Structure and expression of genes of GM-CSF and G-CSF in blast cells from patients with acute myeloblastic leukemia

The hematopoietic growth factors granulocyte/macrophage colony- stimulating factor (GM-CSF) and G-CSF, available as recombinant products, stimulate the growth in culture of blasts from patients with acute myeloblastic leukemia (AML). We used cDNA probes for each gene to study the genomic organization in blast cells of 22 patients and expression in the blast cells of 18 patients. Alteration in the structure of G-CSF (two instances) and GM-CSF (two instances) was found. In two patients in whom it was possible to study DNA from bone marrow obtained at remission, the new bands detected in the leukemic cells were not found. Fifteen of 18 patients showed no RNA expression of either growth factor. Both patients with GM-CSF abnormalities as seen by Southern analysis expressed an abnormally large GM-CSF message but no G-CSF messages. One patient with an abnormal Southern pattern with G-CSF expressed normal-sized G-CSF and GM-CSF messages. The biologic significance of these findings remains to be determined. Nonetheless, the abnormal Southern patterns may prove to be useful clonal markers in the study of AML.     

Stage-specific expression of c-kit protein by murine hematopoietic progenitors

We have analyzed c-kit expression by hematopoietic progenitors from normal and 5-fluorouracil (5-FU)-treated mice by staining with monoclonal anti-c-kit antibody ACK-4. Marrow cells that were enriched for progenitors by a combination of metrizamide density separation and negative immunomagnetic selection with lineage-specific monoclonal antibodies (MoAbs) were separated into three populations based on the level of c-kit expression, c-kit(high), c-kit(low), and c-kit-. The majority of colony-forming cells from normal mice were in c-kit(high) population, whereas most of the progenitors from 5-FU-treated mice were in the c-kit(low) population. Optimal colony formation from c-kit(low) cells from 5-FU-treated mice required the interactions of at least two factors among interleukin-3 (IL-3), IL-11 and steel factor (SF) whereas colony formation from c-kit(high) cells of normal mice was supported well by IL-3 alone. Blast cells that were derived from 5-day culture of c-kit(low) post 5-FU cells were c-kit(high). These observations suggest that the primitive hematopoietic progenitors in cell cycle dormancy are c-kit(low) whereas actively cell cycling maturer progenitors are c- kit(high). Mature cells, with the exception of mast cells, derived from secondary culture of the c-kit(high) blast cells expressed little, if any, c-kit. These results are consistent with a model in which c-kit expression progresses from low levels on primitive, dormant multipotent progenitors to high levels on later, actively cycling progenitors, and finally, decreases to very low or undetectable levels on most mature blood cells, with the exception of mast cells.     

Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments

Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.Trace amounts of DNA can occasionally survive the decomposition of organic matter for long periods of time after the death of an organism. However, the retrieval of these ancient DNA molecules is severely impeded by their small size. DNA fragmentation is at least partly driven by depurination (1, 2), a continually occurring process. It is thus predicted that the degree of DNA fragmentation increases with sample age. This correlation has, in fact, been established in a recent study that analyzed samples of different ages from the same archeological sites (3), but the correlation vanishes in comparisons across different sites (4). The important role of environmental conditions, especially temperature, in DNA preservation is well recognized and reflected—for example, in the concept of thermal age (5). Unsurprisingly, permafrost environments have yielded the oldest credible records of DNA survival, including short stretches of plant and invertebrate DNA with an estimated age of up to 800,000 y that were amplified by PCR from Artic ice cores (6, 7) and the genome sequence of a 700,000-y-old horse published recently (8). More temperate environments have yielded many DNA sequences from the Holocene and the Late Pleistocene, some as old as ∼100,000 (9) or ∼120,000 y (10), but only a single study has convincingly raised the possibility of DNA survival extending far into the Middle Pleistocene outside of permafrost (11). In this study, short PCR products of ∼50 bp were retrieved from several bone samples of Middle Pleistocene cave bears from European caves, the oldest coming from the site of Sima de los Huesos (Atapuerca, Spain) and estimated to be >300,000 y old.It is important to note that direct PCR amplification provides limited power to reconstruct sequences from short DNA fragments, because only fragments that are long enough to allow for the hybridization of two PCR primers around a stretch of informative sequence are amenable to direct amplification and sequencing. If, as in the study above (11), amplicon size is decreased to ∼50 bp, only ∼10 bp of informative sequence remain between the priming sites, which compromises the security of sequence identification while at best allowing genotyping of single nucleotide polymorphisms. In contrast, with current library-based techniques, even shorter DNA fragments can, in principle, be sequenced in their entirety because the priming sites required for amplification and sequencing are added externally by attaching artificial adaptor sequences to the molecule ends. As an additional benefit, this approach allows the determination of damage patterns unique to ancient molecules, thus providing a framework for verifying the authenticity of ancient sequences (4, 12).The preparation of DNA libraries and high-throughput sequencing have, without doubt, greatly advanced the scope of sequence retrieval from ancient DNA in recent years, as is documented by the generation of entire genome sequences (e.g., refs. 8 and 13–17). However, the possibility remains that not all sequence information residing in ancient specimens is optimally recovered with these methods. This possibility becomes apparent when inspecting the size distributions of sequences reported from ancient DNA (e.g., refs. 3, 8, and 15), which consistently show a mode of ∼40 bp or larger. It is unclear whether the deficit of shorter molecules is due to poor preservation in ancient biological specimens or their exclusion during sample preparation. This question is of importance because it is expected that the number of DNA fragments in an ancient sample increases exponentially as length decreases and, hence, that most information resides in very short molecules (3, 8). An obvious step in which loss of short molecules is expected to occur is in library preparation, because commonly used techniques require size-selective DNA purification to remove excess adaptor molecules after ligation. However, such purification steps are absent in a single-stranded library preparation method that was recently developed to sequence the genome of an archaic Denisovan individual to high coverage (16). A direct comparison of fragment size distributions obtained from sequencing the same DNA extracts following single- and double-stranded library preparation indeed revealed an improved recovery of short sequences with the single-stranded method, but Denisovan sequences of <40 bp still remained underrepresented (16).Here we present improvements to a widely used silica-based DNA extraction technique (18) that, in combination with single-stranded library preparation, allows ancient DNA molecules as short as 30 bp to be efficiently recovered and sequenced. We describe the results from applying these methods to a bone sample of a Middle Pleistocene cave bear (Ursus deningeri) from Sima de los Huesos, representing the same type of material for which the longest DNA survival outside of permafrost has been proposed based on genotyping 17 mitochondrial positions where Late Pleistocene cave bears differ from brown bears (Ursus arctos) and American black bears (Ursus americanus) (11). These data support the existence of a monophyletic cave bear clade, in congruence with morphological analyses, but the exact genetic relationships within this clade remain to be determined.     

闭合性输尿管损伤的早期诊断

０　引言　腹部钝性伤所致闭合性输尿管损伤罕见,且早期缺乏典型症状,往往延误诊断［１］,造成严重后果．我们采用电视屏幕动态观察下大剂量静脉肾盂造影或逆行造影的方法,早期诊断闭合性输尿管损伤,取得较满意效果．１　临床资料　本组男性５例,女性１例,年龄３岁～３６岁,右侧４例,左侧２例．２例合并肾损伤,１例合并骨盆骨折,右骶髂关节分离,尿道损伤．４例为撞伤、压伤,２例坠落伤．６例均无典型输尿管损伤症状．４例在电视屏幕动态观察下行大剂量静脉肾盂造影,其中１例同时在电视屏幕动态观察下行逆行造影．本组６例,早…     

Integration of local inputs in visual cortex

In mammalian visual cortex, local connections are ubiquitous, extensively linking adjacent neurons of all types. In this study, optical maps of intrinsic signals and responses from single neurons were obtained from the same region of cat visual cortex while the effectiveness of the local cortical circuitry was altered by focally disinhibiting neurons within a column of known orientation preference. Maps of intrinsic signals indicated that local connections provide strong and functional subthreshold inputs to neighboring columns of other orientation preferences, altering the observed orientation preference to that of the disinhibited column. However, measuring the suprathreshold response using single-cell recordings revealed only mild changes of preferred orientation over the affected region. Because strongly tuned subthreshold inputs from cortex only marginally affect the tuning of a cortical cell's output, it is concluded that local cortical inputs are integrated weakly compared to geniculate inputs. Such circuitry potentially allows for the normalization of responses across a wide range of input activity through local averaging.