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PubMed2006

[Host factors that regulate the intercellular dynamics of HIV-1 genome during the early phase of infection].

An interplay or battle between virus and its host has been observed within a single cell. Upon an infection with retroviruses including human immunodeficiency virus type 1 (HIV-1), the viral genome is subjected to several processes that include uncoating, reverse transcription of the viral genomic RNA into a cDNA copy, transport of this cDNA into the nucleus, and integration of the cDNA into the host chromosome. Antiretroviral restriction factors such as TRIM5 alpha and APOBEC3G have been recently identified. In addition, nuclear membrane protect host chromosomal DNA against incoming viral genome. For successful retroviral infection, viral genome must overcome these cellular barriers to establish proviral state, in which viral cDNA was stably integrated into host chromosomal DNA. In this review, I would summarize the host factors that regulate the intercellular dynamics of HIV-1 genome during the early phase of infection, especially focusing on factors interacting with HIV-1 integrase and the preintegration complex.

APOBEC-3G Deaminase↗
PubMed1995

Medicare program; uniform electronic cost reporting system for hospitals--HCFA. Final rule.

This final rule responds to comments on the May 25, 1994, final rule with comment period that implemented a standardized electronic cost reporting system for all hospitals under the Medicare program. In that rule, we solicited comments on the requirement that cost reporting software be able to detect changes made to the electronic file after the provider has submitted it to the fiscal intermediary. This final rule responds to comments on that requirement and clarifies that although changes to the "as-filed" electronic cost report are prohibited, an intermediary makes a working copy of the as-filed electronic cost report for use in the settlement process.

Accounting↗
PubMed2000

Imprinting of insulin-like growth factor 2 is modulated during hematopoiesis.

The transcription of insulin-like growth factor 2 (IGF-2) is affected by genomic imprinting, a multistep process through which the parental origin of a gene influences its transcription. The maternal copy of IGF-2 is silenced in most human tissues, but in the choroid plexus and the adult liver both alleles of IGF-2 are expressed. This study shows that though in peripheral blood mononuclear cells IGF-2 shows paternal allele-specific expression, in total bone marrow both alleles are transcribed. This modulation of imprinting is not attributable to use of the P1 promoter, because transcription from the P3 promoter occurred from both alleles. These results suggest that transcriptional recognition of the IGF-2 imprint can be modulated during hematopoiesis and may facilitate the development of in vitro model systems to study the transcriptional recognition of a genomic imprint.

Adolescent↗
PubMed1992

Molecular anatomy of the transcription complex of Escherichia coli during initiation.

Transcription is the foremost event in gene expression in which the enzyme RNA polymerase copies the genetic information from DNA to RNA. Much of our understanding of this process have come from studies carried out in Escherichia coli. A faithful and efficient transcription machinery of E. coli can be reconstituted in vitro with purified RNA polymerase and promoter-containing DNA. It is generally believed that in E. coli and most other organisms, the control of gene expression lies with the initiation of transcription. In this review, an attempt has been made to understand the mechanistic details of the initiation of transcription from the structural point of view of the promoter and the RNA polymerase. Allosteric nature of the enzyme has also been discussed at the end.

Allosteric Regulation↗
PubMed1965

MECHANIZATION OF CATALOGING PROCEDURES.

The Columbia-Harvard-Yale Medical Libraries Computerization Project has put into operation its mechanized procedure for the production of catalog cards. Cards produced are in final form ready to be filed into a card catalog. Catalogers prepare copy on a worksheet from which punched cards are punched. An IBM 1401 computer processes the decklets of punched cards on magnetic tape to produce the expanded decklets of punched cards needed to print the various packs of catalog cards required to go into different catalogs. Next, the computer punches the expanded decklets of cards to operate an 870 Document Writer, which types out the catalog cards in final form. Cost of cards ready to file is 12.5 cents per card.

Cataloging↗
PubMed1990

Nerve growth factor-induced differentiation of human neuroblastoma and neuroepithelioma cell lines.

