Carbohydrates engineered at antibody constant domains can be used for site-specific conjugation of drugs and chelates

To improve the efficiency of site-specific conjugation of chelates and drugs to antibodies, and to minimize the incidence of immunoreactivity perturbation to the resultant immunoconjugates, Asn-linked oligosaccharide moieties were designed and engineered into the constant domains of a humanized anti-CD22 monoclonal antibody, hLL2. From 10 potential glycosylation mutants, two CH1 domain glycosylation sites, HCN1 and HCN5, were identified that were positioned favorably for glycosylation. The carbohydrate (CHO) chains attached at these sites were differentially processed so that HCN5-CHOs were physically larger than HCN1-CHOs. Although both the CH1-appended CHOs, and the LL2 Vkappa-appended CHOs conjugated efficiently with small chelates, the HCN5-CHOs, due to the structural and positional superiority, appear to be a better conjugation site for large drug complexes, such as 18 kDa doxorubicin (DOX)-dextran.     

Electrospray ionisation analysis of human fibrinogen

Fibrinogen, a 340 kDa glycoprotein, was purified from human plasma, separated into its constituent polypeptide chains and analyzed by electrospray ionization mass spectrometry. Six individual plasmas were examined, and whilst the A alpha chain appeared homogeneous, the Bbeta and gamma chains were heterogeneous with the predominant form of each lacking a single sialic acid residue. The mean molecular masses of the dominant gamma and Bbeta isoforms were 48,366 and 54,200 Da with standard deviations of 10 and 12 Da respectively compared to predictions, based on amino acid and carbohydrate sequences, of 48,368 and 54,213 Da. The mean mass of the A alpha chain was 66,196 Da but this showed significantly more variation with a standard deviation of 64 Da. This probably reflects genetic and/or post-translational differences, since there is non-stoichiometric phosphorylation at Ser 3 and 345 and the expected residue weight of the non-phosphorylated chain is 66,132 Da. After treatment with thrombin the fibrin beta chain showed a decrease in mass of 1542 Da in good agreement with the expected decrease of 1535 Da resulting from loss of the B peptide. Loss of the A peptide from the alpha chain resulted in a decrease of 1546 Da compared to an expected loss of 1519 Da for non-phosphorylated A peptide. On prolonged thrombin incubations factor XIII induced gamma-gamma dimers were observed at 96,896 Da.     

Divergence towards a dead end? Cleavage of the divergent domains of ribosomal RNA in apoptosis

In several cases of apoptotic death the large ribosomal subunit 28S rRNA is specifically cleaved. The cleavages appear at specific sites within those domains of the rRNA molecule that have shown exceptional high divergence in evolution (D domains). The cleavages accompany rather than precede apoptosis, and there is a positive, but not complete, correlation between rRNA cleavage and internucleosomal DNA fragmentation. Most cell types studied so far show two alternative cleavage pathways that are mutually exclusive. Cleavage can either start in the D8 domain with secondary cuts within a subdomain of D2 (D2c), or in the D2 domain with subsequent excision of the D2c subdomain. The latter pathway is of particular interest since D2 (unlike D8) is normally inaccessible for RNase attack. That apoptosis specifically affects the ribosomal divergent domains suggests that these domains, which make up roughly 25% of total cellular RNA, might have evolved to serve functions related to apoptosis. Future studies will be directed to test the hypothesis that rRNA fragmentation may be part of an apoptotic program directed against the elimination of illegitimate (viral?) polynucleotides.     

Anejaculation: its etiology and pathogenesis, classification and clinical aspects

43 patients suffering from anejaculation were treated in 1983-1993 in the department of andrology of the N. A. Semashko Medical Institute urological clinic. Most commonly anejaculation is caused by prostatic and bladder neck surgery (25.6%), diabetes mellitus (18.6%) and presents as retrograde ejaculation, impaired sperm emission into the urethra, aspermatism (51.2, 27.9, 20.9% of patients, respectively). The leading pathogenetic factors are peripheral neuropathy, surgical injury to the bladder internal sphincter, psychosexual disorders. The authors propose classification and algorithm of the patients' examination to facilitate and enhance diagnosis. Special attention should be given to the presence or absence of orgasm, after-orgasm occurrence of spermatozoa in urine. Anejaculation treatment outcomes remain unsatisfactory. Good responses were achieved with selective adrenomimetic gutron, laser therapy and electrovibration. Prevention of anejaculation should be given more attention.     

