Large-Scale Conformational Transitions and Dimerization Are Encoded in the Amino-Acid Sequences of Hsp70 Chaperones

Hsp70s are a class of ubiquitous and highly conserved molecular chaperones playing a central role in the regulation of proteostasis in the cell. Hsp70s assist a myriad of cellular processes by binding unfolded or misfolded substrates during a complex biochemical cycle involving large-scale structural rearrangements. Here we show that an analysis of coevolution at the residue level fully captures the characteristic large-scale conformational transitions of this protein family, and predicts an evolutionary conserved–and thus functional–homo-dimeric arrangement. Furthermore, we highlight that the features encoding the Hsp70 dimer are more conserved in bacterial than in eukaryotic sequences, suggesting that the known Hsp70/Hsp110 hetero-dimer is a eukaryotic specialization built on a pre-existing template.     

On the effect of temperature and pH on the settling behaviour of a flocculent strain ofZymomonas mobilis

Summary In this paper the effect of temperature and pH on the settling behaviour of a flocculent strain ofZymomonas mobilis is studied by using the old fashioned batch settling technique. Plots are given to show the influence of the above mentioned parameters on the settling curve behaviour.     

Fruit infesting tephritids [Dipt.: Tephritidae] and associated parasitoids in Chiapas,Mexico

A total of 1,302 parasitoids representing 8 species and 4 families were recovered from 9,818 fruit fly host fruits sampled. The most common parasitoid species wasDiachasmimorpha longicaudata (Ashmead). Average percent parasitism ranged between 0.44 and 29.23%. Parasitoid emergence data indicate thatAnastrepha ludens (Loew),A. obliqua (Sein),A. serpentina (Wiedeman),A. striata (Schiner) andToxotrypana curvicauda (Gerstaecker) were subject to parasitism. We provide information on the population fluctuation ofAnastrepha ludens, A. obliqua, A. serpentina, A. distincta (Greene),A. striata, A. fraterculus (Wiedeman),A. chiclayae (Greene),A. montei (Costa Lima),A. leptozona (Hendel) andA. tripunctata (Wulp).Anastrepha ludens andA. obliqua were the most common species, representing 95.3% of all fruit fly species caught in McPhail traps.      

Phase labeling of C−H and C−C spin-system topologies: Application in PFG-HACANH and PFG-HACA(CO)NH triple-resonance experiments for determining backbone resonance assignments in proteins

Summary Triple-resonance experiments can be designed to provide useful information on spin-system topologies. In this paper we demonstrate optimized proton and carbon versions of PFG-CT-HACANH and PFG-CT-HACA(CO)NH straight-through triple-resonance experiments that allow rapid and almost complete assignments of backbone H, ¹³C, ¹⁵N and H^N resonances in small proteins. This work provides a practical guide to using these experiments for determining resonance assignments in proteins, and for identifying both intraresidue and sequential connections involving glycine residues. Two types of delay tunings within these pulse sequences provide phase discrimination of backbone Gly C and H resonances: (i) C–H phase discrimination by tuning of the refocusing period _{a_f}; (ii) C–C phase discrimination by tuning of the ¹³C constant-time evolution period 2T_c. For small proteins, C–C phase tuning provides better S/N ratios in PFG-CT-HACANH experiments while C–H phase tuning provides better S/N ratios in PFG-CT-HACA(CO)NH. These same principles can also be applied to triple-resonance experiments utilizing ¹³C-¹³C COSY and TOCSY transfer from peripheral side-chain atoms with detection of backbone amide protons for classification of side-chain spin-system topologies. Such data are valuable in algorithms for automated analysis of resonance assignments in proteins.     

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.     

Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor

Pinocytosis was measured in monkey aortic smooth muscle cells (SMC), bovine aortic endothelial cells, and Swiss 3T3 cells in culture as cellular uptake of [U-(14)C]sucrose and horseradish peroxidase (HRP) from the tissue culture medium. Monkey arterial SMC and Swiss 3T3 cells were maintained in a quiescent state of growth at low cells density in medium containing 5 percent monkey plasma-derived serum (PDS). Replacement of PDS with 5 percent monkey whole blood serum (WBS) from the same donor, or addition to PDS of partially purified platelet-derived growth factor(s) (PF), resulted in a marked stimulation of pinocytosis as well as of cellular proliferation. In SMC, enhancement of the rate of pinocytosis occurred 4-6 h after exposure to WBS or PF, and the rate was up to twofold higher than the rate in medium containing PDS. In contrast, [(3)H]thymidine uptake by SMC did not increase until 12-16 h after exposure to PF. In endothelial cells the presence of PF or WBS did not enhance either the rate of pinocytosis or the rate of proliferation over that in PDS. Thus, endothelial cells did not become quiescent at subconfluent densities in PDS but maintained rates of proliferation and pinocytosis that were equivalent to those in WBS. By autoradiography, the fraction of labeled nuclei in SMC cultures 24 h after change of medium increased from 0.061 +/- 0.004 in quiescent cultures to 0.313 +/- 0.028 after exposure to WBS or PF. In contrast, labeling indices of endothelial cells were similar for cultures grown in PDS, WBS, or PF at any single time point after change of medium. These findings suggest that the rate of pinocytosis maybe be coupled in some fashion to growth regulation, which may be mediated in part by specific growth factors, such as that derived from the thrombocyte.     

Cyclic AMP and intracellular ionic activities innecturus gallbladder

Summary Open-tip and liquid ion-exchanger microelectrodes were used to study the effects of cAMP (6mm, added to the serosal medium) on apical membrane potential (E _m ) and intracellular sodium, potassium, and chloride activities (a _Na ⁱ ,a _K ⁱ ,a _Cl ⁱ ) inNecturus gallbladder under open-circuit conditions. Transepithelial potential difference (E _Tr ) was also measured. In the presence of cAMP,a _Cl ⁱ fell from about 1.5 times its equilibrium value to a level that corresponded to electrochemical equilibrium across the apical and basolateral cell membranes. Under these conditionsa _Na ⁱ decreased anda _K ⁱ increased,E _m was unchanged andE _Tr increased from virtually zero to a small but significant serosal positive value. The cAMP-induced increase ina _K ⁱ was abolished when Cl^–-free incubation media were used. Addition of the Ca⁺⁺-ionophore A23187 (0.5 g/ml) to the serosal medium had no effect onE _m ,E _Tr , ora _Cl ⁱ . When A23187 was added to the mucosal medium,E _m and the basolateral membrane potential hyperpolarized by about 20 mV and an increase in the outwardly directed electrochemical driving force for Cl^– was observed. These results indicate that cAMP inhibits coupled transapical Na–Cl entry into epithelial cells ofNecturus gallbladder and suggest that this inhibition may not be mediated by an increase in intracellular Ca⁺⁺ concentration.     

Diet crude protein reduction on follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows

This study evaluated the effect of crude protein (CP) reduction in four diets (156, 139, 132, and 127 g Kg^-1 DM) maintaining constant metabolizable protein (188 g/day) on the follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows. Twenty-two Girolando cows with average of 21.55 ±3.19 L daily milk yield, 105.30 ±22.62 days in lactation and 3.22 ±0.03 body condition score were selected. To reduce CP in diets and maintain constant metabolizable protein, urea and soybean meal were gradually replaced by lignosulfonate-treated soybean meal (SoyPass^®, Cargill), resulting in an increase in rumen-undegradable protein and a reduction in rumen degradable protein. A linear and quadratic reduction was observed in the plasma and follicular fluid urea nitrogen concentration following CP reduction, with the most intense reduction occurring in the 127 g Kg^-1 DM group (p<0.001). As CP reduced, there was a tendency for a linear increase in the follicular growth rate (P=0.0696), on the number and proportion of viable oocytes (P<0.09), and also a linear increase for the number (P=0.0397) and proportion (P<0.09) of grade I viable oocytes. Plus, there was a linear effect for the number of cumulus oophorus cells. Cows fed with the lowest amount of CP had cumulus-oocyte complexes with higher numbers of cumulus oophorus cells (P=0.0238). Also, the reduction of diet crude protein was followed by a decrease in the probability of oocytes’ DNA degradation. In conclusion, the reduction of CP in the diet of mid-lactating Girolando cows, reduces urea nitrogen concentration in both blood plasma and follicular fluid, and, as a consequence, increases the viability of oocytes and the number of cumulus oophorus cells while reducing oocytes’ DNA degradation of follicular included cumulus-oocyte complex. The reduction on dietary CP may improve in vivo oocytes’ embryo development impacting fertility of lactating dairy cows.