A series of neuroepithelioma and neuroblastoma cell lines were screened for nerve growth factor (NGF)-induced differentiation. All three neuroepithelioma cell lines and all nine neuroblastoma cell lines with amplified N-myc oncogene did not show any apparent NGF-induced differentiation. However, neurite extension was observed for three of six neuroblastoma cell lines with single-copy N-myc oncogene. The three responsive lines had a neuronal phenotype (short processes) which was enhanced by the addition of NGF. The three nonresponsive cell lines were flat without any processes. The addition of NGF to the responsive cell lines resulted in an up-regulation of neurofilament mRNA expression. Peripherin and synapsin, two markers of terminal neuronal differentiation, were not induced. There was little effect of NGF on the rate of cell growth or colony formation on soft agar. Binding of NGF to eight of the cell lines was analyzed by the method of Scatchard. Two responsive neuroblastoma cell lines and one nonresponsive neuroepithelioma cell line expressed both low- and high-affinity binding sites. Two nonresponsive neuroblastoma cell lines expressed only a small number of high-affinity binding sites, and two other nonresponsive neuroblastoma cell lines did not detectably bind NGF. Hence, NGF-induced differentiation is confined to a particular class of neural-related tumors, and, even for these cell lines, differentiation is incomplete.

Cell Differentiation↗
PubMed2005

Evidence that low-level viremias during effective highly active antiretroviral therapy result from two processes: expression of archival virus and replication of virus.

Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.

Amino Acid Sequence↗
PubMedDate not supplied

Process reengineering for the filmless environment.

Facilities that are converting to filmless operations can learn from the University of Kentucky Chandler Medical Center's (UKMC) experience: that dramatic reengineering processes must take place before radiologists' productivity can increase. To convert a radiology department to soft copy interpretation, a piece-by-piece implementation of new systems or upgrades is customary. The first step may be to link each modality that is already digital into modality-specific mini-PACS, then to link the mini-PACS. Bringing other modalities on-line as new equipment is purchased and expanding the image information system (IIS) to the remainder of the healthcare facility rounds out the transition and may take several years. The downside of this kind of evolutionary approach is that the radiology department temporarily operates in two environments, the traditional film and the new filmless environments. To make the move from film to filmless, an administrator and the radiology staff must reengineer nearly every departmental process. Total quality management (TQM) techniques offer tools that are ideal for the task. Other recommendations include using a multidisciplinary team of staff members who are familiar with film-handling to create flow charts of all departmental processes. Each step should be validated to show its value to the overall process, the department or the institution. Next, flow charts of the expanded or new processes should be developed with input from the IIS manager, referring physicians and key IS personnel. Follow with estimates of staffing requirements that meet the needs of the completed flow charts and, finally, train staff members for the implementation of the new processes.

Efficiency, Organizational↗
PubMed1994

Dorsoventral axis formation in Drosophila depends on the correct dosage of the gene gurken.

The Drosophila gene gurken participates in a signaling process that occurs between the germ line and the somatic cells (follicle cells) of the ovary. This process is required for correct patterning of the dorsoventral axis of both the egg and the embryo. gurken produces a spatially localized transcript which encodes a TGF-alpha-like molecule (Neuman-Silberberg and Schupbach, Cell 75, 165-174, 1993). Mutations in gurken cause a ventralized phenotype in egg and embryo. To determine whether the gurken gene product plays an instructive role in dorsoventral patterning, we constructed females containing extra copies of a gurken transgene. Such females produce dorsalized eggs and embryos, which is expected if gurken acts as a limiting factor in the dorsoventral patterning process. In addition, the expression pattern of the gene rhomboid in the follicle cells is altered in ovaries of females containing extra copies of gurken. Our results indicate that changing gurken dosage in otherwise wild-type ovaries is sufficient to alter the number of somatic follicle cells directed to the dorsal fate. Therefore the gurken-torpedo signaling process plays an instructive role in oogenesis. It induces dorsal cell fates in the follicle cell epithelium and it controls the production of maternal components that will direct the embryonic dorsoventral pattern after fertilization.

Animals↗
PubMed2005

DNA replication during sporulation in Myxococcus xanthus fruiting bodies.

During the developmental process of the Gram-negative soil bacterium Myxococcus xanthus, vegetatively growing rod cells differentiate to ultimately become metabolically quiescent and environmentally resistant myxospores encased within fruiting bodies. This program, initiated by nutrient deprivation, is propagated by both cell-autonomous and cell-nonautonomous signals. Our goal was to determine whether M. xanthus, like many other developmental systems, uses cell-cycle cues to regulate and control its developmental program. To address this question, the DNA replication cycle was used as a marker to monitor progression through the cell cycle in vegetative, stationary, and developing M. xanthus populations. Using flow cytometry, quantitative fluorescence microscopy, and FISH to establish the chromosome copy number of myxospores, it was determined that vegetatively growing cells contain one to two copies of the genome, but upon entry into stationary phase, the chromosome copy number drops to a single copy. Of particular interest, fruiting body-derived myxospores contain a specific two-chromosome DNA complement with both origin and terminus regions localized to the periphery of the myxospore. We speculate that this duplication of genetic information in the myxospore would help assure viability during germination by providing a second copy of each gene. The results of this study imply that not only is DNA replication tightly regulated during the developmental process of M. xanthus, but that there are also regulatory mechanisms to ensure that all myxospores acquire two copies of the chromosome.

