Consequences of terbium (111) binding on the conformation and enzymatic activity of Guinea pig liver transglutaminase

Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10–200 fold molar excess of calcium ions (Ki for Tb(III)=60 µM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl₂ only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl₂ solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.     

Growth dynamics of a ctenophore (Mnemiopsis) in relation to variable food supply. I. Carbon biomass, feeding, egg production, growth and assimilation efficiency

This paper contains new experimental data on the growth dynamicsof a lobate coastal ctenophore, Mnenmiopsis mccradyi, whichadd significantly to our understanding of the nutritional ecologyof ctenophores and their role as opportunistic predators. Theseexperimental observations were necessary to refine the dynamiccarbon budget presented as a simulation model in another report.The ratio of carbon biomass to dry weight may vary several-folddepending on the nutritional state and size from >12% inwell-fed larvae to <1% in starved adults. Feeding effort(clearance rate) is higher for previously starved animals, fallingsharply within a few hours after re-exposure to food. Directvisual observations of feeding activity of animals confirmedthis. Assimilation efficiency can be high (72%) in these animalsbut if they continue to feed at high food concentrations, incomingfood displaces material which is only partially digested andassimilation efficiency decreases substantially. Except at verylow food concentrations, growth efficiency ranges between 20and 45%. Mnemiopsis, begins to produce eggs at a size much lessthan its maximum. Egg production is very sensitive to food supply,and somatic growth is favored over egg production at low fooddensities. Even though several thousand eggs may be producedover a few days, they represent <2% day^–1 of the carbonbiomass of the ctenophore and several-fold less than respiratorycarbon.     

Characterization of a monoclonal antibody against active pectate lyase from Erwinia carotovora

A monoclonal antibody (2E2) produced against pectate lyase from Erwinia carotovora ssp. carotovora reacted with a 41- and a 44-kilodaltion protein on Western blots of concentrated Erwinia culture supernatants resolved by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. It was unequivocally shown that monoclonal 2E2 reacted with an active form of pectate lyase by affinity purifying the antigen with the monoclonal. The affinity-purified antigen was enzymatically active and moved as a single protein band in a nonequilibrium isoelectric focusing gel. Monoclonal 2E2 reacted with the pectate lyases of a diverse range of E. carotovora ssp. carotovora, ssp. atroseptica, and ssp. betavasculorum strains, as well as with one of three strains of E. chrysanthemi. The electrophoretic mobility of the major protein (44 kilodaltons) that reacted with 2E2 was identical within a subspecies but differed among subspecies.     

DNA loop domains in mammalian spermatozoa

The highly condensed and tightly packaged DNA of hamster spermatozoa was found to be organized into topologically constrained DNA loop domains attached at their bases to a nuclear matrix. The loop domains of the sperm nuclei differed from somatic cell loop domains from the same animal in two aspects. Sperm loop domains were 60% smaller than somatic cell loop domains, with an average DNA length of 46±7 kb in sperm as compared with 16±11 kb in brain. Secondly, unlike virtually all somatic cell DNA known which is negatively supercoiled, sperm DNA was devoid of detectable supercoiling. The presence of the loop domain structure in the highly condensed DNA of mammalian spermatozoa suggests that this motif is a fundamental aspect of eukaryotic DNA organization.     

Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis

Spermiogenesis in Caenorhabditis elegans involves the conversion of spherical, sessile spermatids into bipolar, crawling spermatozoa. In males, spermiogenesis is induced by mating, while in hermaphrodites, spermiogenesis occurs before the first oocytes are fertilized. Alternatively, spermiogenesis can be induced in vitro by treatment with monensin triethanolamine, or pronase. Treatment with the calmodulin inhibitors, trifluoperazine, chlorpromazine, or W7, also induces spermiogenesis in vitro with a half maximal effect at 20 microM. Upon initial activation, spermatids extend long, thin spikes and undergo extensive cellular movements. Eventually, a single motile pseudopod forms through the restructuring of one or more of these spikes. These transient spikes can be prolonged in vitro by removing triethanolamine as soon as the spermatids first form spikes. Spermatids from spe-8 and spe-12 spermatogenesis-defective (spe) mutants activate in vivo with male but not hermaphrodite sperm activator. In vitro, the mutant spermatids arrest spermiogenesis at the spike stage when activated with pronase, but form normal spermatozoa if subsequently or initially treated with monensin or triethanolamine. We present a model of spermiogenesis in which the mutant defects and the action of the pharmacological agents are ordered relative to one another.     

Level of protein kinase C activity correlates directly with resistance to adriamycin in murine fibrosarcoma cells

In this report, we demonstrate a direct correlation between protein kinase C (PKC) activity and adriamycin (ADR) resistance in mouse fibrosarcoma cells. PKC activity was measured in four murine UV-2237M fibrosarcoma cell lines that differed in the degrees to which they expressed resistance to ADR, which is an inhibitor of PKC. A comparison of the four cell lines revealed a positive correlation between the level of PKC activity and resistance to ADR. Incubation of the cells with the PKC inhibitor H-7 produced a partial reversal of ADR resistance. Taken together, these results suggest a role for PKC in the mechanism of ADR resistance.     

A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.     

Prenatal diagnosis of Duchenne muscular dystrophy: prospective linkage analysis and retrospective dystrophin cDNA analysis.

The accuracy of DNA-based prenatal diagnosis of Duchenne muscular dystrophy (DMD) was determined by study of 174 families. Only 60% of families had a living affected male, and 63% had history of a single affected male. Prenatal diagnosis was declined by 47% of mothers whose DNA studies predicted a carrier risk below 2%, and none have had affected sons. Fetal risk was estimated prospectively by linkage analysis using intragenic and flanking RFLPs and retrospectively using dystrophin cDNA analysis for families whose linkage estimates lacked precision. Diagnostic accuracy was determined by comparing predictions with 40 male pregnancy outcomes. On the basis of linkage analysis, we anticipated 3.2 DMD males and observed 3.0. Retrospective cDNA analysis identified deletions in 2 of these 3 males. The combined use of linkage and cDNA deletion analysis provided a highly accurate method for prenatal diagnosis of DMD.     

Stimulation of microtubule assembly by recombinant human interferon-alpha and interferon-gamma

Interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) have been demonstrated to stimulate microtubule assembly measured in the in vitro assembly system. The process is substoichiometric occurring when the interferon concentrations are below that of tubulin. IFN-gamma is a more potent effector than IFN-alpha. The critical tubulin concentration describing microtubule assembly decreases from 1.5 mg/ml measured in the absence of added effector to 1.05 mg/ml and 1.3 mg/ml when measured in the presence of 2.16.10(-6) M IFN-gamma and 3.06.10(-6) M IFN-alpha, respectively.