Isolation of Extracellular Cobalt-free Corrinoid from Methanosarcina barkeri

Cobalt-free corrinoids (CFCs) were isolated from Methanosarcina barkeri Fusaro cells growing on a methanol minimum medium. The methanogen cells excreted a trace of CFCs (9.1 μg/I) into the culture medium when cobalt-deficient methanol medium was used. Several CFCs were separated by column chromatographies on ion exchangers and paper electrophoresis, where a major CFC showed a similar characteristic to that of nucleotide-free corrinoid, Factor B (cobinamide), suggesting to be hydrogenobinamide. By chemical insertion of Co^{2 +}, Cu^{2 +}, and Zn²⁺ into CFCs, the corresponding corrinoid and its metal analogues were observed. Bioassay using Escherichia coli 215 revealed that the major CFC (a yellow product obtained after alkaline treatment) and its copper and zinc analogues were inactive as cobalamin but were active as antimetabolites of cobalamin. However, the CFC greatly stimulated the cell growth of M. barkeri grown under cobalt-deficient conditions.     

Photosynthesis of Euglena gracilis under Cobalamin-Sufficient and -Limited Growing Conditions

Cobalamin is essential for growth of Euglena gracilis and photosynthesis. Methylcobalamin in Euglena chloroplasts (Y Isegawa, Y Nakano, S Kitaoka, 1984 Plant Physiol 76: 814-818) functions as a coenzyme of methionine synthetase. The requirement of cobalamin for photosynthesis appeared remarkably high in Euglena grown under the dark-precultured condition. The required amount of cobalamin for normal photosynthetic activity was 7.4 × 10⁻¹¹ molar, while 7.4 × 10⁻¹⁰ molar cobalamin was required for normal growth. The lowered photosynthetic activity in cobalamin-limited cells was restored 20 hours after feeding cyanocobalamin or methionine to cobalamin-limited cells. Lowering of photosynthetic activity was due to loss of photosystem I activity. This photosynthetic activity was recovered after supplementation by methionine or cobalamin. The results suggest that methionine serves for the stabilization of photosystem I. This paper is the first report of the physiological function of cobalamin in chloroplasts of photosynthetic eukaryotes.     

The assay, properties, and kinetics of human T cells forming clusters with sheep erythrocytes (CFC) in stimulated cultures.

This study describes a new method of detecting the in vitro stimulation of lymphocytes based on the appearance of cells having the property to cluster several layers of SRBC around themselves (CFC). The formation of multilayer rosettes is temperature dependent and requires trypan-blue-excluding, metabolically active blastoid cells. Non-separated cells as well as purified T cells stimulated with allogeneic leucocytes (MLR), specific antigens, or polyclonal mitogens (PWM, Con A) gave rise to CFC. Multilayer rosettes were not formed by separate B cells. In the MLR, CFC were detected 48 hr after the beginning of cultures and increased in number thereafter just like thymidine incorporation. The response to Con A and PWM was detected earlier and gave rise to two or three peaks, the first rise in the number of CFC coinciding with the peak of thymidine incorporation but the maximum increase occuring two or three days later. Treatment of blastoid cells with a serum specific for T cells, but not with an anti-immunoglobulin serum, abolished their ability to form ordinary or multilayer rosettes. When separated, however, on a nylon wool column, CFC were more adherent than the bulk of T cells. It is suggested that CFC form a subpopulation of T cells with distinct characteristics, allowing a direct assessment of membrane changes which take place when T lymphocytes are activated in vitro.     

Effect of a corrinoid on <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis> DNA synthesis

Methanosarcina barkeri is capable of synthesizing large amounts of corrinoids, compounds of the vitamin B₁₂ group, although not cobalamin. In the present work, exogenous cobalamin was demonstrated to upregulate DNA synthesis in M. barkeri cell suspensions incubated under air. The effect is similar to the one in Propionibacterium freudenreichii cells, though less pronounced. The growth of the archaeon under anaerobic conditions was shown to be suppressed by cobalamin and 5,6-dimethylbenzimidazole. The data obtained suggest the presence of a corrinoid-dependent ribonucleotide reductase in the archaeal cells which provides for deoxyribose precursors for DNA biosynthesis independently of the presence of molecular oxygen in the medium. Growth suppression under anoxic conditions by cobalamin and 5,6-dimethylbenzimidazole may be due to a decrease in the concentration of factor III, a polyfunctional corrinoid dominating in M. barkeri cells.     

