Asymptotically efficient two-step estimators of the hazards ratio for follow-up studies and survival data

Asymptotically efficient estimators of a common hazard rate ratio (for follow-up studies) and the proportional hazards ratio (for survival studies) are obtained by a single iteration of the "Mantel-Haenszel" estimator appropriate for each setting. Estimators of their variance are also developed. The two-step estimator for survival data and its variance estimator are shown by simulation to be minimally biased and the estimator is shown to be efficient relative to the Cox partial likelihood estimator in small samples.     

Evidence for multiple catalytic sites of human acrosin from kinetic evaluation of fructose-induced acrosin inhibition

Acrosin (acrosomal proteinase; EC 3.4.21.10) is a sperm-specific serine proteinase implicated in sperm penetration of the mammalian oocyte. Previously, we had shown that human acrosin, unlike human trypsin (EC 3.4.21.4), was inhibited by beta-D-fructose and related carbohydrates. The present study was undertaken to more fully elucidate the mechanism of action of fructose as an acrosin inhibitor, and to further differentiate the kinetic properties of acrosin from those of trypsin. Fructose produced a complex pattern of inhibition. At relatively low concentrations (10-60 mM), fructose acted as a competitive inhibitor with an apparent inhibition constant of 13 mM. In contrast, at high concentrations (80-320 mM), fructose behaved as a noncompetitive inhibitor, with an apparent inhibition constant of 205 mM. A Hill plot of enzyme activity as a function of fructose concentration suggested only a single binding site for fructose (slope = -0.90). The pattern of inhibition is not consistent with an enzyme containing only a single catalytic site, based either upon steady-state or rapid equilibrium assumptions; however, good agreement between observed and simulated data were obtained based upon the assumption of two catalytic sites with equal or similar binding and catalytic constants. The data suggested that fructose interacts with a single binding site (Ki = 8 mM) which alters both catalytic sites to produce an enzyme species having a higher apparent Michaelis constant and lower kcat as compared to the uninhibited enzyme. Fructose had no effect upon the rate of acrosin inactivation by either diisopropylfluorophosphate or tosyl-lysine-chloromethylketone, suggesting that neither substrate binding nor acylation were altered by this agent. The above data indicate substantial differences between the catalytic properties of human acrosin and those of trypsin.     

Isolation of S-100 binding proteins from brain by affinity chromatography

S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vice versa, suggesting both proteins may regulate common targets. However these two regulatory proteins also bind few other proteins specific for each. These include cyclic nucleotide phosphodiesterase, 55k, and 220k proteins for calmodulin and 24k, 42k, and 90k proteins for S-100. Certain proteins also specifically bound to S-100 both in Ca2+-dependent and independent ways. In glial cells S-100 protein may replace calmodulin in regulating Ca2+-influenced functions.     

Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance

Eight anionic disaccharide precursors of lipid A accumulate at 42 degrees C in 3-deoxy-D-manno-octulosonic acid-deficient temperature-sensitive mutants of Salmonella typhimurium. These compounds comprise a series of lipids based on the minimal structure, O-[2-amino-2-deoxy-N2,O3-bis(3-hydroxytetradecanoyl)-beta-D-glucopyranos yl] -(1----6)-2-amino-2-deoxy-N2, O3-bis(3-hydroxytetradecanoyl)-alpha-D-glucopyranose 1,4'- bisphosphate (designated lipid IVA) that differ from each other by the presence of an additional phosphoethanolamine moiety (IIIA), or an aminodeoxypentose moiety (IIA), or both (IA). A homologous set of metabolites is further derivatized with a palmitoyl function; these are designated IVB, IIIB, IIB, and IB (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). The attachment of the palmitoyl moiety, known to be on the reducing terminal GlcN residue by mass spectrometry, was determined to be O-beta of the N2-linked beta-hydroxymyristoyl group of that residue of IVB by 13C NMR and two-dimensional 1H chemical shift correlation spectroscopy experiments. 31P NMR indicated the presence of diphosphodiester moieties in IIIA, IIIB, and IA and monophosphodiester moieties in IIA and IA. Selective 1H decoupling of the 31P spectrum of IIIA demonstrated that the O-diphosphoethanolamine moiety is attached to the O4' position in IIIA. On the basis of the observed 31P chemical shifts it was concluded that the aminodeoxypentose is located at position 1 in IIA and IA, while diphosphoethanolamine is most likely located at O-4' in IA and IIIB, as in IIIA.     

