Casein-like immunoreactivity in the male rat

A substance presenting a beta-casein-like immunoreactivity (beta-cas-LI) was found in male serum and tissues. This beta-cas-LI was measured by a specific radioimmunoassay using a purified beta-casein and an anti beta-casein kindly supplied by L. M. Houdebine. Serial dilutions of serum or tissues samples showed a good parallelism to the standard curve. In male rat, beta-cas-Li was found in all sera and in some tissues = mammary gland, prostate and testis which contained respectively 6,200, 408 and 38 ng per organ. beta-cas-LI value in the serum was regulatable. Hypophysectomy and long-term castration strongly reduced basal levels. Insertion of testosterone silastic capsules (25 mg/capsule) was able to restore control values, while estradiol (E2) (4 mg/kg E2 valerianate) to castrated or intact rats increased serum beta-cas-LI above basal values. In conclusion, these results showed, for the first time, the presence in male rat serum and tissues of a beta-casein immunoreactive-like substance. Moreover, this beta-cas-LI seemed to be under pituitary and gonad regulation.     

Mesangial cell biology

Mesangial cells originate from the metanephric mesenchyme and maintain structural integrity of the glomerular microvascular bed and mesangial matrix homeostasis. In response to metabolic, immunologic or hemodynamic injury, these cells undergo apoptosis or acquire an activated phenotype and undergo hypertrophy, proliferation with excessive production of matrix proteins, growth factors, chemokines and cytokines. These soluble factors exert autocrine and paracrine effects on the cells or on other glomerular cells, respectively. MCs are primary targets of immune-mediated glomerular diseases such as IGA nephropathy or metabolic diseases such as diabetes. MCs may also respond to injury that primarily involves podocytes and endothelial cells or to structural and genetic abnormalities of the glomerular basement membrane. Signal transduction and oxidant stress pathways are activated in MCs and likely represent integrated input from multiple mediators. Such responses are convenient targets for therapeutic intervention. Studies in cultured MCs should be supplemented with in vivo studies as well as examination of freshly isolated cells from normal and diseases glomeruli. In addition to ex vivo morphologic studies in kidney cortex, cells should be studied in their natural environment, isolated glomeruli or even tissue slices. Identification of a specific marker of MCs should help genetic manipulation as well as selective therapeutic targeting of these cells. Identification of biological responses of MCs that are not mediated by the renin-angiotensin system should help development of novel and effective therapeutic strategies to treat diseases characterized by MC pathology.     

Identification of Linear Epitopes in Bacillus anthracis Protective Antigen Bound by Neutralizing Antibodies

