Understanding men's health and illness: a gender-relations approach to policy, research, and practice.

Men's health has emerged as an important public concern that may require new kinds of healthcare interventions and increased resources. Considerable uncertainty and confusion surround prevailing understandings of men's health, particularly those generated by media debate and public policy, and health research has often operated on oversimplified assumptions about men and masculinity. A more useful way of understanding men's health is to adopt a gender-relations approach. This means examining health concerns in the context of men's and women's interactions with each other, and their positions in the larger, multidimensional structure of gender relations. Such an approach raises the issue of differences among men, which is a key issue in recent research on masculinity and an important health issue. The gender-relations approach offers new ways of addressing practical issues of healthcare for men in college environments.

1.85 A structure of anti-fluorescein 4-4-20 Fab.

The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti-fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1.

Multivalent Fvs: characterization of single-chain Fv oligomers and preparation of a bispecific Fv.

Single-chain Fv proteins are known to aggregate and form multimeric species. We report here that these molecules represent a new class of molecular assembly, which we have termed multivalent Fvs. Each binding site in a multivalent Fv comprises the variable light-chain (VL) domain from a single-chain Fv, and the variable heavy-chain (VH) domain from a second single-chain Fv. Each single-chain Fv in a multivalent Fv is part of two binding sites. We have characterized the multivalent forms of the 4-4-20, CC49 and B6.2 sFvs. The degree of multivalent Fv formation is linker-dependent. Multivalent Fvs cannot form in the absence of an intact linker. Multivalent Fvs can be stabilized by their antigen. The conversion between different forms of the multivalent Fvs can be catalyzed by disassociating agents such as 0.5 M guanidine hydrochloride with 20% ethanol. Multivalent Fvs have significantly different stabilities depending on the specific variable domains from which they are constructed. Two models have been proposed for the structure of a multivalent Fv. We have tested each model by attempting to produce a heterodimer from the anti-fluorescein 4-4-20 and anti-tumor CC49 variable regions. We successfully produced a 4-4-20/CC49 heterodimer that comprises two mixed sFvs. The first mixed sFv is composed of the 4-4-20 VL domain, a 12 residue linker and the CC49 Vh domain. The second mixed sFv is composed of a CC49 VL domain, a 12 residue linker and the 4-4-20 VH domain. The 4-4-20/CC49 heterodimer bound both fluorescein and the tumor-associated glycoprotein-72 antigen. These results support a VH/VL 'rearrangement' model in which each variable domain of a multivalent Fv binding site comes from a different polypeptide chain.

Crystallization of single-chain Fv proteins.

Single-chain Fv (sFv) proteins consist of the variable heavy chain (VH) and variable light chain (VL) domains of an antibody, covalently joined by an engineered polypeptide linker. We report the crystallization of single-chain Fv's with specificities for fluorescein (4-4-20 sFv) and the TAG-72 pan-carcinoma glycoprotein antigen (CC49 sFv). Concentration of these proteins, preliminary to crystallization, results in a monomer-multimer equilibrium, causing aggregation which interferes with crystallization. Aggregation has been observed to depend primarily on an intact linker between VL and VH domains, although other factors are likely to modulate this phenomenon as well, including the specific identity of Fv and ligand, presence or absence of the ligand, linker length and possibly sequence. We have found two methods to overcome sFv aggregation, both of which yield X-ray diffraction quality crystals. The first, discovered serendipitously, is by introducing a proteolytic clip into the linker region (effectively yielding an Fv fragment). The second is the purification of the sFv dimer form, with linker regions intact, from an equilibrium mixture of aggregates. The sFv molecular association in a dimer is believed to be unusual in that each VL/VH interface may not be formed by the two linker-connected VL and VH domains, but rather by interaction of VL and VH domains from two distinct sFv monomers. Structure determination of the CC49 sFv dimer, with the 14-residue linker designated 212, is underway to test this model. Increasing linker length, to relieve steric strain on the monomer, and inclusion of the appropriate antigen, to slow transitions between monomeric and multimeric forms, may prove valuable strategies with sFv proteins less amenable to crystallization.

An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability.

