New butenolides from two marine streptomycetes.

Chemical examination of two marine Streptomycetes has resulted in the isolation of four new butenolides, namely 4, 10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), two diastereomeric 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olides (2/3), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (4). The structures were identified by interpretation of the 2D NMR and mass spectral data.

Validation of a rat pheochromocytoma (PC12)-based cell survival assay for determining biological potency of recombinant human nerve growth factor.

A method has been validated, according to the Guidelines of the International Conference on Harmonization (ICH), for precise quantitation of the biological activity of recombinant human nerve growth factor (rhNGF) for lot release testing. The assay is based on the survival of a subclone of rat pheochromocytoma PC12 cells (PC12-CF) in response to rhNGF. Cell survival is measured by monitoring the reduction, by living cells, of the alamarBlue dye into a red form which is highly fluorescent. The assay is simple, has high throughput (performed in 96-well microtiter plates) and shows reproducible dose-response curves in the concentration range of 0.2-50 ng/ml. The method was validated for its linearity, accuracy, precision, robustness, and to meet current regulatory requirements. The assay demonstrated good linearity, yielding a coefficient of determination of 0.9902. Sample recovery studies demonstrated an accuracy ranging from 96 to 98%. The repeatability of the assay and intermediate precision had coefficients of variation (CV) of <9%. The assay was stability-indicating since it was able to detect changes in rhNGF samples degraded by protease treatment and in a number of isolated rhNGF variants. Robustness was demonstrated by the relative insensitivity of the assay to small deliberate changes in key method parameters. The validation data, provided in this manuscript, indicate that the newly described bioassay for rhNGF is robust, accurate, precise, and suitable for lot release potency testing of rhNGF.

A cell-based potency assay for insulin-like growth factor-I.

The authors developed a cell-based bioassay for determining the potency of recombinant human insulin-like growth factor I (IGF-I) using HU-3 human megakaryoblastic cell line. Cell proliferation was measured using the alamarBlue fluorescence method. The addition of IGF-I resulted in a dose-dependent growth response after 48 hours under serum-free conditions. The effective range was 0.1-25 ng/ml with half-maximal response at approximately 2 ng/ml IGF-I. The assay is simple, requiring just three steps, performed in 96-well microtitre plates and is able to detect changes in activity of truncated analogues of IGF-I (such as des-Gly-IGF-I, des-Gly-Pro-IGF-I and des-Gly-Pro-Glu-IGF-I) as well as IGF-I samples that had been subjected to proteolytic or disulfide reduction treatments. This assay is precise, with interassay variability of less than 10% and accurate, with percentage recoveries of nearly 100%. The relative efficacies of other insulin-related peptides in stimulating cell growth of the cell line were examined. IGF-II was 5-fold less potent than IGF-I and insulin had little or no proliferative activity. In addition, the growth-promoting activity correlated well with IGF-I stimulation of glucose consumption in this system. In conclusion, the HU-3 human megakaryoblastic cell line constitutes a simple system for measuring the biological activity of recombinant IGF-I in quality control set-up. The safety, convenience and precision of the assay make it an attractive alternative to radioactive and other colorimetric methods.

Maintenance of myonuclear domain size in rat soleus after overload and growth hormone/IGF-I treatment.

