In vitro OKT3-induced mitogenesis in selenium-deficient patients on a diet for phenylketonuria

Patients with phenylketonuria (PKU) are frequently deficient in the essential trace element selenium (Se), because of their very low protein diet. Using two approaches to investigate T-cell response to proliferative signaling, viz, mitogenesis caused by the monoclonal antibody OKT3 and the plant lectin phytohaemagglutinin (PHA), we demonstrated significantly reduced responses to optimal concentrations of OKT3 in a group of PKU patients with reduced serum Se compared with a normal group (p = 0.0005) and with a group of PKU patients whose serum Se was normal (p = 0.0023). The response of the Se-deficient group to optimal levels of PHA did not differ from that of the normal controls or from that of Se-normal PKU patients. A dose-dependent relationship between serum Se levels and mitogenic response was evident for OKT3 (r = 0.34, p = 0.0154), but not for PHA (r = -0.02, p = 0.9086). We suggest that the reduced response to OKT3 mitogenesis in Se-deficient PKU patients is possibly the consequence of impaired Se-dependent metabolic activity, which affects mitogenic signaling via the T cell antigen receptor (TCR/CD3) complex.     

Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum (pea) chlorophyl a/b binding protein gene AB80: identification of a repeated heptamer motif

Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene AB80 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF-2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essential for binding. ABF-2 is present in both green and etiolated tissue and in roots and factors analogous to ABF-2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes.     

Molecular cloning in Lactobacillus helveticus by plasmid pSA3::pVA797 co-integrate formation and conjugal transfer

Summary A gene encoding -glucanase activity from Bacillus amyloliquefaciens was subcloned in both orientations into plasmid shuttle vector pSA3. In only one orientation could a co-integrate be generated with the conjugative plasmid pVA797. The plasmid co-integrate was conjugated into Lactobacillus helveticus strain CNRZ450, where it was stably maintained without antibiotic selection and exhibited -glucanase activity. This method of introducing cloned DNA into thermophilic lactobacilli will facilitate the study of heterologous gene expression in non-transformable species. Offprint requests to: J. K. Thompson     

Functional levels of mitochondrial anion transport proteins in non-insulin-dependent diabetes mellitus

The effect of non-insulin-dependent diabetes mellitus (i.e., NIDDM; type 2 diabetes) on the levels of functional mitochondrial anion transport proteins has been determined utilizing a chemically-induced neonatal model of NIDDM. We hypothesized that moderate insulin deficiency exacerbated by the insulin resistance, which is characteristic of NIDDM, would cause changes in mitochondrial anion transporter function that were similar to those we have previously shown to occur in insulin-dependent diabetes mellitus (i.e., IDDM; type 1 diabetes) (Arch. Biochem. Biophys. 280: 181–191, 1990). Our experimental approach consisted of the extraction of the pyruvate, dicarboxylate and citrate transport proteins from the mitochondrial inner membrane with Triton X-114 using rat liver mitoplasts (prepared from diabetic and control animals) as the starting material, followed by the functional reconstitution of each transporter in a proteoliposomal system. This strategy permitted the quantification of the functional levels of these three transporters in the absence of the complications that arise when such measurements are carried out with intact mitochondria (or mitoplasts). We found that experimental NIDDM did not cause significant changes in the extractable and reconstitutable specific (and total) transport activities of the pyruvate, dicarboxylate, and citrate transporters. These results are in marked contrast to our previous findings obtained using rats with IDDM and negated our hypothesis. The present results, in combination with our earlier findings, allow us to conclude that insulin plays an important role in the regulation of mitochondrial anion transporter function. Accordingly, in this model of NIDDM, where the level of insulin is not profoundly deficient, transporter function is unaltered, whereas in IDDM, where a profound insulinopenia exists, transporter function is altered. Furthermore, the present studies suggest that in the neonatal model of NIDDM the three mitochondrial transporters investigated are neither affected by, nor are they the sites of the well documented hepatic post-receptor insulin resistance which is characteristic of this disease.     

