by the vacuolar membrane transport systems. Recent studies, including proteomic analyses for several plant species, have revealed the tonoplast transporters and their involvement in the nitrogen storage, salinity tolerance, heavy metal homeostasis, calcium signalling, guard cell movements, and the cellular pH homeostasis. It is clear that vacuolar transporters are an integrated part of a complex cellular network that enables a plant to react properly to changing environmental conditions, to save nutrients and energy in times of plenty, and to maintain optimal metabolic conditions in the cytosol. An overview is given of the main features of the transporters present in the tonoplast of plant cells in terms of their function, regulation, and relationships with the microheterogeneity of the vacuome.

Biological Transport↗

Effects of two protein hydrolysates on growth, nitrogen balance and small intestine adaptation in growing rats.

The effects of feeding 2 protein hydrolysates, one prepared by controlled pepsin and pancreatic protease (including elastase II) hydrolysis of milk proteins (PPPH) and the other a di- and tripeptide bacterial protease hydrolysate of bovine albumin (DTPH), on the growth, nitrogen balance and small intestine adaptation of growing rats were analyzed. Two groups of 3-week-old rats (8 rats/group) were fed the liquid diets ad libitum for 2 weeks. The diets had the same caloric, nitrogen, carbohydrate and lipid contents. The amino acid compositions fulfilled the needs of growing rats. The diet differed in the original proteins, the hydrolysis technique used and the molecular weights of the peptides. Nitrogen intakes were similar. Although there was no difference in weight gain, nitrogen balance was significantly higher in the rats fed the PPPH diet (day 4-day 6:PPPH, 60 +/- 4%, DTPH, 25 +/- 5%; day 12-day 15: PPPH, 58 +/- 3%; DTPH, 30 +/- 5%). The stool nitrogens were identical, suggesting improved nitrogen storage in the rats fed the PPPH diet. Small intestine adaptation showed that the rats on the PPPH diet had significantly more protein (mg) and DNA (microgram) per 10 cm of the jejunum (PPPH, 25.6 +/- 2, 393 +/- 20; DTPH: 15.7 +/- 2, 258 +/- 23) and sucrase-specific activity and per microgram of DNA (PPPH, 133 +/- 5.7, 9.7 +/- 0.5; DTPH, 113 v 5, 7 +/- 1). The N-aminopeptidase-specific activity was the same in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

Tumorigenicity of human lymphoblastoid cell lines, acquired during in vitro culture and associated with chromosome gains.

Tumorigenicity of human lymphoma and lymphoblastoid B-cell lines was assessed by their ability to form growing and transplantable masses on subcutaneous inoculation into neonatally thymectomized, Ara-C-protected, total-body-irradiated mice. By these criteria, 12 lines of known malignant origin were tumorigenic, 11 lymphoblastoid lines, tested after less than one year of in vitro growth, were non-tumorigenic and 8/18 long-established lymphoblastoid lines produced transplantable tumours. All of the long-established lines had acquired karyotypic changes on prolonged culture, the predominant characteristic being a gain of whole chromosomes or of major chromosome segments. None showed the classical 8:14 translocation associated with Burkitt's lymphoma. Comparisons with nontumorigenic precursors (recovered from liquid nitrogen storage) and with other non-tumorigenic but chromosomally abnormal, lymphoblastoid lines suggest that imbalance of the dosage of genes carried on chromosomes 7,8 and 9 may be important in determining the tumorigenic phenotype.

Alleles↗

Comparative toxicity of four chlorinated dibenzo-p-dioxins (CDDs) and their mixture. IV. Determination of liver concentrations.

Groups of male Sprague-Dawley rats were administered orally the following chlorinated dibenzo-p-dioxins (CDDs) in corn oil/acetone (95/5; v/v): 30-60 micrograms/kg 2,3,7,8-tetrachlorodibenzo-dioxin (tetra-CDD), 160-270 micrograms/kg1,2,3,7,8-pentachlorodibenzo-p-dioxins (penta-CDD), 630-1249 micrograms/kg 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (hexa-CDD) and 5000-8000 micrograms/kg 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (hepta-CDD) or a mixture of the four homologues such that each was present in the mixture at one quarter of its dose as a single compound. Animals were killed at 2 and 8 days after dosing. Livers were immediately removed, and aliquots frozen in liquid nitrogen. Storage occurred at -80 degrees C until further use. About 0.2 g of each lyophilized rat liver was extracted, the extract purified by column chromatography and analyzed by GC/MS for CDD content. Results obtained suggest that the absorption of CDDs after oral administration decreases in the order of tetra-CDD > or = penta-CDD > hexa-CDD > hepta-CDD, indicating that the dose was an incomplete surrogate of exposure in parts I-III of this publication series (Stahl et al. 1992; Weber et al. 1992a,b). Moreover, data also support the notion that the pharmacokinetics of CDD mixtures at high doses are somewhat different from those expected based on single compound exposures. Our findings suggest that the intrinsic relative potency in terms of toxic equivalents (TEQ) of the higher chlorinated homologues is slightly greater (about a factor of 2) than suggested by Stahl et al.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Allograft semilunar cardiac valves processing and cryopreservation - morphology in scanning electron microscope.