The effects of linker length on binding affinity and degree of aggregation have been examined in the antifluorescein 4-4-20 and anticarcinoma CC49 single-chain Fvs. Longer linkers in the antifluorescein sFvs have higher affinities for fluorescein and aggregate less. A proteolytically susceptible site between Lys8 and Ser9, in the previously reported 212 linker has been identified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG) was designed in which a proline was placed at the C-terminal side of the proteolytic clip site in the 212 linker. The CC49 sFv containing the 218 linker showed reduced aggregation and was found to be more stable to proteolysis in vitro, when compared to the CC49/212 sFv. The CC49 sFv with the longer 218 linker had higher affinity than CC49/212 sFv. An aggregated CC49/212 sFv sample had higher affinity than CC49/218 sFv. The CC49/218 and CC49/212 sFvs had similar blood clearances in mice, while the aggregated CC49/212 sFv remained in circulation significantly longer. In mice bearing LS-174T human colon carcinoma xenografts, the CC49/218 sFv showed higher tumor uptake than the CC49/212 sFv and lower tumor uptake than the aggregated CC49/212 sFv. The higher tumor uptake of the CC49/218 is most likely a result of its higher resistance to proteolysis. The higher affinity and higher tumor uptake of the aggregated CC49/212 sFv are most likely due to the repetitive nature of the TAG-72 antigen and the higher avidity of multivalent aggregates. When the sFvs were radiolabeled with a lutetium-chelate the CC49/218 sFv showed a lower accumulation in the liver and spleen compared to the aggregated CC49/212 sFv.

Microautoradiographic analysis of the normal organ distribution of radioiodinated single-chain Fv and other immunoglobulin forms.

In previous studies, we have compared the immunochemical properties, the in vivo pharmacokinetics, and the tumor penetrance of a radioiodinated single-chain Fv (sFv) in comparison with other immunoglobulin (Ig) forms (intact IgG, F(ab')2, and Fab') (Cancer Res., 51: 6363-6371, 1991). Biodistribution studies demonstrated a higher percent injected dose/g in the liver and spleen for the intact IgG and F(ab')2. Renal uptake was observed with the Fab' and F(ab')2, whereas the sFv demonstrated no specific localization in either of these organs. The 125I-labeled sFv also demonstrated a more even distribution throughout the tumor xenografts as compared to the other Ig forms (Cancer Res., 52: 3402-3408, 1992). Subsequent studies utilizing the sFv conjugated with a radiometal (177Lu) demonstrated that the sFv was being metabolized by the kidney, and a significantly higher percent injected dose/g was obtained with a 177Lu-labeled sFv as compared to a 125I-labeled sFv (Cancer Res., 52: 6413-6417, 1992). These previous studies indicated the potential utility of radioiodinated sFv and other Ig fragments for use in radioimmunoguided surgery with a hand-held probe, diagnostic imaging, and possibly therapy. The present study compares the distribution in normal tissues of the 4 Ig forms of monoclonal antibody (MAb) CC49, which is directed against a pancarcinoma antigen (tumor-associated glycoprotein-72). 125I-labeled sFv, Fab', F(ab')2, and IgG of MAb CC49 were administered to athymic mice either bearing or not bearing the tumor-associated glycoprotein-72 positive human colon carcinoma xenograft (LS-174T). At various intervals following the i.v. injection of the Ig forms, the liver, spleen, kidneys, and lungs were removed for autoradiographic analyses. Dramatic differences were observed in the kidney; the IgG was found only in the renal vasculature, whereas the Fab', F(ab')2, and sFv showed a high density of grains in the cortical tubules. In the liver, the IgG and F(ab')2 were found in association with hepatocytes, Kupffer cells, and in the sinusoids; the Fab' and sFv were primarily associated with the Kupffer cells. In the spleen, the Ig forms localized to the marginal zones surrounding the lymphoid follicles. No specific accumulation of grains for any of the Ig forms was observed in the lung. In each of the tissues, the clearance rates were related to the size of the Ig form. The localization in the liver and spleen was determined to be antigen-mediated.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential metabolic patterns of iodinated versus radiometal chelated anticarcinoma single-chain Fv molecules.

Genetically engineered single-chain Fvs (sFv) are defined as recombinant proteins composed of a variable light chain amino acid sequence of an immunoglobulin tethered to a variable heavy chain sequence by a designed peptide. Previous studies using iodine-labeled sFv, derived from the anticarcinoma monoclonal antibody CC49, showed that the 125I-sFv could efficiently target antigen-positive tumors in a human tumor xenograft model while demonstrating rapid plasma clearance and minimal uptake in normal organs. One of the issues we raised in the analysis of the iodinated sFv metabolic studies was whether similar metabolic patterns would be observed if the sFv were labeled with a radiometal. In the studies reported here, 125I-CC49 sFv and 177Lu-CC49 sFv were co-injected in mice bearing antigen-positive carcinoma xenografts. Both sFv forms showed similar tumor targeting and plasma clearance pharmacokinetics. The 177Lu-sFv, however, showed a greater uptake in liver and spleen and a much higher uptake in kidney. These studies thus demonstrate that despite their small size (M(r) 27,000), the metal-chelated sFv shows a metabolic pattern very different than that of the iodinated sFv, which is most likely due to retention of the metal by organs metabolizing the sFv.

