Action of a cell wall proteinase from Lactococcus lactis subsp. cremoris SK11 on bovine α s1-casein

Summary The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris SK11 was partially purified and incubated with _s1-casein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the aminp acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of _s1-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the _s1-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No cleat consensus sequence of amino acid residues surrounding the cleavage sites could be identified.Offprint requests to: G. G. Pritchard     

Localization of a 64-kDa phosphoprotein in the lumen between the outer and inner envelopes of pea chloroplasts

The identification and localization of a marker protein for the intermembrane space between the outer and inner chloroplast envelopes is described. This 64-kDa protein is very rapidly labeled by [gamma-32P]ATP at very low (30 nM) ATP concentrations and the phosphoryl group exhibits a high turnover rate. It was possible to establish the presence of the 64-kDa protein in this plastid compartment by using different chloroplast envelope separation and isolation techniques. In addition comparison of labeling kinetics by intact and hypotonically lysed pea chloroplasts support the localization of the 64-kDa protein in the intermembrane space. The 64-kDa protein was present and could be labeled in mixed envelope membranes isolated from hypotonically lysed plastids. Mixed envelope membranes incorporated high amounts of 32P from [gamma-32P]ATP into the 64-kDa protein, whereas separated outer and inner envelope membranes did not show significant phosphorylation of this protein. Water/Triton X-114 phase partitioning demonstrated that the 64-kDa protein is a hydrophilic polypeptide. These findings suggest that the 64-kDa protein is a soluble protein trapped in the space between the inner and outer envelope membranes. After sonication of mixed envelope membranes, the 64-kDa protein was no longer present in the membrane fraction, but could be found in the supernatant after a 110,000 x g centrifugation.     

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.     

Effects of temperature and photoperiod upon adult eclosion of the sweetpotato whitefly, Bemisia tabaci

A series of experiments were conducted to characterize patterns of eclosion by Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) to their adult stage and to determine how these patterns are influenced by certain environmental parameters. Under a constant temperature of 29.5±0.6°C and a photoperiod of 14:10LD, 90% of the adults emerged from their pupal cases between 0600 and 0930 h (with lights on occurring at 0600 h). Few emerged during hours of darkness. The peak time of adult emergence was delayed when temperatures were fluctuated. Under a series of constant temperatures, a significant inverse correlation was found between the time of median emergence (i.e., eclosion of 50% of the total number of adults) and temperature (P<0.001). No emergence was observed at temperatures below 17±0.3°C. Emergence patterns persisted under conditions of continuous light and continuous darkness, suggesting the presence of a circadian system.

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Zusammenfassung Um das Verständnis über den Lebenslauf von Bemisia tabaci zu ergänzen, wurde eine Serie von Experimenten durchgeführt, deren Zweck die Charakterisierung des Ausschlüpfvorgangs in das Endstadium war und die Feststellung, wie dieser Vorgang von gewissen Umweltparametern beeinflusst wird. Bei einer konstanten Temperatur von 29.5±0.6°C und einem Beleuchtungszyklus von 14: 10 LD (Licht/Dunkelheit) schlüpften 90% der Ausgewachsenen zwischen 0600 Uhr and 0930 Uhr (ab 0600 Uhr mit Licht) aus ihren Puppenhüllen aus. Wenig Ausschlüpfen geschah während der unbeleuchteten Stunden. Der Höhepunkt des Ausschlüpfens wurde bei wechselnden Temperaturen verschoben. Bei einer Serie von gleichbleibenden Temperaturen wurde eine bedeutende inverse Korrelation zwischen der medianen Ausschlüpfzeit (d.h. 50% der gesamten Ausgewachsenen schlüpften aus) und der Temperatur festgestellt (P<0.001). Kein Ausschlüpfen wurde beobachtet bei Temperaturen unter 17°C. Das Ausschlüpfschema war gleichbleibend bei dauerndem Licht oder dauernder Dunkelheit, was auf das Vorhandensein eines circadianen Systems hinweist.

