Growth of cultured cells from patients with Fanconi anemia.

Fibroblast cultures derived from skin biopsies of patients with Fanconi anemia had doubling times (mean of five lines: 30.3 +/- 0.2 hours) significantly longer than randomly selected normal controls (mean of nine lines: 22.9 +/- 0.4 hours). Control cultures grew more slowly in the enriched media RPMI 1640 and McCoy's 5A than in MEM; while a culture from a patient with Fanconi anemia grew more slowly only in McCoy's 5A. Differences in growth characteristics between Fanconi anemia and normal cell cultures may be useful in analyzing the metabolic error determined by the Fanconi anemia gene.     

Growth of cultured cells from patients with ataxia-telangiectasia

Fibroblast cultures derived from skin biopsies of patients with ataxia-telangiectasia had doubling times (mean of five lines: 29.9 ± 0.6 hours) significantly longer than normal controls (means of ten lines: 22.4 ± 0.5 hours).     

Studies on Down's syndrome in tissue culture. I. Growth rates and protein contents of fibroblast cultures

Skin fibroblast cultures from six patients with Down's syndrome (Trisomy 21) were compared with four in vitro age-matched normal fibroblast cultures. Growth rates were calculated from increases in cell number and total protein during exponential growth, early in culture lifetime (less than 20 doublings). The Down's syndrome (D.S.) cultures had an average population doubling time of 35.6 ± 1.1 hours and average mass doubling time of 38.6 ± 3.2 hours, significantly lower (p<0.005) than the corresponding normal culture values of 23.0 ± 0.7 hours, and 23.3 ± 1.9 hours. D. S. Cells also contained 4.46 ± 0.19 ± 10^?4 μg protein/cell as compared to 3.06 ± 0.13 × 10^?4 μg/cell (p<0.001) for normal fibroblasts. Similar in vitro observations of increased doubling time and protein content have been reported in normal fibroblasts from older donors, and from individuals with premature aging syndromes, as well as in normal fibroblasts near the end of their in vitro lifetime. The present results, obtained from cultures young in vitro, may therefore suggest that D.S. fibroblast cultures age prematurely. This hypothesis is consistent with clinical manifestations of premature aging in D.S. patients and points to a defect in growth regulation, both in vivo and in vitro, resulting from an extra copy of chromosome 21.     

Reduced uptake and incorporation of 3H-thymidine in fanconi anemia fibroblasts

Summary Uptake of ³H-thymidine and its incorporation into DNA was studied in fibroblastic cell lines derived from normal individuals, patients with Fanconi anemia, and those heterozygous for this genetic trait. Uptake and incorporation for the normal cells were about five and seven times higher, respectively, than for Fanconi anemia fibroblasts; mean values for heterozygotes were intermediate. This effect was dependent on the duration of cell exposure to ³H-thymidine and was not observed with other labeled compounds. Thus, a genetically-determined metabolic defect may exist in Fanconi anemia patients which can be readily studied at the cellular level. This finding may be relevant to the observed clinical, cytogenetic, biochemical, and biologic properties related to expression of the Fanconi anemia gene.     

Alteration of a nuclease in Fanconi anemia.

Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a pI of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.     

类风湿关节炎拇长屈肌腱病变的超声特点

目的:总结类风湿关节炎(RA)患者拇长屈肌腱(FHL)病变的超声特点及二者间的相关性。方法:回顾性分析2010年2月至2011年6月因足部疼痛于我院就诊的60例(120足)RA患者资料。应用彩色多普勒超声诊断仪探查拇长屈肌腱,根据超声探查拇长屈肌腱病变情况的结果将患者分为3组:肌腱完整组(A组,45足),肌腱周围炎症组(B组,49足),肌腱断裂组(C组,26足)。对拇长屈肌腱病变的特点及部位进行描述,并将拇长屈肌腱病变严重程度与患者年龄及病程进行相关性分析。结果:45足肌腱完整,49足出现不同部位的肌腱周围炎症(内踝转折处17足,henrry’s结节处11足,第一跖趾关节跖侧21足),26足发生肌腱断裂(9足发生于内踝转折处,17足发生于跖趾关节跖侧)。肌腱病变的发生率71%。A组平均年龄49.9±9.2岁,病程4.7±2.6年;B组平均年龄56.2±9.2岁,病程16.2±7.4年;C组平均年龄54.7±8.0岁,病程20.9±4.4年。三组间上述参数差异具有统计学意义(P0.05)。结论:拇长屈肌腱是类风湿关节炎足部结构的常见受累部位,其病变多发生于内踝转折处,henrry's结节处及第一跖趾关节跖侧,但henrry’s结节处的断裂少见。在对类风湿关节炎平足患者进行肌腱转位手术时,应充分考量拇长屈肌腱的病变。     