Construction, binding properties, metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma monoclonal antibody CC49.

CC49 is a "second generation" monoclonal antibody to B72.3, which reacts with the pancarcinoma antigen TAG-72. CC49 has been shown to efficiently target human colon carcinoma xenografts and is currently being evaluated in both diagnostic and therapeutic clinical trials. We describe here the construction and characterization of a recombinant single-chain Fv (sFv) of CC49. The sFv was shown to be a Mr 27,000 homogeneous entity which could be efficiently radiolabeled with 125I or 131I. Comparative direct binding studies and competition radioimmunoassays using CC49 intact IgG, F(ab')2, Fab', and sFv revealed that the monomeric CC49 Fab' and sFv had relative binding affinities 8-fold lower than the dimeric F(ab')2 and intact IgG. Nonetheless, the 131I-labeled sFv was shown to bind biopsies of TAG-72-expressing tumors. Metabolism studies in mice, using radiolabeled CC49 IgG, F(ab')2, Fab', and sFv, demonstrated an extremely rapid plasma and whole body clearance for the sFv. CC49 sFv plasma pharmacokinetic studies in rhesus monkeys also showed a very rapid plasma clearance (T1/2 alpha of 3.9 min and T1/2 beta of 4.2 h). Tumor targeting studies with all four radiolabeled Ig CC49 forms, using the LS-174T human colon carcinoma xenograft model, revealed a much lower percentage injected dose/g tumor binding for the CC49 monomeric sFv and Fab' as compared to the dimeric F(ab')2 and intact IgG. However, tumor:normal tissue ratios (radiolocalization indices) for the sFv were comparable to or greater than those of the other Ig forms. High kidney uptake with 125I-labeled Fab' and F(ab')2 was not seen with 125I-sFv. Gamma scanning studies also showed that 131I-CC49 sFv could efficiently localize tumors. The CC49 sFv may thus have utility in diagnostic and perhaps therapeutic applications for a range of human carcinomas.

Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli.

A fluorescein-binding single-chain Fv (scFv) was chosen as a model for the study of the physicochemical parameters associated with synthetic IgG fragments. Three such scFv proteins were designed from the primary sequences of one anti-fluorescyl monoclonal antibody (Mab 4.4.20). These were constructed with varying-length interdomain peptide linkers of between 12 and 25 residues, expressed in Escherichia coli, and the protein folding, stability, and antigen-binding characteristics were assessed. Efficient renaturation could be accomplished in vitro to yield approximately 26 mg of active scFv/L of fermentation. Scatchard analysis for fluorescein ligand binding revealed that the scFv designs come within 2-fold of the Ka = 1.99 (+/- 0.18) x 10(9) observed for the parental 4.4.20 Fab and have identical stoichiometries (n approximately 0.99). Reversible solvent denaturation studies demonstrated that the unfolding/refolding equilibria for the scFv proteins can be fit to a simple two-state model and that two of the scFv designs were found to be slightly more stable than single IgG domains (VL and CL) when assessed in terms of the free energy of unfolding, delta Gon-u, or nearly identical to other multiple domain immunoglobulin proteins such as light chains and Fab's when relative transition midpoints, Cm, are compared. Linkers which conferred conformational flexibility beyond the minimally required length of 12 residues were found to have a stabilizing effect. By these criteria of ligand-binding function and protein stability, the scFv proteins were found to be bona fide minimal replicas of their parental IgG molecules.

Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding.

Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)

89Sr therapy: strontium plasma clearance in disseminated prostatic carcinoma.

Strontium plasma clearance is an important factor determining the absorbed dose to metastases and bone marrow in patients receiving 89Sr radionuclide therapy for metastatic bone disease. Amongst male patients with disseminated prostatic carcinoma, the renal component of strontium clearance is frequently greatly reduced compared with values reported for healthy middle aged men. We report a study of renal and gut strontium plasma clearance, renal function, calcium urinary excretion, parathyroid function and extent of skeletal osteoblastic metastatic disease in patients referred for radiostrontium therapy for metastasised prostatic malignancy. The wide variation in net strontium clearance was principally due to variation in the renal component. Low values of strontium renal clearance were found to correlate with the elevation of serum PTH and nephrogenous cyclic AMP, which in turn correlated with extent of skeletal metastatic disease. This suggests that the osteosclerotic metastases characteristic of prostatic carcinoma induce secondary hyperparathyroidism due to the high avidity of the skeleton for calcium. The resulting reduction in strontium excretion may be beneficial to the objectives of radiostrontium therapy.

The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test case.

