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BACKGROUND Bovine hide underlayer is used to manufacture collagen casings for sausage production. Edible collagen casings and natural ovine casing (as a reference) were chosen for analysis. The collagen casings (intended for: A - breakfast sausages, B - scalded sausages and C - dried sausages) were manufactured with the use of glyoxal as a cross-linking agent. The casings properties along with the quality of the sausages manufactured in those casings was analyzed. RESULTS Casing A had a higher swelling capacity and water solubility, a* and b* color parameters, and wet elongation at break values, as well as a lower shrinking temperature. The remaining mechanical and water parameters were comparable to other collagen casings. The ovine casing contained more water, and was redder and less yellow compared to the collagen casings, whereas swelling, solubility and water vapor permeability were higher. The sausages in casing A were the softest and had the highest a* and b* color parameters. The results of the sensory analysis indicate that the sausages in casing B exhibited the highest quality on a three-point scale among the tested sausages and were also the most acceptable for judges. The ovine casing was graded as the hardest during cutting and also when in the mouth. CONCLUSION By contrast to the producer's descriptions, the collagen casings were similar with respect to many properties. The differences between sausages in collagen casings were not so distinct as might be expected. Despite our expectations, the sausages in the natural casing were not rated as the best compared to sausages in collagen casings in the sensory analysis. © 2021 Society of Chemical Industry.

Mitotic arrest can result in cell death through the process of apoptosis. We have shown by live-cell imaging that the ubiquitin-proteasome dependent proteolysis of the apoptotic regulator Mcl-1 under the control of the anaphase-promoting complex or cyclosome (APC/C) provides a timing mechanism that distinguishes prolonged mitotic arrest from normal mitosis.

INTRODUCTION Lysosomes are degradative organelles that break down diverse materials delivered from inside and outside the cell by specialized vesicles called endosomes. Collectively, these membrane-enclosed compartments constitute the endolysosomal network. Endolysosomes can be ruptured or otherwise damaged by materials that they transport or accumulate. Damage can occur intentionally as in the case of incoming pathogens that seek to access the cytoplasm. Alternatively, damage can arise incidentally by membrane destabilizing molecules or by particulates such as crystals and protein aggregates that can puncture the lipid bilayer. To guard against these toxic or harmful substances and preserve pathway function, cells must be able to maintain and restore the integrity of their endolysosomal membranes. The mechanisms responsible for this vital function remain unclear. RATIONALE Extensively damaged compartments can be sequestered and degraded by a form of selective autophagy called lysophagy, which is facilitated by cytosolic damage sensors such as galectins that bind to luminal glycans exposed on injured organelles. More limited damage is likely to require alternative responses for efficient resolution and repair. The endosomal sorting complex required for transport (ESCRT) machinery comprises a collection of proteins that form polymeric filaments to promote budding and fission of membranes in numerous contexts, notably during the formation of multivesicular endosomes. Recent studies highlight an additional role for ESCRT proteins in resolving small wounds on the plasma membrane and tears in the nuclear envelope. We investigated whether ESCRT machinery might also be recruited to damaged endolysosomes to promote their repair. RESULTS Using common peptide reagents that accumulate within acidic endolysosomes and selectively trigger their disruption, we demonstrate that ESCRT machinery rapidly and coherently assembles on the limiting membrane of injured endolysosomal organelles. This response was observed in multiple types of cells, including phagocytes, and was especially prominent on endolysosomes damaged by internalized silica crystals. Notably, damage-triggered ESCRT recruitment required calcium as well as known ESCRT-nucleating factors including TSG101 and ALIX, and was distinct from lysophagy. To investigate the role played by ESCRT machinery on damaged endolysosomes, we used live-cell imaging of fluorescently tagged ESCRT proteins together with probes to dynamically monitor compartmental integrity. These experiments established that ESCRT recruitment correlates with the onset of small perforations permeable to protons, but not with larger ruptures that allow exchange of high molecular weight material, including internalized dextrans and cytoplasmic glycan-sensing galectins. Imaging ESCRT dynamics during a pulse of transient membrane disruption further revealed that ESCRT recruitment precedes recovery of compartmental function, monitored with a fluorogenic indicator of lysosomal protease activity. Accordingly, depleting cells of relevant ESCRT recruitment factors impaired both the reacidification and functional recovery of transiently injured organelles. CONCLUSION Our kinetic and functional data reveal a role for ESCRTs in the repair of small perforations in endolysosomes. This activity enables a restorative response to limited membrane damage that is likely to be protective in pathological contexts involving endolysosomal leakage and may help counter damage-induced inflammation. Transiently pacifying this response could additionally benefit efforts aimed at maximizing targeted drug and nanoparticle delivery through endocytosis. ESCRT participation in endolysosomal repair shares similarities with previously described roles for this machinery in mending nanometer-sized wounds at the plasma membrane and resealing the nuclear envelope, suggesting that ESCRT-promoted membrane repair may constitute a generic cellular response to limited membrane disruption. Based on the geometric constraints of endolysosomes and the type of damage they incur, we speculate that repair could proceed through closure of the membrane wound by ESCRT-containing filaments.

