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PubMed2005

In vivo transcriptome of Plasmodium falciparum reveals overexpression of transcripts that encode surface proteins.

Infections with the human parasite Plasmodium falciparum continue to present a great challenge to global health. Fundamental questions regarding the molecular basis of virulence and immune evasion in P. falciparum have been only partially answered. Because of the parasite's intracellular location and complex life cycle, standard genetic approaches to the study of the pathogenesis of malaria have been limited. The present study presents a novel approach to the identification of the biological processes involved in host-pathogen interactions, one that is based on the analysis of in vivo P. falciparum transcripts. We demonstrate that a sufficient quantity of P. falciparum RNA transcripts can be derived from a small blood sample from infected patients for whole-genome microarray analysis. Overall, excellent correlation was observed between the transcriptomes derived from in vivo samples and in vitro samples with ring-stage P. falciparum 3D7 reference strain. However, gene families that encode surface proteins are overexpressed in vivo. Moreover, this analysis has identified a new family of hypothetical genes that may encode surface variant antigens. Comparative studies of the transcriptomes derived from in vivo samples and in vitro 3D7 samples may identify important strategies used by the pathogen for survival in the human host and highlight, for vaccine development, new candidate antigens that were not previously identified through the use of in vitro cultures.

Adaptation, Physiological↗
PubMed1996

Early phases of HIV type 1 infection.

A workshop entitled "Early Phases of HIV-1 Infection" was held to review current research on the immunological and virological aspects of early phases of HIV infection in humans and in animal models, to identify studies for future research, and to foster collaborations among investigators in the biomedical community. In infections of adults, the appearance of cytotoxic T lymphocyte activity, when present, coincides with a decrease in viral load as measured by plasma viremia. In neonatal infections, however, an initial decrease in viral load has been observed months before cytotoxic T lymphocytes are detected. Immunological data, from a limited number of patients, indicated that CD8+ cytotoxic T lymphocytes detected early after HIV-1 infection may recognize epitopes in any of several HIV-1 proteins: Env, Gag, Pol, Tat, and Nef. With regard to the humoral antibody response, anti-Env binding antibodies appear before neutralizing antibodies and do not predict the appearance of neutralizing activity. The time at which neutralizing antibody appears is variable and unpredictable. Preliminary data indicate that early viral peak load does not predict disease progression in many cases, and the phenotype or virulence of the virus appears to be a critical variable. However, the quantity of HIV-1 RNA in plasma is a strong CD4+ T cell-independent predictor of outcome following HIV-1 seroconversion in homosexual men. Early, high virus load with sustained viremia is often accompanied, in both adults and infants, by the inability to mount an effective immune response, resulting in rapid disease progression.

Animals↗
PubMed1991

Determining transcript number using the polymerase chain reaction: Pgk-2, mP2, and PGK-2 transgene mRNA levels during spermatogenesis.

We describe a technique that uses reverse transcription and the polymerase chain reaction (pcr) to rapidly quantitate numbers of specific mRNA transcripts from nanogram quantities of total cellular RNA. Linearity of input molecules to output signal was maintained by limiting the cycle number and the amount of input RNA and by minimizing the number of manipulations. Absolute levels of specific transcripts were determined by the inclusion of a separate standard curve composed of serially diluted in vitro transcribed RNA run alongside the experimental samples. This allowed rapid quantitation of many samples simultaneously. We applied this technique to measuring the expression of phosphoglycerate kinase 2 (Pgk-2) transgenes in the mouse testis during development. A human PGK-2 transgene, a PGK-2/CAT transgene, and the endogenous mPgk-2 gene all displayed similar patterns and levels of expression, consistent with the conclusion that peak RNA accumulation occurs in pachytene spermatocytes. Mouse protamine 2 (mP2) is expressed at a level approximately tenfold higher than Pgk-2 and displays a different pattern of expression consistent with initiation of transcription occurring in haploid round spermatids.

Animals↗
PubMed2006

Pilot study of mucosal genetic differences in early smokers and nonsmokers.

