The heat shock response is transcriptionally regulated by an evolutionarily conserved protein termed heat shock factor (HSF). We report the purification to homogeneity and the partial peptide sequence of HSF from HeLa cells.

With increased utilization of platelet-rich plasma (PRP), it is important for clinicians to understand the United States, the Food and Drug Administration (FDA) regulatory role and stance on PRP. Blood products such as PRP fall under the prevue of FDA's Center for Biologics Evaluation and Research (CBER). CBER is responsible for regulating human cells, tissues, and cellular and tissue-based products. The regulatory process for these products is described in the FDA's 21 CFR 1271 of the Code of Regulations. Under these regulations, certain products including blood products such as PRP are exempt and therefore do not follow the FDA's traditional regulatory pathway that includes animal studies and clinical trials. The 510(k) application is the pathway used to bring PRP preparation systems to the market. The 510(k) application allows devices that are ?substantially equivalent? to a currently marketed device to come to the market. There are numerous PRP preparation systems on the market today with FDA clearance; however, nearly all of these systems have 510(k) clearance for producing platelet-rich preparations intended to be used to mix with bone graft materials to enhance bone graft handling properties in orthopedic practices. The use of PRP outside this setting, for example, an office injection, would be considered ?off label.? Clinicians are free to use a product off-label as long as certain responsibilities are met. Per CBER, when the intent is the practice of medicine, clinicians ?have the responsibility to be well informed about the product, to base its use on firm scientific rationale and on sound medical evidence, and to maintain records of the product's use and effects.? Finally, despite PRP being exempted, the language in 21 CFR 1271 has caused some recent concern over activated PRP; however to date, the FDA has not attempted to regulate activated PRP. Clinicians using activated PRP should be mindful of these concerns and continued to stay informed.

Schizosaccharomyces pombe is becoming an increasingly useful organism for the study of cellular processes, since in certain respects, such as the cell cycle and splicing, it is similar to metazoans. Previous biochemical studies have shown that the DNA binding ability of S. pombe heat shock factor (HSF) is fully induced only under stressed conditions, in a manner similar to that of Drosophila melanogaster and humans but differing from the constitutive binding by HSF in the budding yeasts. We report the isolation of the cDNA and gene for the HSF from S. pombe. S. pombe HSF has a domain structure that is more closely related to the structure of human and D. The mechanism by which eukaryotic cells respond to heat shock is highly conserved (reviewed in references 21 and 24). This response, which also protects cells from numerous other forms of stress, is presumed to have been conserved throughout evolution because the ability of a cell to survive stress is critical to its viability in a natural setting. A major aspect of the heat shock response is the transcriptional induction of the genes that encode the heat shock proteins. This induction is regulated by heat shock factor (HSF), a transcription factor whose DNA binding site (the heat shock element [HSE]) is similar in all studied eukaryotes. This binding site is found in the promoter regions of heat shock genes and consists of inverted repeats of the sequence nGAAn (3,27,45). Binding of HSF to this site results in 20-to 1,000-fold transcriptional stimulation, depending on the specific promoter and cell type. The potent transcriptional stimulatory properties of HSF, the conserved nature of the heat shock response, and the importance of this response to cellular physiology have caused HSF to be an intensively studied factor.The regulation of HSF following stress has been compared in several eukaryotic organisms. In the budding yeast Saccharomyces cerevisiae, HSF is constitutively bound to the HSE, and heat shock increases the degree of transcriptional stimulation attributed to HSF (14,37,39,40). This increase in transcriptional stimulatory ability is believed to be regulated by posttranslational modification of S. cerevisiae HSF (37, 40). There is a correlation between increased phosphorylation of the molecule and increased transcription of the heat shock genes, and it has therefore been proposed that phosphorylation might regulate transcriptional stimulation * Corresponding author. t Present address: Immunogen, Cambridge, MA 02139.by this HSF. Study of a related budding yeast, Kluyveromyces lactis, identified a conserved seven-amino-acid sequence in HSF that is necessary for appropriate regulation of the transcriptional stimulatory properties of S. cerevisiae and K lactis HSFs (15). The precise role that this seven-amino-acid sequence plays in regulating HSF has not been elucidated. Budding yeast HSF is one of the factors responsible for maintaining basal (nonstressed) expression from certain heat shock promoters, in part because of its ability to constitutively bin...