BACKGROUND Gentiana lutea root is a medicinal herb that contains many active compounds which contribute to physiological effects, and it has recently attracted much attention as a natural source of antioxidants. The aim of this study was to evaluate the effects on the colour, pH, microbial activities, sensory quality and resistance to lipid oxidation (through the thiobarbituric acid method) during storage of beef patties containing different concentrations of G. lutea. Fresh beef patties were formulated with 0–5 g kg−1 of G. lutea and 0 or 0.5 g kg−1 of ascorbic acid and packed in two different atmospheres, Modified Atmosphere 1 (MAP1) and Modified Atmosphere 2 (MAP2), and stored at 4 ± 1 °C for 10 days. MAP1 contained 20:80 (v/v) O2:CO2 and MAP2 contained 80:20 (v/v) O2:CO2. RESULTS G. lutea extracts possessed antioxidant activity measured by the ferric reducing antioxidant power and the oxygen radical absorbance capacity assays. Beef patties containing 2 g kg−1 of lyophilised G. lutea were stable towards lipid oxidation in both atmospheres (P

Artemisia annua is currently the only commercial source of the sesquiterpene lactone artemisinin. Although artemisinin is a major bioactive component present in this Chinese herb, leaf flavonoids have shown a variety of biological activities. The polyphenolic profile of extract from leaves of A. annua was assessed as a source of natural antioxidants. Total phenolic content and total flavonoid content were established and three assays were used to measure the antioxidant capacity of the plant extract. The measurement of scavenging capacity against the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid #ABTS# radical cation, radicals, the oxygen radical absorbance capacity #ORAC# and the ferric reducing antioxidant power #FRAP# were 314.99 µM Trolox equivalents #TE#/g DW, 736.26 µM TE/g DW and 212.18 µM TE/g DW, respectively. A. annua extracts also showed good antioxidant properties in 10% sunflower oil-in-water emulsions during prolonged storage #45 days# at 32 °C. Artemisia extract at 2 g/L was as effective as butylated hydroxyanisole #BHA# at 0.02 g/L in slowing down the formation of hydroperoxides as measured by peroxide value and thiobarbituric acid reactive substances. The results of this study indicate that extract of A. annua may be suitable for use in the food matrix as substitutes for synthetic antioxidants.

The polyphenolic profile of leaves and stalks of Perilla frutescens, was assessed as a source of natural antioxidants. The amount of caffeic and rosmarinic acids, determined by high-performance liquid chromatography (HPLC), were 0.51 mg/g dry weight (DW) and 2.29 mg/g DW, respectively. The measurement of scavenging capacity against the 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation, the oxygen radical absorbance capacity (ORAC), and the ferric reducing antioxidant power (FRAP) were 65.03 mg Trolox equivalents (TE)/g DW, 179.60 mg TE/g DW and 44.46 mg TE/g DW, respectively. P. frutescens extracts also showed good antioxidant properties in 10% sunflower oil-in-water emulsions during storage at 32 °C. Perilla extract at 320 ppm was as effective as butylated hydroxyanisole (BHA) at 20 ppm in slowing down the formation of hydroperoxides as measured by peroxide value, thiobarbituric acid reactive substances and hexanal content. The results of this study indicate that extract of P. frutescens may be suitable for use in the food matrix to help achieve potential health benefits.