BACKGROUND: Global gene expression analysis is proving to be an important means of assessing human tumors and may identify key components of carcinogenesis or clinical prognosis. This technique has been successfully applied to head and neck squamous cell carcinoma (HNSCC) and thyroid carcinomas; however, little has been done to evaluate premalignant states. METHODS: Human buccal mucosal cells were sampled from smokers and nonsmokers using a noninvasive brush technique. The method was validated by assessing the quantity and quality of RNA obtained. The purified RNA was then assayed using cDNA microarrays containing 27,323 cDNA clones to examine the buccal mucosa in these patients for differences in gene expression patterns. Using unsupervised and supervised hierarchical clustering methods, we developed a gene profile signature for an initial training set of smokers and nonsmokers and then used this to predict smoking status in a subsequent test set of subjects. Selected genes were then cross-referenced with previously published gene sets found in HNSCC identified by our group. RESULTS: Nineteen subjects were used in this pilot analysis, 9 smokers and 10 nonsmokers. Smoking among the study group ranged from 1 to 60 pack years. RNA purified from buccal mucosal brushing demonstrated a high degree of similarity in gene expression profiles among independent samples. Through the application of supervised clustering techniques, we were able to identify 113 genes whose expression differed significantly between samples from smokers and nonsmokers (t test, P < .001). This expression signature was able to accurately predict who within the second set of subjects were smokers, with the exception of one person who had a minimal tobacco history and clustered with the nonsmokers. Cross-referencing data with that found in HNSCC, we were able to identify a tumor suppressor gene involved in the c-myc pathway (Mxi1) that was similarly under-expressed in smokers and cancer patients with progressive disease. CONCLUSIONS: Although the sample size was small in this preliminary dataset, our analysis revealed several groups of genes that were either over- or under-expressed in the smokers and which could be used to predict smoking exposure. Many of these represent genes of possible interest as early molecular markers for head and neck carcinogenesis.

Adult↗
PubMed1995

Rapid and sensitive analysis of mRNA polyadenylation states by PCR.

A rapid and sensitive technique is described that measures the length of the poly(A) tail on a specific mRNA within subnanogram quantities of total cellular RNA [the Poly(A) test (PAT)]. In a single-tube reaction, a poly(dT) primer is synthesized in situ on the poly(A) tail of mRNAs using oligo(dT) and DNA ligase. By modulating the annealing temperature and primer concentrations, a GC-rich adapter sequence is targeted to the 5' end of the poly(dT) primer. This ligated poly(dT)-anchor is then used to prime reverse transcription of the mRNA, yielding a library of PAT cDNAs. The length of a poly(A) tail is determined by PCR amplification using the oligo(dT)-anchor primer and a message-specific primer. Comparison of PCR products from different samples allows quantitative determination of changes in polyadenylation of a given mRNA. This technique overcomes many of the pitfalls associated with conventional poly(A) tail length assessments and should prove useful in studying a variety of processes relating to polyadenylation.

Animals↗
PubMedDate not supplied

Laser-assisted microdissection applied to frozen surgical pathologic specimens - methodological aspects on RT-PCR.

Laser-assisted microdissection has become a unique technique for an accurate gene-expression profiling analysis in human tissues. The introduction of this approach requires the development of a reliable, efficient, and reproducible procedure for tissue processing. We report a systematic evaluation of the different relevant steps required to obtain sufficient quantity and good quality RNA for reverse transcriptase-polymerase chain reaction when using frozen surgical pathologic tissues as starting material. We propose an optimized and very efficient method with respect to time and effort that can easily be put into practice in research laboratory as well as in any pathology laboratory.

Colon↗
PubMed2001

Polymerase chain reaction as a diagnostic adjunct in herpesvirus infections of the nervous system.

Polymerase chain reaction (PCR) is a powerful technique that allows detection of minute quantities of DNA or RNA in cerebrospinal fluid (CSF), vesicle and endoneurial fluids, blood, fresh-frozen, and even formalin-fixed tissues. Various infectious agents can be detected with high specificity and sensitivity, including bacteria, parasites, rickettsia and viruses. PCR analysis of CSF has revolutionized the diagnosis of nervous system viral infections, particularly those caused by human herpesviruses (HHV), and has now replaced brain biopsy as the gold standard for diagnosis of herpes simplex virus (HSV) encephalitis. PCR analysis of both CSF and nervous system tissues has also broadened our understanding of the spectrum of disease caused by HSV-1 and -2, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), and HHV-6. Nonetheless, positive tissue PCR results must be interpreted cautiously, especially in cases that lack corroborating clinical and neuropathologic evidence of infection. Moreover, positive PCR results from tissues do not distinguish latent from productive (lytic) viral infections. In several neurological diseases, negative PCR results have provided strong evidence against a role for herpesviruses as the causative agents. This review focuses on the use of PCR tests to diagnose HSV and VZV infections of the nervous system.