The molecular morphology of bovine heart mitochondrial NADH-ubiquinone reductase. Cross-linking with dithiobis(succinimidyl propionate)

The molecular morphology of NADH-ubiquinone reductase (complex I) was investigated by cross-linking with the cleavable bifunctional reagent, dithiobis(succinimidyl propionate). Cross-linking inhibits the following activities of the complex--NADH----3-acetylpyridine adenine dinucleotide (oxidized), NADH----2,6-dichloroindophenol, NADH----ferricyanide, and NADH----menadione--to different degrees with the greatest inhibition occurring with either ferricyanide or 3-acetylpyridine adenine dinucleotide as electron acceptor. Addition of 150 microM NADH affords partial protection from inhibition. Cross-linking quenches the FMN fluorescence of complex I (288 nm excitation/515 nm emission), and addition of 150 microM NADH greatly reduces the quenching. Treatment of complex I (1 mg/ml) for 2 min with dithiobis(succinimidyl propionate) (0.2 mg/ml) at 4 degrees C revealed a cross-linked product consisting of the following seven subunits: 75-80, 53-57, 42, 33-35, 24-27, 17-18, and 12.5-15.5 kDa. Five minutes of treatment cross-linked the unidentified polypeptides of 69 and 51 kDa to six of the seven complex I subunits, but the 12.5-15.5-kDa subunit may be missing from this cross-linked product, while 15 min of treatment cross-linked additional unidentified polypeptides of 177, 107, 72, and 63 kDa. Since longer times of cross-linking result in a larger number of unidentifiable polypeptide spots, the shorter cross-linking time results are taken as a more accurate picture of the native enzyme conformation. This would indicate that within complex I the following subunits are within 12 A of each other at one or more points in space: 75-80, 53-57, 42-45, 33-35, 24-27, 17-18, and, perhaps, 12.5-15.5 kDa. These subunits represent portions of all three fractions of the enzyme, i.e. flavoprotein, iron-protein, and insoluble or hydrophobic fractions.     

Production of insulin-like growth factor-II (MSA) by endoderm-like cells derived from embryonal carcinoma cells: possible mediator of embryonic cell growth

The present study was carried out to determine if an insulin-like growth factor (IGF) type activity might be produced by embryonal carcinoma-derived cells. The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm-like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid. When growth medium conditioned by Dif 5 cells is chromatographed on Sephadex G-75 in 1 M acetic acid two peaks of activity are observed which compete for specific [125I]iodo multiplication stimulating activity (MSA) binding to PYS cells. MSA is the rat homologue of human IGF-II. The high molecular weight fraction (Mr approximately 60K) apparently corresponds to IGF-binding protein as determined by its ability to bind [125I]iodo-MSA. The low molecular weight fraction (Mr approximately 8K) is biologically active as this fraction stimulates [3H]thymidine incorporation into serum-starved chick embryo fibroblasts. Radioimmunoassay data indicate that the IGF-like activity produced by Dif 5 cells is more closely related to IGF-II than to IGF-I. Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA-like activity, while PYS-2 (parietal endoderm-like) cells produced about 16 ng MSA/10(6) cells/24 hr as determined by radioimmunoassay. Dif 5 and PSA-5E (visceral endoderm-like) cells, are found to secrete significant amounts of MSA into the growth medium (30-50 ng MSA/10(6) cells/24 hr). These findings offer further support to a proposal that MSA (IGF-II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic growth factor.     

Blocks to polyspermy in Chaetopterus

Blocks to polyspermy may act either at the level of the egg plasma membrane to prevent gamete fusion or at the level of egg surface coats to prevent gamete attachment. The present study was undertaken to determine what type(s) of block(s) to polyspermy exist in Chaetopterus. The results showed the existence of both types. A rapid block acts at the plasma membrane level based on independence from detectable changes in the vitelline layer and is dependent on external sodium ions. A vitelline layer block had been predicted on morphological evidence and is supported here by demonstrating an increase in polyspermy following chemical disruption of the vitelline layer. However, the vitelline layer of the fertilized egg retained its ability to initiate the acrosome reaction in sperm and attach sperm which had undergone the acrosome reaction. The vitelline layer block resulted from the retraction of egg microvilli from the vitelline layer, and not from elevation of the vitelline layer per se. Thus the vitelline layer of the fertilized egg could be involved in preventing sperm penetration into the egg without being altered structurally or functionally.     

