Human pro-tumor necrosis factor: molecular determinants of membrane translocation, sorting, and maturation.

Human pro-tumor necrosis factor (pro-TNF) is a type II transmembrane protein with a highly conserved 76-residue leader sequence. We have analyzed the behavior, both in a microsomal translocational system and by transfection, of a series of mutants with deletions from the cytoplasmic, transmembrane, and linking domains. Cytoplasmic deletions included the Arg doublet at -49 and -48 and/or the Lys doublet at -58 and -57; additional mutants included deletion of residues -73 to -55 and -73 to -55, -49, and -48. The transmembrane and linking domain mutants included deletions in the -42 to -35 region, combined with the deletion of residues -32 to -1. Two hybrid mutants combined the cytoplasmic deletions with the deletion of residues -32 to -1. All of the cytoplasmic deletion mutants were properly translocated, as were the transmembrane deletion mutants with deletions up to residues -36, -35, -32 to -1, although the last one exhibited reduced efficiency; further incremental deletions, including deletions of residues -38 to -35 and -32 to -1, completely blocked translocation. Both hybrid mutants were effectively translocated; furthermore, transfection analysis revealed competent expression and maturation of both the cytoplasmic and hybrid mutants. Thus, proper expression and maturation of human pro-TNF can be accomplished with as few as approximately 12 of the 26 residues of the native transmembrane domain and with a net negative charge in the cytoplasmic domain flanking the transmembrane region.

Effects of truncation of human pro-tumor necrosis factor transmembrane domain on cellular targeting.

Human tumor necrosis factor is initially synthesized as a transmembrane prohormone anchored by a hydrophobic region of the leader sequence. This hydrophobic domain has been previously localized to extend from Leu-46 to Ile-21 based on hydropathy calculations. To functionally determine the nature of this domain, we have generated a series of pro-TNF mutant cDNAs in which either half or both halves of this encoding domain is deleted. These cDNAs were analyzed both by the ability of their mRNAs to direct translation in a microsomal system and by cellular localization of their encoded TNFs following transfection of NIH/3T3 cells. We determined that the mutant protein with deletion of the periluminal region of the transmembrane domain (Thr-32 to Ile-21) was translocated into microsomes and localized on the inner surface of the microsomal membrane in a fashion identical to that of the parental TNF. In contrast, the mutants with deletion of either the pericytoplasmic aspect (Leu-46 to Gly-34) or of virtually the entire transmembrane domain were not localized in the microsomes. Transfection experiments indicated that only the cDNAs whose peptide products were translocated across microsomal membranes gave rise to transmembrane prohormones and matured TNFs. Thus, the functions of membrane targeting and orientation prior to proteolytic processing can be fulfilled by the sequence Leu-46 to Ala-33 of the transmembrane domain, but not by the sequence Ala-33 to Ile-21.

Hypnotizability and hypnoanalgesia: hypnotizability of patients using hypnoanalgesia during surgery.

We administered the Stanford Hypnotic Suggestibility Scale (Form A) (SHSS:A) to 10 patients from a population of 20 who had undergone surgery in the previous 10 years using hypnoanalgesia as the sole or principal analgesic agent. Time since surgery ranged from 2 days to 10 years. Scores on the SHSS ranged from 5 (medium susceptibility) to 12 (high susceptibility) with a mean of 8.6, significantly higher than the SHSS:A normative group (p less than .001). The relationship between severity of surgery and the use of hypnoanalgesia as the sole or principal analgesia was significant for our patient population (N = 20) but not for our patient sample (N = 10).

The use of hypnosis with cancer patients.

Hypnosis has proven to be extremely valuable in the treatment of cancer patients. Specific applications include: establishing rapport between the patient and members of the medical health team; control of pain with self-regulation of pain perception through the use of glove anesthesia, time distortion, amnesia, transference of pain to a different body part, or dissociation of the painful part from the rest of the body; controlling symptoms, such as, nausea, anticipatory emesis, learned food aversions, etc.; psychotherapy for anxiety, depression, guilt, anger, hostility, frustration, isolation, and a diminished sense of self-esteem; visualization for health improvement; and, dealing with death anxiety and other related issues. Hypnosis has unique advantages for patients including improvement of self-esteem, involvement in self-care, return of locus of control, lack of unpleasant side effects, and continued efficacy despite continued use.

Hypnosis in the 1990s--and beyond.

For this Presidential Address, I accepted the challenge to discuss my perception of the future directions of hypnotherapy. I believe that the next decade will bring increased attention to the mind/body relationship and how hypnosis can be most effectively employed in this area. As an oncologist, one of the most exciting areas of current research is in psychoneuroimmunology. The role of communication in the practice of the health sciences is receiving more emphasis. Training in hypnosis is helpful in recognizing spontaneous trance states which may modify the effects of "nocebos." The use of hypnoanesthesia for surgery would be ideal in third world and developing countries. I believe there will be increased interest in the use of hypnosis in self-care, in the forensic area, and in the use of self-hypnosis by the general population. Finally, I expect that in the next decade the ASCH and SCEH will cooperate more closely in many areas of significance to hypnosis.

Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells.

The insulin receptor is an integral transmembrane glycoprotein comprised of two alpha-(approximately 135 kDa) and two beta-(approximately 95 kDa) subunits, which is synthesized as a single polypeptide chain precursor (alpha beta). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into alpha- and truncated beta-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (alpha beta)2 dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain binds insulin with an apparent dissociation constant comparable to that of the wild-type hIR. However, the secreted dimer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR is curvilinear.(ABSTRACT TRUNCATED AT 250 WORDS)