The purpose of this study was to determine the effects of functional overload (FO) combined with growth hormone/insulin-like growth factor I (GH/IGF-I) administration on myonuclear number and domain size in rat soleus muscle fibers. Adult female rats underwent bilateral ablation of the plantaris and gastrocnemius muscles and, after 7 days of recovery, were injected three times daily for 14 days with GH/IGF-I (1 mg/kg each; FO + GH/IGF-I group) or saline vehicle (FO group). Intact rats receiving saline vehicle served as controls (Con group). Muscle wet weight was 32% greater in the FO than in the Con group: 162 +/- 8 vs. 123 +/- 16 mg. Muscle weight in the FO + GH/IGF-I group (196 +/- 14 mg) was 59 and 21% larger than in the Con and FO groups, respectively. Mean soleus fiber cross-sectional area of the FO + GH/IGF-I group (2,826 +/- 445 microm2) was increased compared with the Con (2,044 +/- 108 microm2) and FO (2,267 +/- 301 microm2) groups. The difference in fiber size between the FO and Con groups was not significant. Mean myonuclear number increased in FO (187 +/- 15 myonuclei/mm) and FO + GH/IGF-I (217 +/- 23 myonuclei/mm) rats compared with Con (155 +/- 12 myonuclei/mm) rats, although the difference between FO and FO + GH/IGF-I animals was not significant. The mean cytoplasmic volume per myonucleus (myonuclear domain) was similar across groups. These results demonstrate that the larger mean muscle weight and fiber cross-sectional area occurred when FO was combined with GH/IGF-I administration and that myonuclear number increased concomitantly with fiber volume. Thus there appears to be some mechanism(s) that maintains the myonuclear domain when a fiber hypertrophies.

Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting.

The role of apoptosis in the elimination of myonuclei during hindlimb unloading-induced atrophy and the inhibition of apoptosis in the prevention of muscle atrophy were examined. The number of nuclei demonstrating double-stranded DNA fragmentation seen by terminal deoxynucleotidyl transferase (TDT) histochemical staining, an indicator of apoptosis, was significantly increased after 14 days of suspension. Double staining with TDT and antilaminin immunohistochemistry revealed that some TDT-positive nuclei were within the fiber lamina and were most likely myonuclei. The number of fibers containing morphologically abnormal nuclei was also significantly greater in suspended compared with control rats. Combined treatment with growth hormone and insulin-like growth factor I (GH/ IGF-I) and resistance exercise attenuated the increase in TDT-positive nuclei (approximately 26%, P > 0.05) and significantly decreased the number of fibers with morphologically abnormal nuclei. The data suggest that 1) "programmed nuclear death" contributes to the elimination of myonuclei and/or satellite cells from atrophying fibers, and 2) GH/IGF-I administration plus muscle loading ameliorates the apoptosis associated with hindlimb unloading.

The stability of recombinant human growth hormone in poly(lactic-co-glycolic acid) (PLGA) microspheres.

PURPOSE: The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained. METHODS: rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37 degrees C. RESULTS: rhGH PLGA formulations were produced with a low initial burst (< 20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37 degrees C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occurred at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH. CONCLUSIONS: rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37 degrees C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.

A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody.

A simple and non-radioactive complement-dependent cytotoxicity assay was developed to determine the relative potency of an anti-CD20 mAb, IDEC-C2B8. The assay measures the relative number of viable cells based on the uptake and metabolism of the redox dye, Alamar blue. A linear relationship between the relative fluorescence unit generated and the number of viable cells was demonstrated. The assay is simple, has high throughput (performed in 96-well microtiter plates), and shows reproducible dose-response curves in the concentration range of 0.02-3.3 micrograms/ml. With intra-assay variability of 5-12%, interassay variability of 6-10% and spike recoveries of 101-109%, the assay has high precision and accuracy. Specificity was demonstrated by the lack of activity of immunoglobulins that do not bind CD20, or anti-CD20 antibody isotype (gamma 4) which does not bind complement. The assay is able to detect degradative changes in the molecule caused by heat, light and proteolytic treatments, suggesting its use as a stability-indicating method. Finally, the Alamar blue method compared favorably with other more conventional methods used to assess cell viability. The assay has the desired properties for use as a potency assay for quality control testing of anti-CD20 mAb.

Growth hormone/IGF-I and/or resistive exercise maintains myonuclear number in hindlimb unweighted muscles.

