Angiotensin-(1-7) reverts the stimulatory effect of angiotensin II on the proximal tubule Na(+)-ATPase activity via a A779-sensitive receptor.

Recently, we demonstrated that the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubules is reversed, in a dose-dependent manner, by Ang-(1-7) [Biochim. Biophys. Acta 1467 (2000) 189]. In the present paper, we characterized the receptor involved in this phenomenon. The preincubation of the Na(+)-ATPase with 10(-8) M Ang II increases the enzyme activity from 7.50+/-0.02 (control) to 12.40+/-1.50 nmol Pi mg(-1) min(-1) (p<0.05). Addition of 10(-9) M Ang-(1-7) completely reverts this effect returning the ATPase activity to the control level. This effect seems to be specific to Ang-(1-7) since Ang III (10(-12)-10(-8) M) does not modify the stimulation of the renal proximal tubule Na(+)-ATPase activity by Ang II. Saralasin abolishes the Ang-(1-7) effect in a dose-dependent manner being the maximal effect obtained at 10(-11) M. The increase in A779 concentration (from 10(-12) to 10(-7) M), a specific Ang-(1-7) antagonist, also abolishes the Ang-(1-7) effect. On the other hand, PD123319 (10(-8)-10(-6) M), an AT(2) antagonist receptor, and losartan (10(-12)-10(-7) M), an AT(1) antagonist receptor, does not modify the effect of Ang-(1-7). Taken together, these data indicate that Ang-(1-7) reverts the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubule through a A779-sensitive receptor.     

Age-related decrease in lysosomal β-D-galactosidase activity of human liver cell lines: Argument against post-traductional modifications

Aging of human liver cell lines is accompanied by a decrease in the lysosomal β-D-galactosidase activity. In six cell lines, both catalytic assay using 4-methylumbelliferyl-β-D-galactopyranoside and immunochemical titration by a monospecific anti-β-D-galactosidase anti-serum were performed. The reduced catalytic activity was not associated with the presence of cross-reaating material and probably did not result from a modification of the biosynthesis, nor from a post-translational modification of the enzyme.     

In Vitro Assembly of the T=13 Procapsid of Bacteriophage T5 with Its Scaffolding Domain

