Characterization of a backbone cleavage product of BMS-196854 (Oncostatin M), a recombinant anti-inflammatory cytokine.

PURPOSE: BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. METHODS: SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. RESULTS: Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. CONCLUSION: Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin inhibitor and/or controlling the solution pH and storage temperature.

Hypothyroidism: its incidence and prevalence in adults older than 55 years of age in an acute rehabilitation unit.

OBJECTIVE: To investigate the incidence and prevalence of hypothyroidism in the acute rehabilitation unit. DESIGN: Retrospective chart review. SETTING: Inpatient rehabilitation unit. PATIENTS: Thirty-five men and 91 women older than the age of 55 years (average, 74 years) separated into postsurgical (PS) and nonsurgical (NS) groups. Twenty-two men and 76 women were PS, 21 of whom had a history of hypothyroidism. Thirteen men and 15 women were NS, and 4 in this group had a history of hypothyroidism. MAIN OUTCOME MEASURES: Levels of thyroid-stimulating hormone and free thyroxine. RESULTS: There were 34 cases of hypothyroidism, a prevalence rate of 27%. The incidence of newly diagnosed cases was 9% (9 of 101). Six of the newly diagnosed cases were PS patients and three were NS patients. Eleven cases of undertreated hypothyroidism were found, in 9 PS patients and 2 NS patients. The rate of undertreated hypothyroidism in the PS population was 43% (9 of 21); in the nonsurgical population, it was 50% (2 of 4). The overall rate of undertreated hypothyroidism for both PS and NS groups was 44% (11 of 25). CONCLUSION: There is a high prevalence of hypothyroidism on an inpatient rehabilitation unit and a high rate of undertreated hypothyroidism in PS patients. Screening high-risk patients is recommended.

Comparison between men and women admitted to an inpatient rehabilitation unit after cardiac surgery.

OBJECTIVE: To determine whether there are differences in several factors between men and women who undergo inpatient post-cardiac surgery rehabilitation. DESIGN: A retrospective chart review. Information was collected on a variety of factors: age; previous myocardial infarction; number of days from surgery to admission to rehabilitation; postsurgery, prerehabilitation complications; length of stay on the rehabilitation unit; living arrangements before surgery; disposition; and postdischarge recommendations. SETTING: Community hospital rehabilitation unit associated with a university hospital. PATIENTS: One hundred thirty-eight patients (54 men, 84 women) admitted to an inpatient rehabilitation unit after cardiac surgery. RESULTS: There was a significant relationship between sex and preadmission living arrangements; 56% of women lived alone versus 26% of men (p < .01). There was a statistically significant difference in length of stay on the rehabilitation unit (p < .02). Men stayed longer, with a median stay of 16 days (95% confidence interval, 15 to 20) versus 15 days for women (95% confidence interval, 14 to 15). Ninety-three percent of men were discharged from rehabilitation at 30 days versus 98% of women. No relationship was noted between men and women in age, previous myocardial infarction, number of days from surgery to rehabilitation admission, length of stay on the rehabilitation unit, postsurgery-prerehabilitation complications, complications on the rehabilitation unit, presurgery living arrangements, disposition, and postdischarge therapy recommendations. CONCLUSION: Men and women showed comparable courses after cardiac surgery. Before surgery, women lived alone more frequently than men.

The transferrin receptor cytoplasmic domain determines its rate of transport through the biosynthetic pathway and its susceptibility to cleavage early in the pathway.

The soluble human transferrin receptor (TfR) found in blood is the result of a proteolytic cleavage occurring in the ectodomain of the receptor close to the transmembrane domain at Arg-100. We have discovered another cleavage site between Gly-91 and Val-92 even closer to the transmembrane domain. Cleavage at Gly-91 differs markedly from the normal cleavage site. It occurs when the entire cytoplasmic portion or the proximal 31 amino acids of the transmembrane domain are deleted. A soluble disulfide-bonded dimer of the TfR is released into the medium in contrast to the cleavage at Arg-100 where a dimer lacking intersubunit disulfide bonds is released. Whereas the cleavage at Arg-100 is generated by cycling through the endosomal system, pulse-chase experiments indicate that cleavage at Gly-91 occurs predominantly during the biosynthesis of the receptor. Pulse-chase analysis of the biosynthesis of mutant TfRs that lack the membrane-proximal cytoplasmic domain show that they exit the endoglycosidase H-sensitive compartment at a slower rate than the wild type TfR. These results suggest that the cytoplasmic domain influences the trafficking of the TfR either by influencing the folding of the ectodomain or by providing a positive signal for its transport through the biosynthetic pathway.

Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions.

The residue C221 on pyruvate decarboxylase (EC. 4.1.1.1) from Saccharomyces cerevisiae has been shown to be the site where the substrate activation cascade is triggered [Baburina et al. (1994) Biochemistry 33, 5630-5635] and is located on the beta domain [Arjunan et al. (1996) J. Mol. Biol. 256, 590], while the active-center thiamin diphosphate is located > 20 A away, at the interface of the alpha and gamma domains. The reactivity of all three exposed cysteines (152, 221, and 222) was examined under the influence of known activators and inhibitors. Protein chemical methods, in conjunction with [1-14C] and [3-3H] analogues of the mechanism-based inhibitor p-ClC6H4CH=CHCOCOOH, demonstrated that the holoenzyme bound approximately 2-3 atoms of tritium/atom of C-14. However, when the labeled enzyme was subjected to trypsinization, followed by sequencing of the labeled peptide, only the tritium label was in evidence at C221, with a stoichiometry of 2 atoms of tritium/tetrameric holoenzyme. Apparently, the product of decarboxylation bonded to the enzyme survived the limited proteolysis and sequencing, but the bound 2-oxoacid was released during the protocol. Surprisingly, the C221S or C222A variants, although they still possess 20-30% specific activity compared to the wild-type enzyme, could still be inhibited by the XC6H4CH=CHCOCOOH class of inhibitors/substrate analogues, as well as by the product of decarboxylation from such compounds, cinnamaldehydes. Other potential nucleophilic sites for the inhibitor [C152 (the third exposed cysteine), residues D28, H114, H115, and E477 at the active center and H92 at the regulatory site] were also substituted by a nonnucleophilic side chain. All variants were still subject to inhibition by p-ClC6H4CH=CHCOCOOH, the active-center variants being inactivated even faster than the wild-type enzyme, suggesting that the active center is involved in the inactivation process. It appears that C221 is one of only two sites of interaction with such compounds (perhaps the result of a Michael addition across the C=C bond), yet the bound [1-14C]-labeled inhibitor could no longer be detected after peptide mapping at this site or at the catalytic site. Upon combining the tritiated inhibitor with [2-14C]-thiamin diphosphate, no evidence could be found for a thiamin-inhibitor-protein ternary complex, suggesting that the thiamin-bound enamine intermediate did not react further with the protein. It is likely that the second form of inhibition is at the active center, with the inhibitor cofactor-bound, which would have been released during the proteolytic protocol. Among other known activators, ketomalonate was found to react at C221 only. Glyoxalic acid, a mechanism-based inhibitor, on the other hand, could react at both the regulatory and the catalytic center. The high reactivity of C221 is consistent with it being in the thiolate form at the optimal pH of the enzyme [forming a Cys221S(-) + HHis92 ion pair; see Baburina et al. (1996) Biochemistry 35, 10249-10255, and Baburina et al. (1998) Biochemistry 37, 1235-1244]. Several additional compounds were tested as potential regulatory site-directed reagents: iodoacetate, 1,3-dibromoacetone, and 1-bromo-2-butanone. All three compounds reduced the Hill coefficient and hence appear to react at C221. It was concluded that either substitution of C221 by a nonnucleophilic residue or large groups attached to C221 in the wild-type enzyme lead to a distortion of domain interactions, interactions which are required for both optimal activity and substrate activation.

Family interaction and migration decision making.

Using data from the Residential Preference and Migration Survey, a panel study of Pennsylvania households on migration intention and behavior, the authors examine some aspects of the decision to migrate. "We first examine the impact of the presence or absence of household interaction on the desire to move, migration expectations, and actual migration behavior. We hypothesize that the presence of household interaction is associated with migration behavior, while the absence of household interaction on migration-related issues is a predictor of staying in the present residence. Second, we test an expanded residential satisfaction migration decision model.... The expanded model permits us to test the thesis that household interaction frequency and consensus/conflict are moderating factors in explaining and predicting the outcome of migration decision making."

"Phantom" carpal tunnel syndrome.

Phantom sensation is ubiquitous among persons who have had amputation; however, if it develops into phantom pain, a thorough clinical investigation must ensue. We illustrate this with the case of a 49-year-old woman, 14 years after traumatic amputation of her left 2nd through 5th fingers, and 10 years after traumatic left transfemoral amputation. She had had phantom sensation in her absent fingers for years and developed progressive pain in her phantom fingers 3 months before presentation. Nerve conduction study revealed a high-normal distal motor latency of the left median nerve and a positive Bactrian test (sensitivity 87%). She was diagnosed with "phantom" carpal tunnel syndrome and treated with a resting wrist splint, decreased weight bearing on the left upper limb, and two corticosteroid carpal tunnel injections with marked improvement. Clinicians should recognize that phantom pain may be referred from a more proximal region and may be amenable to conservative management.

Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor.

The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron-transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

Acquisition of the functional properties of the transferrin receptor during its biosynthesis.

The properties of the newly synthesized and partially glycosylated forms of the transferrin receptor were examined to determine which co- and post-translational modifications are necessary for the acquisition of transferrin binding activity and transport of the receptor to the cell surface. The nascent transferrin receptor containing core-glycosylated asparagine-linked oligosaccharides does not possess complete intersubunit disulfide bonds, sediments predominantly as a monomer in sucrose density gradients, and shows reduced binding to transferrin-agarose. Within 20-30 min after synthesis, the transferrin receptor acquires the ability to bind to a transferrin-linked affinity column. Intersubunit disulfide bond formation occurs slowly throughout the transit of the receptor to the cell surface. These results indicate that core glycosylation of the receptor may be necessary but is not sufficient for the acquisition of the ability of the receptor to bind transferrin and that intersubunit disulfide bond formation is a post-translational event. Inhibition of complex carbohydrate synthesis by either swainsonine (1 micrograms/ml) or deoxynojirimycin (4 mM) does not inhibit the ability of this receptor to form intersubunit disulfide bonds or to be transported to the cell surface. The partially glycosylated receptor, however, does show an approximately 3-fold reduced affinity for transferrin.

Characterization of proteins that associate with an unglycosylated form of the transferrin receptor in A431 cells.

A protein doublet (Mr = 135,000/130,000) was found to coprecipitate with an unglycosylated form of the transferrin receptor in tunicamycin-treated A431 cells. This doublet is not detected with either a monoclonal or polyclonal antibody to the transferrin receptor on Western blots indicating that these proteins do not interact directly with transferrin receptor antibody. Proteolytic digestion patterns of the individual proteins of the Mr = 135,000/130,000 doublet suggest that they are related to one another and are distinct from the transferrin receptor. Further characterization of these proteins indicates that they form a high molecular weight complex with the unglycosylated but not the glycosylated form of the transferrin receptor. Pulse-chase experiments demonstrate that the proteins post-translationally associate with the receptor.

Modulation of the transferrin receptor during DMSO-induced differentiation in HL-60 cells.

When HL-60 cells are induced to differentiate by dimethyl sulfoxide along a granulocytic pathway there is a fivefold decrease in the total number of transferrin receptors within 3 days, as compared to untreated cells. This decrease is due primarily to a rapid decline in the synthesis of the receptor rather than an increase in the degradation of the receptor. The decrease in transferrin receptor synthesis is a specific and early event that precedes the cessation of cell proliferation, differentiation, and the decrease in total protein synthesis.

Family reunification and Philippine migration to the United States: the immigrants' perspective.

PIP: This article focuses on the impact of the family reunification provisions in the US immigration policy for legal immigration from the Philippines. Immigration and Naturalization Service data on the changing pattern of Philippine immigration to the US between 1971 and 1984 show an increase of nearly 2 1/2 times in the number of immediate family members exempt from numerical limitations, a doubling in the number of immigrants entering under family preference categories, but a marked decline in the number of occupational preference immigrants. Immigration-related plans, behavior, and characteristics from the immigrants' perspective are also analyzed. A family unification policy-based typology has been constructed to categorize intended and actual immigrants to the US. Using this typology, systematic differences are reported for out-migration plans, family contacts, the immigration process, and the characteristics of intended and actual immigrants. While political and economic system competition and inequality are contextual factors for international migration, from the immigrants' perspective, joining family members by means of the family reunification provisions of the US immigration policy is the dominant explanation for legal immigration to the US in a sample of 1340 adults in Philippine households in 1982.^ieng

Gas embolism death after laparoscopy delayed by "trapping" in portal circulation.

A young woman died suddenly about 1 hour after instillation of CO2 for diagnostic larparoscopy. Post-mortem x-rays revealed large volumes of gas in the portal system, the heart, and the brain. In addition, autopsy revealed gas bubbles in the coronary arteries, pulmonary hemorrhage and edema, and a probe-patent foramen ovale. We postulated the "trapping" of gas in the portal circulation and affirmed this by experiments in 6 dogs. We further postulate the delayed and intermittent release of this gas and of platelet aggregates into the systemic circulation would occur in volumes which would be insufficient to produce hemodynamic signs yet sufficient to produce serious pulmonary insult.