Fanconi anemia complementation group C is required for proliferation of murine primordial germ cells

Fanconi anemia is a polygenic trait hypothesized to be a DNA damage repair disease. We show that all three Fanconi anemia loci that have been cloned are expressed in the embryonic gonad during the period of primordial germ cell proliferation. Mice mutant for the Fanconi anemia complementation group C locus (Fancc) have reduced germ cell numbers as early as embryonic day E12.5, suggesting the Fancc protein functions prior to meiosis in both sexes. Depletion in the mutant occurs at a time when all three loci would be expressed in a wild-type gonad, implying a function in the early germline. Determination of the mitotic index of primordial germ cells by BrdU incorporation shows that germ cells in Fancc(-/-) mice proliferate significantly more slowly than littermate controls. This study demonstrates Fancc is required for mitotic proliferation of primordial germ cells.     

Normal Monocular and Binocular Visual Acuity in Seven-Year-Old Children

The purpose of this study was to obtain age-specific normative data for visual acuity (VA) in sevenyear- old children. The sample included a total of 127 healthy children (mean age 7.53 ± 0.025 years) without ophthalmic disorders. Monocular and binocular VAs were determined to the threshold level at two distances, 5 m and 0.5 m, using eye charts with eight intermediate levels between 1.0 and 2.4 (in the decimal notation system) with widely spaced tumbling Snellen’s E optotypes randomly distributed in four orientations. Since the optotypes were located in lines at intervals that were twice as large as the size of the optotype itself, they could be considered as single stimuli. The normal monocular and binocular VAs were 1.279 ± 0.025 and 1.491 ± 0.028 at 5 m and 1.256 ± 0.024 and 1.388 ± 0.025 m at 0.5 m, respectively (mean ± standard error of mean). Interocular differences in VA are also discussed. Our findings show that the normal VA in seven-yearold children is higher than the clinically accepted norm of 1.0; therefore, VA measurement to the threshold level is necessary for earlier visual disorders to be detected.     

Conditionally lethal mutations in Chinese hamster cells. I. Isolation of a temperature-sensitive line and its investigation by cell cycle studies

The isolation of a temperature sensitive cell line from the Chinese hamster line CCL39 of the American Type Culture Collection is described. At the nonpermissive temperature (39°C) the cells become attached to the surface of tissue culture dishes, but no microscopically observable colonies are formed upon prolonged incubation. Exposure to the high temperature for more than 24 hours leads to an almost complete loss in viability. A karyotypic analysis showed that this new line has lost one of the medium-sized metacentric chromosomes, although no proof is available so far to show that this loss is not simply coincidental. In nonsynchronized cultures transferred to 39°C DNA synthesis stops first, RNA synthesis shortly thereafter, while protein synthesis (turnover) continues for a longer time. After such a shift the cell number increases by less than 15% as measured with the Coulter counter. Studies with synchronized cultures give the following results: (1) one round of DNA synthesis can occur at 39°C when the cells are released from serum starvation or a hydroxyurea block, or when mitotic cells are placed at 39°C; (2) the entry of cells into metaphase of mitosis at 39°C is almost normal when the preceding time interval at 39°C is only eight hours (release of cells from G₁/S boundary), but considerably reduced when the cells spend an additional 12 to 15 hours at 39°C in G₁ (release from serum starvation). Infection by SV40 virus temporarily induces DNA synthesis after it has come to a stop at the nonpermissive temperature, but cells permanently transformed by SV40 still exhibit the temperature-sensitive phenotype.     

Interactions between a seed‐eating neotropical rodent,the Azara's agouti (Dasyprocta azarae), and the Brazilian ‘pine’ Araucaria angustifolia

We studied the seed predation and scatter‐hoarding behaviour of Azara's agoutis Dasyprocta azarae (Rodentia: Dasyproctidae) in relation to the seeds of the Brazilian ‘pine’, Araucaria angustifolia (Araucariaceae), the rodent's main winter food source. We compared seed‐removal rates, seed‐caching rates, cache distances and recovery rates between a summer period of food abundance (with a low demand for A. angustifolia seeds and no such seeds naturally available) and a winter period of food scarcity (with a high demand for A. angustifolia seeds). We investigated whether the relative seed value affected the rodent's seed‐handling behaviour. We predicted that during the high seed‐demand period (winter): (1) cache distances would be greater; (2) fewer seeds would be stored; (3) more seeds would be recovered and the seed‐recovery time would be lower. In support of our first two predictions, the caching distances were greater in winter (mean ± SE = 15.67 ± 5.11 m) than in summer (9.40 ± 1.59 m), and agoutis hoarded >9 times more seeds in summer (55) than in winter (6). Our third prediction was not supported, and the proportion of unrecovered caches and buried seed recovery times did not differ between winter (mean ± SE = 3.00 ± 0.00 days, n = 5 seeds) and summer (11.05 ± 3.68 days, n = 20 seeds). The high resource density (during summer) rather than the density of A. angustifolia seeds likely influenced seed fate. Agoutis acted mainly as predators, leaving few intact seeds, caching a low proportion of handled seeds (? 8%) and rapidly consuming the caches. Agoutis may cache seeds to keep them safe from competitors on a short‐term basis rather than maintaining medium‐ or long‐term reserves for use during food‐scarcity periods.     