Identification of an insertion sequence, IS1081, in Mycobacterium bovis

Abstract: An insertion sequence, IS1081, in the genome of Mycobacterium bovis has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. bovis and the element is also present in Mycobacterium tuberculosis . IS1081 is not closely related to other DNA elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus . IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.     

The gene for the alpha i1 subunit of human guanine nucleotide binding protein maps near the cystic fibrosis locus.

The gene for the alpha i1 subunit of human guanine nucleotide binding (G) protein was mapped by in situ hybridization to chromosome 7 at band q21. The regional chromosomal location of the human alpha i1 gene was confirmed using human/mouse somatic-cell hybrid lines containing portions of human chromosome 7. Because the alpha i1 gene mapped near the cystic fibrosis locus and because an abnormal G protein might be expected to contribute to the pathophysiology of this disease, the alpha i1 gene was mapped with respect to the cystic fibrosis locus as defined by the Met oncogene and anonymous DNA marker pJ3.11. The location of the alpha i1 gene proved to be distinct from that of the cystic fibrosis locus.     

Nitrate depletion in the riparian zone of a small woodland stream

Field enrichments with nitrate in two spring-fed drainage lines within the riparian zone of a small woodland stream near Toronto, Ontario showed an absence of nitrate depletion. Laboratory experiments with riparian substrates overlain with nitrate enriched solutions revealed a loss of only 5–8% of the nitrate during 48 h incubation at 12°C. However, 22–24% of the initial nitrate was depleted between 24 and 48 h when a second set of substrate cores was incubated at 20°C. Short-term (3 h) incubations of fresh substrates amended with acetylene were used to estimate in situ denitrification potentials which varied from 0.05–3.19 g N g^–1 d^–1 for organic and sandy sediments. Denitrification potentials were highly correlated with initial nitrate content of substrate samples implying that low nitrate levels in ground water and riparian substrates may be an important factor in controlling denitrification rates. The efficiency of nitrate removal in spring-fed drainage lines is also limited by short water residence times of < 1 h within the riparian zone. These data suggest that routes of ground water movement and substrate characteristics are important in determining nitrate depletion within stream riparian areas.     

The genetics and expression of an esterase locus inAnopheles gambiae

The main polymorphic system of esterase isoenzymes in adults of the G3 laboratory strain ofAnopheles gambiae consists of two to five major bands of activity per individual. The bands are designated 5S, 5F, 13, 14, and 15. In genetic crosses, the genes which coded for the bands assorted as three codominant alleles, Est A, Est B, and Est C, at a single autosomal locus. Homozygotes for the Est C allele were significantly underrepresented among backcross progeny. The developmental pattern of esterase expression was examined. Esterase gene expression in embryos was first detectable between 2 and 12 hr after oviposition. The initiation or termination of expression of some of the bands corresponded to boundaries between developmental stages. Most of the esterase fractions were not specifically localized within the tissues tested, with the exception of a series of bands which were restricted largely to adult male testes.     

Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T

The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. Upon photolysis, TnI and/or TnT formed covalent cross-links with TnC. The cross-linked TnC-TnT heterodimer obtained from the binary complex was digested into progressively smaller cross-linked peptides that were purified by HPLC and then characterized by amino acid analysis and sequencing. An initial cross-linked CNBr fraction contained the expected peptide CB9 (residues 84-135) of TnC, plus CNBr peptides spanning residues 152-230 of TnT. Results from a peptic digest of the CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilisin digest yielded a heterogeneous cross-linked fraction, which suggested that an especially high degree of cross-links was formed in the vicinity of residues 175-178 (Met-Lys-Lys-Lys) of TnT. Although this region of TnT had previously been implicated in binding, we show here for the first time that it is close to Cys-98 of TnC. In an analogous study on the binary complex of TnC and TnI [Leszyk, J., Collins, J. H., Leavis, P. C., & Tao, T. (1987) Biochemistry 26, 7042-7047], we previously showed that Cys-98 of TnC was cross-linked mainly to CN4, the "inhibitory region", of TnI.(ABSTRACT TRUNCATED AT 250 WORDS)