In the present study of rats, we examined the role, during 2 wk of hindlimb suspension, of growth hormone/insulin-like growth factor I (GH/IGF-I) administration and/or brief bouts of resistance exercise in ameliorating the loss of myonuclei in fibers of the soleus muscle that express type I myosin heavy chain. Hindlimb suspension resulted in a significant decrease in mean soleus wet weight that was attenuated either by exercise alone or by exercise plus GH/IGF-I treatment but was not attenuated by hormonal treatment alone. Both mean myonuclear number and mean fiber cross-sectional area (CSA) of fibers expressing type I myosin heavy chain decreased after 2 wk of suspension compared with control (134 vs. 162 myonuclei/mm and 917 vs. 2,076 micron2, respectively). Neither GH/IGF-I treatment nor exercise alone affected myonuclear number or fiber CSA, but the combination of exercise and growth-factor treatment attenuated the decrease in both variables. A significant correlation was found between mean myonuclear number and mean CSA across all groups. Thus GH/IGF-I administration and brief bouts of muscle loading had an interactive effect in attenuating the loss of myonuclei induced by chronic unloading.

Long-acting growth hormones produced by conjugation with polyethylene glycol.

Derivatives of human growth hormone (hGH) of increasing size were produced by reaction with the N-hydroxysuccinimide ester of polyethylene glycol-5000 (PEG5000), a 5-kDa reagent that selectively conjugates to primary amines. By adjusting the reaction conditions and purification procedure, it was possible to isolate hGH derivatives containing up to seven PEG moieties that altered the Stokes radius and thereby the effective molecular masses of the unmodified hormone from 22 to 300 kDa. Fortunately, the most reactive amines were ones that did not lie in either of the two sites important for receptor binding. Nonetheless, increasing the level of PEG modification linearly reduced the affinity of hGH for its receptor and increased the EC50 in a cell-based assay up to 1500-fold. Most of the reduction in affinity was the result of slowing the association rate for the receptor. The clearance rate of hGH in rats was inversely proportional to effective molecular weight and closely fit a filtration model. We have tested the potency of these analogs by injecting them daily or every 6 days into hypophysectomized rats and determining the effects on body and organ growth. The efficacy of these analogs was optimal for hGH conjugated with 5 eq of PEG5000, and the potency was increased by about 10-fold compared with unmodified hGH. Such PEG-hGH derivatives show promise as long-acting alternatives to daily injections of hGH. More generally these studies show that improving hormone clearance properties, even at the expense of reducing receptor binding affinity, can lead to dramatic increases in hormone efficacy.

IGF-I, growth hormone, and/or exercise effects on non-weight-bearing soleus of hypophysectomized rats.

The effects of insulin-like growth factor (IGF-I) or growth hormone (GH) with and without exercise on predominantly slow muscles of hypophysectomized hindlimb-suspended (HS) rats were determined. HS resulted in a 21, 23, and 30% decrease in soleus, adductor longus, and vastus intermedius masses, respectively, compared with ambulatory rats. Compared with values in HS rats, IGF-I increased the vastus intermedius mass and GH or exercise alone increased both the soleus and vastus intermedius masses. There was a strong interactive effect between GH, but not IGF-I, and exercise in all three muscles of HS rats. The soleus fiber type distribution of HS rats was not affected by any treatment. HS resulted in a 24, 18 (P > 0.05), 32, and 20% (P > 0.05) decrease in the size of soleus fibers containing type I, IIa, I + IIa, and IIa + IIx myosin heavy chains, respectively, compared with ambulatory hypophysectomized rats. Hormone or exercise alone had no effect on fiber size in HS rats. However, all fiber sizes (except for type IIa + IIx in IGF-I with exercise rats) were larger in HS rats treated with GH or IGF-I and exercise than those in HS rats. These data indicate an interactive effect of both GH and IGF-I with exercise in maintaining fiber size of chronically non-weight-bearing predominantly slow muscles. Furthermore, the results suggest that the myosin heavy-chain phenotype in rats deficient in all pituitary factors is unresponsive to short-term administration of either GH or IGF-I or to exercise or HS.