OBJECTIVE: The most important factors of long term clinical performance of biological heart valve prostheses are methods of processing and cryopreservation. That is why we decided to evaluate the impact of current Allograft Heart Valves (AHV) Bank protocol on valve tissue morphology. Scanning electron microscope (SEM) is a valuable tool for investigation of biological surfaces. In case of cardiac valves it is especially suitable for detection of fine changes in endothelial covering and underlying layers. MATERIAL AND METHODS: "Fresh" aortic and pulmonary AHV samples, harvested from "heart-beating" cadaveric donors, were compared with (1) tissue from AHV obtained from non heart-beating donors, (2) samples stored in 4 degrees C saline for 24 h, (3) antibiotic treated tissue for 24 h at 37 degrees C and finally (4) cryopreserved valves, stored in liquid nitrogen (-196 degrees C) for 6-38 months. All samples were dissected, dried with hexamethyldisilazane (HMDS), gold coated, studied and photographed by SEM (Tesla BS 301). RESULTS: Our alternative method of drying samples by the HMDS method proved to be suitable for thin membranes of human semilunar valves. We were able to detect early changes in the endothelium after harvesting, and denudation of the endothelial covering during preservation with and without freezing. CONCLUSION: SEM (using HMDS drying) along with other methods may be helpful for the morphological control of processing, cryopreservation and liquid nitrogen storage of AHV. According to the current findings we have to avoid washing of AHV in saline after harvesting.

Cryopreservation↗

Experimental intraocular proliferation and neovascularization.

An experimental model of massive periretinal proliferation and intraocular neovascularization, produced in rabbits by the intravitreal injection of 250,00 cultured heterologous fibroblasts, showed no significant difference in the detachment rate (69% to 100%) or neovascularization rate (45% to 88%) between the animals injected with autologous cells and those injected with heterologous cells. Dermal fibroblasts produced a slightly higher detachment rate than conjunctival fibroblasts and were equally effective after reconstitution and subculture from liquid nitrogen storage in 7% dimethyl sulfoxide. Heterologous cells produced no clinical or histologic evidence of rejection when compared with autologous cells in the same animal and had the following advantages: (1) elimination of several biopsies and extended cell culture time; (2) a ready source of cryopreserved cells is available; (3) multiple injections of many animals can be performed within a short time; (4) in vivo and in vitro drug testing can be correlated on the same cell line.

Animals↗

A gas-flow cryostat for use in freeze-quench studies: design and application to discontinuous pre-steady-state spectral analyses.

A design for an improved freeze-quench apparatus is presented. A freeze-quench apparatus has two main parts: the apparatus which mixes the reactants and after a set time sprays the reacting mixture into the quencher, and the quenching apparatus itself. The quenching apparatus is the novel feature of our design and it comprises a gas-flow cryostat mounted directly onto a liquid nitrogen storage Dewar. The gas flow maintains the temperature of a small volume of isopentane, which acts as the freeze-quench agent, at -140 degrees C. (The apparatus can maintain the quenching temperature over the range -190 degrees C to room temperature.) Because of the small size of the cryostat and the much reduced volume of solvent used by this method it is more convenient to use than its predecessors, can be used in the open laboratory, and is safer. Our apparatus is designed for application to electron paramagnetic resonance studies but could be easily modified for use with other spectroscopic techniques.

Biochemistry↗

The use of rat mixed-thymocyte culture-conditioned medium for hybridoma production, cloning and revival.

Allogeneic rat mixed-thymocyte 48 h culture-conditioned medium (MTM) was used successfully in place of feeder cells for hybridoma production with the NS-1 and NS-0 plasmacytoma lines. It permitted lower concentrations of fused cells to be seeded, and supported the transition from 96 to 24 well plates. MTM improved the performance of poor sera during cloning. It also assisted the survival of cells that were sensitive to thawing from liquid nitrogen storage, and cells that had inadvertently been allowed to overgrow. Two rats could produce the equivalent of 1500-5000 ml feeder cell suspension according to the dilution used; 150-500 mice would be required to produce such a quantity of cells. Thus use of MTM entailed a considerable saving in mice and provided a secure supply of 'reagent', since a batch could be prepared, checked for sterility, frozen and stored indefinitely.

Animals↗

Effects of liner composition, cooling profile and time of glycerolization on survival of bovine spermatozoa.

Bovine spermatozoa from five bulls (158 ejaculates) were collected into one-piece combination liner-collection cones in an artificial vagina. Spermatozoal motility and differential staining were evaluated at each sequential step of semen processing. Linear 30-min and nonlinear 150-min cooling profiles resulted in equivalent postthaw spermatozoan survival rates. Preglycerolation holding times of 60 and 150 min, after 30-min linear cooling, were associated with increased, but nonsignificant, postthaw survival rates for bovine spermatozoa collected in polyethylene liner-collection cones. Highly significant differences (P<0.0001) were found to be associated with the composition of the liner-collection cones used. Motility estimates made at collection and after thawing from liquid nitrogen storage indicated that at least 50% of motile spermatozoa collected in polyethylene liner-collection cones survived; whereas, not more than 30% of the cells survived when they had been collected in rubber liner-collection cones.

Journal Article↗