Aquatic metagenomes implicate Thaumarchaeota in global cobalamin production

Cobalamin (vitamin B₁₂) is a complex metabolite and essential cofactor required by many branches of life, including most eukaryotic phytoplankton. Algae and other cobalamin auxotrophs rely on environmental cobalamin supplied from a relatively small set of cobalamin-producing prokaryotic taxa. Although several Bacteria have been implicated in cobalamin biosynthesis and associated with algal symbiosis, the involvement of Archaea in cobalamin production is poorly understood, especially with respect to the Thaumarchaeota. Based on the detection of cobalamin synthesis genes in available thaumarchaeotal genomes, we hypothesized that Thaumarchaeota, which are ubiquitous and abundant in aquatic environments, have an important role in cobalamin biosynthesis within global aquatic ecosystems. To test this hypothesis, we examined cobalamin synthesis genes across sequenced thaumarchaeotal genomes and 430 metagenomes from a diverse range of marine, freshwater and hypersaline environments. Our analysis demonstrates that all available thaumarchaeotal genomes possess cobalamin synthesis genes, predominantly from the anaerobic pathway, suggesting widespread genetic capacity for cobalamin synthesis. Furthermore, although bacterial cobalamin genes dominated most surface marine metagenomes, thaumarchaeotal cobalamin genes dominated metagenomes from polar marine environments, increased with depth in marine water columns, and displayed seasonality, with increased winter abundance observed in time-series datasets (e.g., L4 surface water in the English Channel). Our results also suggest niche partitioning between thaumarchaeotal and cyanobacterial ribosomal and cobalamin synthesis genes across all metagenomic datasets analyzed. These results provide strong evidence for specific biogeographical distributions of thaumarchaeotal cobalamin genes, expanding our understanding of the global biogeochemical roles played by Thaumarchaeota in aquatic environments.     

Effects of a dietary crude fibre concentrate on growth in weaned piglets

Many fibre sources can help the adaptation of piglets at weaning, improving the growth. In this study, the effects of a dietary crude fibre concentrate (CFC) on piglet’s growth was investigated. From 31 to 51 days of age, 108 weaned piglets (D×(Lw×L)), had access to two isofibrous, isoenergetic and isonitrogenous diets, supplemented with 1% of CFC (CFC group) or not (control (CON) group). From days 52 to 64 all piglets received the same starter diet. During the dietary treatment period the CFC group showed higher average daily gain, average daily feed intake and feed efficiency (P<0.001) than CON group. At 64 days of age, BW was higher in CFC group compared with CON group (P<0.001). Blood samples were collected at days 31, 38, 45 and 52 of age. From days 31 to 52 significant differences in the somatotropic axis between groups were observed. In particular, growth hormone levels were higher only at the end of the 1^st week of dietary treatment (P<0.05) in CFC group animals compared with CON group animals. The IGF-I trend was similar between groups even if the IGF-I levels were higher in the CFC group than CON group 1 week after starting treatment (P<0.01). The IGF-binding protein 3 (IGFBP-3) levels were higher in the first 2 weeks of dietary treatment and lower in the 3^rd week in CON group compared with CFC group (P<0.01). Specifically, the IGFBP-3 profile was consistent with that of IGF-I in CFC group but not in CON group. At the same time, an increase of leptin in CFC compared with CON group was observed (P<0.05). Piglets fed the CFC diet showed a lower diarrhoea incidence (P<0.05) and a lower number of antibiotic interventions (P<0.05) than CON diet from 31 to 51 days of age. Pig-major acute-phase protein plasma level (P<0.01) and interleukin-6 gene expression (P<0.05) were higher in CON group than CFC group at the end of 1^st week of dietary treatment. In conclusion, this study showed that CFC diet influences the hormones related to energy balance enhancing the welfare and growth of piglets. Furthermore, the increase in feed intake during 3 weeks of dietary treatment improved the feed efficiency over the entire post-weaning period.     