BACKGROUND The successful replacement of some synthetic food antioxidants by safe natural antioxidants has fostered intensive search for new vegetable sources of antioxidants. In our study the phenol and flavonoid content of extracts of tara pods was determined. The antioxidant activity was also studied by three different analytical assays: the measurement of scavenging capacity against a radical ABTS+, the oxygen radical absorbance capacity (ORAC) and the ferric reducing antioxidant power (FRAP). RESULTS All analyzed samples showed a good antioxidant capacity, but the use of a solution of ethanol 75% in a 1 h ultrasonic process allowed achieving the greatest quantity of phenolics (0.464 mg gallic acid equivalent (GAE) g−1 dry weight (DW) ) and the highest antioxidant activity measured by the ABTS+ and ORAC methods (10.17 and 4.29 mmol L−1 Trolox equivalents (TE) g−1 DW, respectively). The best method for efficient extraction of flavonoids (3.08 mg catechin equivalent (CE) g−1 DW) was a 24 h maceration in cold water. Two extracts obtained with ethanol 75% and water were added to a model food system (oil-in-water emulsion) and the oxidative stability was studied during storage at 38 °C. Oxidation was monitored by determination of the peroxide value. The addition of 48 µg mL−1 ethanol extract to the emulsion delayed oxidation to the same extent as 17.8 µg mL−1 of Trolox, while water extract was only effective in the early stages of the oxidation process. CONCLUSION The results of this study indicate that ethanolic tara extracts may be suitable for use in food, cosmetic and nutraceutical applications. © 2013 Society of Chemical Industry

Galactofuranose (Galf) is the five-membered ring form of galactose. Although it is absent from mammalian glycans, it occurs as a structural and antigenic component of important cell surface molecules in a variety of microbes, ranging from bacteria to parasites and fungi. One such organism is Cryptococcus neoformans, a pathogenic yeast that causes lethal meningoencephalitis in immunocompromised individuals, particularly AIDS patients. C. neoformans is unique among fungal pathogens in bearing a complex polysaccharide capsule, a critical virulence factor reported to include Galf. Notably, how Galf modification contributes to the structure and function of the cryptococcal capsule is not known. We have determined that Galf is β1,2-linked to an unusual tetrasubstituted galactopyranose of the glucuronoxylomannogalactan (GXMGal) capsule polysaccharide. This discovery fills a longstanding gap in our understanding of a major polymer of the cryptococcal capsule. We also engineered a C. neoformans strain that lacks UDP-galactopyranose mutase; this enzyme forms UDP-Galf, the nucleotide sugar donor required for Galf addition. Mutase activity was required for the incorporation of Galf into glucuronoxylomannogalactan but was dispensable for vegetative growth, cell integrity, and virulence in a mouse model.

Caesalpinia decapetala (Roth) Alston (Fabaceae) (CD) is used in folk medicine to prevent colds and treat bronchitis. This plant has antitumor and antioxidant activity. The antioxidant effects of an extract from Caesalpinia decapetala (Fabaceae) were assessed by storage of model food oil-in-water emulsions with analysis of primary and secondary oxidation products. The antioxidant capacity of the plant extract was evaluated by the diphenylpicrylhydrazyl (DPPH), Trolox equivalent antioxidant capacity (TEAC), oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays and by electron paramagnetic resonance (EPR) spectroscopy. Lyophilized extracts of CD were added at concentrations of 0.002%, 0.02% and 0.2% into oil-in-water emulsions, which were stored for 30 days at 33 ± 1 °C, and then, oxidative stability was evaluated. The CD extract had high antioxidant activity (700 ± 70 µmol Trolox/g dry plant for the ORAC assay), mainly due to its phenolic components: gallic acid, quercetin, catechin, 4-hydroxybenzoic acid and p-coumaric acid. At a concentration of 0.2%, the extract significantly reduced the oxidative deterioration of oil-in-water emulsions. The results of the present study show the possibility of utilizing CD as a promising source of natural antioxidants for retarding lipid oxidation in the food and cosmetic industries.

