Degradation of intracellular protein in Salmonella typhimurium peptidase mutants

Multiply peptidase-deficient mutant strains of Salmonella typhimurium fail to carry out normal protein degradation during starvation for a carbon source. In these mutants, the extent of protein breakdown during starvation is about fourfold less than in the wild type. The products of protein breakdown in the mutant are mainly small, trichloroacetic acid-soluble peptides, not free amino acids as in the wild type. The carbon-starved mutant strain produces only about one thirtieth as much free amino acid from protein as the wild type. As a result, protein synthesis during starvation is reduced in the mutant compared to the wild type and the mutant strain shows a greatly prolonged lag phase after a nutritional shift-down.     

Peptide accumulation during growth of peptidase deficient mutants

Mutant strains of Salmonella typhimurium simultaneously lacking peptidases N, A, B and D accumulate a heterogeneous mixture of small, trichloroacetic acid-soluble peptides during growth in minimal medium. Approximately 20% of the labelled leucine supplied to a growing culture of the mutant strain is converted to peptides. These peptides accumulate inside the cells before being released into the growth medium. Although the origin of these peptides has not been established, there are several processes that might contribute peptides to this pool. These include (1) turnover of signal sequences, (2) turnover of attenuator peptides, and (3) degradation of prematurely terminated proteins. These results indicate that the same family of peptidases that catabolizes exogenously supplied peptides and functions in carbon-starvation-induced protein turnover also hydrolyzes peptides generated during normal exponential growth.     

Crystal structure studies and inhibition kinetics of tripeptide chloromethyl ketone inhibitors with Streptomyces griseus protease B

The bacterial serine protease, SGPB, was inhibited by two specific tripeptide chloromethyl ketones, N-t-butyloxycarbonyl-l-alanylglycyl-l-phenylalanine chloromethyl ketone (BocAGFCK) and N-t-butyloxycarbonyl-glycyl-l-leucyl-l-phenylalanine chloromethyl ketone (BocGLFCK). Crystals of the inhibited complexes were grown and examined by X-ray crystallographic methods. The peptide backbone of each inhibitor is bound by three hydrogen bonds to the main chain of residues Ser214 to Gly216. There are two well-characterized hydrophobic pockets, S₁ and S₂, on the surface of SGPB which accommodate the P₁ and P₂ side-chains of the BocGLFCK inhibitor. A conformational change of Tyr171 is induced by the binding of this inhibitor. Both inhibitors make two covalent bonds to the SGPB enzyme. The imidazole ring of His57 is alkylated at the N^?2 atom and O^γ of Ser195 forms a hemiketal bond with the carbonyl-carbon atom of the inhibitor. Comparison of the binding modes of the two tripeptides in conjunction with the differences in their inhibition constants (K_I) allows one to estimate the binding energy of the leucyl side-chain as ?2.6 kcal mol^?1. The importance of an electrophilic component in the serine protease mechanism, which involves the polarization of the susceptible carbonyl bond of a substrate or inhibitor by the peptide NH groups of Gly193 and Ser195 is discussed.     

Lipoxygenase activity in apples in relation to storage and physiological disorders

Different methods for the preparation of active lipoxygenase (LOX) extracts from apples were compared. Highest activities were obtained using a 0.25 M phosphate buffer (pH 7) containing 1% Triton X-100 and 10^?2 M metabisulphite as extraction solvent. LOX activity during storage was investigated in the core, flesh, and peel. Activity was always highest in the core and peel. On storage, activity was increased in each part of the fruit but especially in the core and peel. Increase in LOX preceded the browning of the core. LOX may be responsible for the browning and may be concerned in the induction of superficial scald.     

Effets des fluctuations rapides de la lumiere sur la photosynthese du phytoplancton

