Substrate specificity of acyl-lipid Δ9-desaturase from <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacterium producing myristoleic acid

Chlorophyll b-containing cyanobacterium Prochlorothrix hollandica is characterized by a high content of esterified fatty acids (FA) with 14 and 16 carbon atoms in the membrane lipids. Depending on the conditions of cultivation, the relative amount of myristic (C_14:0) and myristoleic (C_14:1) acids can reach 35%, and palmitic (С_16:0) and palmitoleic (С_16:1) acids can reach 60% of the sum of all fatty acids in cells. Monounsaturated FAs are represented by C_14:1, and C_16:1 with an olefinic bond presumably located in the Δ⁹ position. We cloned the gene of acyl-lipid Δ9-desaturase, desC1, from Prochlorothrix hollandica and characterized its specificity to the length of the substrate using the heterologous expression in Escherichia coli cells adding C_14:0 or stearic (C_18:0) acids as exogenous substrates. The results show that DesC1 Δ⁹ desaturase generates olefinic bonds in the FAs with a length of 14 to 18 carbon atoms with an approximately equal efficiency. This indicates that the length of the FA chain in P. hollandica is determined by the activity of the FA synthase, and the chain is desaturated at the Δ⁹ position nonspecifically relatively to its length.     

Erythrocyte profile of circulating blood of Neogobius melanostomus (Pallas, 1814) under conditions of experimental hypothermia

The influence of hypothermia on erythrocyte profile of thermophile teleost species round goby, Neogobius melanostomus (Pallas, 1814), has been studied. Fish were acclimated to temperature 1-2^оС, 15-16^оС and 19-20^оС (control group) and held at given conditions for 5 days. The number of red blood cell precursors (pronormoblasts, basophilic and polychromatophilic normoblasts) in circulating blood has been estimated. Also, the number of abnormal erythrocytes, i.e. cells with micronuclei, nuclei invaginations, red blood cell shades, dacryocytes and cells undergoing amitosis has been determined on smears. The number of immature erythrocytes increased more than two times (p < 0,001) at 1-2^оС. The number of low-differentiated precursors, pronormoblasts and early basophilic normoblasts, increased for the most part. The number of abnormal erythrocytes did not change substantially, The changes in cellular blood composition were accompanied with the increase of plasma lactate concentration, indicating hypoxic state of fish. The results of the present work indicate that hematopoietic tissue remains sensitive to controlling factors at hypothermia, such as hypoxia, and may enhance proliferation and differentiation of erythroid cells.     

Isotope composition of carbon and nitrogen in tissues and organs of <Emphasis Type="Italic">Betula pendula</Emphasis>

Ratios of ¹³С/¹²C and ¹⁵N/¹⁴N isotopes were identified in different parts and organs of drooping birch (Betula pendula Roth) in preforest-steppe and pine-birch forests of the Middle Urals by mass spectrometry. The data were analyzed and interpreted from the perspective of biochemical processes of carbon and nitrogen metabolism in the leaf, cambial tissue, trunk wood, branches, roots, and in the soil. The lighter isotopic composition of carbon is characteristic for the leaves, trunk cambium as well as fine (<2 mm) roots. The trunk wood is characterized by the basal trend for ¹³C enrichment. The heavier carbon isotopic composition inversely related to metabolic activity of organs and tissues, in addition, ¹³С/¹²C ratio corresponds to the nitrogen content in the organs and tissues, indicating the metabolic control of carbon fractionation in woody plants. The isotopic composition of nitrogen in the aboveground parts of the plant (leaves, trunk cambium, wood) and in the medium and fine roots was significantly depleted in ¹⁵N (δ¹⁵N varies from 0 to–3‰), while main roots (δ¹⁵N = 0.6 ‰) and soil (δ¹⁵N = 2.4–6.7‰) were more enriched. The ratio of stable isotopes of carbon and nitrogen is an integrating index of carbon and nitrogen metabolism in plants.     