Novel assays based on human growth hormone receptor as alternatives to the rat weight gain bioassay for recombinant human growth hormone.

Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as 'receptor' in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyzed the resulting complex, which forms in a 2:1 receptor to rhGH ratio. The 2:1 complex is assayed at a concentration near the Kd (approximately 0.4 nM), providing high specificity for rhGH and detection of rhGH variants with reduced activity. In the CP assay, a mouse myeloid leukaemia cell line (FDC-P1) transfected with the full-length receptor is exposed to varying levels of rhGH for 16-20 h. The incorporation of 3H-thymidine into DNA is used as an index of cell proliferation. The results show that the HPRBC assay provides significantly improved precision with a relative standard deviation (RSD) of < or = 5% vs. an RSD of 23% for the rat bioassay. The CP assay has RSDs of 4-16%. Analysis of rhGH variants and mutants shows that the potencies measured by both the HPRBC and CP assays are in general agreement with the rat weight gain bioassay. Both of the HPRBC and CP assays are sufficiently rugged for operating in a Good Manufacturing Practices (GMP) routine batch release testing environment. In vitro alternatives such as the HPRBC and CP assays build a foundation for replacing the hypophysectomized rat weight gain bioassay by correlating receptor dimerization, binding specificity and signal transduction with the biological activity of rhGH.

Affinity purification and microcharacterization of recombinant DNA-derived human growth hormone isolated from an in vivo model.

A procedure has been developed for the isolation and purification of trace amounts of unlabeled proteins from biological solutions. Using a combination of affinity chromatography and reversed-phase HPLC, microgram amounts of recombinant DNA-derived human growth hormone (rhGH) were purified from an in vivo rat model. Microcharacterization techniques were developed, and picomole amounts of the recovered protein were digested with trypsin and characterized using capillary HPLC peptide mapping. The described procedures were used to study the chemical changes that occur in rhGH following intravenous administration. The study demonstrated that both deamidation and oxidation can occur in vivo, although the former would occur to a significant extent only in proteins with an extended half-life.

Stimulation of myofibrillar protein synthesis in hindlimb suspended rats by resistance exercise and growth hormone.

The objective of this study was to determine the ability of a single bout of resistance exercise alone or in combination with recombinant human growth hormone (rhGH) to stimulate myofibrillar protein synthesis (Ks) in hindlimb suspended (HLS) adult female rats. Plantar flexor muscles were stimulated with resistance exercise, consisting of 10 repetitions of ladder climbing on a 1 m grid (85 degrees), carrying an additional 50% of their body weight attached to their tails. Saline or rhGH (1 mg/kg) was administered 30' prior to exercise, and Ks was determined with a constant infusion of 3H-Leucine at 15', 60', 180', and 360' following exercise. Three days of HLS depressed Ks approximately 65% and 30-40% in the soleus and gastrocnemius muscles, respectively (p < or = 0.05). Exercise increased soleus Ks in saline-treated rats 149% 60' following exercise (p < or = 0.05), decaying to that of non-exercised animals during the next 5 hours. Relative to suspended, non-exercised rats rhGH+exercise increased soleus Ks 84%, 108%, and 72% at 15', 60' and 360' following exercise (p < or = 0.05). Gastrocnemius Ks was not significantly increased by exercise or the combination of rhGH and exercise up to 360' post-exercise. Results from this study indicate that resistance exercise stimulated Ks 60' post-exercise in the soleus of HLS rats, with no apparent effect of rhGH to enhance or prolong exercise-induced stimulation. Results suggests that exercise frequency may be important to maintenance of the slow-twitch soleus during non-weightbearing, but that the ability of resistance exercise to maintain myofibrillar protein content in the gastrocnemius of hindlimb suspended rats cannot be explained by acute stimulation of synthesis.

