[en] The immune response during infectious diseases leads to a rise in antibody titre to the various different antigenic determinants of the causative organism. The response is further complicated by the fact that it is relatively unusual for one individual to respond to all antigenic components of an organism. Demonstration of the specific immune response of an infected host by serological tests is often hampered by the broad cross-reactivity between several bacterial antigens. The authors report on a serodiagnostic application of murine monoclonal antibodies (MAB), specific for a human pathogen, M. tuberculosis by a technique which is applicable in principle to the serodiagnosis of many other infectious diseases. The serum diagnostic test is based on the competitive inhibition by human sera of the binding of ¹²⁵I-labelled murine monoclonal antibodies to M. tuberculosis-coated polyvinyl plates. Five monoclonal antibodies binding to distinct antigenic determinants of the organism were used as structural probes which conferred their stringent combining site specificities to the polyclonal mixture of antibodies from patients' sera. When compared with healthy controls, increased titres of inhibitory antibodies were found in about 70% of patients with active tuberculosis. The diagnostic value of the individual monoclonal antibodies as well as the benefit from the use of multiple specificity probes has been qualified

[en] Cadmium can replace zinc and magnesium in alkaline phosphatase from Escherichia coli, which permits the characterization of the catalytically important metal-binding sites by ¹¹³Cd NMR. At pH 6.5, in the absence of phosphate, two equivalents of cadmium are bound in identical sites (A), one in each monomer. Either raising the pH or phosphorylation of Cd₂AP (AP is apoalkaline phosphatase) results in migration of Cd(II) from the site A in one monomer to the opposite monomer to occupy a second site (B) adjacent to the A site in the first monomer, a site stabilized by phosphorylation or high pH. At pH 6.5 in the presence of phosphate, the ¹¹³Cd NMR spectrum of Cd₆AP consists of three narrow resonances from three pairs of fully occupied sites, A, B, and C. The resonances at 153 and 70 ppm represent two metal sites (A and B) 3.9 angstrom apart at each active center and adjacent to the serine phosphorylated during turnover. At this pH the enzyme exists almost exclusively as the covalent phosphoseryl form E-P with a ³¹P resonance at approx. 9 ppm. As the pH is raised a ³¹P signal from the noncovalent E.P complex appears at approx. 13ppm. This is reflected in the ¹¹³Cd spectrum by a split of both the A- and B-site resonances into pairs, a set at 137 and 65 ppm for E.P, and 153 and 70 ppm for the E-P species. ¹¹³Cd-O-³¹P coupling of 30 Hz on the ³¹P resonance of E.P shows the noncovalently bound phosphate to be coordinated to one but not both metal ions at each active site

[en] Conflicting evidence exists as to whether the gluconeogenetic process is active in the late gestation fetal lamb. In vitro evidence based on measurements of enzyme activity and substrate flux into glucose indicates that the capacity for gluconeogenesis exists in fetal liver. The in vivo conversion of [¹⁴C]lactate and [¹⁴C]alanine into glucose in the lamb fetus has been demonstrated. Lactate and alanine account for 49 and 2.3% of the fetal glucose pool, respectively. Although gluconeogenesis can occur in the fetal lamb, alterations in net rates of umbilical uptake of glucose or lactate, fetal blood glucose concentrations, fetal or maternal glucose replacement rates, or maternal nutrition may alter the observed rates of fetal gluconeogenesis

[en] Nuclear magnetic resonance (NMR) of metalloproteins is briefly discussed, and a summary of the properties of several NMR active nuclei is given. In addition, the use of these NMR properties in solving biochemical problems is presented

[en] ¹¹³Cd NMR has been used to determine the structures of the multiple metal-binding sites in the two major isoproteins of metallothionein from mammalian livers (rabbits, calf, and human) and from Scylla serrata hepatopancreas. The native protein isolated from the livers of rabbits that had been subjected to repeated injections of ¹¹³CdCl₂ contains an appreciable amount of Zn²⁺ in addition to ¹¹³Cd²⁺, ranging from 2 to 3 g-atoms of a total metal content of 7 g-atoms/mol of protein. The native Zn²⁺ can be replaced in vitro with ¹¹³Cd²⁺ to give a ¹¹³Cd NMR spectrum consisting of eight distinct multiplets in the chemical shift range of 604-670 ppm. The multiplet structure is due to ¹¹³Cd- ¹¹³Cd scalar coupling arising from two-bond interactions between ¹¹³Cd²⁺ ions linked to one another by bridging cysteine thiolate ligands. Analysis of the ¹¹³Cd spectra by selective homonuclear ¹¹³Cd decoupling techniques showed that both isoproteins of rabbit liver metallothionein contain two separate metal clusters, one containing four Cd²⁺ ions (cluster A) and the other containing three (cluster A) and the other containing three (cluster B)

[en] Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized (¹²⁵I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis

[en] Our recent studies with tritium and ¹⁴C-labeled lactate and alanine in starved rats revealed that the sites of tracer administration and sampling have a profound effect on the kinetics of the specific activity curves and the calculation of metabolic parameters. The importance of the sites of tracer administration and sampling for the experimental design and interpretation of tracer data in vivo is discussed

[en] High-dose fractionated total lymphoid irradiation (TLI) is a safe, routine regimen used to treat patients with lymphoid malignancies. Although few side effects are associated with the regimen, a profound suppression of cell-mediated immunity is observed for several years after therapy, as judged by both in vivo and in vitro assays. A profound immunosuppression has also been observed in mice and rats given TLI. Recently, we have achieved similar results using TLI in nonmatched bone marrow transplantation in outbred dogs. The experimental work in animals and underlying cellular mechanisms are reviewed here

[en] Methods designed for estimation of the synthesis of plasma glucose are based on the transfer of ¹⁴C atoms from a selected precursor (substrate) such as lactate or alanine. This approach was shown to lead to an underestimation of the true synthesis of glucose because of the metabolic exchange of ¹⁴C atoms for ¹²C atoms in the hepatic oxaloacetate pool. From the incorporation of ¹⁴C atoms from intravenously infused [2-¹⁴C]acetate into plasma glucose, the extent of the metabolic exchange has been estimated. In normal dogs, metabolic exchange leads to an under-estimation of plasma glucose synthesis from plasma lactate or alanine by a factor of 2.2 +/- 0.07, i.e., by 55%. In insulin-deprived diabetic dogs, the factor was found to be 1.8 +/- 0.05. In long-term fasted dogs, the factor may be higher than in the postabsorptive state, whereas treatment with methylprednisolone has no effect. The assumptions and sources of possible errors in the estimation of the extent of metabolic exchange are reviewed

[en] Our recent studies with tritium and ¹⁴C-labeled lactate and alanine in starved rats revealed that the sites of tracer administration and sampling have a profound effect on the kinetics of the specific activity curves and the calculation of metabolic parameters. The importance of the sites of tracer administration and sampling for the experimental design and interpretation of tracer data in vivo is discussed