Context: Betula pendula Roth (Betulaceae) exhibits many pharmacological activities in humans including anticancer, antibacterial, and antiviral effects. However, the antioxidant activity of BP towards lipid degradation has not been fully determined. Objective: The BP ethanol and methanol extracts were evaluated to determine antioxidant activity by an in vitro method and lyophilized extract of BP was added to beef patties to study oxidative stability. Materials and methods: Antioxidant activities of extracts of BP were determined by measuring scavenging radical activity against methoxy radical generated by Fenton reaction 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (TEAC) radical cation, the oxygen radical absorbance capacity (ORAC) and the ferric reducing antioxidant power (FRAP) assays. The lipid deterioration in beef patties containing 0.1% and 0.3% (w/w) of lyophilized extract of BP stored in 80:20 (v/v) O2:CO2 modified atmosphere (MAP) at 4 °C for 10 days was determined using thiobarbituric acid reacting substances (TBARS), % metmyoglobin and colour value. Results: The BP methanol extract revealed the presence of catechin, myricetin, quercetin, naringenin, and p-coumaric acid. The BP ethanol (50% w/w) extract showed scavenging activity in TEAC, ORAC and FRAP assays with values of 1.45, 2.81, 1.52 mmol Trolox equivalents (TE)/g DW, respectively. Reductions in lipid oxidation were found in samples treated with lyophilized BP extract (0.1% and 0.3% w/w) as manifested by the changes of colour and metmyoglobin concentration. A preliminary study film with BP showed retard degradation of lipid in muscle food. Conclusion: The present results indicated that the BP extracts can be used as natural food antioxidants.

The effect of dried pods of Caesalpinia spinosa, known as tara, on pH, cooking loss, lipid oxidation, colour stability and texture of model meat systems stored at 4 °C for 21 days was investigated. Tara pod powder showing a potential antioxidant activity was added at 0.02, 0.04 and 0.08% (by mass) directly to the pork batter and compared with a synthetic antioxidant, butylated hydroxyanisole (BHA) and control (no added antioxidants). The addition of tara pod powder at 0.02% was as effective as BHA (0.02%) in retarding lipid oxidation in pork products during storage. Results showed that redness increased after the addition of tara pod powder. Specifically, 0.02% of tara pod powder was effective in keeping the red colour of meat batter stored under illumination at 4 °C for 48 h. Hardness of pork products was the lowest in samples manufactured with tara pod powder compared with control. Results highlight the potential of using tara pod powder as natural functional ingredient in the development of pork products with enhanced quality and shelf life.

The Met4 transcriptional activator of methionine biosynthesis is negatively regulated by the SCFMet30 ubiquitin ligase in response to accumulation of methionine. This mechanism requires polyubiquitination, but not proteolysis. We report that a previously unappreciated mechanism involving growth control regulates Met4. Unless methionine is present in the growth medium, polyubiquitinated Met4 is stabilized in late exponential cultures, correlating with transcriptional repression. Polyubiquitinated Met4 becomes destabilized in a proteasome-dependent manner upon reentry into exponential growth, correlating with transcriptional activation. Met4 stabilization is regulated at the level of SCFMet30 binding and requires transcriptional cofactors. These lock Met4 and SCFMet30 into a tight complex active in ubiquitination but incapable of binding the proteasome. Release of polyubiquitinated Met4 from SCFMet30 is sufficient for degradation, and specific sulfur amino acids can promote the degradation by destabilizing Met4 binding to cofactors and SCFMet30. Thus, destabilization of cofactors and SCFMet30 binding is the rate-limiting regulatory step in Met4 proteolysis.