High affinity binding of the transcobalamin II–cobalamin complex and mRNA expression of haptocorrin by human mammary epithelial cells

Little is known about the acquisition of cobalamin by the mammary gland and its secretion into milk. Human milk and plasma contain at least two types of cobalamin binding proteins: transcobalamin II (TC) and haptocorrin (HC). In plasma, TC is responsible for the transport of cobalamin to tissues and cells; however, cobalamin in milk is present exclusively bound to HC. We show that human mammary epithelial cells (HMEC) exhibit high affinity for TC; Scatchard analysis revealed a single class of binding sites for the TC–[⁵⁷Co]cyanocobalamin complex with a dissociation constant (K_d) of 4.9×10⁻¹¹ M. Uptake of the TC–[⁵⁷Co]cyanocobalamin complex at 37°C was saturable by 24 h. Binding of free [⁵⁷Co]cyanocobalamin to HMEC was not saturable and very limited binding of the HC–[⁵⁷Co]cyanocobalamin complex was observed. Expression of the haptocorrin gene by HMEC was confirmed by Northern blot and PCR analysis. Thus, a specific cell surface receptor for the TC–cobalamin complex exists in the mammary gland and once cobalamin is internalized, it may be transferred to HC and subsequently secreted into milk as a HC–cobalamin complex.     

The generation of colony-forming cells (CFC) and the expansion of hematopoiesis in cultures of human cord blood cells is dependent on the presence of stem cell factor (SCF)

We have analyzed the effect of stem cell factor (SCF), alone or in combination with other growth factors, on the generation of colony-forming cells (CFC) and on the expansion of hematopoiesisin vitro from light density, soybean agglutinin^–, CD34⁺ cord blood cells under serum-deprived conditions. The growth factors were either added only once at the onset of the culture or added every few days when the cultures were demidepopulated and refed with fresh medium. No growth factor, alone, generated CFC or expanded hematopoiesis under these conditions. However, SCF, in combination with interleukin 3 (IL-3) or with late-acting factors (granulocyte colony-stimulating factor (G-CSF) or erythropoietin (Epo)), generated large numbers of mature cells as well as CFC. The number of CFC generated depended on the refeeding procedure adopted. In cultures never refed, the CFC numbers increased from > 160 CFC/culture at day 0 to > 3000 CFC at day 10. The CFC numbers stayed above the input levels for 25 days before declining. Almost no CFC were detectable after one month. In contrast, in cultures regularly refed, CFC were detectable for at least 40 days. The lineages of the mature cells and the types of CFC generated varied with the different growth factors. In the presence of SCF plus IL-3, erythroid burst-forming cells (BFU-E) and granulocyte/macrophage colony-forming cells (GM-CFC) were generated and erythroid as well as myelomonocytic precursors were present among the differentiated cells. In contrast, in the presence of SCF and G-CSF or Epo, the progenitor cells as well as the differentiated cells were dictated by the late-acting growth factor (i.e. mostly G-CFC and myeloid cells in the presence of SCF and G-CSF vs. BFU-E, erythroid colony-forming cells (CFU-E) and erythroblasts in the presence of SCF and Epo). Thus, marked expansion of erythropoiesis and granulopoiesis can be achievedin vitro by as few as two factors — SCF acting as the early factor along with the appropriate late-acting factor.Paper presented in part at the World Congress on Cell Cultures, Washington D.C., 21–24 June 1992.     

Radiosensitivity increases with differentiation status of murine hemopoietic progenitor cells selected using enriched marrow subpopulations and recombinant growth factors

The radiosensitivity of populations of colony-forming cells (CFC) in murine bone marrow was investigated using different recombinant colony-stimulating factors (CSFs; murine IL-3 and granulocyte-macrophage CSF and human granulocyte CSF), or purified murine macrophage CSF. With unfractionated normal bone marrow the CFC increased in radiosensitivity as they progressed through the granulocyte lineage. The D0 values ranged from 129 +/- 12 cGy for CFC stimulated with GM-CSF down to 42 +/- 2 cGy after stimulation with G-CSF. IL-3 stimulated a CFC population which gave the only survival curve with a shoulder (n = 1.9 +/- 0.3). With semipurified populations of primitive or bipotential CFC, D0 values were generally lower with respect to the equivalent values for unpurified bone marrow (range 62 +/- 7 cGy to 135 +/- 7 cGy). Changes in cluster/colony ratio and colony morphology together possibly with products of accessory cells influence the interpretation of the radiosensitivity parameters.     