The Siphoviridae coliphage T5 differs from other members of this family by the size of its genome (121 kbp) and by its large icosahedral capsid (90 nm), which is organized with T=13 geometry. T5 does not encode a separate scaffolding protein, but its head protein, pb8, contains a 159-residue aminoterminal scaffolding domain (Δ domain) that is the mature capsid. We have deciphered the early events of T5 shell assembly starting from purified pb8 with its Δ domain (pb8p). The self assembly of pb8p is regulated by salt conditions and leads to structures with distinct morphologies. Expanded tubes are formed in the presence of NaCl, whereas Ca²⁺ promotes the association of pb8p into contracted tubes and procapsids. Procapsids display an angular organization and 20-nm-long internal radial structures identified as the Δ domain. The T5 head maturation protease pb11 specifically cleaves the Δ domain of contracted and expanded tubes. Ca²⁺ is not required for proteolytic activity but for the organization of the Δ domain. Taken together, these data indicate that pb8p carries all of the information in its primary sequence to assemble in vitro without the requirement of the portal and accessory proteins. Furthermore, Ca²⁺ plays a key role in introducing the conformational diversity that permits the formation of a stable procapsid. Phage T5 is the first example of a viral capsid consisting of quasi-equivalent hexamers and pentamers whose assembly can be carried out in vitro, starting from the major head protein with its scaffolding domain, and whose endpoint is an icosahedral T=13 particle.The self assembly of the icosahedral capsid of double-stranded DNA (dsDNA) viruses is a highly regulated unidirectional process that proceeds by steps, starting with the association of the full-length capsid protein subunits into a precursor capsid form. Several maturation steps follow (proteolytic cleavage, conformational reorganization of capsid subunits, and stabilization processes) that take place before, during, or after the packaging of the viral genome (34). Finally, the mature capsid has to withstand the high internal pressure imposed by the densely packed genome as well as environmental stresses after exit from the bacterial host. Capsid assembly thus relies on highly regulated protein-protein interactions that transition from the dynamic association and maturation phases to the stable and robust endpoint.The architectural principles that govern icosahedral capsids comprising several hundred identical protein subunits were formulated by Caspar and Klug (2), who introduced the concept of quasi-equivalence. Accordingly, capsid proteins have enough flexibility in shape and binding interfaces to assemble into hexamers (hexons) or pentamers (pentons) and to form hexon-penton and hexon-hexon contacts that are nearly equivalent to each other. In addition, assembly is tightly controlled to yield a single size in most cases, with the precise geometry of the capsid being described by the lattice triangulation number T. Viruses have developed various strategies to control the proper assembly of their capsid. The T4 bacteriophage (26) and human adenoviruses (14), for example, use distinct proteins to form hexons and pentons, while others, including herpesviruses and phage λ, use the same protein. Capsid assembly may rely on scaffolding proteins that subsequently are expelled from the capsid during maturation and DNA encapsidation (6, 15). If the scaffolding protein is mutated or absent, incorrect structures are formed, which can be open tubes (T4) or smaller particles (P22, φ29, herpesvirus) (1, 3, 10, 27, 28, 35). Capsid assembly also may rely only on the major head protein, as shown in the case of the lambdoid coliphage HK97 (T=7). Its major head protein, gp5, contains a mature domain and a 102-residue amino-terminal Δ domain that is cleaved after initial assembly by a viral protease and thus is considered to play the role of the scaffolding protein in other phages (5, 7). Furthermore, in vitro and in vivo studies have shown that preformed pentamers and hexamers of uncleaved gp5 accurately assemble into T=7 icosahedral shells without the requirement of the portal protein (5, 7, 9, 36). Gp5 thus carries by itself all the information needed to assemble into procapsids.The Siphoviridae coliphage T5 differs from other members of this family, like phage λ or HK97, by the large size of its dsDNA genome, 121,750 bp (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"AY587007","term_id":"59897142","term_text":"AY587007"}}AY587007, {"type":"entrez-nucleotide","attrs":{"text":"AY692264","term_id":"51511954","term_text":"AY692264"}}AY692264, and {"type":"entrez-nucleotide","attrs":{"text":"AY543070","term_id":"45774915","term_text":"AY543070"}}AY543070). Similarly to HK97, the T5 genome does not encode a scaffolding protein. The structure of the T5 icosahedral mature capsid was solved by cryoelectron microscopy (cryo-EM) and image reconstruction revealing the first capsid of a wild-type single-layered virus to have the triangulation number T=13 (11) (Fig. ​(Fig.1).1). The 90-nm-diameter capsid shell consists of the major head protein pb8 (39), whose subunits form 11 pentons located at the vertices and 120 hexons located at the faces. The hexons are decorated by an accessory protein, pb10 (11, 32). The 12th vertex is occupied by the connector, which comprises a dodecamer ring of the portal protein pb7. pb8 is a 50-kDa protein with two domains in its full-length form (called pb8p). pb8p undergoes proteolytic cleavage during morphogenesis, leaving a 32-kDa mature protein (pb8m) in the viral particle (38). N-terminal sequencing indicated that the severed region of pb8m corresponds to the 159 aminoterminal residues (11). This N-terminal extension, called the Δ domain by analogy with the HK97 major head protein, is predicted to have a high α-helical content mostly organized as a coiled-coil formation, and it may function as a scaffold domain (11). The mechanism by which the Δ domain is cleaved is unknown but likely involves pb11, a head maturation protease identified on the genome and located upstream of the gene encoding pb8 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY692264","term_id":"51511954","term_text":"AY692264"}}AY692264). A schematic of the T5 head assembly pathway is presented in Fig. ​Fig.1.1. Compared to that of HK97, the T5 capsid exhibits a higher level of complexity. Several constraints must be fulfilled to assemble its T=13 shell. A unique protein must associate into pentons occupying the vertices and into two kinds of hexons, one interacting both with pentons and hexons and the other exclusively with hexons. This raises the question of whether a T=13 procapsid can be assembled in vitro with the unique contribution of the major head protein carrying both structural and scaffold functions. To address these questions, we have expressed and purified pb8 and searched for physicochemical conditions allowing its assembly. We demonstrate that the precursor form of pb8 with its Δ domain (pb8p) assembles in vitro into diverse structures, including expanded and contracted tubes, and that the conformational flexibility introduced in pb8p by Ca²⁺ allows it to assemble into procapsids with internal radial structures corresponding to the Δ domain.Open in a separate windowFIG. 1.Bacteriophage T5 head assembly and characteristics of pb8 constructs. (A) Schematic of the head assembly pathway. The precursor form of the major head protein, pb8p, includes a scaffolding Δ domain (residues 1 to 159; represented as a white triangle) and a mature domain, pb8m (residues 160 to 458; represented as a gray rectangle). Pb8p is coassembled with the dodecameric portal complex (pb7) to form prohead I. The head maturation protease (pb11) cleaves the Δ domain, yielding prohead II. In vivo, DNA encapsidation through the portal triggers capsid expansion and the binding of the decoration protein (pb10) to the capsid exterior. In the present work, prohead I (or procapsid I) refers to the portalless assembly, and prohead II (or procapsid II) refers to the purified particles isolated from a T5stAmN5 mutant. (B) The T5 head structure calculated from cryo-EM data displays icosahedral T=13 symmetry, as shown previously (11). Vertices are indicated by the number 5 in the schematic at the top, and the T=13 pattern of hexamer positions is indicated in the schematic by the number 6. Scale bar = 20 nm. (C) Characteristics of pb8 constructs. The schematics at the left represent pb8p with its mature domain (in gray) and scaffolding domain (in white). The position of the His tag is indicated by H₆. The fifth construct encompasses both pb8p and the protease pb11 (in black). Details of the purification procedures are given in Materials and Methods. pCDF refers to the pCDF-2 Ek/LIC plasmid.     