Background The S73/S97/loop motif is a hallmark of the Cdc34 family of E2 ubiquitin-conjugating enzymes that together with the SCF E3 ubiquitin ligases promote degradation of proteins involved in cell cycle and growth regulation. The inability of the loop-less Δ12Cdc34 mutant to support growth was linked to its inability to catalyze polyubiquitination. However, the loop-less t riple m utant (tm) Cdc34, which not only lacks the loop but also contains the S73K and S97D substitutions typical of the K73/D97/no loop motif present in other E2s, supports growth. Whether tmCdc34 supports growth despite defective polyubiquitination, or the S73K and S97D substitutions, directly or indirectly, correct the defect caused by the loop absence, are unknown. Results tmCdc34 supports yeast viability with normal cell size and cell cycle profile despite producing fewer polyubiquitin conjugates in vivo and in vitro. The in vitro defect in Sic1 substrate polyubiquitination is similar to the defect observed in reactions with Δ12Cdc34 that cannot support growth. The synthesis of free polyubiquitin by tmCdc34 is activated only modestly and in a manner dependent on substrate recruitment to SCFCdc4. Phosphorylation of C-terminal serines in tmCdc34 by Cka2 kinase prevents the synthesis of free polyubiquitin chains, likely by promoting their attachment to substrate. Nevertheless, tm CDC34 yeast are sensitive to loss of the Ubp14 C-terminal ubiquitin hydrolase and DUBs other than Ubp14 inefficiently disassemble polyubiquitin chains produced in tm CDC34 yeast extracts, suggesting that the free chains, either synthesized de novo or recycled from substrates, have an altered structure. Conclusions The catalytic motif replacement compromises polyubiquitination activity of Cdc34 and alters its regulation in vitro and in vivo, but either motif can support Cdc34 function in yeast viability. Robust polyubiquitination mediated by the S73/S97/loop motif is thus not necessary for Cdc34 role in yeast viability, at least under typical laboratory conditions.

Rapid and reliable tailoring of the dose of controlled release tablets to suit an individual patient is a major challenge for personalized medicine. The aim of this work was to investigate the feasibility of using a fused deposition modelling (FDM) based 3D printer to fabricate extended release tablet using prednisolone loaded poly(vinyl alcohol) (PVA) filaments and to control its dose. Prednisolone was loaded into a PVA-based (1.75 mm) filament at approximately 1.9% w/w via incubation in a saturated methanolic solution of prednisolone. The physical form of the drug was assessed using differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). Dose accuracy and in vitro drug release patterns were assessed using HPLC and pH change flow-through dissolution test. Prednisolone loaded PVA filament demonstrated an ability to be fabricated into regular ellipse-shaped solid tablets using the FDM-based 3D printer. It was possible to control the mass of printed tablet through manipulating the volume of the design (R(2) = 0.9983). On printing tablets with target drug contents of 2, 3, 4, 5, 7.5 and 10mg, a good correlation between target and achieved dose was obtained (R(2) = 0.9904) with a dose accuracy range of 88.7-107%. Thermal analysis and XRPD indicated that the majority of prednisolone existed in amorphous form within the tablets. In vitro drug release from 3D printed tablets was extended up to 24h. FDM based 3D printing is a promising method to produce and control the dose of extended release tablets, providing a highly adjustable, affordable, minimally sized, digitally controlled platform for producing patient-tailored medicines.