Identification of the major sites of phosphorylation in IGF binding protein-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) is the major carrier of insulin-like growth factor I and II in the circulation. IGFBP-3 is secreted by various tissues and cell lines as a glycosylated phosphoprotein. We have identified two major serine phosphorylation sites located at amino acids 111 and 113 of the human protein. These serine residues and neighboring amino acids potentially involved in defining a protein kinase recognition sequence were mutated to alanine using PCR. Single and double point mutants were stably transfected into CHO-cells and analyzed for their level of phosphorylation. Mutation of both serines reduced phosphorylation by > 80% in the full-length protein and completely abolished phosphorylation in a 17 kDa IGFBP-3 fragment, derived from digestion with EndoProteinase Lys-C. The 17 kDa fragment contained serines 111 and 113. S111A/S113A, a double serine-to-alanine mutant at positions 111 and 113, showed a strongly reduced glycosylation pattern that appears to be the result of amino acid substitutions rather than lack of phosphorylation. Mutant S111A/S113A, despite being non-phosphorylated and non-glycosylated, is functionally similar to the wild-type IGFBP-3 in terms of IGF-I binding. These results enhance our understanding on the functional role of glycosylation and phosphorylation of IGFBP-3.

Resistance exercise and growth hormone as countermeasures for skeletal muscle atrophy in hindlimb-suspended rats.

Unweighting of rat hindlimb muscles results in skeletal muscle atrophy, decreased protein synthesis, and reduced growth hormone (GH) secretion. Resistance exercise (ladder climbing) and GH treatment partially attenuate skeletal muscle atrophy in hypophysectomized hindlimb-suspended rats. It was hypothesized that a combination of multiple bouts of daily resistance exercise and GH (1 mg.kg-1.day-1) would prevent skeletal muscle atrophy in growing nonhypophysectomized hindlimb-suspended rats. Hindlimb suspension decreased the absolute (mg/pair) and relative (mg/100 g body wt) weights of the soleus, a slow-twitch plantar flexor, by 30 and 21%, respectively, and the absolute and relative weights of the gastrocnemius, a predominantly fast-twitch plantar flexor, by 20 and 11%, respectively (P < 0.05). Exercise did not increase soleus mass but attenuated loss of relative wet weight in the gastrocnemius muscles of hindlimb-suspended rats (P < 0.05). Hindlimb suspension decreased gastrocnemius myofibrillar protein content and synthesis (mg/day) by 26 and 64%, respectively (P < 0.05). The combination of exercise and GH attenuated loss of gastrocnemius myofibrillar protein content and synthesis by 70 and 23%, respectively (P < 0.05). Results of the present investigation indicate that a combination of GH and resistance exercise attenuates atrophy of unweighted fast-twitch skeletal muscles.

Interactive effects of growth hormone and exercise on muscle mass in suspended rats.

Measures to attenuate muscle atrophy in rats in response to stimulated microgravity [hindlimb suspension (HS)] have been only partially successful. In the present study, hypophysectomized rats were in HS for 7 days, and the effects of recombinant human growth hormone (GH), exercise (Ex), or GH+Ex on the weights, protein concentrations, and fiber cross-sectional areas (CSAs) of hindlimb muscles were determined. The weights of four extensor muscles, i.e., the soleus (Sol), medial (MG) and lateral (LG) gastrocnemius, and plantaris (Plt), and one adductor, i.e., the adductor longus (AL), were decreased by 10-22% after HS. Fiber CSAs were decreased by 34% in the Sol and by 17% in the MG after HS. In contrast, two flexors, i.e., the tibialis anterior (TA) and extensor digitorum longus (EDL), did not atrophy. In HS rats, GH treatment alone maintained the weights of the fast extensors (MG, LG, Plt) and flexors (TA, EDL) at or above those of control rats. This effect was not observed in the slow extensor (Sol) or AL. Exercise had no significant effect on the weight of any muscle in HS rats. A combination of GH and Ex treatments yielded a significant increase in the weights of the fast extensors and in the CSA of both fast and slow fibers of the MG and significantly increased Sol weight and CSA of the slow fibers of the Sol. The AL was not responsive to either GH or Ex treatments. Protein concentrations of the Sol and MG were higher only in the Sol of Ex and GH + Ex rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal and metabolic responses of hypophysectomized rats with head-down suspension.

