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.

There is an increasing interest in nootropic drugs for the treatment of CNS disorders. Since the last meta-analysis of the clinical efficacy of piracetam, more information has accumulated. The primary objective of this systematic survey is to evaluate the clinical outcomes as well as the scientific literature relating to the pharmacology, pharmacokinetics/pharmacodynamics, mechanism of action, dosing, toxicology and adverse effects of marketed and investigational drugs. The major focus of the literature search was on articles demonstrating evidence-based clinical investigations during the past 10 years for the following therapeutic categories of CNS disorders: (i) cognition/memory; (ii) epilepsy and seizure; (iii) neurodegenerative diseases; (iv) stroke/ischaemia; and (v) stress and anxiety. In this article, piracetam-like compounds are divided into three subgroups based on their chemical structures, known efficacy and intended clinical uses. Subgroup 1 drugs include piracetam, oxiracetam, aniracetam, pramiracetam and phenylpiracetam, which have been used in humans and some of which are available as dietary supplements. Of these, oxiracetam and aniracetam are no longer in clinical use. Pramiracetam reportedly improved cognitive deficits associated with traumatic brain injuries. Although piracetam exhibited no long-term benefits for the treatment of mild cognitive impairments, recent studies demonstrated its neuroprotective effect when used during coronary bypass surgery. It was also effective in the treatment of cognitive disorders of cerebrovascular and traumatic origins; however, its overall effect on lowering depression and anxiety was higher than improving memory. As add-on therapy, it appears to benefit individuals with myoclonus epilepsy and tardive dyskinesia. Phenylpiracetam is more potent than piracetam and is used for a wider range of indications. In combination with a vasodilator drug, piracetam appeared to have an additive beneficial effect on various cognitive disabilities. Subgroup 2 drugs include levetiracetam, seletracetam and brivaracetam, which demonstrate antiepileptic activity, although their cognitive effects are unclear. Subgroup 3 includes piracetam derivatives with unknown clinical efficacies, and of these nefiracetam failed to improve cognition in post-stroke patients and rolipram is currently in clinical trials as an antidepressant. The remaining compounds of this subgroup are at various preclinical stages of research. The modes of action of piracetam and most of its derivatives remain an enigma. Differential effects on subtypes of glutamate receptors, but not the GABAergic actions, have been implicated. Piracetam seems to activate calcium influx into neuronal cells; however, this function is questionable in the light of findings that a persistent calcium inflow may have deleterious impact on neuronal cells. Although subgroup 2 compounds act via binding to another neuronal receptor (synaptic vesicle 2A), some of the subgroup 3 compounds, such as nefiracetam, are similar to those of subgr...

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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 ...