Human plasma immunoreactive beta-lipotropin: correlation with basal and stimulated plasma ACTH concentrations.

A sensitive radioimmunoassay for human β-lipotropin (LPH) has been developed utilizing an N-terminal antibody which exhibits no cross-reactivity with β_h-MSH and appears to be species specific, with less than 10% crossreactivity with rat, ovine or bovine LPH. 0800-0900 mean plasma LPH concentrations were 47.9±5.7 pg/ml (5 normal subects), 100.5±13.2 pg/ml (Cushing's Disease (CD) n=6), 769.3±390.4 pg/ml (Nelson's Syndrome (NS) n=5). Mean plasma ACTH/plasma LPH ratios were: 1.96±0.13 (normal subjects), 1.69±0.11 (CD) and 1.16±0.07 (NS) Plasma ACTH and LPH rose in parallel in response to insulin-induced hypoglycemia in 4 normal subjects. There was a 375% increase in plasma ACTH concentration, a 474% increase in plasma LPH concentration. Plasma ACTH/LPH ratios in specimens obtained following attainment of peak concentrations were significantly lower than those in either control or peak specimens.     

Nuclease modification in Chinese hamster cells hypersensitive to DNA cross-linking agents--a model for Fanconi anemia.

Fanconi anemia is a human inherited disease that is characterized by cellular hypersensitivity to DNA cross-linking agents. A number of potential experimental models for that disorder have been developed by selecting mutants that are hypersensitive to bifunctional mutagens. The six mutants of that class in Drosophila, all of which map to the mus308 locus, express an alteration in a mitochondrial nuclease. A recent extension of that observation to cell lines from complementation group A of Fanconi anemia has established a new cellular phenotype for that disorder. In the current study an analogous enzyme has been analyzed in eight recently isolated Chinese hamster cell lines that are hypersensitive to cross-linking agents. Among these lines. V-H4 and V-B7 are shown to exhibit an enzyme modification analogous to that observed in the mutant Drosophila and human cells. These results validate the nuclease assay as an indicator of the Fanconi defect and further establish the V-H4 cell line as a valuable cellular model for analysis of the Fanconi A defect.     

An investigation into the reproductive characteristics ofMicroctonus hyperodae (Hym.: braconidae), a parasitoid ofListronotus bonariensis (Col.: Curculionidae)

The reproductive tract of the parasitoidMicroctonus hyperodae was found to comprise 6.1±0.2 ovarioles containing a total of 40–60 oöcytes. After oviposition into its hostListronotus bonariensis, the parasitoid's egg volume increased by 205 times prior to hatching. At 19.1°C ovipositingM. hyperodae survived for a mean 21±4 days and laid a mean of 48±8 eggs.M. hyperodae collected from Colonia, Uruguay laid a mean of 62±15 eggs which was significantly more than the other ecotypes. Under caging conditions with an ample supply of hosts,M. hyperodae laid 51% of its eggs in the first 72 hours and on average 41% of the species' life-span occurred after the exhaustion of its egg supply. Minimum temperature forM. hyperodae oviposition was found to be c. 5°C; beyond this the rate of increase in egg-laying was approximately linear until 30°C whereafter the rate fell abruptly. There was effectively no egg-laying at 39°C. The parasitoid showed no preference for one host sex or the other either in the laboratory or under field conditions. Compared to otherMicroctonus spp.,M. hyperodae appeared to show low fecundity and high longevity. The adaptive implications of this are discussed.     

Separation of Two Forms of Anthranilate Synthetase from 5-Methyltryptophar Susceptible and -Resistant Cultured Solarium tuberosumCells

Potato cell suspension cultures (Solanum tuberosumL. cv. Merrimack) have been selected which are resistant to growth inhibition by D,L-5-methyltryptophan. Anthranilate synthetase activity in crude extracts from resistant cells was less sensitive to feedback inhibition by L-tryptophan and D,L-5-methyltryptophan than the activity from the sensitive line. This altered feedback control apparently accounts for the cell's resistance to growth inhibition since there is a 48-fold increase in free tryptophan in one of the resistant cell lines. Preparative polyacrylamide gel electro-phoresis separated feedback-sensitive and -resistant forms of anthranilate synthetase in extracts from both 5-methyltryptophan-susceptible and -resistant cells, with a predominance of the corresponding form in the respective cell type. The anthranilate synthetase activity from the 5-methyltryptophan-resistant line was inactivated more slowly by incubation of crude extracts at 50°C than the activity from the sensitive line. These results suggest the presence of two isoenzymes of anthranilate synthetase in cultured potato cells.     