Proteome analysis of circulating exosomes in health and breast cancer

Microvesicles were isolated from blood plasma and total blood of healthy females and breast cancer patients by filtration and ultracentrifugation. According to flow cytometry, different subpopulations of exosomes were represented in blood of healthy donors and cancer patients at different levels with median fluorescence intensity (MFI) values in both groups arranged in the following order: CD24/СD9 > СD9/СD81 > CD9/CD63 = CD24/CD63. Concentration of exosomes in blood plasma of healthy females estimated by nanoparticle tracking analysis (NTA) did not exceed (3.71 ± 1.15) × 10⁷ particles/mL of blood and did not differ from that in plasma of breast cancer patients, which averaged (3.99 ± 1.03) × 10⁷ particles/mL of blood. Concentration of total exosomes in blood (including exosomes from plasma and blood cell surface-bound exosomes) did not depend on the presence/absence of a tumor; the values were (7.66 ± 0.7) × 10⁷ particles/mL of healthy blood and (9.4 ± 1.24) × 10⁷ particles/mL of blood from cancer patients. Comparative analysis of exosomes using 2-D electrophoresis with subsequent analysis of 2-D proteomic maps revealed proteins missing in blood or differentially expressed in healthy females and breast cancer women. The data presented provide the possibility for identification of exosomal proteomic markers and isolation of tumor-specific exosomes, which contributes to the development of breast cancer diagnostics.     

Peculiarities of development in the marine brown alga <Emphasis Type="Italic">Alaria angusta</Emphasis> Kjellman, 1889 (Alariaceae: Ochrophyta) under laboratory-controlled conditions

This paper describes the formation of gametophytes and the early stages of the development of sporophytes in the kelp seaweed Alaria angusta from Kamchatka. To establish laboratory cultures we used zoospores obtained from A. angusta sporophytes collected on October 29, 2014. The gametophytes were grown under different conditions: at 6–7°С and natural light and at 10°С and illumination with cool white fluorescent bulbs, 30 μmol photon m^–2 s^–1, 12: 12 h L: D cycle. In the first case (natural light, 6–7°С), the vegetative growth of male and female gametophytes lasted for more than 4 months; maturation of sexual products occurred 144 days after germination of the embryospores. In the second case (artificial light, 10°С), rapid development of the gametophytes occurred; the first juvenile sporophytes appeared on the 10th day after the zoospores settled onto the substrate. Our data contribute to understanding of the regulatory effect of temperature and light on the peculiarities of species vegetation in the natural environment and formation of the age structure of the species population, one particular feature of this process is the continuous appearance of juvenile sporophytes in the warm period of the year.     

Stability of kappa phage (Serratia marcescens) and its c-mutant at different pH

Устойчивость каппа-фага (Serratia marcescens) и его мутанта «С» исследовалась в буферных растворах при pH от 2.5 до 12 с целью подтверждения, или опровержения, возможности их инактивирования в результате кратковременного доведения кислотности основной суспензии в течение приготовления концентрата фага до pH 4. Было установлено, что каппа-фаг и его с-мутант в среде с pH 4 и выше остаются относительно устойчивыми и что в этих пределах pH между обоими фагами нет существенных различий. Однако при низких pH (2,5–3,5) титр обоих фагов вскоре довольно резко падает, причем с-мутантинакмпбпруемся бысмрее, чем каппа-фаг.     

Weekly work hours and clinical activities of Canadian family physicians: results of the 1997/98 National Family Physician Survey of the College of Family Physicians of Canada