A study to establish relationship between the petal colour, extraction solvent, phenolic, flavonoid, anthocyanin content and antioxidant activity in three varieties of Viola × wittrockiana (yellow, red and violet) is reported in this article. Identification and quantification of flavonoids and anthocyanins using HPLC-DAD-ESI-MS is also presented. The antioxidant activity was studied by four different analytical assays: the measurement of scavenging capacity against ABTS + and DPPH free radicals, oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP). The violet petals were the most active in all solvents employed for extraction and also showed higher total phenolic, flavonoid and anthocyanin content in comparison with red and yellow ones. Fourteen constituents were identified by HPLC coupled with diode-array detection (DAD) and mass spectrometry (ESI-MS), ten of them were flavonoids and four anthocyanins. The major compounds found in violet pansy were rutin, violanthin and violanin. Aqueous extract of violet pansy can be regarded as a suitable candidate to serve as a radical scavenging agent that could be used in functional foods.

Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro . We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.

DnaK, DnaJ, and GrpE heat shock proteins of Escherichia coli activate site-specific DNA binding by the RepA replication initiator protein of plasmid P1 in a reaction dependent on ATP and Mg2+. We previously showed that GrpE is essential for in vitro RepA activation specifically at about 1 μM free Mg2+. In this paper, we demonstrate that GrpE lowers the requirement of DnaK ATPase for Mg2+, resulting in a large stimulation of ATP hydrolysis at about 1 μM Mg2+ with and without DnaJ and RepA. In contrast to its effect on the Mg2+ requirement, GrpE increases the ATP requirement for DnaK ATPase and dramatically lowers the affinity of DnaK for ATP in the absence of Mg2+. We propose that GrpE not only lowers the affinity of DnaK for nucleotide but, by increasing affinity of DnaK for Mg2+, also weakens the interactions of Mg2+ with nucleotide prior to its release.

The heat shock protein 70 molecular chaperone system of DnaK, DnaJ and GrpE is necessary for the replication of several E. coli phage and plasmids. For plasmid P1 DNA replication, this system activates DNA binding by the initiator protein, RepA. Activation exposes the DNA binding domain of RepA through the conversion of RepA dimers to monomers. In apparent contrast, the chaperones act after the assembly of the preprimosomal complex for phage λ DNA replication. They remodel and rearrange the proteins within the complex to activate the DnaB helicase. The mechanism of action of DnaK, DnaJ and GrpE is, however, very likely similar in both of these systems. Members of a new chaperone family, ClpA and ClpX, activate the DNA replication initiator proteins of plasmid P1 and phage λ, respectively. They also function in the ATP-dependent degradation of these proteins in conjunction with ClpP peptidase.

This work is supported by the The Scottish Universities Life Sciences Alliance (SULSA) Single-stranded DNA-binding proteins (SSBs) play vital roles in all aspects of DNA metabolism in all three domains of life and are characterized by the presence of one or more OB fold ssDNA-binding domains. Here, using the genetically tractable euryarchaeon Haloferax volcanii as a model, we present the first genetic analysis of SSB function in the archaea. We show that genes encoding the OB fold and zinc finger-containing RpaA1 and RpaB1 proteins are individually non-essential for cell viability but share an essential function, whereas the gene encoding the triple OB fold RpaC protein is essential. Loss of RpaC function can however be rescued by elevated expression of RpaB, indicative of functional overlap between the two classes of haloarchaeal SSB. Deletion analysis is used to demonstrate important roles for individual OB folds in RpaC and to show that conserved N- and C-terminal domains are required for efficient repair of DNA damage. Consistent with a role for RpaC in DNA repair, elevated expression of this protein leads to enhanced resistance to DNA damage. Taken together, our results offer important insights into archaeal SSB function and establish the haloarchaea as a valuable model for further studies. Publisher PDF

Cryptococcal meningoencephalitis is an AIDS-defining illness caused by the opportunistic pathogen Cryptococcus neoformans. This organism possesses an elaborate polysaccharide capsule that is unique among pathogenic fungi, and the glycobiology of C. neoformans has been a focus of research in the field. The capsule and other cellular glycans and glycoconjugates have been described, but the machinery responsible for their synthesis remains largely unexplored. We recently discovered Xpt1p, an enzyme with the unexpected activity of generating a xylose-phosphate-mannose linkage. We now demonstrate that this novel activity is conserved throughout the C. neoformans species complex, localized to the Golgi apparatus, and functions in the O-glycosylation of proteins. We also present the first survey of O-glycans from C. neoformans.