Protective antigen (PA), the binding subunit of anthrax toxin, is the major component in the current anthrax vaccine, but the fine antigenic structure of PA is not well defined. To identify linear neutralizing epitopes of PA, 145 overlapping peptides covering the entire sequence of the protein were synthesized. Six monoclonal antibodies (mAbs) and antisera from mice specific for PA were tested for their reactivity to the peptides by enzyme-linked immunosorbent assays. Three major linear immunodominant B-cell epitopes were mapped to residues Leu¹⁵⁶ to Ser¹⁷⁰, Val¹⁹⁶ to Ile²¹⁰, and Ser³¹² to Asn³²⁶ of the PA protein. Two mAbs with toxin-neutralizing activity recognized two different epitopes in close proximity to the furin cleavage site in domain 1. The three-dimensional complex structure of PA and its neutralizing mAbs 7.5G and 19D9 were modeled using the molecular docking method providing models for the interacting epitope and paratope residues. For both mAbs, LeTx neutralization was associated with interference with furin cleavage, but they differed in effectiveness depending on whether they bound on the N- or C-terminal aspect of the cleaved products. The two peptides containing these epitopes that include amino acids Leu¹⁵⁶–Ser¹⁷⁰ and Val¹⁹⁶–Ile²¹⁰ were immunogenic and elicited neutralizing antibody responses to PA. These results identify the first linear neutralizing epitopes of PA and show that peptides containing epitope sequences can elicit neutralizing antibody responses, a finding that could be exploited for vaccine design.Bacillus anthracis is a Gram-positive, facultatively anaerobic, rod-shaped bacterium that secretes a variety of toxins, including anthrax toxin. This toxin is made up of three proteins as follows: protective antigen (PA),³ edema factor (EF), and lethal factor (LF). Like other binary toxins, anthrax toxin follows the same pattern where distinct subunits are involved in the different steps at which it can act. The B subunit (PA) is involved in receptor binding and cellular internalization into the cytoplasm, whereas the A subunit (EF and/or LF) bears the enzymatic activity (1). Anthrax can occur naturally in animals by spore transmission via ingestion, inhalation, or an open skin wound, but it can also be a result of bioterrorism and biological warfare (2).PA is the component of the currently licensed anthrax vaccine that elicits protective antibodies. Recent studies have demonstrated that a strong humoral response to truncated recombinant PA contributes to a protective immune response to anthrax (3, 4). Accordingly, there is considerable interest in ascertaining the location and immunogenicity of B-cell epitopes on the PA molecule. The development of numerous monoclonal antibodies (mAbs) to different epitopes on the PA molecule that influence PA functions, in conjunction with epitope mapping, has provided an opportunity to study the fine antigenic structure of PA. Most of these epitopes have been defined in mice (5–8), in macaques (9), in rabbits (10), as well as in vaccinated humans (11). Consequently, the epitopes described thus far are localized to three discrete regions within the PA. These regions correspond to the 2β2–2β3 loop of domain 2, the domain 3-domain 4 boundary, and the small loop of domain 4. The 2β2–2β3 loop of domain 2 is responsible for mediating membrane insertion, and many neutralizing murine mAbs target this region (5, 8). The boundary between domains 3 and 4, which does not have a known functional activity, has been suggested as a region recognized by polyclonal antibodies from vaccinated humans and rabbits (6, 12). The cellular receptor binding region is localized to the small loop of domain 4, and this region has been described to be recognized by two neutralizing mAbs (7, 9). With the exception of a neutralizing mAb that bound to PA₂₀ (13), no B-cell epitopes have been reported in domain 1. Therefore, identification of further dominant antigenic epitopes is pivotal for understanding the minimal immunogenic region of PA that will allow for precise direction of potent immune responses.Two mAbs to PA have been reported previously by our laboratory, one known as 7.5G binds to domain 1 and can neutralize the cytotoxic activity of lethal toxin (LeTx) (13). The other, mAb 10F4, binds to domain 4 and has weak neutralizing activity. In addition, we now describe four new anti-PA mAbs, of which only one neutralizes LeTx. The characterization of the B-cell epitopes in PA recognized by protective and nonprotective mAbs is important to better understand the antigenic structure of this toxin, and such information is potentially useful for the design of vaccines and passive immune therapies against B. anthracis. Because PA has been identified as an effective subunit vaccine, we wanted to identify the specific epitopes that provide the protection and use them as immunogens. Using mAbs and 145 overlapping peptides covering the entire sequence of PA, we identify the first linear neutralizing epitopes in domain 1 of PA, and we demonstrate that two peptides containing epitopes in domain 1 were capable of inducing strong LeTx-neutralizing antibody responses.     

Steroid regulation of gonadotropins in genetically hypoprolactinemic females (IPL nude rats)

IPL nude females present an absence of lactation with hypoprolactinemia. While males present a slight but significant decrease in serum testosterone and gonadotropins, females show normal values of estradiol, progesterone, LH and FSH during all estrus cycle stages. In this work, we observed that the postovariectomy rise of LH and FSH was significantly lower in the IPL nude females. We studied also the effect of acute (1 injection of 25 micrograms/rat E2Bz) or long-term (E2Bz capsule for 8 days) estradiol benzoate (E2Bz) treatment, with or without progesterone injection (5 mg/rat) in ovariectomized (OVX) IPL and normal females. The sensitivity of gonadotropins to E2 negative feedback is decreased in the IPL nude rats, result in agreement with previous reports and which could be linked to both hypoprolactinemia and decreased beta-endorphin observed in the IPL nude rat. The responsiveness of LH to LHRH was also tested in OVX and OVX + E2Bz or OVX + E2B + P treated. In OVX females responsiveness of LH to LHRH was similar in IPL nude to that of normal females. However, LH responsiveness in acute and long-term steroid-treated OVX IPL nude was significantly depressed. Since the mechanism whereby PRL interacts with steroids to modify gonadotropin secretion is still unexplained, IPL nude rat could be a good model to study it.     

Evaluation of the impedance technique for cryosurgery in a theoretical model of the head

Bioimpedance is a noninvasive technique that produces information on the electrical characteristics of tissue inside the body from currents injected and electrical potentials measured on the surface of the body. Because freezing causes a large increase in tissue electrical impedance we thought that it may also cause significant changes in the surface electrical potential making the bioimpedance technique suitable for noninvasive monitoring and imaging of cryosurgery. To evaluate the feasibility of the bioimpedance technique in cryosurgery we examined, as a case study, a theoretical model for the electrical potentials during brain cryosurgery. A three-dimensional spherical model was used to calculate the change in the electrical potential distribution in the head as a function of the current source location and the size of the frozen lesion in the brain. The numerical calculations were executed using the finite volume method and the iterative successive over relaxation method. The results demonstrate that, indeed, freezing inside the head produces measurable changes in the electrical potential on the outer surface-the scalp.     