Body Fluids↗
PubMed1975

Rapid purification of 70S RNA from media of cells producing RNA tumor viruses.

Media from cells producing RNA tumor viruses, when treated with sodium dodecyl sulfate and polyvinyl sulfate, yield 70S RNA as the major species binding oligo(dT)-cellulose. The procedure described for purifying 70S RNA requires no special equipment and is suitable for rapidly processing large quantities of media or for purifying RNA from commercially avialable virus, with a 5- to 10-fold higher yield than was obtained using existing methods.

Cell Line↗
PubMed2003

Gene cloning and functional analysis of a second delta 6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus.

We demonstrated that Mortierella alpina 1S-4 has two delta 6-desaturases, which are involved in the desaturation of linoleic acid to gamma-linolenic acid. For one of the two delta 6-desaturases, designated as delta 6I, gene cloning and its heterologous expression in a fungus, Aspergillus oryzae, has previously been reported. In addition, we indicated in this paper that there is an isozyme of the two delta 6-desaturases, designated as delta 6II, in M. alpina 1S-4. The predicted amino acid sequences of the Mortierella delta 6-desaturases were similar to those of ones from other organisms, i.e. borage and Caenorhabditis elegans, and had a cytochrome b5-like domain at the N-terminus, being different from the yeast delta 9-desaturase, which has the corresponding domain at the C-terminus. The full-length delta 6II cDNA was expressed in A. oryzae, resulting in the accumulation of gamma-linolenic acid (which was not detected in the control Aspergillus) up to 37% of the total fatty acids. The analysis of real-time quantitative PCR (RTQ-PCR) showed that the quantity of delta 6I RNA was 2.4-, 9-, and 17-fold higher than that of delta 6II RNA on 2, 3, and 4 days in M. alpina 1S-4, respectively. M. alpina 1S-4 is the first fungus to be confirmed to have two functional delta 6-desaturase genes.

Amino Acid Sequence↗
PubMed2006

beta-Glucuronidase is an optimal normalization control gene for molecular monitoring of chronic myelogenous leukemia.

Quantitative monitoring of breakpoint cluster region (BCR)-Abelson kinase (ABL) transcripts has become indispensable in the clinical care of patients with chronic myelogenous leukemia. Because quantity and quality of RNA in clinical samples are highly variable, a suitable internal normalization control is required for accurate BCR-ABL quantification. However, few studies have examined suitability of the control genes using criteria relevant to residual disease testing. In this study, we evaluated a number of control genes with the application of several novel criteria, including control gene performance on serial patient sample testing and in a residual disease model. We also examined expression of the control genes in BCR-ABL-positive K562 cells in response to Gleevec treatment. We found that beta-glucuronidase is the best control gene among those studied. Importantly, ABL, a widely used control gene, generates misleading BCR-ABL changes that potentially affect the clinical management of chronic myelogenous leukemia patients.

Benzamides↗
PubMed1991

Developmental capacity acquisition requires exposure of uncondensed chromosomes before maturation of bovine oocytes.

Immature bovine oocytes are forming an heterogeneous population since only one third of all morphologically normal cumulus-oocyte-complexes will result in a developing embryo after in vitro culture. To increase developmental competence, it is essential to improve the quality of the 2/3 not responding to the in vitro maturation context. To achieve this goal we can address the cytoplasmic domain or the nucleus capacity to control future events leading to normal fertilization and development. To influence for example the quantity and quality of RNA present at meiotic resumption (MR), we need access to uncondensed chromatin before that period. Maintenance of the germinal vesicle for a minimum of 24 h can be achieved with protein synthesis or phosphodiesterase (PI) inhibitors or with cAMP accumulation at a level higher that the one required in the cumulus-granulosa portion to stimulate MR. It is suspected that in vivo, the granulosa layer is responsible for the arresting signal since follicular fluid (BFF) alone is not sufficient for a complete effect in vitro. Low concentrations of granulosa cells in vitro (10(6)/ml) stimulate MR but monolayers with their conditioned medium provide inhibitory conditions (33% GV) to oocytes adhering to the monolayers. To achieve a complete meiotic arrest for 24 h (greater than 75% GV), a high concentration of granulosa cells must be used (10(7)/ml). The embedding of cumulus-enclosed oocytes in agar prior to culture with the cells prevents the significant effect of the cells on MR indicating either a need for contact stimulation or a very labile product.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Monocytes, dendritic cells, and Langerhans cells in human immunodeficiency virus infection.