Genetic and biochemical studies have established that the sole function of the Escherichia coli DnaJ, DnaK, and GrpE heat shock proteins in plasmid P1 DNA replication is to convert RepA dimers to monomers. Monomers bind avidly to oriP1 DNA and initiate DNA replication. However, with purified heat shock proteins, only DnaJ, DnaK, and ATP were required for the monomerization of RepA; GrpE was not required. We have found reaction conditions that mimic the physiological situation. GrpE function is absolutely necessary for RepA activation in vitro with DnaJ and DnaK when the free Mg2+ concentration is maintained at a level of approximately 1 microM by a metal ion buffer system. EDTA or physiological metabolites, including citrate, phosphate, pyrophosphate, and ATP, all elicit the GrpE requirement. With these metal ion-buffering systems, GrpE specifically lowers the concentration of Mg2+ required for the RepA activation reaction. The absence of Mg2+ blocks activation and high levels of Mg2+ in solution bypass the requirement for GrpE but not for the other two heat shock proteins. Our results imply that GrpE facilitates the utilization of Mg2+ for an essential step in RepA activation.

In this work we show that the GroEL (Hsp60 equivalent) chaperone protein can protected purified Escherichia coli RNA polymerase (RNAP) holoenzyme from heat inactivation better than the DnaK (Hsp70 equivalent) chaperone can. In this protection reaction, the GroES protein is not essential, but its presence reduces the amount of GroEL required. GroEL and GroES can also reactivate heat-inactivated RNAP in the presence of ATP. The mutant GroEL673 protein, with or without GroES, is incapable of reactivating heat-inactivated RNAP. GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES. The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK's ability to reactivate heat-inactivated RNAP GroEL673 has also been investigated. We found that the DnaJ protein substantially reduces the levels of DnaK protein needed in this reactivation assay. However, the observed lag in reactivation is diminished only in the additional presence of the GrpE protein. Hence, DnaJ and GrpE are involved in both steps of this reactivation reaction (recognition of substrate and release of chaperone from the substrate-chaperone complex) while, in the case of the GroEL-dependent reaction, GroES is involved only during the release of chaperone from the substrate-chaperone complex.

We have found that three Escherichia coli heat shock proteins, DnaK (the hsp70 homolog), DnaJ, and GrpE, function in oriP1 DNA replication in vitro solely to activate DNA binding by the replication initiator protein RepA. Activation results from the conversion of P1 or P7 RepA dimers to monomers that bind with high affinity to the origin of replication of plasmid P1. Thus, the essential role of these three heat shock proteins in this replication system is to change the quaternary structure of a single protein, RepA.

It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys(48), but not through Lys(63), target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys(48) chains (E6AP) or Lys(63) chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys(48)-Ub chain or, surprisingly, to a Lys(63)-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys(63) chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys(63)-Ub chains from proteasomal degradation.

There are many similarities in the replication mechanisms employed by bacteriophage λ and its host E. coli (for reviews, see Bramhill and Kornberg, 1988, Keppel et al., 1988, and McMacken et al., 1987). In both cases, the host-coded primase enzyme (the dnaG gene product) is mostly responsible for synthesizing the RNA primers for both the leading and lagging strand, to be extended into DNA by the DNA polymerase III holoenzyme. A major problem in both systems is that the primase enzyme is normally unable to make RNA primers by itself (except in the case of a few single-stranded bacteriophages). Instead, it must first make contact with the dnaB helicase, properly positioned on single-stranded DNA. The dnaB helicase unwinds DNA by traveling in the 5’→3’ direction (LeBowitz and McMacken, 1986). To do so, the dnaB helicase must somehow “climb” onto DNA at a single-stranded region. Such a single-stranded DNA region is created at the E. coli oriC site by the binding of the dnaA•ATP protein form (Bramhill and Kornberg, 1988). To enter the single-stranded region at oriC, the dnaB helicase must be escorted by the dnaC initiation protein (Bramhill and Kornberg, 1988).