BackgroundHealth systems planning is a challenging task, exacerbated by a lack of detailed information on the role played by family physicians, as indicated by practice variations across regions and demographic characteristics. Outcome measures used in past studies of family physician practice patterns were not uniform. Furthermore, past research has generally been limited to narrowly defined geographic regions. A national study of family physician practice patterns was undertaken to allow regional-level comparisons of clinical workload and range of medical services offered.MethodsThe 1997/98 National Family Physician Survey was mailed to a sample of 5198 Canadian family physicians and general practitioners (FP/GPs); the overall response rate was 58.4% (3036 questionnaires returned, of which 3004 were analyzable). Sampling strata were based on College of Family Physicians of Canada (CFPC) membership status and regions of Canada.ResultsClinical workload varied considerably across the demographic categories studied. Male physicians reported 8.9 more total weekly work hours than female physicians, but the mean number of medical and clinical services offered did not differ between the sexes. Solo practitioners reported 53.8 (95% confidence interval [CI] 52.7–55.0) total weekly work hours, whereas those practising in multidisciplinary clinics reported 45.0 (95% CI 43.2–46.8) hours. FP/GPs in the Atlantic and Prairie provinces reported 5.6 and 5.1 more weekly work hours, respectively, than the national average of 51.4 (95% CI 50.8–52.0) hours. Finally, FP/GPs who served inner-city populations reported 48.6 (95% CI 46.8–50.5) total weekly work hours, whereas those serving rural populations reported 57.0 (95% CI 54.7–59.2) hours. Mean weekly work hours were similar for all age cohorts less than 65 years. FP/GPs practising in less populated provinces and in rural areas reported the highest numbers of work hours, medical services offered and clinical procedures performed. InterpretationThese data suggest significant variations in FP/GP clinical workload in relation to key demographic variables. Physician resource planning in Canada is a challenging and inexact science. Past attempts have resulted in variable estimates of the ultimate need for physician services. There is a clear recognition that we need more information than simple head counts of physicians. We need to know, for example, what physicians do and how they work with other physicians, and we need to identify regional variations and differences in practice patterns.Past studies of family physician practice patterns have measured workload in terms of hours worked,^1,2,3 number of patient visits,^2,3 billings to health insurance plans^4,5 and range of clinical procedures performed.^5,6,7 These outcomes have been analyzed in relation to practice setting,^1,2,6,7 geographic physician density,^4,5 sex,^{1,2,3,4,5,6,7} age,^1,2,4,5,7 years in practice^3,6 and type of practice.^1,2,3,6,7Although past studies have proven useful in describing the significant relations that exist between physician workloads and demographic characteristics, they have not addressed the broader issue of access to family physicians'' services throughout Canada. By gathering uniform information from family doctors across the country, the College of Family Physicians of Canada (CFPC) National Family Physician Survey (NFPS) addresses this information gap. In this report we present the results of the 1997/98 NFPS, describing physician workload measures in relation to a comprehensive set of demographic variables.     

Fructose is the most uptaken sugar for (-)-carvone biosynthesis in Mentha spicata L.

Mentha spicata L. cuttings were fedin vivo with ¹⁴C-palmitate, ¹⁴C-acetate, ¹⁴C-sucrose, ¹⁴C-glucose, and ¹⁴C-fructose. No labelled (-)-carvone was detected when cuttings were fed with ¹⁴C-palmitate and/or ¹⁴C-acetate, whereas feeding of cuttings with ¹⁴C-sucrose, ¹⁴C-glucose, and ¹⁴C-fructose resulted in labelled (-)-carvone biosynthesis. Among sugars, fructose feeding caused the highest (-)-carvone biosynthesis. The lack of incorporation of exogenous ¹⁴C-palmitate and/or ¹⁴C-acetate into (-)-carvone and the incorporation of sugars into this monoterpene, also confirm for this species the presence of an alternative, non-mevalonate biosynthetic pathway for monoterpene production.     

Biodegradation of 2,4,5-trichlorophenoxyacetic acid in liquid culture and in soil by the white rot fungus Phanerochaete chrysosporium

Summary Extensive biodegradation of [¹⁴C]-2,4,5-trichlorophenoxyacetic acid ([¹⁴C]-2,4,5-T) by the white rot fungus Phanerochaete chrysosporium was demonstrated in nutrient nitrogen-limited aqueous cultures and in [¹⁴C]-2,4,5-T-contaminated soil inoculated with this fungus and supplemented with ground corn cobs. After incubation of [¹⁴C]-2,4,5-T with aqueous cultures of the fungus for 30 days, 62.0%±2.0% of the [¹⁴C]-2,4,5-T initially present was degraded to ¹⁴CO₂. Mass balance analysis demonstrated that water soluble metabolites were formed during degradation, and HPLC and thin layer chromatography (TLC) of methylene chloride-extractable material revealed the presence of polar and non-polar [¹⁴C]-2,4,5-T metabolites. It was also shown that only 5% of the [¹⁴C]-2,4,5-T initially present in cultures remained as undegraded [¹⁴C]-2,4,5-T. In incubations composed of [¹⁴C]-2,4,5-T-contaminated soil, ground corn cobs, and 40% (w/w) water, 32.5%±3.6% of the [¹⁴C]-2,4,5-T initially present was converted to ¹⁴CO₂ after 30 days of incubation. These results suggest that it may be possible to develop practical systems based on the use of this fungus to detoxify 2,4,5-T-contaminated water and soil.     

