Gene dosage effect for fumarate hydratase (FH; E.C. 4.2.1.2) in partial trisomy 1

Summary A strain of fibroblasts partially trisomic for the larger part of 1q (Norwood and Hoehn, 1974) contains about 1.5 times as much fumarate hydratase (FH) as various control-strains. This gene dosage effect was ascertained by (1) comparative measurements of the specific activity; (2) relating the specific activity of FH to that of reference enzymes, not influenced by the chromosomal anomaly; and (3) by immunoprecipitation methods, using a rabbit antiserum against pig heart FH which cross-reacts with the human enzyme. Among others, this gene dosage effect can be demonstrated numerically by the following parameters:Ratio of the average specific activity of FH in the trisomic strain to that of the control strains: 1.53.Corresponding ratio after dividing FH activity by that of reference enzymes; for acid phosphatase: 1.58, for glutamate dehydrogenase: 1.53.Average ratio of the immunoprecipitation areas obtained upon radial immunodiffusion according to Mancini et al. (1965): 1.56.This paper is dedicated to the painter and philosopher Helmut Berninger on the occasion of his 50th birthday     

Analytical isoelectric focusing of R factor-determined beta-lactamases: correlation with plasmid compatibility.

R factor-determined beta-lactamases have been investigated by analytical isoelectric focusing. The enzymes such as those specified by the R6K and RP4 plasmids (TEM-type enzymes) are notably homogenous in biochemical tests (Hedges et al., 1974), but two subclasses can be distinguished by isoelectric focusing. Three subclasses can be distinguished among the oxacillin-hydrolyzing enzymes, in good agreement with the classification based upon biochemical characteristics (Dale and Smith, 1974). The TEM-type beta-lactamases are promiscuously distributed among plasmids of a wide variety of compatibility groups, whereas the various oxacillin-hydrolyzing enzymes show some degree of correlation with compatibility.     

Common and rare genetic variants of human red blood cell enzymes in Italy

In the present paper we report on new data of the frequency of common and rare variants in the Italian population for ADA, AK-1, 6-PGD, EsA, EsB, EsD, PGM-1, PGM-2, SOD-A, AcP, GPT, and PGI. Moreover we present a comprehensive review of the available data on the electrophoretic variants of red cell enzymes in Italians. We find a considerable degree of genetic heterogeneity between the various populations living in the Peninsula and between the population of the Peninsula and of Sardinia. We also find that the estimates of the average heterozygosity are considerably smaller for the population of Sardinia as compared to Peninsula and Sicily. Finally, we report on the occurrence of several uncommon enzyme variants, which overall frequency is very similar to previously reported estimates for North European populations (Harris et al. 1974).     

Effect of a partial trisomy in the mouse upon cellular and nuclear RNA

In a previous paper (Rake and Edwards, 1987) it was shown that the majority of the chromatin from trisomic mouse cells has nucleosomes with a smaller repeat length of DNA than the nucleosome repeat length of normal cells. Here it is shown that the RNA content of the total cell and of the nuclei is the same in all tissues studied, in both normal and trisomic cells. However, the amount per unit time or rate of RNA synthesis is depressed in the trisomic liver and brain nuclei. The depression of RNA synthesis could not be specified to the small trisomic section of the chromatin but instead must reflect the overall nuclear activity. These results, along with those of Devlinet al. (1988), indicate that the trisomic condition alters a substantial part of nuclear organization and activity, not just the small trisomic part.     

Potassium stimulated calcium dependent release of immunoreactive somatostatin from incubated rat hypothalamus

S omatostatin (somatotropin release inhibiting factor, SRIF) is present in the median eminence of the hypothalamus in high concentration (K ronheim et al., 1976), is visualized in nerve endings (H ökfelt et al., 1974) and has been found to be concentrated in the synaptosome fraction of hypothalamic homogenates (E pelbaum et al., 1977; B erelowitz et al., 1978), suggesting a true neurosecretory role. To further explore this possibility we have studied the release of immunoreactive SRIF from the incubated rat hypothalamus (B radbury et al., 1974: R otsztein et al., 1977), basally and in response to depolarising concentrations of potassium, and have assessed the calcium dependence of this release.     