     

Purification and comparison of the receptors for insulin and insulin like growth factor I

The insulin-like growth factors are structurally and biologically similar to insulin. The receptor sites for insulin-like growth factor-I have recently been shown to have a sub-unit structure very similar if not identical to insulin. We have compared the behavior of insulin and insulin-like growth factor-I receptors during purification using gel filtration, lectin affinity chromatography, insulin affinity chromatography, and gel electrophoresis. We demonstrate the remarkably similar physicochemical characteristics of these two receptors, but have achieved complete separation by the use of insulin affinity chromatography.     

The effect of nalidixic acid on expression from related E. coli promoters

The effect of the DNA gyrase inhibitor, nalidixic acid, on expression from E. coli promoters was studied using the pKO-1, galactokinase expression vector system. Expression from a series of related hybrid promoters, tet promoter variants and the trp promoter flanked by oligonucleotide blocks was measured after incubation with nalidixic acid. Expression from the pBR322 tet promoter and tet promoter mutants within the -10 region was reduced after the drug treatment. The lacUV5, trp, and tettrp promoters were essentially unaffected while the trplac and the trptet promoters were stimulated. Studies of the trp promoter flanked by upstream or downstream oligonucleotide blocks revealed similar responses to the trp promoter parent control plasmids.     

Serum steroid levels in intact and endocrine ablated BALB/c nude mice and their intact littermates

An investigation was made of the serum steroid levels found in intact and endocrine ablated nude mice of both sexes and in their intact homozygous littermates. The results showed that nude mice have a normal steroidogenesis, but with decreased levels of circulating steroids compared to those of the littermates. The efficacy of the endocrine ablations was confirmed by the reduction in serum oestrone following oophorectomy, and by the reduction in serum testosterone and progesterone following orchiectomy. The normal steroidogenesis in nude mice, and the similarities between mouse and man with regard to changes in serum steroids following oophorectomy and orchiectomy, support the usefulness of human tumor xenograft models for the study of hormone-tumor interactions.     

Polar mobilization of the Escherichia coli chromosome by the ColE1 transfer origin

Summary Mobilization of the plasmid ColE1 from cells containing a conjugative plasmid (such as F) requires the synthesis of ColE1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of ColE1 containing the origin of transfer (oriT). The process of ColE1 transfer is thought to resemble that of the conjugative plasmid F, although the plasmids share little sequence homology. In F, conjugation is preceded by a strand-specific nicking event at oriT. The nicked strand is then conducted to the recipient with the 5 end leading. This is believed also to occur with ColE1, but direct biochemical confirmation has been precluded by its small size (6.65 kb). To test this hypothesis genetically, a novel method, using a dv-based vector, has been devised to site-specifically integrate bom (or any other cloned sequence) into the chromosome of Escherichia coli. When provided with suitable mobilizing plasmids, such strains were found to transfer the chromosome in a polar way. From these data, the orientation of transfer of ColE1 was deduced and shown to be analogous to F.     

Sensitivity to X-irradiation of peripheral blood lymphocytes from ageing donors

The proliferation of human blood lymphocytes from ageing donors, responding to concanavalin A, showed greater sensitivity to inhibition by X-rays than similar cells from younger donors. This increased sensitivity was associated with deficiency in repair of X-ray-induced damage to nuclear material, as measured by density in sucrose gradients, and with increased incidence of chromosomal damage following exposure of freshly isolated lymphocytes. There was also an increased frequency of spontaneous chromosomal aberrations in ageing subjects whose lymphocytes were deficient in repair of DNA damage.     

Transglutaminase and the Neuronal Cytoskeleton in Alzheimer''s Disease

Transglutaminase [EC 2.3.2.13, (R)-glutaminyl-peptide:amine gamma-glutamyltransferase], an enzyme that catalyzes the introduction of glutamine-lysine cross-links into proteins, was purified. Neurofilament and microtubule proteins were substrates for this enzyme but the insoluble neurofibrillary tangles (NFT) isolated from Alzheimer's disease brain were not substrates. In vitro cross-linking of neurofilaments and microtubules by the enzyme did not produce paired helical filaments (PHF), which are the major ultrastructural component of NFT. These results make it unlikely that PHF are formed by the straightforward cross-linking of neurofilaments or microtubules.