The heat shock response appears to be universal. All eucaryotes studied encode a protein, heat shock factor (HSF), that is believed to regulate transcription of heat shock genes. This protein binds to a regulatory sequence, the heat shock element, that is absolutely conserved among eucaryotes. We report here the identification of HSF in the fission yeast Schizosaccharomyces pombe. HSF binding was not observed in extracts from normally growing S. pombe (28C) but was detected in increasing amounts as the temperature of heat shock increased between 39 and 45°C. This regulation is in contrast to that observed in Saccharomyces cerevisiae, in which HSF binding is detectable at both normal and heat shock temperatures. The S. pombe factor bound specifically to the heat shock element, as judged by methylation interference and DNase I protection analysis. The induction of S. pombe HSF was not inhibited by cycloheximide, suggesting that induction occurs posttranslationally, and the induced factor was shown to be phosphorylated. S. pombe HSF was purified to near homogeneity and was shown to have an apparent mobility of approximately 108 kDa. Since heat-induced DNA binding by HSF had previously been demonstrated only in metazoans, the conservation of heat-induced DNA binding by HSF among S. pombe and metazoans suggests that this mode of regulation is evolutionarily ancient.

The late promoter of simian virus 40 is transcriptionally activated, in trans, by large T antigen, the primary viral early gene product. Although large T antigen is a well-characterized DNA-binding protein, a variety of data suggest that its trans-activation function does not require direct interaction with DNA. We demonstrate that defined late promoter elements, omega (w), tau (T), and delta (8), necessary for T-antigen-mediated trans-activation, are binding sites for simian cellular factors, not T antigen. Two of the late promoter elements (w and T) are shown to bind the same factor or family of factors. These factors bind to a site very similar to that for the HeLa cell factor AP1. We refer to these factors as the simian APl-sequence recognition proteins (sAP1-SRPs). Compared with normal simian CV-1P cells, the sAPl-SRPs from T-antigen-producing COS cells, or from 14-h simian virus 40-infected CV-1P cells, showed altered binding patterns to both the w and T binding sites. In addition, the sAPl-SRPs from T-antigen-containing cells bound to the T site more stably than did the analogous factors from normal CV-1P cells. The altered pattern of binding and the increased stability of binding correlated with the presence of T antigen in the cell. Additionally, the alteration of the binding pattern within 14 h of infection in CV-1P cells is temporally correct for late promoter activation. Overall, the data show (i) that the late promoter elements necessary for T-antigen-mediated trans-activation contain binding sites for simian cellular DNA-binding proteins; (ii) that the presence of T antigen causes alterations in the binding characteristics of specific simian cellular DNA-binding factors or families of factors; and (iii) that factors which bind to the late promoter elements required for activation have altered and more stable binding characteristics in the presence of T antigen. These points strongly suggest that T antigen mediates trans-activation indirectly through the alteration of binding of at least one specific simian cellular factor, sAPl-SRP, or through the induction of a family of sAPl-SRP factors.It is clear from many recent reports that numerous eucaryotic transcription factors must interact with specific elements within viral and cellular promoters for the ultimate transcription of a gene by RNA polymerase II (for review, see reference 39). It also appears quite likely that modification, induction, or repression of individual factors may account, in large part, for modulation of transcription. Evidence in support of such transcriptional control is indicated by studies of the DNA tumor viruses. These viruses, in a broad sense, follow a relatively similar strategy for regulating the temporal order of their gene expression. Specifically, the proteins first expressed by the virus (for example, the adenovirus Ela protein and the papovavirus large T antigens) or first introduced as part of the virion (for example, the herpesvirus VP16 or alpha trans-inducing factor) mediate trans-activation mechanisms which ca...