Immunocytochemical studies of relaxin in ovaries of pregnant and cycling mice

Immunocytochemical staining for relaxin in ovarian sections of pregnant mice from day 11 through day 18 of gestation revealed that only corpora lutea (CL) of pregnancy are stained. Evaluation of serial sections of ovaries from a day 16 pregnant mouse revealed that the only luteal structures present are CL of pregnancy. The number of CL present in each ovary equaled the number of implantation sites in each related horn (7 on the right side and 8 on the left side). These large CL varied in shape, being round in some profiles to very elongate in others. All CL were immunochemically stained for relaxin using the peroxidase-antiperoxidase method of L. Sternberger (Immunocytochemistry, 2nd ed. Wiley, New York, 1979). The intensity of the strain varied from cell to cell within each CL. Small luteal structures that were observed to be immunochemically stained for relaxin were demonstrated to represent the periphery of CL of pregnancy. No luteinized follicles were observed and interstitial cells and follicles were not immunochemically stained in any of the day 16 serial ovarian sections or in any of the ovarian sections from pregnant mice on the other days of gestation studied. CL of previous cycles were not observed to be present in the ovaries at days 15, 16, or 18 of gestation. However on day 14 and before, CL of previous cycles were observed and they did not exhibit any relaxin immunostaining. Immunocytochemical studies using the biotin-avidin system revealed that no relaxin immunostaining could be demonstrated in the ovaries of cycling mice at any stage of the estrous cycle. In conclusion, this study revealed that the only ovarian structures demonstrating relaxin immunocytochemical staining in the mouse were CL of pregnancy.     

Epithelial heterogeneity in the murine thymus: fucose-specific lectins bind medullary epithelial cells

By means of histochemical techniques, two lectins with nominal specificity for L-fucose, Tetragonolobus purpureas agglutinin (TPA), and Ulex europeus agglutinin (UEA) were found to specifically label the medullary area of murine thymuses. Although the binding of both lectins was restricted to the medullary area of the thymus, each staining pattern was unique. Cells binding UEA formed a reticular network throughout the medulla, whereas cells binding TPA occurred as single cells or small clumps of cells and resembled Hassall's corpuscles. The cells binding either lectin were identified as epithelial on the basis of ultrastructural features (tonofilaments, desmosomes, and keratohyalin bodies) and resembled Ia+ medullary epithelial cells described previously. An age-related decline in UEA binding was observed, whereas labeling with TPA remained unchanged. On the basis of the labeling patterns obtained with UEA and TPA and the reported specificities of these two lectins, it is suggested that the majority of the fucose detected is associated with type 1 carbohydrate chains.     

Studies of in vitro activation and differentiation of human B lymphocytes. I. Phenotypic and functional characterization of the B cell population responding to anti-Ig antibody

Investigation of the activation of splenic B cells by anti-immunoglobulin (Ig) antibody has enabled us to characterize the anti-Ig-responsive B cell and to analyze the phenotypic changes which accompany proliferation and differentiation. The anti-Ig antibody-responsive B cell population was characterized by the expression of high levels of the B2 antigen and represented approximately 40% of splenic B cells. Brisk mitogenesis which peaked at 3 to 4 days was induced by anti-Ig antibody. The proliferative phase was characterized phenotypically by a dramatic decline in B2 antigen expression, with most cells showing no detectable B2 by 4 days post-activation. The other hallmark of this phase was de novo expression of a group of "activation antigens." These included the B cell-restricted antigens B-LAST 1, BB1, and B5, and the T cell-associated interleukin 2 receptor and T12 antigens. Concomitantly, B1, B4, and Ia expression increased, the increase being roughly proportional to the increase in cell size. After day 4, the mitogenic response progressively diminished, while Ig synthesis increased. During this differentiation phase, cell surface antigens again displayed a distinct sequence of changes. The five activation antigens and the B1, B4, and Ia antigens began to decrease. However, two markers, T10 and PCA-1, which are found on plasmacytomas, appeared and their level of expression steadily increased. These changes and the appearance of morphologically identifiable plasma cells required the presence of T cells in this system. T cell supernatants alone induced Ig secretion but did not induce expression of PCA-1 or the appearance of cells with plasma cell morphology. The culture system developed in this study has allowed us to analyze the antigenic changes following activation by anti-Ig antibody. This sequence of changes has not only permitted the identification of antigens which, by their appearance at distinct stages may have an important role in proliferation and differentiation of B cells, but also provides us with the means of studying the function of each antigen.