Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli

Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.     

Specificity of the heat-stable protein inhibitor of the branched-chain alpha-keto acid dehydrogenase phosphatase

A potent, heat-stable protein inhibitor of branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase has been identified and purified to near homogeneity from bovine kidney mitochondria (Damuni, Z., Humphreys, J. S., and Reed, L. J., Proc. Natl. Acad. Sci. U.S.A., in press). This protein is a noncompetitive inhibitor of BCKDH phosphatase, with a Ki about 0.13 nM. By contrast, this protein inhibitor did not affect the activity of the cytosolic protein phosphatase-1 and phosphatase-2A or the mitochondrial pyruvate dehydrogenase (PDH) phosphatase at concentrations up to 10 nM. The cytosolic protein phosphatase inhibitor-1 and inhibitor-2 had no effect on the activity of BCKDH phosphatase or PDH phosphatase at concentrations up to 50 and 300 nM respectively. These results, together with previous evidence, demonstrate that BCKDH phosphatase and its inhibitor protein are distinct from the cytosolic protein phosphatase-1 and phosphatase-2A and from protein phosphatase inhibitor-1 and inhibitor-2, respectively.     

Geographical variability of alpha-1-antitrypsin alleles in China: A study on six Chinese populations

Summary Alpha-1-antitrypsin phenotypes of six Chinese Han populations (20°–45°N. latitude) were determined. The frequency of allele M2 increases from North to South China. The north-south change of M2 appears to be mainly at the expense of alleles M1 and M3. Geographical variability of other variants was also observed. In the northern populations, the most common variants are M4 and Etokyo; in the southern populations, Pweishi, a variant which can not be distinguished electrophoretically from Pyasugi. These results form a distribution pattern of alpha-1-antitrypsin alleles in China.     

Sulfmyoglobin. Resonance Raman spectroscopic evidence for an iron-chlorin prosthetic group

The green heme protein sulfmyoglobin (SMb) has been suggested to contain a sulfur-modified iron chlorin prosthetic group. To evaluate this hypothesis, we have obtained high-frequency (greater than 1000 cm-1) resonance Raman spectra of both oxidized and reduced SMb with 457.9-, 488.0-, 514.5-, 568.2-, and 647.1-nm excitation. The SMb spectra are compared to those of native met- and deoxymyoglobin (Mb). Vibrational frequencies for SMb are generally similar to those of Mb, suggesting a high-spin state for both the Fe(III) and Fe(II) SMb species, as is typical of native Mb. However, major differences between SMb and Mb occur both for patterns of relative spectral intensities and for depolarization ratios. In particular, all B1g-depolarized porphyrin modes in the Mb spectra have become polarized, totally symmetric vibrational modes in the SMb spectra. These contrasts reflect a dramatic lowering of the effective symmetry for the SMb prosthetic group. Several new bands are observed in SMb spectra that are not present in spectra of either native Mb or iron protoporphyrin IX complexes. The observation of additional polarized bands flanking the oxidation state marker, V4, is of particular interest. In a parallel study, we compared the resonance Raman spectral properties of iron protoporphyrin IX-derived chlorins and metallo-octaethylchlorins with those of the analogous porphyrins: the chlorin spectra exhibited altered intensity patterns, an increased number of totally symmetric (polarized) vibrational bands, and several new vibrational bands, including one or two in the region of the oxidation state marker, V4. Thus, the resonance Raman spectral characteristics of SMb and metallo-chlorins are complementary and strongly support a chlorin prosthetic group for SMb. Furthermore, they establish testable criteria for investigating the prosthetic group structures of other green heme proteins by resonance Raman spectroscopy.     

Oxytocin potentiates the ACTH-releasing activity of CRF(41) but not vasopressin

The effects of CRF(41), oxytocin (OT), and arginine vasopressin (AVP) on ACTH secretion were studied alone and in combination in an in vitro system of superfused rat hemipituitaries. CRF(41) (10(-9)M) and AVP (10(-8)M) alone produced a significant increase in ACTH secretion while OT (10(-8)M) alone had no effect. However the same concentration of OT markedly potentiated the ACTH response to CRF(41) while having no effect on the ACTH response to AVP. The data support a physiologic role for OT in the regulation of ACTH secretion.     

克山病是一种“心肌线粒体病(Mitochondrial Cardiomyopathy)”

亚急克病人心肌线粒体内膜电子传递链的琥珀酸氧化酶系,琥珀酸脱氢酶和细胞色素氧化酶活性明显低于对照。H~ -ATP酶的活性及其对寡霉素的敏感性都明显下降。ATP能量化后线粒体膜电位的变化也比对照明显降低。膜脂流动性低于对照。亚急克病人心肌线粒体内观察到较多的电子致密无定形物质,经电镜X射线微区等方法分析,认为这些物质不是Ca_3(PO_4)_2,而可能是一种蛋白质凝聚物。此外,心肌线粒体的硒含量远低于对照,而Ca含量明显高于对照。上述结果都反映亚急克病人心肌线粒体明显损伤。根据克山病患者心肌细胞线粒体结构与功能方面呈现的如此广泛与明显的异常,可将克山病称为“心肌线粒体病(Mitochondrial Cardiomyopathy)”。     

High-performance liquid chromatography of uroporphyrinogen and coproporphyrinogen isomers with amperometric detection.

A reversed-phase h.p.l.c. system, with an ODS-Hypersil column with acetonitrile or methanol in ammonium acetate buffer as mobile phase, is described for the separation of uro-and copro-porphyrinogen isomers. The porphyrinogens are detected amperometrically with sensitivity comparable with that of the fluorescent detection of porphyrins. The effects of pH, buffer concentration and organic modifiers on retention and resolution were studied. The method is suitable for both analytical and preparative separation of porphyrinogens.