The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats

The concentration-dependent metabolism of 1-¹⁴C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [¹⁴C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-¹⁴C]20:5n-3 to [3-¹⁴C]22:6n-3 was more efficient than that of [1-¹⁴C]20:4n-6 to [3-¹⁴C]22:5n-6. At low substrate concentration (4 μM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 μM). The conversion of [1-¹⁴C]22:5n-3 to [1-¹⁴C]22:6n-3 was 1.7 times more efficient than that of [1-¹⁴C]22:4n-6 to [1-¹⁴C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-¹⁴C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-¹⁴C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-¹⁴C]20:4n-6 or [1-¹⁴C]22:4n-6 to [¹⁴C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-¹⁴C]20:5n-3 and [1-¹⁴C]22:5n-3 to [¹⁴C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.     

Does delivery volume of family physicians predict maternal and newborn outcome?

Background

The number of births attended by individual family physicians who practice intrapartum care varies. We wanted to determine if the practice–volume relations that have been shown in other fields of medical practice also exist in maternity care practice by family doctors.

Methods

For the period April 1997 to August 1998, we analyzed all singleton births at a major maternity teaching hospital for which the family physician was the responsible physician. Physicians were grouped into 3 categories on the basis of the number of births they attended each year: fewer than 12, 12 to 24, and 25 or more. Physicians with a low volume of deliveries (72 physicians, 549 births), those with a medium volume of deliveries (34 physicians, 871 births) and those with a high volume of deliveries (46 physicians, 3024 births) were compared in terms of maternal and newborn outcomes. The main outcome measures were maternal morbidity, 5-minute Apgar score and admission of the baby to the neonatal intensive care unit or special care unit. Secondary outcomes were obstetric procedures and consultation patterns.

Results

There was no difference among the 3 volume cohorts in terms of rates of maternal complications of delivery, 5-minute Apgar scores of less than 7 or admissions to the neonatal intensive care unit or the special care unit, either before or after adjustment for parity, pregnancy-induced hypertension, diabetes, ethnicity, lone parent status, maternal age, gestational age, newborn birth weight and newborn head circumference at birth. High- and medium-volume family physicians consulted with obstetricians less often than low-volume family physicians (adjusted odds ratio [OR] 0.586 [95% confidence interval, CI, 0.479–0.718] and 0.739 [95% CI 0.583–0.935] respectively). High- and medium-volume family physicians transferred the delivery to an obstetrician less often than low-volume family physicians (adjusted OR 0.668 [95% CI 0.542–0.823] and 0.776 [95% CI 0.607–0.992] respectively). Inductions were performed by medium-volume family physicians more often than by low-volume family physicians (adjusted OR 1.437 [95% CI 1.036–1.992].