Mode of Action of Nisin Z against Listeria monocytogenes Scott A Grown at High and Low Temperatures

Nisin Z, a natural nisin variant, was recently isolated from Lactococcus lactis subspecies lactis NIZO 22186. The gene for this lantibiotic, designated nisZ, has been cloned, and its nucleotide sequence was found to be identical to that of the precursor nisin gene with the exception of a single mutation resulting in the substitution of Asn-27 for His-27 in the mature polypeptide (J. W. M. Mulders, I. J. Boerrigter, H. S. Rollema, R. J. Siezen, and W. M. de Vos, Eur. J. Biochem. 201:581-584, 1991). A K⁺ electrode was used to investigate the effect of various environmental parameters on the action of nisin Z against Listeria monocytogenes. Addition of nisin Z resulted in immediate loss of cell K⁺, depolarization of the cytoplasmic membrane, inhibition of respiratory activity, and hydrolysis and partial efflux of cellular ATP. The action of nisin Z was optimal at pH 6.0 and was significantly reduced by di- and trivalent cations. The lanthanide gadolinium (Gd³⁺) was an efficient inhibitor and prevented nisin Z activity completely at a concentration of 0.2 mM. Nisin Z-induced loss of cell K⁺ was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. In cells grown at 30°C, the action of nisin Z was prevented below 7°C, whereas in cells grown at 4°C nisin Z was able to induce K⁺ leakage at this low temperature.     

Protective outcomes of low-dose doxycycline on renal function of Wistar rats subjected to acute ischemia/reperfusion injury

Renal ischemia-reperfusion injury (IRI) is a major cause of acute renal failure. Doxycycline (Dc) belongs to the tetracycline-class of antibiotics with demonstrated beneficial molecular effects in the brain and heart, mainly through matrix metalloproteinases inhibition (MMP). However, Dc protection of renal function has not been demonstrated. We determined whether low doses of Dc would prevent decreases in glomerular filtration rate (GFR) and maintain tubular Na⁺ handling in Wistar rats subjected to kidney I/R. Male Wistar rats underwent bilateral kidney ischemia for 30 min followed by 24 h reperfusion (I/R). Doxycycline (1, 3, and 10 mg/kg, i.p.) was administered 2 h before surgery. Untreated I/R rats showed a 250% increase in urine volume and proteinuria, a 60% reduction in GFR, accumulation of urea-nitrogen in the blood, and a 60% decrease in the fractional Na⁺ excretion due to unbalanced Na⁺ transporter activity. Treatment with Dc 3 mg/kg maintained control levels of urine volume, proteinuria, GFR, blood urea-nitrogen, fractional Na⁺ excretion, and equilibrated Na⁺ transporter activities. The Dc protection effects on renal function were associated with kidney structure preservation and prevention of TGFβ and fibronectin deposition. In vitro, total MMP activity was augmented in I/R and inhibited by 25 and 50 μM Dc. In vivo, I/R augmented MMP-2 and -9 protein content without changing their activities. Doxycycline treatment downregulated total MMP activity and MMP-2 and -9 protein content. Our results suggest that treatment with low dose Dc protects from IRI, thereby preserving kidney function.     