Loss of Cytosolic Phosphoglucose Isomerase Affects Carbohydrate Metabolism in Leaves and Is Essential for Fertility of Arabidopsis

Carbohydrate metabolism in plants is tightly linked to photosynthesis and is essential for energy and carbon skeleton supply of the entire organism. Thus, the hexose phosphate pools of the cytosol and the chloroplast represent important metabolic resources that are maintained through action of phosphoglucose isomerase (PGI) and phosphoglucose mutase interconverting glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. Here, we investigated the impact of disrupted cytosolic PGI (cPGI) function on plant viability and metabolism. Overexpressing an artificial microRNA targeted against cPGI (amiR-cpgi) resulted in adult plants with vegetative tissue essentially free of cPGI activity. These plants displayed diminished growth compared with the wild type and accumulated excess starch in chloroplasts but maintained low sucrose content in leaves at the end of the night. Moreover, amiR-cpgi plants exhibited increased nonphotochemical chlorophyll a quenching during photosynthesis. In contrast to amiR-cpgi plants, viable transfer DNA insertion mutants disrupted in cPGI function could only be identified as heterozygous individuals. However, homozygous transfer DNA insertion mutants could be isolated among plants ectopically expressing cPGI. Intriguingly, these plants were only fertile when expression was driven by the ubiquitin10 promoter but sterile when the seed-specific unknown seed protein promoter or the Cauliflower mosaic virus 35S promoter were employed. These data show that metabolism is apparently able to compensate for missing cPGI activity in adult amiR-cpgi plants and indicate an essential function for cPGI in plant reproduction. Moreover, our data suggest a feedback regulation in amiR-cpgi plants that fine-tunes cytosolic sucrose metabolism with plastidic starch turnover.Starch and Suc turnover are major pathways of primary metabolism in all higher plants. As such, they are essential for carbohydrate storage and the energy supply of sink tissues and as building blocks for amino acid, fatty acid, or cell wall biosynthesis (Stitt and Zeeman, 2012).A core reaction in both starch and Suc biosynthesis is the reversible interconversion of the hexose phosphate pool metabolites Fru 6-phosphate (Fru6P) and Glc 6-phosphate (Glc6P), which is mediated by phosphoglucose isomerase (PGI). Arabidopsis (Arabidopsis thaliana) contains two isoforms of PGI, one in the plastids and one in the cytosol (Caspar et al., 1985).During the light period, the plastid isoform of PGI (PGI1) is involved in starch biosynthesis by generating Glc6P from the primary photosynthetic product Fru6P. Glc6P is further converted to Glc 1-phosphate (Glc1P) and ADP-glucose via action of phosphoglucomutase (PGM) and ADP-glucose pyrophosphorylase (AGPase), respectively (Stitt and Zeeman, 2012). Finally, transfer of the glucosyl moiety of ADP-glucose to the growing carbohydrate chain of starch is mediated by starch synthases. Any of the enzymatic reactions of this linear pathway is essential for starch synthesis, as illustrated by the virtual absence of transitory starch in chloroplasts of mutant plant lines with impaired function of PGI1 (Yu et al., 2000; Kunz et al., 2010), PGM (Caspar et al., 1985; Kofler et al., 2000), or AGPase (Lin et al., 1988). Interestingly, in a few specific cell types, e.g. leaf guard cells and root columella cells, loss of PGI1 activity can be bypassed by the presence of the plastid Glc6P/phosphate translocator GPT1 (Niewiadomski et al., 2005; Kunz et al., 2010).The cytosolic isoform of PGI (cPGI) is involved in anabolism and catabolism of Suc, the major transport form of carbohydrates in plants. Glc6P and Fru6P interconversion is necessary for both Suc synthesis during the day and during the night. During the day, Suc synthesis in source leaves is fueled mainly by triose phosphates exported from chloroplasts that are eventually converted to Fru6P in the