The primary purpose of this investigation was to secure select anatomical and physiological measurements from hypophysectomized rats and their sham-operated control to determine how various endocrine influences could be modified by conditions of simulated microgravity. The focal point of the study was the exercise responses after head-down suspension; however, we were also interested in obtaining insights on nonexercise-related mechanisms. Since more details and information concerning this study will be published elsewhere, we will highlight those findings which warrant further research.

Recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3): effects on the glycemic and growth promoting activities of rhIGF-1 in the rat.

The effects of IGFBPs on IGF-1 bioactivity in vivo remain largely unknown. We have tested the ability of rhIGFBP-3, made in 293 cells or CHO cells, to affect the glycemic and anabolic activities of co-administered rhIGF-1. For glycemic studies female dw/dw rats were anesthetized, a jugular catheter inserted, basal blood samples taken and i.v. injections given of rhIGF-1 (0.25 mg/kg), rhIGFBP-3 (0.50 mg/kg), or rhIGF-1 plus rhIGFBP-3 (0.25 plus 0.50 mg/kg, respectively). The blood glucose (20 min later, as a percent of initial, mean +/- SD) reached its nadir for rhIGF-1 alone (58 +/- 5%) but was not changed by rhIGFBP-3 alone (99 +/- 4%) or rhIGF-1 plus rhIGFBP-3 (93 +/- 5%). In growth studies, young female hypophysectomized rats (90-105 g) were injected s.c. twice daily for 3-4 days, or infused s.c. for 7 days, with excipient, rhIGF-1 or rhIGF-1 plus rhIGFBP-3 at doses similar to those used in the hypoglycemia studies. Weight gain induced by rhIGF-1 was either unchanged, or enhanced, when the rhIGF-1 was delivered with rhIGFBP-3. The hypoglycemic activity of IGF-1 was greatly reduced if IGF-1 was administered bound to 293-cell rhIGFBP-3 but anabolic activity was unchanged or enhanced.

Chemical heterogeneity as a result of hydroxylamine cleavage of a fusion protein of human insulin-like growth factor I.

Recombinant DNA techniques were used to biosynthesize human insulin-like growth factor I (hIGF-I) as a fusion protein wherein the fusion polypeptide is an IgG-binding moiety derived from staphylococcal protein A. This fusion protein is produced in Escherichia coli and secreted into the fermentation broth. In order to release mature recombinant-derived hIGF-I (rhIGF-I), the fusion protein is treated with hydroxylamine, which cleaves a susceptible Asn-Gly bond that has been engineered into the fusion protein gene. Reversed-phase h.p.l.c. was used to estimate the purity of the rhIGF-I preparations, especially for the quantification of the methionine sulphoxide-containing variant. It was determined that hydroxylamine cleavage of the fusion protein produced, as a side reaction, hydroxamates of the asparagine and glutamine residues in rhIGF-I. Although isoelectric focusing was effective in detecting, and reversed-phase h.p.l.c. for producing enriched fractions of the hydroxamate variants, ion-exchange chromatography was a more definitive procedure, as it allowed quantification and facile removal of these variants. The identity of the variants as hydroxamates was established by Staphylococcus aureus V8 proteinase digestion, followed by m.s., as the modification was transparent to amino acid and N-terminal sequence analyses. The biological activity of rhIGF-I was established by its ability to incorporate [3H]thymidine into the DNA of BALB/c373 cells and by a radioreceptor assay utilizing human placental membranes. Both assays demonstrate that the native, recombinant and methionine sulphoxide and hydroxamate IGF-I variants are essentially equipotent.