Cells of the immune system are the target of infection with HIV. CD4 + T cells latently carry much of the viral burden in the blood and ultimately are depleted by infection with HIV. In contrast, infected tissue macrophages are long-lived and may serve as a viral reservoir. They are productive of relatively greater quantities of viral message RNA and its transcriptional product, infectious virions. Viral production by both cell types is modulated by environmental cytokines, the availability of which may be modified by the virus itself or by abnormally functioning HIV-infected immune cells. Not all susceptible cells are equally infected; although this phenomenon is not well understood, it has been related in vitro to maturation or differentiation. Blood DC and LC, antigen-presenting cells bearing many similarities to cells of the monocyte-macrophage lineage, are susceptible to HIV infection in vitro. Some evidence clearly indicates that, in vivo, epidermal LC may be infected with and productive of HIV and may be depleted or phenotypically altered in the HIV-infected individual. We, and others, have been unable to substantiate these findings by routine techniques used in the identification of HIV-infected macrophages in susceptible tissues, such as the brain, lungs, and lymph nodes (in situ hybridization for HIV-specific mRNA, electron microscopy for typical viral particles, recovery of infectious virus onto target cells, immunohistochemical staining of surface proteins in tissue, and polymerase chain reaction amplification of viral DNA). Evidence for the presence of HIV within the dermis of patients is clear; however, dermis contains a great variety of cell types as well as cells from the peripheral blood. We feel strongly that were the epidermis to harbor virus to any significant degree, it would have been identified by at least some of the methods described earlier. Although it is difficult to reconcile these reported differences, it appears that LC must be infected rarely. LC from lesional and apparently normal skin of HIV-infected individuals do not serve as an important reservoir of infectious HIV. Additionally, the diverse cutaneous manifestations seen in this population cannot be attributed directly to viral presence within the lesions but are more likely to result from the multifacted immunologic disregulation occurring systemically.

Dendritic Cells↗
PubMed1991

In vitro analysis of polychlorinated biphenyls (PCBs) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) cellular toxicity in PLC/PRF/5 cell proliferation--the effect of ursodeoxycholic acid, inchin-gorei-san and shou-saiko-to on cell toxicity.

We investigated the cell toxicity of polychlorinated biphenyls (PCBs) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) as an indicator of the quantity of m-RNA, which synthesizes the HBV core antigen region, and secreting protein, which is an HBV surface antigen in PLC/PRF/5 cells. The determination of m-RNA was conducted according to the methods of reverse transcriptase and polymerase chain reaction. Furthermore, the reductive action of ursodeoxycholic acid, shou-saiko-to and inchin-gorei-san on the PCBs and PCDF toxicity was investigated. The cell number of 1 x 10(5)/ml and concentrations of 200 micrograms/ml for PCBs and 500 microM for PCDF were used in these experiments. The titer of HBsAg was gradually increased from 1 in 1 x 10(3)/ml of cell numbers to 100 in 1 x 10(6)/ml of cell numbers. The curve became a plateau in 1 x 10(6)/ml of cell numbers. The cell number of 1 x 10(5)/ml was used in the experiments. The titer of HBsAg decreased following in the increase of concentration of PCBs. The HBsAg, even in the PCBs concentration of 1000 micrograms/ml showed a titer of 22.5%. However, the highest concentration of PCDF in this study, that is, 500 microM of PCDF, did not show any decrease of HBsAg activity. The concentrations of 200 micrograms/ml for PCBs and 500 microM for PCDF were used in the investigation of drug effects. A high titer HBsAg was observed in high concentrations of shou-saiko-to in comparison with a control group. Ursodeoxycholic acid and inchin-gorei-san exhibited a similar tendency.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzofurans↗
PubMed1994

[Examination in clinical course of HCV in interferon treatment].

