Application of the threshold of toxicological concern approach for the safety evaluation of calendula flower (Calendula officinalis) petals and extracts used in cosmetic and personal care products.

Calendula flower (Calendula officinalis) (CF) has been used in herbal medicine because of its anti-inflammatory activity. CF and C. officinalis extracts (CFE) are used as skin conditioning agents in cosmetics. Although data on dermal irritation and sensitization of CF and CFE's are available, the risk of subchronic systemic toxicity following dermal application has not been evaluated. The threshold of toxicological concern (TTC) is a pragmatic, risk assessment based approach that has gained regulatory acceptance for food and has been recently adapted to address cosmetic ingredient safety. The purpose of this paper is to determine if the safe use of CF and CFE can be established based upon the TTC class for each of its known constituents. For each constituent, the concentration in the plant, the molecular weight, and the estimated skin penetration potential were used to calculate a maximal daily systemic exposure which was then compared to its corresponding TTC class value. Since the composition of plant extracts are variable, back calculation was used to determine the maximum acceptable concentration of a given constituent in an extract of CF. This paper demonstrates the utility and practical application of the TTC concept when used as a tool in the safety evaluation of botanical extracts.

Differential subcellular localization of SNAP-25a and SNAP-25b RNA transcripts in spinal motoneurons and plasticity in expression after nerve injury.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in the molecular regulation of neurotransmitter release. SNAP-25 exists in two isoforms, which arise from alternative splicing of exon 5. In situ hybridization was used to examine whether SNAP-25 isoform mRNA expression may be altered by experimental manipulations. The effect of unilateral nerve injury on SNAP-25 mRNA levels was studied in motoneurons of the rat lumbar spinal cord. In all animals, SNAP-25a RNA transcripts were demonstrated in the nucleus of motoneurons, whereas SNAP-25b mRNA was present mainly in the cytoplasm. Cloning of the rat Snap gene intron spacing the alternative exon 5a and 5b sequences and generation of an intron-specific oligonucleotide probe used for in situ hybridization did not point to the presence of unspliced variants of SNAP-25b mRNA. After unilateral sciatic nerve transection (axotomy), SNAP-25a and SNAP-25b expression decreased in axotomized motoneurons compared with corresponding motoneurons on the unlesioned side. A significant decrease was demonstrated 2 days after axotomy, which reached a maximum after 7 days (62% for SNAP-25a and 67% for SNAP-25b), while levels had slightly recovered by 14 and 28 days. Ventral root avulsion also induced a decrease in levels of SNAP-25 RNA transcripts, suggesting that the axonal injury in itself was responsible for the down-regulation of Snap gene expression. This study shows that, in spinal motoneurons, SNAP-25a and SNAP-25b RNA transcripts have different subcellular localization and that levels of SNAP-25 RNA transcripts are down-regulated after axonal injury.

Differential expression of SNAP-25 protein isoforms during divergent vesicle fusion events of neural development.

The presynaptic plasma membrane protein SNAP-25 (synaptosome-associated protein of 25 kDa) has been implicated as one of several neural-specific components that direct constitutive fusion mechanisms to the regulated vesicle trafficking and exocytosis of neurotransmitter release. There exist two alternatively spliced isoforms of SNAP-25, a and b, which differ in a putative membrane-interacting domain. We show that these two isoforms have distinct quantitative and anatomical patterns of expression during brain development, in neurons, and in neuroendocrine cells and that the proteins localize differently in neurites of transfected PC12 pheochromocytoma cells. These findings indicate that alternative isoforms of SNAP-25 may play distinct roles in vesicular fusion events required for membrane addition during axonal outgrowth and for release of neuromodulatory peptides and neurotransmitters.

Regulated vesicular fusion in neurons: snapping together the details.

In the past year major strides have been made toward our understanding of the molecular mechanisms involved in regulated vesicle fusion and exocytosis in neurons and neuroendocrine cells. Much of this advance has come from the identification of proteins participating in these events and of their potential roles mediated by interactions with each other, the constituent membranes, and, in some cases, Ca2+ signaling. The involvement of vesicle fusion in elongation of neuronal processes during development and release of transmitters and neuromodulatory peptides in the mature nervous system indicates, however, that refinements in the fusion machinery may be required for each of these acts. For many of the participants in synaptic membrane fusion, variant isoforms have been identified that exhibit modifications that might alter interactive properties of these proteins. We discuss the idea that diversification of isoforms, as illustrated by the expression of alternatively spliced variants of SNAP-25, is likely to be an important component in providing the detail necessary to differentiate the physiology of regulated fusion of different classes of vesicles employed in development, neurotransmission, and secretion.

Human cDNA clones encoding two different isoforms of the nerve terminal protein SNAP-25.

Two distinct cDNA sequences, corresponding to alternative isoforms of the human nerve terminal protein SNAP-25 (synaptosomal associated protein of 25 kDa), were cloned and characterized. Sequence analysis demonstrated that the two isoforms are generated by alternative splicing between two distinct but homologous exons 5, a and b each encoding 39 amino acids (aa). Although the two isoforms, SNAP-25a and SNAP-25b, differ by only 9 aa, this domain encodes the portion of the protein that is a substrate for post-translational fatty acylation, and therefore might be important for regulating subcellular localization and membrane targeting.

Structure of the chicken gene for SNAP-25 reveals duplicated exon encoding distinct isoforms of the protein.

SNAP-25 (synaptosomal associated protein of 25 kDa) is a neuronal-specific but non-uniformly expressed protein. Expression is correlated to the time of synaptogenesis and SNAP-25 protein is found predominantly in presynaptic fields. Here we report the cloning and characterization of the SNAP-25 chicken gene. The gene spans more than 65 kb of genomic DNA and is interrupted into nine different exons. Identification of the 5'-end of the mRNA showed that transcriptional initiation is heterogeneous, although a major transcriptional start site is present 30 bp downstream of a putative TATA-box. Analysis of the exon-intron organization revealed that exon 5 exists in two homologous but distinct versions separated by 140 base-pairs. Both exons 5 are expressed as mRNA, indicating that alternative splicing regulates the expression of two isoforms, a and b, of the SNAP-25 protein. The alternative splicing results in a difference of nine amino acid residues between the proteins in a domain demonstrated to be subject to palmitoylation. This implies that the two isoforms differ in their capacity or efficiency to be modified by fatty acylation, suggesting divergent abilities to interact with neuronal membranes.