Point mutations were introduced into the overlapping trans-regulatory genes (tat-III and trs) of human immunodeficiency virus type 1 (HIV-1), and the mutants were evaluated for virus expression. The results showed that tat-III has a positive transacting role and is required for transcriptional activation. A chain terminating mutation early in the trs gene resulted in an increase in transcription of viral messenger RNA as measured by nuclear transcription experiments, but only one major species of viral messenger RNA (1.8 kilobases) was detected, and little or no viral structural proteins were made. Thus, the trs gene product is essential for expression of virus structural proteins but, at the same time, may have a negative trans-regulatory role in transcription. Cotransfection of the point mutant proviruses defective in tat or trs with each other or with a complementary DNA clone containing tat and trs sequences restored the normal transcription pattern and subsequent virus production.
We report on a patient with long-standing severe autonomic failure that affected his sympathetic and parasympathetic nervous systems. Antibodies against the ganglionic acetylcholine receptors were detected in the serum. Removal of the antibodies by means of plasma exchange resulted in a dramatic clinical improvement.e is a pink complexioned person, except when he has stood for a long time, when he may get pale and faint. His handshake is warm and dry. . . . He is thin because his appetite is modest; he never feels hunger pains and his stomach never rumbles. . . . As old age comes on he will suffer from retention of urine and impotence but frequency, precipitancy, and strangury will not worry him." Paton's description of the "hexamethonium man" 1 illustrates the consequences of a pharmacologic blockade of ganglionic transmission. Hexamethonium blocks ganglionic nicotinic acetylcholine receptors, resulting in dysfunction of the efferent sympathetic and parasympathetic pathways. Disruption of these pathways causes severe orthostatic hypotension, anhydrosis, decreased production of saliva and tears, impaired bladder emptying, and erectile dysfunction. Patients with severe autonomic neuropathy have similar symptoms. Autonomic neuropathy can result from diabetes mellitus or amyloidosis, but in many patients no cause is evident. Occasional reports that intravenous immunoglobulins can be effective in autonomic neuropathy 2-5 suggest an immune-mediated mechanism. 6 The serum of some patients with subacute autonomic neuropathy contains autoantibodies against the ganglionic acetylcholine receptor, 7 and autonomic dysfunction develops in rabbits that are immunized against the ganglionic acetylcholine receptor. 8 These findings suggest that autoimmune autonomic neuropathy is an antibody-mediated neurologic disorder. However, a causal relation between anti-ganglionic acetylcholine receptor antibodies and autonomic failure has not been convincingly demonstrated in humans.A 43-year-old man presented with recurrent orthostatic syncope. His symptoms had developed gradually over a 20-year period. Recently, he was unable to stand upright for more than a few seconds before fainting. He also reported decreased sweating, dry mouth, and urinary retention. He had difficulty focusing on near objects. Early satiety,
This pictorial essay will review and discuss the aspects of differential diagnosis with splenic sonography, including recent literature and exemplary pictorial sonographic cases. Although the spleen is well evaluated by computed tomography and magnetic resonance imaging, sonography has certain advantages, including its ubiquitous availability, lack of ionizing radiation, and low cost. Sonography of the spleen plays an important role in emergency diagnosis of splenic rupture and hemorrhage. The additional use of contrast-enhanced sonography can improve the diagnostic validity. Depending on the indication, sonography of the spleen is especially important for oncologic differential diagnosis of focal lesions, follow-up examinations, and image guidance of therapeutic interventions.
Single nucleotide alterations were introduced into an infectious clone of human imnmunodeficiency virus type 1 to create a series of missense mutants in the tat coding region. Although mutations in a proline-rich region and a basic lysine-arginine-rich region resulted in wild-type phenotypes, five of six mutations in a cysteine-rich domain completely abolished tat activity and virus replication. One cysteine mutant retained tat activity but was negative for virus expression. Surprisingly, this mutant could not be complemented by tat, and virus expression was restored only by cotransfection with a plasmid expressing the rev gene. Another mutant with an alteration toward the C-terminal region showed significantly reduced tat activity and required complementation by a combination of tat and rev for virus replication. Further analysis revealed that a previously unrecognized splice acceptor site within this region, apparently used to generate the rev mRNA, had been altered. We provide evidence suggesting that tat and rev proteins are encoded by distinct mRNA species.Human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS) contains three major structural genes (gag, pol, and env) in common with all retroviruses (1). In addition, HIV contains several accessory genes-namely, vif (virion infectivity factor) (2), vpr (viral protein R) (3), tat (trans-activator) (4), rev (regulator of expression of virion proteins) (5), and nef (negative factor) (6); these designations were proposed recently to standardize the nomenclature for known human retrovirus accessory genes (7). The tat-and rev-encoded proteins have both been shown to be required for virus replication. Tat activates transcription from the viral long terminal repeat (LTR), resulting in increased levels of viral RNA and proteins (8). The rev gene product allows accumulation of steady-state levels of gag-pol and env mRNAs (5) and, in addition, has been shown to negatively affect transcription (8). Also rev has been proposed to function as an anti-repressor at the translational level (9). The trans-regulatory genes, tat and rev, are expressed from largely overlapping reading frames. To identify domains of these proteins that are important for function, we have generated a series of HIV-1 proviruses containing altered coding sequences in the tat and rev genes. Site-directed mutagenesis was targeted to the three major structural motifs of the functional first coding exon of the tat gene-the N-terminal proline-rich region, the middle cysteine-rich region, and the C-terminal basic amino acid stretch. Alterations in the last of these regions affected the first rev coding exon as well.The mutants were evaluated for phenotypic changes by transfection into COS-1 cells and assaying (i) their ability to trans-activate the homologous LTR by chloramphenicol acetyltransferase (CAT) assay, (ii) viral mRNA synthesis by Northern (RNA) blot, (iii) viral protein synthesis by radioimmunoprecipitation (RIP) assay, (iv) virus production ...
