Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Synthesis of rhodanese in Hep 3B cells

Summary The biosynthesis of rhodanese was studied in human hepatoma cell lines by immunoblotting and pulselabeling experiments using polyclonal antibodies raised against the bovine liver enzyme. Rhodanese, partially purified from human liver, showed an apparent molecular weight of 33,000 daltons, coincident with that of rhodanese from Hep 3B cells. After pulse labeling of Hep 3B cells both at 37°C and 25°C, rhodanese in the cytosol fraction exhibited the same molecular weight as the enzyme isolated from the particulate fraction containing mitochondria. Moreover, newly synthesized rhodanese from total Hep 3B RNA translation products showed the same electrophoretic mobility as rhodanese from Hep 3B cells. These results suggest that rhodanese, unlike most mitochondrial proteins, is not synthesized as a higher molecular weight precursor.     

Biosynthesis of coenzyme F420 and methanopterin in Methanobacterium thermoautotrophicum

Growing cultures of Methanobacterium thermoautotrophicum were supplemented with [U-¹⁴C]adenosine or [1-¹⁴C]adenosine. 7,8-Didemethyl-8-hydroxy-5-deazariboflavin (factor F₀) and 7-methylpterin were isolated from the culture medium. Hydrolysis of cellular RNA yielded purine and pyrimidine nucleotides. The ribose side chain of proffered adenosine is efficiently incorporated into cellular adenosine and guanosine nucleotide pools but not into pyrimidine nucleotides. Thus, M. thermoautotrophicum can utilize exogenous adenosine by direct phosphorylation without hydrolysis of the glycosidic bond, and AMP can be efficiently converted to GMP. Factor F₀ and 7-methylpterin had approximately the same specific activities as the purine nucleotides. It follows that the ribityl side chain of factor F₀ is derived from the ribose side chain of a nucleotide precursor by reduction. The pyrazine ring of methanopterin is formed by ring expansion involving the ribose side chain of the precursor, GTP.Abbreviations Factor F₀ 8-hydroxy-6,7-didemethyl-5-deazariboflavin - APRT adenine phosphoribosyltransferase - GPRT guanine phosphoribosyltransferase - PRPP phosphoribosylpyrophosphate - HPLC high performance liquid chromatography     

The myelodysplastic syndrome: prognostic factors in relation to the "in vitro" clonogenic assay.

The object of this study was to determine whether the "in vitro" parameters of medullary and blood granulopoiesis in patients with MDS, furnish information of either prognostic or diagnostic value. This study covered 94 patients with MDS. All patients were studied at the onset of disease. In order to identify the factors related to patients' survival, Cox Multiple Regression analysis was performed by the BMD P2L program. When analyzing by means of actuarial curves the survival probability of patients with benign development versus those of malignant development (those who developed ANLL), the significance between both groups was p = 0.0001. Different variables of patients included in this study were analyzed and all showed great significances. Fab: p = 0.0022, disease evolution: p = 0.0001 and presence of blastic aggregates: p = 0.0011. Cox's regression analysis revealed that the only predictable survival variable is the presence of blastic colonies and/or clusters. Accordingly, two groups were constructed: favourable and unfavourable. In the favourable group, 40% of the patients belonged to the RA group, while in the unfavourable group, 55% belonged to the RAEB group. This study shows the validity of the elaboration of prognostic groups in MDS according to the presence of blastic colonies and/or clusters in CFUGM medullary and/or peripheral cultures. The "in vitro" myeloid progenitors culture techniques may therefore be advantageously applied in these disorders for formulating a diagnosis and predicting the patient's short term evolution.     

Structure and function of hemoglobin in antarctic fishes and evolutionary implications

Summary The hematological features of cold-adapted, red-blooded Antarctic teleosts has prompted this study on the relationship between hemoglobin molecular structure and oxygen-binding properties. The hemolysates from 21 species of 5 families contained one component (Hb 1), often accompanied by an additional, minor one (Hb 2, 5%–10% of total). On the other hand, 3 species of Zoarcidae, a non-endemic family, had 4–5 components. All purified hemoglobins from the former group, but only 1–2 of the 4–5 hemoglobins of Zoarcidae, showed a strong Root effect (pH regulation of oxygen binding). Globins from each hemoglobin have been purified and characterised with respect to molecular structure in several species. The similarity between the complete amino acid sequence of one -chain and those of non-Antarctic -chains is lower than that among the latter sequences, suggesting independent pathways of evolution.Presented at the 5th SCAR Symposium on Antarctic Biology, Hobart, Australia (August 29th-September 3rd, 1988)     

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.     

Angiotensin converting enzyme inhibitor potentiated the vasorelaxant response of atrial natriuretic peptide in toad aortic rings

1. The vasorelaxant effect of synthetic atrial natriuretic peptide (ANP) on the vascular response to angiotensin II (A II) and norepinephrine (NE) in aortic rings from Bufo arenarum toad was studied. 2. Pretreatment with ANP partially inhibited the vascular response to A II and NE. 3. Angiotensin converting enzyme inhibitor (ACEI) treatment partially inhibited the contractile response of angiotensin I (A I) and did not affect the A II response. 4. The inhibitory effect of ANP on vascular response to A II and NE were potentiated by pretreatment with ACEI. 5. Results suggest that the angiotensin converting enzyme present in the vascular wall from Bufo arenarum toad may be involved in the metabolism of ANP.     

Importance of some factors on the dimorphism of Candida albicans

The passage between the yeast and mycelial forms of Candida albicans B 311-10 was studied by using the minimal syntehtic medium of Shepherd et al. [19] modified without biotin and with low glucose concentrations. It was observed that biotin, aminoacids and particularly pH are not important factors in the dimorphism of C. albicans. The only factor of notable importance in the passage of yeast form to mycelial form in C. albicans was glucose concentration.     

Thermoregulatory adjustments during continuous heat exposure

Body temperature regulation was studied in 6 male subjects during an acclimation procedure involving uninterrupted heat exposure for 5 successive days and nights in a hot dry environment (ambient temperature = 35 degrees C, dew-point temperature = 7 degrees C; air velocity = 0.2 m.s-1). Data were obtained at rest and during exercise (relative mechanical workload = 35% VO2max). At rest, hourly measurements were made of oesophageal and 4 local skin temperatures, to allow the calculation of mean skin temperature, and of body motility and heart rate. During the working periods these measurements were made at 5 min intervals. Hourly whole-body weight loss was measured at rest on a sensitive platform scale while in the working condition just before starting and immediately after completing the bicycle exercise. The results show that, in both exercise and at rest, the successive heat exposures increased the sweat gland output during the first 3 days. Afterwards, sweat rate decreased without any corresponding change in body temperature. For the fixed workload, the sweat rate decline was associated with a decrease in circulatory strain. Adjustments in both sweating and circulatory mechanisms occur in the first 3 days of continuous heat exposure. The overall sweat rate decline could involve a redistribution of the regional sweating rates which enhances the sweat gland activities of skin areas with maximal evaporative efficiencies.