Cell Cycle↗
PubMed1993

A technique for developing and photographing ridge impressions on decomposed water-soaked fingers.

One of the most challenging tasks confronting a crime laboratory technician is the fingerprinting and subsequent identification of an unknown homicide or drowning victim whose fingers have been subjected to a long period of exposure to water and the effects of decomposition. If the fingers of the individual have not been exposed to the erosive effects of water and decomposition for a long period of time, they may be allowed to dry, and suitable impressions are often obtainable. In other cases the fingers may have to be removed, with the permission of the Medical Examiners Office, and processed by the Crime Laboratory in an attempt to develop suitable ridge structure for inked impressions or an exact photographic copy of the individual's fingers. In extreme cases the effects of water and decomposition make the fragile ridge structure appear to be nonexistent to the naked eye. The procedure used in this case report, combines the use of cyanoacrylate vapor, commonly called "super glue fuming," and the ninhydrin process in conjunction to develop fragile ridge structure into discernable ridges that are easily seen and photographed for the purpose of making an identification of the individual.

Anthropology, Physical↗
PubMed2001

Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum.

Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.

Aspergillosis↗
PubMed1991

[A new system of selecting yeast mutants with disrupted genetic recombination processes].

A new convenient system for isolation of the yeast mutants deficient in the genetical recombination is proposed. The chimeric plasmids constructed to carry the noncomplimenting mutant copy of the yeast ADE2 gene and different selectable yeast markers (LEU2 or TRP1 genes) are the basis for the system. Interplasmid intragenic recombination of ADE2 gene alleles in yeast cells transformed by two chimeric plasmids results in appearance of the secondary white prototrophic clones covering the primary red colony. The number of the clones reflects the recombination processes and is subject to an easy visual control. The proposed technique allows one to reveal both hypo and hyperrecombination mutants. Crossover or the gene conversion events can be distinguished by the simple genetical analysis of the secondary clones. The collection of mutants deficient in the genetical recombination has been obtained by the proposed technique.

Alleles↗
PubMed1996

Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR.

A random sequence library of single stranded DNA was screened to isolate sequences with high affinity for Thermus aquaticus DNA polymerase (Taq pol), a thermostable enzyme commonly used in the polymerase chain reaction (PCR). Selected oligonucleotide sequences bound Taq pol with dissociation constants in the low picomolar range, and efficiently inhibited polymerase activity at room temperature (20 to 25 degrees C), but did not inhibit at temperatures above 40 degrees C. Moreover, inhibition was thermally reversible. A process called "hot start" PCR is commonly used to prevent non-specific PCR products in amplification of low copy number targets. We show that the addition of oligonucleotide inhibitors eliminated the need for "hot start" conditions and improved the efficiency of detection of a low copy number target in PCR.

DNA, Viral↗
PubMed1993

A sensitive and quantitative method for the determination of number of HPV 16 DNA copies by using the competitive polymerase chain reaction.

The infectivity of human papillomavirus (HPV) is closely linked to the characteristics of the epithelial lesions that it induces in the uterine cervix. In a previous study of ours, we reported that the incidence of positivity for HPV DNA increases as lesions progress toward cervical carcinoma. However, the incidence of positivity did not increase during the process of tumorigenesis from severe dysplasia to invasive carcinoma. These findings suggest that patients with invasive carcinoma had much fewer DNA copies of HPV in host cells. Based on these findings, we have prepared a HPV16 DNA standard (DNA standard) with an EcoRI restriction site by using a polymerase chain reaction (PCR) technique and site-directed mutagenesis. We have also developed a sensitive quantitative method based on PCR that uses primers and competition between the patient's HPV16 DNA and an internal DNA standard for the determination of numbers of copies of HPV16 DNA. In this study, uterine cervical tissues obtained from 12 patients with invasive carcinoma were studied using competitive PCR (CPCR) to determine the number of HPV16 DNA copies. We found that the number of copies in the tissues of patients with invasive varied between 5 x 10(6) to 4 x 10(9)/micrograms DNA.