Interpretation

Family physicians'' delivery volumes were not associated with adverse outcomes for mothers or newborns. Low-volume family physicians referred patients and transferred deliveries to obstetricians more frequently than high- or medium-volume family physicians. Further research is needed to validate these findings in smaller facilities, both urban and rural.More than 20 years ago, Luft and associates¹ conducted one of the earliest volume–outcome studies. Since then, many studies addressing the relation between volume of procedures and patient outcomes have been published.^2,3 In some of these studies, either the hospital size or the physician procedural volume was used as a surrogate for physician expertise. Among studies analyzing hospital volumes and outcomes, better outcomes have been associated with higher patient volumes in some instances^4,5,6,7 but not others.^3,8,9 Some studies of individual provider volume have shown a positive relation between volume and outcomes,^10,11 whereas others have shown no relation or inconsistent results.^3,12 Finally, a few studies analyzing both hospital volume and provider volume have reported a positive volume–outcome relation.^13,14Criticism levelled at the methods used in volume–outcome studies have addressed the lack of adjustment for case mix, different cutoff points for volume categories and retrospective design.³ Other factors that have an effect on patient outcomes but that have not been included in previous volume analyses include health maintenance organization status, physician certification and years since graduation, and patient socioeconomic status, age and ethnicity. Furthermore, most of the studies on volume have covered surgical or oncology specialities.The few studies that have been done on volume and outcome in maternity care have shown variable effects. Rural health care is often associated with lower volumes of obstetric procedures. However, no differences in maternal or newborn outcomes have been shown in some comparisons of births in urban and rural locations.^15,16,17,18 Other studies have shown poorer maternal and newborn outcomes in low-volume hospitals, neonatal intensive care units (NICUs) and rural locations.^19,20,21,22 Conversely, higher volume (hospitals with more than 1000 deliveries per year) has been associated with more maternal lacerations or complications.²³When the health care provider has been the unit of analysis, a relation between volume and maternal or newborn outcome has been demonstrated in at least one study²⁴ but not in others.^25,26 Low volume has been defined as 20 to 24 deliveries per year.^24,26 Hass and colleagues²⁴ reported an adjusted odds ratio (OR) of 1.4 for low birth weight for infants delivered by low-volume non-board-certified physicians relative to high-volume non-board-certified physicians; the adjusted OR was 1.56 for low-volume board-certified physicians relative to high-volume board-certified physicians (98.7% of whom were obstetricians).Possible explanations for the differences among studies include differences in health care delivery systems, insurance coverage, experience and training of providers, maternal risk factors, triage or transfer of high-risk cases, choice of outcome measures, and changes over time in access to care, quality assurance and standard of living. Relations have been reported between maternal or newborn outcomes and smoking, maternal history of low birth weight (for previous pregnancies), pregnancy–induced hypertension, diabetes, prepregnancy weight, gestational weight gain, maternal height and age, multiple gestation, previous vaginal birth after cesarean section, history of previous delivery problems, parity, large-for-date fetus, ethnicity and fetal sex.^25,27,28,29 Few studies of the relation between volume of births and obstetric outcome have been able to control for these potentially confounding variables and adjust for maternal risk factors.Our database of detailed accounts of births in one hospital setting allowed us to examine this issue more rigorously. We posed 2 research questions: Is there a relation between the volume of deliveries attended by individual family physicians and maternal and newborn outcomes? If there are differences in outcomes, are they related to different physician practice styles and consultation patterns?     

Characterization of 14-3-3sigma Dimerization Determinants: Requirement of Homodimerization for Inhibition of Cell Proliferation

The seven highly conserved 14-3-3 proteins expressed in mammalian cells form a complex pattern of homo- and hetero-dimers, which is poorly characterized. Among the 14-3-3 proteins 14-3-3sigma is unique as it has tumor suppressive properties. Expression of 14-3-3sigma is induced by DNA damage in a p53-dependent manner and mediates a cell cycle arrest. Here we show that the 14-3-3sigma protein exclusively forms homodimers when it is ectopically expressed at high levels, whereas ectopic 14-3-3zeta formed heterodimers with the 5 other 14-3-3 isoforms. The x-ray structure of 14-3-3sigma?revealed 5 residues (Ser⁵, Glu²⁰, Phe²⁵, Q⁵⁵, Glu⁸⁰) as candidate determinants of dimerization specificity. Here we converted these amino-acids to residues present in 14-3-3zeta at the analogous positions. Thereby, Ser⁵, Glu²⁰ and Glu⁸⁰ were identified as key residues responsible for the selective homodimerization of 14-3-3sigma. Conversion of all 5 candidate residues was sufficient to switch the dimerization pattern of 14-3-3sigma to a pattern which is very similar to that of 14-3-3zeta. In contrast to wildtype 14-3-3sigma this 14-3-3sigma variant and 14-3-3zeta were unable to mediate inhibition of cell proliferation. Therefore, homodimerization by 14-3-3sigma is required for its unique functions among the 7 mammalian 14-3-3 proteins. As inactivation of 14-3-3sigma sensitizes to DNA-damaging drugs, substances designed to interfere with 14-3-3sigma dimerization may be used to inactivate 14-3-3sigma function for cancer therapeutic purposes.     