BackgroundThe hierarchical organization of hematopoiesis with unidirectional lineage determination has become a questionable tenet in view of the experimental evidence of reprogramming and transdifferentiation of lineage-determined cells. Clinical examples of hematopoietic lineage plasticity are rare. Here we report on a patient who presented with an acute B-lymphoblastic leukemia and developed a Langerhans' cell sarcoma 9 years later. We provide evidence that the second neoplasm is the result of transdifferentiation. Design and MethodsB-cell acute lymphoblastic leukemia was diagnosed in an 11-year old boy in 1996. Treatment according to the ALL-BFM-1995 protocol resulted in a complete remission. Nine years later, in 2005, Langerhans' cell sarcoma was diagnosed in a supraclavicular lymph node. Despite treatment with different chemotherapy protocols the patient had progressive disease. Finally, he received an allogeneic peripheral blood stem cell transplant and achieved a continuous remission. Molecular studies of IGH-and TCRG-gene rearrangements were performed with DNA from the Langerhans' cell sarcoma and the cryopreserved cells from the acute B-lymphoblastic leukemia. The expression of PAX5 and ID2 was analyzed with real-time reverse transcriptase polymerase chain reaction. ResultsIdentical IGH-rearrangements were demonstrated in the acute B-lymphoblastic leukemia and the Langerhans' cell sarcoma. The key factors required for B-cell and dendritic cell development, PAX5 and ID2, were differentially expressed, with a strong PAX5 signal in the acute Blymphoblastic leukemia and only a weak expression in the Langerhans' cell sarcoma, whereas ID2 showed an opposite pattern. ConclusionsThe identical IGH-rearrangement in both neoplasms indicates transdifferentiation of the acute B-lymphoblastic leukemia into a Langerhans' cell sarcoma. Loss of PAX5 and the acquisition of ID2 suggest that these key factors are involved in the transdifferentiation from a B-cell phenotype into a Langerhans'/dendritic cell phenotype. Haematologica 2010;95(9):1461-1466. doi:10.3324/haematol.2009 This is an open-access paper.
Some types of cancer have been associated with abnormal DNA fingerprinting. We used random amplified polymorphic DNA (RAPD) to generate fingerprints that detect genomic alterations in human breast cancer. Primers were designed by choosing sequences involved in the development of DNA mutations. Seventeen primers in 44 different combinations were used to screen a total of 6 breast cancer DNA/normal DNA pairs and 6 uveal melanoma DNA/normal DNA pairs. Forty-five percent of these combinations reliably detected quantitative differences in the breast cancer pairs, while only 18% of these combinations detected differences in the uveal melanoma pairs. Fourteen (32%) and 12 (27%) primers generated a smear or did not produce any band patterns in the first and second cases, respectively. Taking into account the ability of RAPD to screen the whole genome, our results suggest that the genomic damage in breast cancer is significantly higher than in uveal melanoma. Our study confirms other reports that the molecular karyotypes produced with random priming, called amplotypes, are very useful for assessing genomic damage in cancer. Genomic instability is a hallmark of neoplastic transformation and a herald of genomic damage. The existence of an additional type of genomic instability has been described, the microsatellite mutator phenotype pathway. Progress in molecular cancer genetics has facilitated the detection of these mutations. In the last decade, representation differential analysis and comparative genomic hybridization (CGH) were added to methods like flow cytometry, restriction fragment length polymorphisms of polymorphic minisatellite loci and allelotyping, a PCR amplification of informative microsatellite loci.Another promising approach has been introduced, arbitrarily primed PCR 1 combined with random amplified polymorphic DNA (RAPD). 2 Both PCR-based techniques, called amplotyping, use fingerprinting to quantitatively and qualitatively evaluate band alterations. 3 This random priming method allows comparison of normal and tumor tissues at a minimum of 20 -40 genome sites in a single PCR, though evidence suggests that the number of compared loci runs into the hundreds if we take into account that a gel band consists of many comigrating fragments. 4,5 It also includes internal controls and detects moderate gains of chromosomal sequences (trisomy/tetrasomy) that would otherwise escape detection.Previously, we addressed the problems of reproducibility and contamination in random priming. In the present work, we used RAPD fingerprinting to assess the genomic damage present in breast cancer and in uveal melanoma cells. For this task, we designed primers based on sequences that are supposed to play an important role in the mechanisms leading to DNA alterations. Human gene mutations appear to be caused by multiple mechanisms whose relative importance is probably governed by local primary and secondary DNA structure. 7 We have searched for consensus sequences located in the immediate vicinity of gene mutations and for "hot s...
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