[¹⁴C]-assimilation rates were measured on cultures of two unicellulargreen algae (Chlamydomonas sp. and Oocystis sp.) as a functionof light intensities (saturation curves), under steady lightand also under rapidly alternating high and low light intensities.Assimilation rates vary according to the frequency of the intermittentlight regime and it falls under two categories: (1) at 0.1 and0.2 Hz, the assimilation rate is equal to the average of therates observed at high and low light intensities under steadylight, and (2) under 1.0, 1.6 and 10 Hz the assimilation rateis equal to the rate observed under a mean steady irradiance.Moreover, the range of assimilation rates at a given frequencydepends on the difference between the high and the low intensities.Batch cultures of Oocystis sp. have been grown under intermittentlight of 0.1, 1.0 and 10.0 Hz (same mean intensity). Growthrate under intermittent light of 0.1 Hz is –40% lowerthan the control under steady light. Photosynthetic potential(P^B_max)and efficiency () change with the growth stages of thecultures. At the end of the logarithmic growth phase, both photosyntheticparameters are maximum at 1.0 Hz and minimum at 0.1 Hz. Averagecell concentrations of chlorophyll a increase as the frequencyof the light regime decreases. During the log phase, concentrationof carotenoids relative to chlorophyll a increases at 1.0 Hz,decreases at 0.1 Hz, and remains constant at 10.0 Hz. Underclear sky conditions, wave-induced light fluctuations in thephotic layer may therefore enhance primary production, especially(1) in the lower part of the photic layer, where low frequencylight changes might cause cell chlorophyll a to increase, and(2) at a depth of 1–4 m, where the main frequencies (ofthe order of 1.0 Hz), might cause a significant increase ofboth the photosynthetic potential (P^B_max)and efficiency (). ¹Contribution au programme du GIROQ (Groupe interuniversitairede recherches océanographiques du Québec) ²Adresse actuelle: Centre de recherches en nutrition, UniversitéLaval, Québec, Qué. G1K 7P4, Canada     

Entamoeba histolytica and Giardia lamblia: Growth responses to reducing agents

The growth responses of Entamoeba histolytica strains HM-1:IMSS and HK-9 to a variety of reducing agents were tested for one subculture in TYI-S-33 medium, prepared with no cysteine or ascorbic acid. Amoebae did not grow in this medium. Addition of l-ascorbic acid, d- or l-cysteine, or l-cystine each permitted the maximum growth observed. Dithiothreitol supported 68% maximum growth of HK-9 amoebae, but only 12% of HM-1. In contrast, growth of both strains was greatly diminished (0–13% growth) with 11 other compounds tested including glutathione, thiomalic acid, thioglycolic acid, and methionine. The growth responses of Giardia lamblia were similarly tested in TYI-S-33, as well as in TP-S-1 media. If l-cysteine was omitted from either medium, trophozoites did not grow, and eventually lysed. In TYI-S-33 medium, the requirement for l-cysteine was specific, whereas in TP-S-1 medium, other sulfhydryl compounds were partially effective and lower concentrations of l-cysteine satisfied the requirement. Ascorbic acid or l-cystine alone was totally ineffective; however, in combination, 30 to 60% of maximum growth was achieved. Once added to either medium, cysteine was rapidly oxidized. Amino acid analysis of the growth media revealed that the broth components of TP-S-1 medium contained 2.8 mM and TYI-S-33 broth 2.1 mM endogenous levels of cysteine (or half-cystine), with an additional 3 mM contributed by 10% serum.     

In vitro uptake of particles by lysosomes

Isolated rat liver lysosomes were incubated with Percoll particles in vitro at 25 and 37 °C. On morphological examination the incubated lysosomes contain vesicles some of which enclose Percoll particles, indicating that invagination of the lysosomal membrane occurs in vitro by means of microautophagy. Vesiculation occurs by formation of flaplike processes or cuplike invaginations. At later time points of incubation Percoll particles can be seen free in the lysosomal matrix indicating rupture or digestion of the vesicular membrane. The uptake of isotopically pre-labelled Percoll particles increases with incubation time and temperature.It is concluded that lysosomes show microautophagic activity in vitro and that this may be a mechanism for degradation of soluble cytoplasmic proteins also in vivo.     

Internally elongated rodent α-crystallin A chain: Resulting from incomplete RNA splicing?

αA^Ins, an elongated α-crystallin A chain previously observed in rat, was present beside the normal αA chain in mouse, gerbil and hamster, which places its origin at least 30 million years ago. Like in rat the sequences of golden hamster αA^Ins and αA were found to be identical, apart from the internal insertion of 22 residues in αA^Ins. The hamster chains only differed from the rat chains by a single substitution in the inserted sequence of αA^Ins. The origin of αA^Ins, by insertion of 22 residues in an otherwise unchanged αA chain, and its rigid evolutionary conservation are most easily explained by assuming the incomplete removal of a putative intervening sequence from the precursor mRNA of αA, leaving an intracistronic insert of 66 nucleotides in part of the eventually translated mRNA.     

Révision systématiquedu genre Steginoporella smitt, 1873 (Bryozoa Cheilostomata)

Systematic revision of the genus Steginoporella: until now about eighty species were described. Only twenty recent species and thirty-four fossil ones are maintained. Several species and subspecies are new.The main interest of this revision is to establish a biostratigraphical scale: the settlement of this scale is based on the known stratigraphical distribution and on an attempt of phylogeny.The second advantage is ecological: all recent species live in marine tropical environment. The Steginoporella are good paleoecological indicators.At last, the establishment of a paleobiogeography, even incomplete and not definitive, allows to understand more easily recent distribution of Steginoporella connected with the great events of earth evolution.