The heat shock or stress response has been universally conserved among organisms (reviewed in Morimoto et al., 1990; Georgopoulos et al., 1990; Gross et al., 1990). When the gram-negative bacterium, E. coli is treated with a heat shock, the cell responds by transiently accelerating the rate of transcription of heat shock genes. This increased transcription is promoted by the Eσ32 RNA polymerase holoenzyme. The genes of approximately half of the 20 or so observed heat shock proteins in E. coli have been cloned and their corresponding proteins purified and characterized. Surprisingly, five of the major heat shock genes, namely dnaK, dnaJ, grpE, groES, and groEL, were previously identified because certain mutations in them block bacteriophage λ growth (reviewed in Friedman et al., 1984; Georgopoulos et al., 1990). The dnaK, dnaJ, and grpE gene products are required for bacteriophage λ growth specifically at the level of DNA replication, whereas the groES and groEL gene products are required specifically at the level of prohead assembly. In this paper, we summarize our previous and current studies on these five genes and suggest models for the mechanism of action of their gene products.

Pelham previously proposed that the hsp70 family of heat shock proteins could prevent the formation and/or allow the dissolution of protein aggregates created during stress conditions. We confirmed this hypothesis by showing that the E. coli hsp70 homolog, the dnaK gene product, protects the host RNA polymerase enzyme from heat inactivation in an ATP-independent reaction. In addition, we show that heat-inactivated and aggregated RNA polymerase is both disaggregated and reactivated following simultaneous incubation with DnaK protein and hydrolyzable ATP. The DnaK756 mutant protein has lost the ability to disaggregate the inactivated RNA polymerase enzyme. Our results demonstrate that the DnaK protein contributes to E. coli's growth not only by protecting some enzymes from denaturation but also by reactivating some once they are misfolded or aggregated.

[Fe(TPC)] 2 O cristallise dans le systeme orthorhombique, groupe d'espace Pnna et sa structure est affinee jusqu'a R=0,0726

We have examined some of the early pre-priming steps of bacteriophage lambda dv DNA replication in vitro. Previous experiments have shown that bacteriophage lambda replication requires host RNA polymerase-dependent RNA synthesis near or at the origin of replication (ori lambda) to initiate DNA synthesis. Using a crude Fraction II enzymatic system we have shown that during RNA polymerase action, at least the bacteriophage lambda O and lambda P replication proteins as well as the host dnaB protein must be present to initiate ori lambda-specific DNA replication. The presence of three other host initiation proteins, dnaG primase, dnaJ, and dnaK, is not required during RNA polymerase action. Because of the apparent absence of a requirement for the dnaJ and dnaK pre-priming proteins during the transcriptional activation step, we propose that the early events of lambda dv DNA replication, prior to action by the dnaG primase, can be divided into two recognizable steps: an early step which requires at least RNA polymerase, lambda O, lambda P, and dnaB, and a subsequent step which requires the action of at least the dnaJ and dnaK proteins.

The DnaK protein of Escherichia coli and its eukaryotic hsp70 analogues are known to bind some polypeptides and to release or dissociate from them following ATP hydrolysis. Here we demonstrate that hydrolysis (and not simply binding) of nucleotide triphosphates leads to a change in the DnaK protein, from the "closed" to the "open" conformation. A conformational change is not observed with the mutant DnaK756 protein, which is always found in the open conformation. Although ATP is the preferred substrate, the hydrolysis of CTP, GTP, UTP, and dATP also results in DnaK's conversion from a closed to an open conformation. The ability of DnaK to hydrolyze various triphosphates correlates perfectly with its ability to release the bound denatured bovine pancreatic trypsin inhibitor polypeptide.

Breve presentation du processus de solubilisation de metaux alcalins dans des solvants organiques faisant intervenir un transfert d'electrons entre le metal et les molecules du solvant