Mitotic Phosphorylation of Cdc25B Ser321 Disrupts 14-3-3 Binding to the High Affinity Ser323 Site

Cdc25B is a key regulator of entry into mitosis, and its activity and localization are regulated by binding of the 14-3-3 dimer. There are three 14-3-3 binding sites on Cdc25B, with Ser³²³ being the highest affinity binding and is highly homologous to the Ser²¹⁶ 14-3-3 binding site on Cdc25C. Loss of 14-3-3 binding to Ser³²³ increases cyclin/Cdk substrate access to the catalytic site, thereby increasing its activity. It also affects the localization of Cdc25B. Thus, phosphorylation and 14-3-3 binding to this site is essential for down-regulating Cdc25B activity, blocking its mitosis promoting function. The question of how this inhibitory signal is relieved to allow Cdc25B activation and entry into mitosis is yet to be resolved. Here, we show that Ser³²³ phosphorylation is maintained into mitosis, but phosphorylation of Ser³²¹ disrupts 14-3-3 binding to Ser³²³, mimicking the effect of inhibiting Ser³²³ phosphorylation on both Cdc25B activity and localization. The unphosphorylated Ser³²¹ appears to have a role in stabilizing 14-3-3 binding to Ser³²³, and loss of the Ser hydroxyl group appears to be sufficient to significantly reduce 14-3-3 binding. A consequence of loss of 14-3-3 binding is dephosphorylation of Ser³²³. Ser³²¹ is phosphorylated in mitosis by Cdk1. The mitotic phosphorylation of Ser³²¹ acts to maintain full activation of Cdc25B by disrupting 14-3-3 binding to Ser³²³ and enhancing the dephosphorylation of Ser³²³ to block 14-3-3 binding to this site.     

Conversion of labeled substrates to sugars, cell wall polysaccharides, and tartaric Acid in grape berries

[U-¹⁴C]Sucrose, myo-[U-¹⁴C]inositol, [6-¹⁴C]- and [U-¹⁴C]glucuronate, UDP-[U-¹⁴C]glucuronate, [U-¹⁴C]gluconate, and l-[1-¹⁴C]ascorbic acid were fed into grape berries, Vitis labrusca L. cv. Delaware, at intervals throughout the ripening process and incorporation of ¹⁴C into several metabolites was studied.     

Influence of temperature on the infradian growth rhythm in Ulva lactuca (Chlorophyta)

The vegetative growth of Ulva lactuca was studied to determine if the growth rate of the alga is driven by infradian rhythmicity. The influence of temperature on the infradian rhythm of growth was also investigated. Discs of Ulva were grown in controlled laboratory conditions at different combinations of temperature (5, 10, 15, 20°С) and irradiance (40 and 60 μmol photons m^?2 s^?1) under 12 : 12 h light : dark cycles. The growth rates exhibited a rhythmic pattern with one major peak every 2 or 3 days. Growth at 5 or 10°С increased the prevalence of 3-day cycles and maintained U. lactuca in the vegetative growth stage. In contrast, growth at 15 or 20°С provoked a predominance of the 2-day cycle and induced reproduction. The 2- or 3-day cycles were combined in longer cycles having a period close to 6 days. We suppose that the 2-, 3- and 6-day rhythms of physiological processes are related to large-scale Rossby and Kelvin waves, which produce oscillations in the geomagnetic field and seawater temperature with the same periods. The predominance of 2-day or 3-day fluctuations of the geomagnetic field and temperature probably determine the prevalence of reproduction and vegetative growth, respectively, in Ulva.     

A SOLUBLE PREPARATION FROM RAT BRAIN THAT INCORPORATES INTO ITS OWN PROTEINS [14C]ARGININE BY A RIBONUCLEASE-SENSITIVE SYSTEM AND [14C]TYROSINE BY A RIBONUCLEASE-INSENSITIVE SYSTEM