cytosol. However, Fru6P is only one substrate for the Suc-generating enzyme Suc phosphate synthase. The second substrate, UDP-glucose, is synthesized from Fru6P via Glc6P and Glc1P by the cytosolic isoenzymes of PGI1 and PGM as well as UDP-glucose pyrophosphorylase.Because Suc is the major long-distance carbon transport form, its synthesis has to continue throughout the night to supply energy and carbohydrates to all tissues. The nocturnal synthesis of Suc is dependent on breakdown and mobilization of transitory starch from chloroplasts (Zeeman et al., 2007) via export of maltose and Glc (Weber et al., 2000; Niittylä et al., 2004; Weise et al., 2004; Cho et al., 2011). Exported maltose is temporarily integrated into cytosolic heteroglycans (Fettke et al., 2005) mediated by disproportionating enzyme2 (DPE2; Chia et al., 2004; Lu and Sharkey, 2004) yielding Glc and a heteroglycan molecule elongated by an α1-4-bound glucosyl residue. Cytosolic Glc can directly be phosphorylated to Glc6P by the action of hexokinase, while temporarily stored Glc in heteroglycans is released as Glc1P mediated by cytosolic glucan phosphorylase2 (PHS2; Fettke et al., 2004; Lu et al., 2006). Both Glc6P and Glc1P can then be converted to UDP-glucose as during the day.Generation of Fru6P, the second substrate for Suc synthesis, can proceed only to a limited extent from triose phosphates during the night. This limitation is caused mainly by the nocturnal inactivation of Fru 1,6-bisphosphatase (Cséke et al., 1982; Stitt, 1990), a key enzyme in Suc biosynthesis during the day. Hence, in contrast to the situation in the light, cPGI activity is now crucial for providing Fru6P from Glc6P.On the catabolic side, degradation of Suc into its monosaccharides in sink tissues yields both Glc6P and Fru6P, of which only Fru6P can be utilized in glycolytic degradation. Therefore, cPGI is also required for Glc6P conversion to Fru6P in glycolysis, which, in combination with respiration, is the major path of energy production in heterotrophic tissues.Impairment or loss of function of enzymes contributing to the cytosolic hexose phosphate pool has recently been investigated for the Glc1P-forming enzyme PGM (Egli et al., 2010). The Arabidopsis genome encodes three PGM isoforms, with PGM1 localized to plastids and PGM2 and PGM3 localized to the cytosol (Caspar et al., 1985; Egli et al., 2010). Analyses of transfer DNA (T-DNA) mutants showed that homozygous pgm2/pgm3 double mutants were nonviable because of impaired gametophyte development. However, pgm2 and pgm3 single mutants grew like ecotype Columbia (Col-0) wild-type plants, indicating overlapping functions of PGM2 and PGM3 (Egli et al., 2010).By contrast, cPGI is encoded only by a single locus in Arabidopsis (Kawabe et al., 2000). Higher plant mutants reduced in cPGI activity have so far been characterized only in ethyl methanesulfonate-mutagenized Clarkia xantiana (Jones et al., 1986a; Kruckeberg et al., 1989; Neuhaus et al., 1989). The C. xantiana genome encodes for two isoenzymes of cPGI, and homozygous point mutations in each individual cPGI led to significant decrease in cPGI enzyme activity, which was further reduced to a residual activity of 18% in cpgi2/cpgi3 double mutants, where the cPGI3 locus was heterozygous for the mutation (Jones et al., 1986a; Kruckeberg et al., 1989). Detailed physiological analyses of these mutants indicated a negative impact on Suc biosynthesis and elevated starch levels when cPGI activity was decreased at least 3- to 5-fold (Kruckeberg et al., 1989).The physiological impact of decreased or even absent cPGI activity has not been characterized in the genetic model organism Arabidopsis. Here, we show that homozygous T-DNA insertion mutants in the cPGI locus are nonviable and present data from analyses of mature Arabidopsis plants constitutively expressing artificial microRNAs (amiRNAs) targeted against cPGI. These mutants reveal altered photosynthesis, a strong impact on nocturnal leaf starch degradation, and impaired Suc metabolism.     