不同体位对严重肥胖者血氧饱和度的影响

目的:探讨不同体位对严重肥胖者血氧饱和度的影响。方法:16名平均体重指数(BMI)为40±5肥胖者和16名年龄匹配的正常体重者被纳入研究。分别在不同体外下(坐位、仰卧位、侧卧位)对所有参与者进行动脉血气监测。结果:肥胖者于坐位时动脉Pa O_2为75±4 mm Hg,Pa CO_2为37±3 mm Hg;仰卧位时动脉Pa O_2为62±5 mm Hg,Pa CO_2为47±5 mm Hg;侧卧位时Pa O_2为73±3 mm Hg,Pa CO_2为39±2 mm Hg;而正常体重者无明显变化。结论:严重肥胖者于平卧位时更容易出现低氧及高碳酸血症。     

Morphological measurement of the SEP using a dynamic time warping algorithm

A dynamic time warping technique was created to align the components of digitally high-pass (300 Hz–2500 Hz) filtered somatosensory evoked potentials evoked by median nerve stimulation recorded with a bipolar cephalic montage. A cost function was assigned related to the amount of warping necessary to match a standard wave derived from 24 normal subjects. Its value ranged from 0.525 to 2.456 (mean 1.305±0.501). This contrasted with a mean of 5.089±4.277 (range 0.701–13.972) derived from 25 patients with definite (n = 24) or possible (n = 1) multiple sclerosis chosen on the basis of having few or no clinical abnormalities at the time of testing. Fourteen (56%) of the patients had cost functions that were 3 or more S.D.s above the normal mean as compared to 3 (12%) having prolonged latency of the N19 peak. When used in combination, the cost function and latency yielded 60% abnormalities; 5 times higher than latency measurement alone.     

Molecular defects identified by whole exome sequencing in a child with Fanconi anemia

Fanconi anemia is a rare genetic disease characterized by bone marrow failure, multiple congenital malformations, and an increased susceptibility to malignancy. At least 15 genes have been identified that are involved in the pathogenesis of Fanconi anemia. However, it is still a challenge to assign the complementation group and to characterize the molecular defects in patients with Fanconi anemia. In the current study, whole exome sequencing was used to identify the affected gene(s) in a boy with Fanconi anemia. A recurring, non-synonymous mutation was found (c.3971C>T, p.P1324L) as well as a novel frameshift mutation (c.989_995del, p.H330LfsX2) in FANCA gene. Our results indicate that whole exome sequencing may be useful in clinical settings for rapid identification of disease-causing mutations in rare genetic disorders such as Fanconi anemia.     

Comparison of the sensitivity of Fanconi's anemia and normal fibroblasts to the induction of sister-chromatid exchanges by photoaddition of mono- and bi-functional psoralens

The induction of sister-chromatid exchanges (SCE) by photoaddition of a monofunctional furocoumarin (pyrido[3,4-c]psoralen, PyPs) and a bifunctional furocoumarin (8-methoxypsoralen, 8-MOP) in a normal and three Fanconi anemia (FA) fibroblastic cell lines was investigated. When compared to normal cells, the three FA cell lines demonstrated: a higher sensitivity to 8-MOP photoaddition; an equal or reduced sensitivity to PyPs photoaddition in the low dose range. Normal cells demonstrated a higher sensitivity to photoaddition of PyPs than to 8-MOP in the range of doses used; this is likely to be related to the higher amount of lesions induced by PyPs in DNA. Since FA cells were almost equally sensitive to 8-MOP and PyPs photoaddition and demonstrated a higher sensitivity to SCE induction by 8-MOP than normal cells, it can be concluded that this latter difference is mainly due to cross-links.     

Diurnal variations of plasma testosterone in men

Plasma testosterone (T) levels were assayed by a Competitive Protein Binding (CPB) technique in a group of 31 healthy males. In 22 subjects a single blood sample was taken between 8:00 and 9:00 A.M. and the mean T concentration was 6.84 ± 2.11 ng/ml. In the other 9 normal men, blood samples were taken every 4 hours. The existence of temporal variations for testosterone was confirmed by finding the highest mean plasma levels at 4:00 A.M. (9.28 ± 1.17 ng/ml) and lowest mean levels at 8:00 P.M. (2.66 ± 0.52 ng/ml).