Abstract— A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G-25-40 was able, after addition of some factors, to incorporate [^I4C]arginine (apparent K_m= 5 μM) and [¹⁴C]tyrosine (apparent K_m= 20 μM) into its own proteins. The factors required for the incorporation of [¹⁴C]arginine were: ATP (optimal concentration = 0-25-2 μM) and Mg²⁺ (optimal concentration 5 mM). For the incorporation of [^I4C]tyrosine the required factors were: ATP (apparent K_m= 0-75 μM), Mg²⁺ (optimalconcentration 8-16 mM) and K⁺ (apparent K_m= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [¹⁴C]arginine and [¹⁴C]tyrosine, respectively, 7-4 and 7-0 in phosphate buffer and 7–9 and 7-3-8-1 in tris-HCl buffer. Pancreatic ribonuclease abolished the incorporation of [¹⁴C]arginine but had practically no effect in the incorporation of [¹⁴C]tyrosine. Furthermore, [¹⁴C]arginyl-tRNA was a more effective donor of arginyl groups than [¹⁴C]arginine, whereas [¹⁴C]tyrosyl-tRNA was considerably less effective than [¹⁴C]tyrosine. The incorporations of [¹⁴C]arginine and [¹⁴C]tyrosine into brain proteins were from 25- to 2000-fold higher than for any other amino acid tested (12 in total). In brain [¹⁴C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [¹⁴C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [¹⁴C]Arginine was bound so that its a-amino group remained free. Maximal incorporation of [¹⁴C]tyrosine in brain of 30-day-old rats was about one-third of that in the 5-day-old rat. The changes with postnatal age in the incorporation of [¹⁴C]arginine were not statistically significant.     

Biosynthesis of C(20) and C(22) Fatty Acids by Developing Seeds of Limnanthes alba: CHAIN ELONGATION AND Delta5 DESATURATION

The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C₂₀ and C₂₂ fatty acids, which contain an unusual Δ5 double bond. When [1-¹⁴C]acetate was incubated with developing seed slices, ¹⁴C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13-docosenoate, and cis-5,cis-13-docosadienoate. The label from [¹⁴C]acetate and [¹⁴C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[¹⁴C]11-eicosenoate, cis-13-[¹⁴C]docosenoate, and cis-5,cis-13-[¹⁴C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C₂₀ and C₂₂ acyl species. The Δ5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-¹⁴C-labeled free fatty acids. Using [1-¹⁴C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate.     

Characteristics of photosynthesis in maize leaves (С<Subscript>4</Subscript> plants) upon changes in the level of illuminance and nitrate nutrition

We studied assimilation of ¹⁴СО₂ and distribution of ¹⁴С among the products of 3-min-long photosynthesis of maize (Zea mays L.) leaves. The day before the experiment, half of the plants were fertilized with Ca(NO₃)₂ (1 g/L of water) at a rate of 6 L/m². Five days before the experiment, some plants were shaded for adaptation (illuminance was reduced by 50%). On the day of the experiment (before the application of ¹⁴СО₂), several shaded plants were exposed to direct sunlight for 3 min, and some plants grown at full light (light plants) were shaded for 3 min (illuminance of 50%). Unfertilized plants adapted for 5 days to shading showed photosynthesis of 75.9% of control level (full light). If light plants were transferred to shading for 3 min, their photosynthesis decreased to 42.1%. In plants shaded for 5 days and then transferred to full light, photosynthesis in 3 min was 96.3% of control level. At full light, fertilization with nitrate boosted photosynthesis to 132.6% as compared with control material, but photosynthesis decreased to 43.5 and 65.4% of control level in plants shaded for 5 days and those shaded for 3 min, respectively. At the same time, the plants shaded for 5 days and then exposed for 3 min to full light restored photosynthesis to almost control level (95.5%). Analysis of ¹⁴С distribution among the products of 3-min-long photosynthesis showed that, the same as in C₃ plants, a decrease in illuminance (especially a sudden one) in maize reduced the ratio between labeled sucrose and hexoses and elevates incorporation of ¹⁴С into malate, which indicated that its consumption in bundle sheath cells was suppressed. A decrease in the ratio between labeled sucrose and hexoses became more pronounced under the influence of nitrates with this effect also occurring in transport products of photosynthesis (20 cm below ¹⁴С-providing leaf area). In plants fertilized with nitrates, radioactivity of sucrose (% of radioactivity of soluble compounds) decreased in all the types of illumination. When illuminance was suddenly reduced for 3 min, incorporation of ¹⁴С into sucrose was 21.5 against 51.2% in light plants, and radioactivity of aspartate and malate sharply rose to 13.7 and 26.1% (against 2.1 and 8.9% in control material). Incorporation of ¹⁴С into compounds of glycolate pathway was low (less than 2.5%), but it was somewhat greater in nitrate plants. We concluded that the same mechanism of interaction between stomatal apparatus of leaf epidermis, invertase of mesophyll apoplast, and photosynthetic metabolism of carbon with electron flux via electron transport chain in chloroplasts of bundle sheath cells, which governs the rate of photosynthesis and assimilate export from the leaf but is triggered by the extent of consumption in the bundle sheath cells of C₄ acids produced in the mesophyll operates in C₄ plants (the same as in C₃ plants).     

