Theoretical design of antisense genes with statistically increased efficacy.

Endogenous expression of antisense RNA represents one major way of applying antisense nucleic acids. To express antisense RNA intracellularly, recombinant antisense genes have to be designed and introduced into cells where the target RNA is encountered. Efficient annealing between the antisense RNA and the target RNA is crucial for efficacy and is strongly influenced by RNA structure. Here we extend structural rules for the design of in vitro transcribed antisense RNAs to the design of recombinant antisense genes. Intracellularly expressed antisense RNA transcripts contain a central antisense portion and additional flanking vector-derived sequences. A computer algorithm was generated to compose large sets of antisense genes, to calculate secondary structures of the transcribed sequences and to select for favorable structures of antisense RNA in terms of annealing and efficacy. The biological test system to measure efficiency of antisense genes was human immunodeficiency virus type 1 (HIV-1) replication in 293T cells. When considering the lower intracellular steady-state levels of favorably structured endogenous transcripts, an antisense effect against HIV-1 replication was observed that was up to 60-fold stronger than that measured for predicted unfavorable species. The computational selection was successful for antisense portions of 300 nt but not 100 nt in length. This theoretical design of antisense genes supports their improved application under time- and labor-saving conditions.

Overtraining and the BCAA hypothesis.

The purpose of this review was to give an answer to the question whether there are convincing data to support the hypothesis of an amino acid imbalance as one possible mechanism to explain overtraining syndrome. Animal studies point to an enhanced synthesis of the neurotransmitter 5-hydroxytryptamine through an amino acid imbalance at the blood-brain barrier with a preferable tryptophan uptake into the brain, resulting in premature fatigue. Human studies, however, show contradictory results, mainly because of nonstandardized methodology, so that a final conclusion cannot be made at present. BCAA supplementation in addition to standard carbohydrate ingestion during sustained exercise seems to be of no eminent advantage to delay fatigue. The overall results concerning the BCAA hypothesis to explain overtraining are inconclusive and require more controlled experimental research.

Training and overtraining: an overview and experimental results in endurance sports.

Overtraining can be defined as "training-competition > > recovery imbalance", that is assumed to result in glycogen deficit, catabolic > anabolic imbalance, neuroendocrine imbalance, amino acid imbalance, and autonomic imbalance. Additional non-training stress factors and monotony of training exacerbate the risk of a resulting overtraining syndrome. Short-term overtraining called overreaching which can be seen as a normal part of athletic training, must be distinguished from long-term overtraining that can lead to a state described as burnout, staleness or overtraining syndrome. Persistent performance incompetence, persistent high fatigue ratings, altered mood state, increased rate of infections, and suppressed reproductive function have been described as key findings in overtraining syndrome. An increased risk of overtraining syndrome may be expected around 3 weeks of intensified/prolonged endurance training at a high training load level. Heavy training loads may apparently be tolerated for extensive periods of time if athletes take a rest day every week and use alternating hard and easy days of training. Persistent performance incompetence and high fatigue ratings may depend on impaired or inhibited transmission of ergotropic (catabolic) signals to target organs, such as: (I) decreased neuromuscular excitability, (II) inhibition of alpha-motoneuron activity (hypothetic), (III) decreased adrenal sensitivity to ACTH (cortisol release) and increased pituitary sensitivity to GHRH (GH release) resulting in a counter-regulatory shift to a more anabolic endocrine responsibility, (IV) decreased beta-adrenoreceptor density (sensitivity to catecholamines), (V) decreased intrinsic sympathetic activity, and (VI) intracellular protective mechanisms such as increased synthesis of heat-shock proteins (HSP 70) represent a complex strategy against an overload-dependent cellular damage.

Spontaneous uptake of biologically active recombinant DNA by mammalian cells via a selected DNA segment.

DNA can be internalized by mammalian cells without taking advantage of helper reagents. Here, we ask whether the spontaneous cellular uptake of double-stranded DNA (dsDNA) occurs in a biologically significant and sequence-dependent way. We describe a combinatorial approach to search for dsDNA sequence segments that are preferentially internalized. A selected dsDNA species was identified and covalently linked to a luciferase expression cassette. The increased apparent cellular uptake of long-chain recombinant DNA accompanied by an increased apparent expression of luciferase provides strong evidence for the view that (i) naked long-chain dsDNA can be taken up spontaneously by mammalian cells, (ii) specific sequences substantially increase this process, and (iii) dsDNA is transported into the nucleus of cells in a bioactive form. Experimental evidence indicates a tissue- or cell-type specificity for this process. This work indicates that, in principle, specific nucleotide sequences can facilitate the introduction of naked dsDNA into target cells of interest, thereby improving existing vector systems and providing a new methodology to study DNA uptake by mammalian cells. The cellular uptake of biologically active genetic material in vivo occurs to be conceivable.