Dynamics and Determinants of Pneumococcal Antibodies Specific against 13 Vaccine Serotypes in the Pre-Vaccination Era

Introduction

Introduction of pneumococcal conjugate vaccines (PCVs) for infants decreased overall invasive pneumococcal disease (IPD), while non-vaccine serotype IPD increased. To fully understand this serotype replacement, knowledge about serotype dynamics in the pre-vaccine era is needed. In addition to IPD surveillance and carriage studies, the serotype replacement can be investigated by serosurveillance studies. The current study compared the results of two Dutch serosurveillance studies conducted in 1995–1996 (PIENTER1) and 2006–2007 (PIENTER2).

Methods

Participants in these studies donated a blood sample and completed a questionnaire. Pneumococcal antibodies of serotypes included in PCV13 were measured with a fluorescent-bead based multiplex immunoassay. Geometric mean antibody concentrations (GMCs) and determinants of pneumococcal antibody levels were investigated.

Results

GMCs were higher in PIENTER2 for serotypes 1, 6A, 6B, 9V, 18C, 19F and 23F and lower for 3 and 5. Age, day care attendance, household size, vaccination coverage, and urbanisation rate were associated with pneumococcal antibodies in children. Education level, ethnicity, age, low vaccination coverage sample, urbanisation rate, and asthma/COPD were associated with pneumococcal antibodies in elderly. The determinants significantly associated with pneumococcal IgG were slightly different for the elderly in PIENTER1 compared to the elderly in PIENTER2.

Conclusion

Although most of the serotype antibody levels remained stable, some of the serotype-specific antibody levels varied during the pre-vaccine era, indicating that exposure of certain serotypes changes without interference of PCVs.     

The measurement of resting metabolic rate in preschool children

Objective: This study examined the repeatability of measuring resting metabolic rate (RMR) in preschool children and the effect of different calculation protocols. Research Methods and Procedures: Eleven children (4 females and 7 males) participated in the project. They were recruited through advertisements in local schools and community centers. Resting metabolic rate was measured on 3 occasions over a 2‐week period, each after an overnight fast and each lasting ～20 to 25 minutes. Results were compared using repeated‐measures ANOVA to check for repeatability, and a number of methods of calculating RMR were assessed. Results: Repeatability of RMR measurements was good (coefficient of variation of replicates, 6.8%), with no significant difference between days of measurement. The lowest RMR measurement was obtained when the first 10 minutes were excluded and periods during which large activity was observed were excluded. This measurement was, on average, 4% lower than averaging the measurements after the first 5 minutes, including body movements. Discussion: This study suggests that RMR can be measured in preschool children and that the best method for calculating RMR in these subjects is to exclude periods when large body movements occur and the first 10 minutes of the measurement period. Only a single measurement of RMR is needed to obtain a reliable estimate.     

Identification of anesthetic binding sites on human serum albumin using a novel etomidate photolabel

We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(+) enantiomer being significantly more potent than the S(-) enantiomer. Its effects on alpha1beta2gamma2s GABA(A) receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (beta2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds approximately 75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABA(A) receptor or other putative targets.     

Caprine articular,meniscus and intervertebral disc cartilage: An integral analysis of collagen network and chondrocytes

Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM. Although it is known that the various (fibro)cartilaginous tissues (articular cartilage, annulus fibrosus, nucleus pulposus, and meniscus) differ from one each other with respect to their molecular make-up, remarkable little quantitative information is available with respect to its biochemical constituents, such as collagen content, or the various posttranslational modifications of collagen. Furthermore, we have noticed that tissue-engineering strategies to replace cartilaginous tissues pay in general little attention to the biochemical differences of the tissues or the phenotypical differences of the (fibro)chondrocytes under consideration. The goal of this paper is therefore to provide quantitative biochemical data from these tissues as a reference for further studies. We have chosen the goat as the source of these tissues, as this animal is widely accepted as an animal model in orthopaedic studies, e.g. in the field of cartilage degeneration and tissue engineering. Furthermore, we provide data on mRNA levels (from genes encoding proteins/enzymes involved in the synthesis and degradation of the ECM) from (fibro)chondrocytes that are freshly isolated from these tissues and from the same (fibro)chondrocytes that are cultured for 18 days in alginate beads. Expression levels of genes involved in the cross-linking of collagen were different between cells isolated from various cartilaginous tissues. This opens the possibility to include more markers than the commonly used chondrogenic markers type II collagen and aggrecan for cartilage tissue-engineering applications.