Long-term effects of cryopreservation on clinically prepared hematopoietic progenitor cell products

Background aimsThe question of how long hematopoietic progenitor cells (HPCs) destined for clinical applications withstand long-term cryopreservation remains unanswered. To increase our basic understanding about the stability of HPC products over time, this study focused on characterizing long-term effects of cryopreservation on clinically prepared HPC products.MethodsCryovials (n = 233) frozen for an average of 6.3 ± 14.2 years (range, 0.003–14.6 years) from HPC products (n = 170) representing 75 individual patients were thawed and evaluated for total nucleated cells (TNCs), cell viability, viable CD34+ (vCD34+) cells and colony-forming cells (CFCs). TNCs were determined by use of an automated cell counter, and cell viability was measured with the use of trypan blue exclusion. Viable CD34 analysis was performed by means of flow cytometry and function by a CFC assay.ResultsSignificant losses in TNCs, cell viability, vCD34+ cells and CFC occurred on cryopreservation. However, once frozen, viable TNCs, vCD34+ cells and CFC recoveries did not significantly change over time. The only parameter demonstrating a change over time was cell viability, which decreased as the length of time that an HPC product was stored frozen increased. A significant negative correlation (correlation coefficient = −0.165) was determined between pre-freeze percent granulocyte content and post-thaw percent viability (n = 170; P = 0.032). However, a significant positive correlation was observed between percent viability at thaw and pre-freeze lymphocyte concentration.ConclusionsOnce frozen, HPC products were stable for up to 14.6 years at <−150°C. Post-thaw viability was found to correlate negatively with pre-freeze granulocyte content and positively with pre-freeze lymphocyte content.     

Ocular,neurologic and hematologic manifestations of an inherited abnormality in vitamin B12 metabolism: case study and review of the literature

A deficiency of vitamin B₁₂ or cobalamin is well documented to cause both neurologic deficits and anemia. These clinical manifestations are particularly extensive when the lack of cobalamin occurs at birth. The profound deficits in Cobalamin C deficiency originate from the inability of the body's cells to transform inactive cobalamin obtained from the diet into an active form necessary for the proper growth and development of the child. The relevant biochemistry and physiology of cobalamin which functions in vital bodily processes is discussed. This serves to explain the laboratory abnormalities and some of the neurologic and ocular manifestations of cobalamin deficiency.     

小鼠胚胎干细胞来源的HPP-CFC体外和体内造血活性分析

小鼠的造血系统起源于胚胎发育7d的卵黄囊胚外中胚层,研究表明胚胎干细胞（Embryonic stem cells, ES cells）体外分化模型能够模拟卵黄囊造血的发生过程;此外,诱导ES细胞体外定向造血细胞分化对于建立治疗性克隆以治愈多种血液病具有重要的研究和应用价值。高增殖潜能集落形成细胞（High proliferative potential colonyforming cells, HPPCFC）是体外培养的最原始的多潜能造血前体细胞之一。本研究发现：小鼠ES细胞在体外分化5～14d形成的拟胚体中含有HPP-CFC。其再生潜能与胚胎期9d的卵黄囊来源的HPP-CFC相似,与骨髓来源则不同。RT-PCR分析表明：ES细胞来源的HPP-CFC表达与造血干细胞增殖相关的特异性转录因子和多种造血生长因子受体。但分化12d的拟胚体细胞和HPP-CFC集落细胞移植受致死剂量照射的小鼠不能产生典型的脾结节。因此,ES细胞来源的HPP-CFC在体外和体内造血活性的差异值得更深入地研究。     

Kinetics of cobalamin repression of the cob operon in Salmonella typhimurium

Abstract The cob operon in Salmonella typhimurium encodes 25 proteins involved in the biosynthesis of cobalamin. Expression of the cob operon is negatively feedback regulated by cobalamin via a translational control mechanism. The concentration of cobalamin required to repress cob expression to half-maximal was determined in vivo and in vitro to 0.4 μM and 0.6 μM, respectively. These results suggest that cob expression in wild-type cells is partially repressed by de novo synthesized cobalamin.     

Interdependence of calcium and cobalamin binding by wild-type and mutant BtuB protein in the outer membrane of Escherichia coli.

The binding of calcium and cobalamin to outer membranes from cells of Escherichia coli that contained amplified levels of wild-type or mutant btuB was studied. The mutant (BBam50) had an aspartyl-prolyl dipeptide inserted after the original 50th amino acid residue of the mature BtuB protein, which is within a region that shows extensive homology with the ferric siderophore receptors. This insertion resulted in cleavage of the BtuB in two places. The larger form retained the insertion but had lost 11 amino acid residues from the amino terminus. The smaller form was cut at the insertion site. Both the wild-type protein and the larger form of mutant BtuB showed calcium-dependent cobalamin binding with the same affinity for cobalamin, although the mutant had a much lower affinity for calcium. The smaller form of the mutant BtuB protein had a greatly reduced affinity for cobalamin, which was probably the result of inactivation of the cobalamin-dependent calcium-binding site. Cobalamin-dependent calcium binding was measured in wild-type BtuB preparations and was found to have the same corrinoid specificity and response to various corrinoid concentrations as shown previously for cobalamin binding. The results are consistent with a role for calcium in the cobalamin pump of the outer membrane of E. coli and show that a conserved part of the BtuB protein is required for the cobalamin-dependent binding of calcium.     