BMP-2 regulates cardiomyocyte contractility in a phosphatidylinositol 3 kinase-dependent manner

Bone morphogenetic protein-2 (BMP-2) regulates development of heart during vertebrate embryogenesis. In vitro BMP-2 induces differentiation of precardiac cells into mature cardiomyocytes by inducing the expression of cardiac-specific genes. However, the role of BMP-2 and its signaling in other cardiac functions have not been studied. We examined the action of phosphatidylinositol (PI) 3 kinase in isolated adult rat cardiomyocytes. Incubation of rat ventricular cardiomyocytes with BMP-2 increased the PI 3 kinase activity. Ly294002, a pharmacological inhibitor of PI 3 kinase, blocked BMP-2-induced PI 3 kinase activity completely. To investigate the contractility of isolated cardiomyocytes, fractional shortening was examined. BMP-2 significantly increased the percent fractional shortening of the cardiomyocytes. Inhibition of PI 3 kinase activity completely abolished this action of BMP-2. These data indicate that PI 3 kinase regulates BMP-2-induced myocyte contractility. To further confirm this observation, we used adenovirus-mediated gene transfer to express a constitutively active myristoylated catalytic subunit of PI 3 kinase in rat cardiomyocytes. Infection of cardiomyocytes with the adenovirus vector increased the expression of constitutively active PI 3 kinase within 24 h. Expression of constitutively active PI 3 kinase significantly increased cardiomyocyte contractility. Together, these data show for the first time that the growth and differentiation factor, BMP-2, stimulates cardiomyocyte contractility. Also we provide the first evidence that BMP-2-induced PI 3 kinase activity regulates this cardiomyocyte function.     

Correlation between bacterial haemoglobin gene (vgb) and aeration: their effect on the growth and alpha-amylase activity in transformed Enterobacter aerogenes

AIMS: To evaluate the effects of bacterial haemoglobin on bacterial growth and alpha-amylase formation under different aeration conditions. METHODS AND RESULTS: Enterobacter aerogenes was transformed with the gene encoding Vitreoscilla (bacterial) haemoglobin, vgb. The growth kinetics and ability to synthesize alpha-amylase enzyme were investigated in this transformed Enterobacter strain as well as in two other Enterobacter control strains that do not harbour the vgb gene. Such comparison was made under variable aeration conditions, using the agitation rate as a measure of aeration. The expression of bacterial haemoglobin-supported cell growth determined as O.D.600 and cell viability in addition to the alpha-amylase production. These positive effects of bacterial haemoglobin were observed under both low and high aerations, but at different extents. CONCLUSIONS: In addition to improving cell growth under low aeration, the bacterial haemoglobin is able to promote bacterial cell tolerance during exposure to high oxygen tension. SIGNIFICANCE AND IMPACT OF THE STUDY: The expression of bacterial haemoglobin is advantageous in reducing the burden of certain toxic conditions such as high oxygen levels. It may have the same impact on some environmental toxic substances. This, haemoglobin biotechnology can be extended to induce enzymes of pollutants degradation or production of some useful industrial substances.     

Mechanism and stereochemistry of enzymic reactions involved in porphyrin biosynthesis.

5-Aminolaevulinate synthetase cataylses the condensation of glycine and succinyl-CoA to give 5-aminolaevulinic acid. At least two broad pathways may be considered for the initial C--C bond forming step in the reaction. In pathway A the Schiff base of glycine and enzyme bound pyridoxal phosphate (a) undergoes decarboxylation to give the carbanion (b) which then condenses with succinyl-CoA with the retention of both the original C2 hydrogen atoms of glycine. In pathway B, loss of a C2 hydrogen atom gives another type of carbanion (c) that reacts with succinyl-CoA. Evidence has been presented to show that the initial C--C bond forming event occurs via pathway B which involves the removal of the pro R hydrogen atom of glycine. Subsequent mechanistic and stereochemical events occurring at the carbon atom destined to become C5 of 5-aminolaevulinate have also been delineated.(Carticle) Several mechanistic alternatices for the formation of the two vinyl groups of haem from the propionate residues of the precursor, coproporphyrinogen III, have been examined. (see article). It is shown that during the biosynthesis both the hydrogen atoms resident at the alpha positions of the propionate side chains remain undisturbed thus eliminating mechanisms which predict the involvement of acrylic acid intermediates. Biosynthetic experiments performed with precursors containing stereospecific labels have shown that the two vinyl groups of haem are formed through the loss of pro S hydrogen atoms from the beta-positions of the propionate side chains. In the light of these results, three related mechanisms for the conversion, propionate leads to vinyl, have been considered. In order to study the mechanism of porphyrinogen carboxy-lyase reaction, stereo-specifically deuterated, tritiated-succinate was incorporated into the acetate residues of uroporphyrinogen III which on decarboxylation generated asymmetric methyl groups in coproporphyrinogen III and then in haem. Degradation of the latter yielded chiral acetate deriving from C and D rings of haem. Configurational analysis of this derivate acetate shows that the carboxy-lyase reaction proceeds with a retention of configuration.