In vivo labeling of myelin lipids and proteolipid protein with [3H]myristate, [14C]linoleate,and [14C]linolenate

To investigate the incorporation of essential fatty acids into myelin components, 24-day-old rabbits were injected intracerebrally with [¹⁴C]linoleate, [¹⁴C]linolenate, or [³H]Myristate for comparison. Animals were killed 22 hr later and myelin was isolated. [³H]myristate labeled all myelin lipids including monogalactosyl diglyceride, with the exception of sulfatides. With¹⁴C-essential fatty acids, only glycerophospholipids were efficiently labeled and their specific activities were in the following decreasing orders: PC>PI>PE>PS with [¹⁴C]linoleate, and PE>PC>PI=PS with [¹⁴C]linolenate. Among myelin proteins, PLP and DM-20 were labeled with all 3 precursors. PLP was purified from myelin labeled with¹⁴C-essential fatty acids. The label was then cleaved from the protein by alkaline methanolysis and was identified as a dienoic ([¹⁴C]linoleate) or a tetraenoic ([¹⁴C]linolenate) fatty acid. MBP was not labeled with [³H]myristate, but was slightly labeled with both¹⁴C-essential fatty acids. The signification of the latter result is discussed.Abbreviations FA fatty acid(s) - HPTLC high-performance thin-layer chromatography - MBP myelin basic protein - PLP proteolipid protein - PC phosphatidylcholine - PE phosphatidylethanolamine and ethanolamine plasmalogens - PI phosphatidylinositol - PS phosphatidylserine - SDS sodium dodecylsulfate     

Stereoselective pharmacokinetics of [14C]-labeled KE-298, a new anti-rheumatic drug,in rats

The stereoselective pharmacokinetics of two enantiomers of [¹⁴C]-labeled KE-298 [2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanonic acid] were investigated in rats. The blood levels of radioactivity after the oral administration of (+)-(S)-[¹⁴C]KE-298 were higher than that for (−)-(R)-[¹⁴C]KE-298; the AUC of the former was approximately twice that of the latter. No significant stereoselectivity was observed in absorption rate. The tissue/plasma level ratios at 30 min after oral administration of (−)-(R)-[¹⁴C]KE-298 in the liver and kidney, the major metabolic and/or excretory organs, were 2 to 3 times higher than those for (+)-(S)-[¹⁴C]KE-298. Neither was evidence of stereoselectivity found in the excretion of radioactivity. During incubation with isolated rat hepatocytes in vitro, the metabolic rates of KE-298 enantiomers were not significantly different. Plasma protein binding 30 min after the oral administration of (+)-(S)-[¹⁴C]KE-298 and (−)-(R)-[¹⁴C]KE-298 was 99.3% and 97.0%, respectively. Comparing the unbound fraction, (−)-(R)-[¹⁴C]KE-298 was approximately 4 times higher than (+)-(S)-[¹⁴C]KE-298. In order to make clear the relationship between stereoselective pharmacokinetics and protein binding for [¹⁴C]KE-298, the comparative pharmacokinetics of (+)-(S)-[¹⁴C]KE-298 and (−)-(R)-[^14CC]KE-298 were investigated in analbuminemic rats. In these animals, no evidence of stereoselectivity was found for either blood level-time profiles or plasma protein binding. These results revealed that the stereoselective pharmacokinetics of KE-298 in rats might be due to enantiomeric differences in binding to plasma albumin. © 1996 Wiley-Liss, Inc.