Adult↗
PubMed2005

Comparative studies of the quantification of genetically modified organisms in foods processed from maize and soy using trial producing.

Seven types of processed foods, namely, cornstarch, cornmeal, corn puffs, corn chips, tofu, soy milk, and boiled beans, were trial produced from 1 and 5% (w/w) genetically modified (GM) mixed raw materials. In this report, insect resistant maize (MON810) and herbicide tolerant soy (Roundup Ready soy, 40-3-2) were used as representatives of GM maize and soy, respectively. Deoxyribonucleic acid (DNA) was extracted from the raw materials and the trial-produced processed food using two types of methods, i.e., the silica membrane method and the anion exchange method. The GM% values of these samples were quantified, and the significant differences between the raw materials and the trial-produced processed foods were statistically confirmed. There were some significant differences in the comparisons of all processed foods. However, our quantitative methods could be applied as a screening assay to tofu and soy milk because the differences in GM% between the trial-produced processed foods and their raw materials were lower than 13 and 23%, respectively. In addition, when quantitating with two primer pairs (SSIIb 3, 114 bp; SSIIb 4, 83 bp for maize and Le1n02, 118 bp; Le1n03, 89 bp for soy), which were targeted within the same taxon specific DNA sequence with different amplicon sizes, the ratios of the copy numbers of the two primer pairs (SSIIb 3/4 and Le1n02/03) decreased with time in a heat-treated processing model using an autoclave. In this report, we suggest that the degradation level of DNA in processed foods could be estimated from these ratios, and the probability of GM quantification could be experimentally predicted from the results of the trial producing.

Animals↗
PubMed2000

Song selectivity and sensorimotor signals in vocal learning and production.

Bird song, like human speech, is a learned vocal behavior that requires auditory feedback. Both as juveniles, while they learn to sing, and as adults, songbirds use auditory feedback to compare their own vocalizations with an internal model of a target song. Here we describe experiments that explore a role for the songbird anterior forebrain pathway (AFP), a basal ganglia-forebrain circuit, in evaluating song feedback and modifying vocal output. First, neural recordings in anesthetized, juvenile birds show that single AFP neurons are specialized to process the song stimuli that are compared during sensorimotor learning. AFP neurons are tuned to both the bird's own song and the tutor song, even when these stimuli are manipulated to be very different from each other. Second, behavioral experiments in adult birds demonstrate that lesions to the AFP block the deterioration of song that normally follows deafening. This observation suggests that deafening results in an instructive signal, indicating a mismatch between feedback and the internal song model, and that the AFP is involved in generating or transmitting this instructive signal. Finally, neural recordings from behaving birds reveal robust singing-related activity in the AFP. This activity is likely to originate from premotor areas and could be modulated by auditory feedback of the bird's own voice. One possibility is that this activity represents an efference copy, predicting the sensory consequences of motor commands. Overall, these studies illustrate that sensory and motor processes are highly interrelated in this circuit devoted to vocal learning, as is true for brain areas involved in speech.

Animal Communication↗
PubMed2005

Comparison of specimen processing and nucleic acid extraction by the swab extraction tube system versus the MagNA Pure LC system for laboratory diagnosis of herpes simplex virus infections by LightCycler PCR.

A total of 563 specimens (234 dermal and 329 genital swabs) from patients suspected of having herpes simplex virus (HSV) infections were processed using two different extraction methods (the MagNA Pure LC system and the swab extraction tube system [SETS]); HSV DNA was amplified by LightCycler PCR. HSV DNA was detected in 157 of 563 specimens (27.9%) processed by the MagNA Pure LC system and in 179 of 563 specimens (31.8%) processed by SETS (P < 0.0001). There was no specimen processed by the MagNA Pure LC extraction method that was positive only for HSV DNA. Of 157 specimens positive by both methods, HSV DNA copy levels were higher (using cycle crossover points [cycle threshold {C(T)}]) with SETS (mean C(T), 25.9 cycles) than with the MagNA Pure LC system (mean C(T), 32.0 cycles) (P < 0.0001). The time to process 32 samples was longer with the MagNA Pure LC extraction system (90 min) than with SETS (35 min). HSV DNA extraction using SETS is faster, less expensive, and more sensitive than the MagNA Pure LC system and could replace the latter for the laboratory diagnosis of HSV infections using LightCycler PCR.

Clinical Laboratory Techniques↗