A weak Ca2+ binding site in the bacterial serine protease subtilisin BPN' (EC 3.4.21.14) was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+ binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN' at 65 degrees C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+ specific binding site with an apparent log Ka = 1.49. A series of refined X-ray crystal structures (R less than or equal to 0.15, 1.7 A) of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2 has allowed a detailed structural characterization of this Ca2+ binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+ by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+ binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein (ionic strength = 0.10). X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 A, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory.(ABSTRACT TRUNCATED AT 250 WORDS)

Incorporation of grass silage, whole cereal grains, cassava and cottonseed meal into diets of rabbits kept in a simulated tropical environment.

The growth and feed conversion of rabbits fed either grass silage or whole grains and supplementary pelleted concentrate of cassava/cottonseed diets were investigated. Poor quality grass silage (pH 4.9) was almost completely rejected by young rabbits initially fed either 17.8 g or 35.5 g DM/day of a supplementary concentrate. Rabbits on the lower level of concentrate provision lost 0.35 g liveweight per day. Rabbits initially weighing 1.77 kg fed complete pelleted diets containing 667 g/kg maize or 667 g/kg sorghum showed improved daily liveweight gains (22.6 g) over rabbits fed whole grains and pelleted supplements (19.4 g) in an experiment lasting 40 days. In a second experiment there were no significant effects of pelleting or type of cereal on liveweight gain or feed conversion ratio. Pelleting significantly improved crude protein digestibility of diets whilst maize diets were superior in DM, organic matter and crude protein digestibilities. The inclusion of cottonseed meal containing 700 mg/kg free gossypol in diets at levels of 150 and 300 g/kg did not affect growth rate or feed conversion in rabbits weighing 0.92 kg initially. These diets contained up to 364 g/kg cassava suggesting that this ingredient can be used in rabbit diets as an energy source in replacement for whole grains.

Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond.

Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers.

A potential role for the extracellular matrix glycoprotein laminin in macrophage-tumor-cell interactions.

Although cell surface molecules are thought to be involved in macrophage (MO)-tumor-cell recognition, the nature of these molecules remains unknown. In this study we have shown that the glycoprotein laminin may facilitate macrophage-tumor-cell binding. Macrophage binding to tumor cells was assessed by measuring the adherence of radiolabelled 3-MCA2 induced malignant fibrosarcoma cells to syngeneic peritoneal MOs. Addition of exogenous laminin promoted the binding of a weakly metastatic subline of these tumor cells by 31-68%. These weakly metastatic tumor cells express negligible endogenous cell-surface laminin but display specific cell-surface receptors for binding soluble laminin. Exogenous laminin promoted MO binding of these tumor cells whether it was present during the assay or whether the tumor cells were pretreated with the laminin. This increase in binding was blocked by anti-laminin antibody. In contrast, MO binding of a strongly metastatic variant of the same tumor was not enhanced by the addition of exogenous laminin. This highly malignant fibrosarcoma line already expressed endogenous cell-surface laminin. Since the MOs were found to specifically bind 125I-laminin, the interaction between laminin-bearing tumor cells and MOs may be mediated via a specific MO plasma membrane receptor. Thus, the expression of cell-surface laminin and its receptors on both tumor cells and MOs may provide a mechanism for promoting MO-tumor-cell binding.

Annual and diurnal cycles in plasma testosterone and thyroxine in the male green sea turtle Chelonia mydas.

Male plasma testosterone (T) and thyroxine (T4) were monitored over several annual cycles in a captive breeding colony of green sea turtles, Chelonia mydas. Daily and annual water temperatures varied by only approximately 1 and 3 degrees, respectively. A pronounced season cycle in plasma T was evident in the population as a whole and in individual animals: plasma T was at a nadir (approximately 3 ng/ml) in September-November and then increased progressively to a peak (27-39 ng/ml) in April; levels began declining immediately thereafter, coincident with the onset of copulatory behavior. By contrast, plasma T4 remained uniform (approximately 9 ng/ml) throughout the year and, thus, could not readily account for the decline in androgen levels. Plasma hormones were relatively stable over a 24-hr period at three times a year, and there was a correlation for individual plasma T levels sampled in April and May. Thus, limited sampling should allow identification of seasonal rhythms and individual variability in plasma T levels. Testis mass and spermatogenic activity were significantly greater in January than in September; i.e., spermatogenesis and androgen secretion were not "uncoupled." Copulatory activity began in April but did not peak until May-June, after plasma T had significantly declined. However, there was a significant (but weak) correlation between individual peak levels of plasma T (i.e., in April) and the quantitative level of mating activity (time spent mounting and number of mates) measured for the entire subsequent season. Thus, green turtles do not exhibit the "postnuptial" type of testis cycle typical of many temperate-zone turtles, and the levels of plasma androgen may be important for initiating and maintaining sex behavior, although they are not tightly linked during the mating season.