Primary care by desire or default? Specialty choices of minority graduates of US medical schools in 1983

This study was undertaken to determine if US medical school students of different racial/ethnic backgrounds demonstrate similar patterns of evolution of specialty choice between their senior year of medical school and their third postgraduate year. The study identified the specialty choices of US medical school seniors in 1983 through their responses to the Association of American Medical Colleges Graduating Medical Student Questionnaire (GQ). The cohort was classified into three groups: underrepresented minorities, non-underrepresented minorities, and whites. Using these AAMC data as baseline, each racial/ethnic background group was tracked through their third residency year. Comparisons were made between anticipated specialty choices as senior medical students and actual specialties as revealed through residency tracking. The study found that more than 95% of the cohort began residencies in specialties compatible with their GQ choices. Unexpectedly, almost 20% of blacks, Commonwealth Puerto Ricans, and other Hispanics were not in graduate medical education in their third postgraduate year. This group needs to be studied further in order to learn the proportion of these physicians who subsequently completed residency training and the reason(s) for attrition in physicians who did not fulfill minimum training requirements for board certification.     

Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human anti-mouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T-lymphocytes

T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')2 fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv18 (overexpressed by ovarian carcinoma cells) as part of a phase I/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10(6)-10(9)) of bsAb-targeted T lymphocytes plus low-dose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10(9) targeted T lymphocytes, 2 mg soluble bsAb, and low-dose IL-2. Using enzyme-linked immunosorbent assays (ELISA), human anti-mouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (1) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18+ or CD3+ cells by absorption of human anti-mouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18+ ovarian carcinoma cell line Igrov-I by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.     

Allergy to bumblebee venom. II. IgE cross-reactivity between bumblebee and honeybee venom

To obtain more information on IgE cross-reactivity between bumblebee venom and honeybee venom, we tested sera from venom-sensitized patients for specific IgE against venoms from the European bumblebee (Bombus terrestris), the North American bumblebee (Megabombus pennsylvanicus), and the honeybee (Apis mellifera). RAST, RAST-inhibition, and immunoblotting experiments indicate that bumblebee venom and honeybee venom contain venom-specific IgE-binding epitopes. These results suggest that immunotherapy using honeybee venom may not be effective in all bumblebee venom-allergic patients. Our experiments also revealed differences in IgE binding for venom from European and American bumblebees.     

Constitutive activation of opsin by mutation of methionine 257 on transmembrane helix 6

Rhodopsin is a member of the large family of G protein-coupled receptors (GPCR's). Constitutive activity of GPCR's, defined as ligand-independent signaling, has been recognized as an important feature of receptor function and has also been implicated in the molecular pathophysiology of a number of human diseases. Rhodopsin has evolved a unique mechanism to minimize receptor basal activity. The chromophore 11-cis-retinal, which acts as an inverse agonist in rhodopsin, is covalently bound to the receptor to ensure extremely low receptor signaling in the dark. In this study, we replaced Met257 in TM helix 6 of opsin with each of the remaining 19 amino acids. Only mutant opsin M257R failed to be expressed in COS-cell membranes. Each of the remaining 18 mutant opsins, with the exception of M257L, was significantly constitutively active. Two mutants in particular, M257Y and M257N, displayed very high levels of constitutive activity. In addition, the double-site mutants with substitutions of both Met257 and Glu113 in TM helix 3 tended to be much more constitutively active than the sums of the activities of the individual single-site mutants. Based on existing structural models of rhodopsin, we conclude that Met257 may form an important and specific interhelical interaction with a highly conserved NPXXY motif in TM helix 7, which stabilizes the inactive receptor conformation by preventing TM helix 6 movement in the absence of all-trans-retinal. Furthermore, we are able to show that the pharmacological properties of the large number (approximately 50) of mutant opsins that we have characterized to date support the two-state model of GPCR function. These results suggest that rhodopsin and other GPCR's share a common mechanism of receptor activation that involves specific changes in helix-helix interactions.