Adaptations to reduction of endometrial surface available for placental development in sheep

On Day 5 of pregnancy, before the blastocyst migrates to the uterus, one uterine horn was ligated to restrict the trophoblast to the lumen ipsilateral to the corpus luteum. The numbers of placentomes (caruncles and cotyledons) were reduced by half, but neither at 120 nor at 140 days of pregnancy (term 147 days) did the weights of placentae and fetuses of treated ewes differ significantly from those of control ewes. Amongst uterus-ligated animals prepared for chronic study, the rate of uterine blood flow (electromagnetic flow transducer, ml/min) to the pregnant horn was higher than in control ewes, as was the concentration of progestagens in maternal peripheral blood. There may be a compensatory response that causes hypertrophy of placentomes and that increases blood flow to the uterine horn containing placental tissue.     

2-(4-Bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Determination of reaction parameters and characterization of an active site peptide

A new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, has been prepared, Reductive amination of ribulose-P2 with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography on quaternary aminoethyl-Sephadex. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido) anilino-2-deoxypentitol 1,5-bisphosphate) which were competitive inhibitors of the carboxylase with Ki values of 705 and 104 microM, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO2 and Mg2+, however, resulted in an irreversible, time-dependent loss of activity, with a Kinact of 125 microM and a minimal half-time of 7.3 min. Covalent incorporation of [14C]reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of [14C]reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. Automated Edman degradation established the sequence as (sequence in text) which is located near the NH2 terminus of the enzyme. The lack of reactivity with the spinach enzyme is explained by the deletion of the histidyl residue and the replacement of cysteine by tryptophan in the eukaryotic species. Although the nonconservation of the modified residues argues against a functional role other than maintenance of structural integrity, the extensive homology in this region among seven different species of carboxylase is compatible with the region comprising a portion of the active site.     

Peptidergic pathways in the avian brain

The results obtained by topographical studies on the immunoreactive peptide systems in the embryonic and adult avian brain (domestic fowl, domestic mallard, pigeon, Japanese quail, and zebra finch) can be realized only by means of phylogenetical comparisons. The comparative studies mainly demonstrate a fascinating constancy of the immunological properties and the spatial distribution of the neuropeptides. Independent of the development of the neopallium, and the increasing cerebral complexity, the spatial distribution of the neuropeptides, the location of their main perikaryal accumulations which are interconnected by immunoreactive fiber projections (and thereby forming widespread but continuous peptide systems) remain nearly unchanged during vertebrate evolution. The recognition of the neuropeptides as integral parts of the central nervous system is demonstrated by the fact that neuropeptidergic structures connect sensory inputs with central nervous areas as well as with the peripheral endocrinium.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

Interferon-induced proteins. Purification and characterization of a 15,000-dalton protein from human and bovine cells induced by interferon

Human interferons induce a protein of 15,000 daltons in human and bovine cells. This protein is located in the cytoplasm in a soluble form and is induced by concentrations of interferon which induce the antiviral state. Messenger RNA prepared from interferon-treated human and bovine cells contains a mRNA which yields on translation in vitro a protein similar in size to the 15-kDa protein induced by interferon in vivo. The human protein has been purified to homogeneity from interferon-treated human cells by ion-exchange chromatography and reverse-phase high-performance liquid chromatography. A comparison of the peptides generated by V8 protease from the human and bovine 15-kDa proteins reveals that the two proteins are similar but not identical.     

Association of fibronectin with the platelet cytoskeleton

Triton-insoluble cytoskeletons prepared from either normal or thrombasthenic platelets were found to contain approximately 1.3 micrograms of fibronectin/10(9) platelets as measured by a radioimmunoassay. Total endogenous platelet fibronectin was quantitatively retained on the platelet cytoskeleton, whereas 70% of exogenously added fibronectin that bound the surface of thrombin-activated platelets was recovered with the Triton-insoluble cytoskeleton. The exogenously added fibronectin specifically bound platelets and cytoskeletons with the same affinity giving an apparent binding constant of 1.47 X 10(-7) M. The possibility that fibrin associated with the platelet cytoskeleton could serve as the fibronectin receptor was investigated by measuring the binding constant of fibronectin for polymerizing fibrin and by measuring the amount of fibronectin associated with cytoskeletons of thrombasthenic platelets which contain 4-fold less fibrin than controls. The binding constant of fibronectin for polymerizing fibrin was 14-fold lower than that for cytoskeletons and cytoskeletons prepared from thrombasthenic platelets contained approximately the same amount of fibronectin as controls. Therefore, it is unlikely that fibrin is the platelet fibronectin receptor. These results support the hypothesis that platelet fibronectin is released from platelet alpha granules upon thrombin stimulation and becomes bound to the platelet surface and cytoskeleton either directly or through some intermediate protein that spans the membrane and interacts both with fibronectin and the internal cell cytoskeleton.     

Distribution of calcium during interphase and mitosis as observed by ion microscopy

The ion microscope, based on secondary ion mass spectrometry, has been used to demonstrate the distribution of calcium in the root tip cells of two plant species, Allium cepa and Vicia faba. Interphase nuclei showed higher intensities of calcium than cytoplasm, while nucleoli exhibited higher calcium intensities than the rest of the nucleoplasm. The chromosomes showed high intensities of calcium at all stages of mitosis. Calcium was also detected in the cell plate and phragmoplast region of dividing cells. It appears that during prophase calcium concentrates in the condensing chromosomes, and during telophase it is transferred to nucleoli. These observations suggest that chromosomes may serve as a reservoir of calcium during mitosis.     

A novel type of human T cell clone with highly potent natural killer-like cytotoxicity divorced from large granular lymphocyte morphology

Interleukin 2-dependent cloned lymphocytes derived from an allogeneic HLA class II-mismatched but class I-matched mixed lymphocyte culture were screened for cytotoxic activity on target cell lines known to be susceptible or resistant to lysis by natural killer (NK) cells. Of 24 clones, eight were found to display NK-like cytotoxicity. Two manifested extremely high cytotoxicity levels (50% lysis of K562 at an effector to target ratio of 1:1), whereas the remainder were only moderately active (about 20% lysis at 25:1). NK-like clones were studied with regard to cell surface markers defined by monoclonal antibodies, as well as for their morphologic and cytochemical characteristics, and were compared with clones displaying different functions. The moderately active NK-like clones exhibited characteristic large granular lymphocyte morphology (many azurophilic granules, indented nuclei, high cytoplasm to nucleus ratio, and a basophilic peripheral cytoplasmic zone). This was, however, also characteristic of the majority of lymphocyte clones displaying functions other than NK. Surprisingly, the two clones with high NK-like activity did not exhibit large granular lymphocyte morphology, with few granules, round nuclei, and low cytoplasm to nucleus ratio. The T3, T9, T10, and T11 markers, as well as HLA-DR determinants, were expressed on their surfaces, but in contrast to the other clones, they did not display OKT4-, OKT8-, or OKM1-defined antigens. No distinction between them was possible on the basis of a cytochemical profile in relation to their function, because all clones were positive for acid phosphatase, either focal or dispersed and negative for nonspecific esterase or chloracetate esterase. The highly active lytic clones were, however, distinguished by an exceptionally rapid growth rate in culture (cell doubling time: 9 hr as compared to 30 to 40 hr, as usually required). These results demonstrate two different types of human NK-active lymphocytes with remarkably disparate lytic capacity, cell surface markers, and morphology.