Age-dependent macrophage functions in New Zealand black mice.

Various functions of macrophage derived from young (2-month-old) and old (14- to 17-month-old) New Zealand Black (NZB) mice with autoimmune disease were studied and compared with macrophage functions of age-matched BALB/c mice. Macrophages from young and old NZB mice demonstrated elevated levels of β-glucuronidase, cathepsin D, lysozyme, and DNase compared with those from age-matched BALB/c. DNase activity in the macrophages of NZB mice significantly increased with age. Macrophages from young and old NZB mice had greater phagocytic capacity for both ¹²⁵I-labeled Shigella flexneri and Staphylococcus albus than did BALB/c macrophages. NZB macrophages from both young and old mice had higher bactericidal activity against S. albus than those from age-matched BALB/c mice. The number of macrophage/granulocyte colony-forming cells (CFC) in both bone marrow and spleen was markedly higher in young and old NZB mice than in BALB/c mice. Colony-stimulating factor (CSF) released by macrophages derived from NZB mice had higher CFC activity than that released from macrophages of age-matched BALB/c mice. In NZB mice, the CSF activity significantly increased with age. It is suggested that potentiation of macrophage number and activity compensates for the deficiency of T cell functions in NZB mice with autoimmune disease.     

Survival of human colony-forming cells (CFC) after freezing

To examine the ability of human macrophage-granulocyte colony-forming cells (CFC) to survive a defined freeze-thaw procedure, we cultured fresh and cryopreserved marrow cells from the same specimens in a methylcellulose system. The cultures were stimulated by two kinds of conditioned medium, fibroblast (FCM) or leukocyte (LCM). We have shown previously that LCM stimulated CFC smaller in size than those stimulated by FCM. Cultures of cryopreserved and fresh cells stimulated by LCM showed no significant difference either in the number of colonies formed or in colony formation kinetics. In contrast with FCM stimulation there was a significant decrease in colony number in cryopreserved specimens, and in addition, the kinetics of colony formation between cryopreserved and fresh specimens were distinctly different. Cryopreserved specimens stimulated with serially diluted FCM or LCM always exhibited their respective kinetic pattern of colony formation. Cell size distribution analysis and morphological examination of fresh and cryopreserved cells revealed that following freeze-thaw there was a marked reduction in cells 9 μm or larger, and that there was a shift in the morphological cell differential from myeloid to lymphoid predominance. From these observations, we concluded that CFC responsive to LCM stimulation survived the freeze-thaw procedure better than CFC responsive to FCM stimulation.     

GROWTH OF STEM CELL CONCENTRATES IN DIFFUSION CHAMBERS (DC)

The growth in diffusion chamber (DC) of normal murine marrow and marrow separated by discontinuous albumin centrifugation was studied. The colony-forming cells (CFC) assayed in soft agar, total cell counts and differentials were measured in the DC over a 19 day period after intraperitoneal implantation into CF₁ mice. Growth of implants of normal marrow or fraction 3 (F3) in which CFC had been concentrated 1.7–3.9-fold were compared at an initial cell concentration of 1 × 10⁵. There was a good correlation between the number of CFC implanted with granulocyte production but not with macrophage production. When higher cell concentrations of normal unfractionated marrow were implanted growth was reduced as was recovery of CFC. In two experiments in which both CFU and CFC concentrations were measured there was a general correlation between the two.     

Molecular characterization of cycloinulooligosaccharide fructanotransferase from Bacillus macerans

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of beta-(2-->1)-linked D-fructofuranose by catalyzing an intramolecular transfructosylation reaction. The CFTase gene was cloned and characterized from Bacillus macerans CFC1. The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculated Mr of 149,563. Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the B. macerans CFC1 cells. The processed CFT107 was similar in its mass to the previously purified CFTase from B. macerans CFC1. The CFT107 enzyme was produced by B. macerans CFC1 but was not detected from the recombinant Escherichia coli cells, indicating that the processing event occurred in a host-specific manner. The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction. However, the thermal stability of CFT107 was slightly higher than that of CFT150. The most striking difference between the two enzymes was observed in their Km values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM). Thus, the specificity constant (kcat/Km) of CFT150 was about fourfold higher than that of CFT107. These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.