We treated 812 patients who had chronic hepatitis C with interferon (IFN) and studied the effects of interferon. We defined complete response as normalization of amino-transferase more than 6 months after termination of IFN. Complete response by means of IFN therapy is dependent to some factors, that is HCV-genotype, quantity of HCV-RNA, histology. Cases of HCV genotype III or IV, CH2A, less than 10(5) copy/ml of HCV-RNA are effective in treatment of IFN. On the other hand, cases of HCV genotype II, CH2B, more than 10(6) copy/ml of HCV-RNA are effective in treatment of IFN.

Genotype↗
PubMed1996

[Cloning genes, specifically expressed in the cerebral cortex and cerebellum of adult rats].

To isolate genes differentially expressed in the rat brain cortex and cerebellum, a subtractive cDNA cloning procedure requiring only small quantities of poly(A+) RNA, followed by differential screening, was used. Two novel genes, MK and 3L7, with cortex- and cerebellum-specific expression were identified. These genes displayed different expression patterns in the brain cortex, cerebellum, and hippocampus during postnatal development.

Animals↗
PubMed1998

Ion transporters and receptors in cDNA libraries from lens and cornea epithelia.

PURPOSE: To construct tissue-specific cDNA libraries containing copies of genes of ion transporting and receptor proteins in lens and cornea epithelia. To screen the libraries for the cDNA of these molecules and to deduce the protein sequence from the cDNA sequence. METHOD: Lens and corneal cDNA libraries are not commercially available and are difficult to prepare from microdissected single animal eyes or cadaver human eyes due to the small amount of tissue. To overcome these problems, we refined an approach for preparing high efficiency, non-PCR amplified plasmid cDNA libraries, where only nanogram quantities of poly (A) RNA are required. Plasmids were electrotransformed into DH10B bacteria and routinely yielded >10(6) independent colonies with typically >90% recombinants. Randomly selected colonies were subjected to colony PCR analysis to determine the libraries average insert size (typically approximately 1-kb). Primers to known genes were used in PCR amplification to check for representation of the genes in the cDNA libraries. RESULTS: Using libraries from rabbit cornea epithelium and endothelium, cultured human lens epithelium, and alphaTN4 cells, we have found the libraries to contain the cDNA for 3 common housekeeping proteins expected to be at high copy numbers in all cells. In addition, we identified 22 rare proteins and for many we determined the complete coding regions. These include K+ channels, Cl- channels, Ca++ channels, Na+ channels, three exchangers, the Na+-HCO3- cotransporter, and three kinds of receptors. CONCLUSION: Cornea and lens libraries have been constructed from single cornea or lens preparations. The libraries often contain the cDNA sequences for ion transporting and receptor proteins which are expected to be relatively rare in these cells. Many of these cDNA's have now been sequenced and the encoded proteins identified.

Animals↗
PubMed1991

Synaptic activity results in increased transcription as detected by the brain-specific probe pEL-48 in Torpedo marmorata.

The cloning of sequences expressed exclusively in neurons that use acetylcholine as a neurotransmitter resulted in multiple probes. One of them, pEL-48, was used to detect changes in transcription patterns in the electric lobes of Torpedo marmorata. Fish were stimulated either electrically or mechanically to induce discharge of the electric organs; they were killed after different poststimulation time intervals. The RNA isolated from the electric lobes was quantified using Northern blots and pEL-48 as a probe; distinct changes in the quantity of pEL-48 complementary RNA could be detected. Partial sequencing of the clone pEL-48 revealed an unusual primary sequence with numerous short open reading frames, coding for hitherto unknown polypeptides.

Acetylcholine↗
PubMed1995

An improved method for isolating RNA from coccidian oocysts.

A method, based on the disruption of eimerian oocysts in a French pressure cell in the presence of guanidine isothiocyanate, has been developed to isolate large quantities of high quality total RNA efficiently. This procedure results in a 12.5-fold greater number of oocysts broken, and a 22-fold greater yield of total RNA than from disruption by conventional grinding in liquid N2. In addition, the RNA isolated by the French pressure cell method was of equal or superior quality when compared to RNA isolated by grinding. This procedure provides a significant improvement in RNA extraction from eimerian oocysts and will greatly facilitate the study of gene expression in this important group of parasites.

Animals↗