Reduced oocyte numbers in tertiary trisomic mice with male sterility

Oocyte counts carried out in 3- to 5-day-old tertiary trisomic Ts(5(12))31H mice revealed a mean reduction of 71% in the number of oocytes as compared with that of normal littermates. The pool of small oocytes was reduced by 75%, and the number of growing oocytes by 8%. The sperm count of the trisomic males was less than 1% of normal, with most spermatozoa being abnormal (Beechey et al., 1980). These results indicate that the presence of the extra 5(12) chromosome, which causes male sterility, also has a marked effect on oogenesis. Possible reasons for the difference in severity of the gametogenic impairment in males and females are discussed.     

Evaluation of mutagenic effect of the fungicide fenaminosulf in Drosophila melanogaster

Fenaminosulf (p-dimethylaminobenzenediazo sodium sulfonate, CAS registry No. 140-56-7) which is an active ingredient in several commercial fungicides was reported to be mutagenic in Salmonella typhimurium (McCann et al., 1975), Bacillus subtilis (Kada et al., 1974) and shown to cause chromosome aberrations in plants (Zutshi and Kaul, 1975). Since fenaminosulf has structural similarity to the potent carcinogen, butter yellow (p-dimethylaminoazobenzene, CAS registry No. 60-11-7), the present studies were undertaken to evaluate the mutagenic potential of this fungicide in Drosophila melanogaster. Fenaminosulf administered at 10 mg/100 ml food medium failed to induce sex-linked recessive mutations in Drosophila. Since Drosophila has drug-metabolizing enzymes similar to those of mammals (Vogel, 1975), it is suggested that the lack of mutagenic activity of fenaminosulf could be due to the conversion of fenaminosulf to non-mutagenic derivatives in Drosophila.     

Giemsa banding patterns in chronic lymphocytic leukaemia.

The banding patterns of chromosomes from 20 patients with chronic lymphocytic leukaemia (C.L.L.) have been analyzed. 97 of 100 metaphases examined had a normal banding pattern. The 3 remaining metaphases, all from one patient had bands similar to those seen after aging. It is concluded that the chromosomes in C.L.L. have normal banding patterns. The majority of cytogenetic studies in chronic lymphocytic leukaemia have reported normal chromosomes (Fitzgerald and Adams 1965; Oppenheim et al., 1965; Lawler et al., 1968). An inherited abnormality of G group chromosome (No. 22) has been reported in a family, three members of whom developed C.L.L. (Fitzgerald and Hamer, 1969), but further investigations of cases of familial leukaemia failed to reveal a similar abnormality (Fitzgerald et. al., 1966). The development of new techniques which allow the positive identification of individual chromosomes (Caspersson et al., 1969; Dutrillaux and Lejeune, 1971; Sumner et al., 1971; Seabright, 1971), has revolutionised human cutogenetics and revealed additional information regarding chromosome abnormalities and leukaemia (Rowley, 1973; Lobb et al., 1972; Milligan and Garson, 1974). The purpose of this investigation was to determine whether the chromosomes in C.L.L. have normal banding patterns.     

SOD-A and chromosome 21

Summary A balanced maternal chromosome translocation (9p24;21q214) resulted in two offspring with unbalanced karyotypes. One of these, a girl trisomic for both segment 9pter9p24 and segment 21pter21q214, was found to have a SOD-A activity not significantly different from those found in a group of five cases with trisomy 21. However, clinical evaluation of this girl revealed no symptoms of the Down syndrome. These findings suggest that, providing the gene dosage theory is correct, the gene for SOD-A is probably localized on chromosome 21 proximal to, or in, band q21.     

Involvement of glutathione and glutathione-related enzymes in the protection of normal and trisomic human fibroblasts against daunorubicin

We measured the glutathione content, and the activity of glutathione-related enzymes and DT-diaphorase in cultured normal (cell line: S-126) and trisomic (cell lines: S-158, S-240) human fibroblasts exposed to daunorubicin (DNR). Determination of reduced and total glutathione levels, and measurement of the activity of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and DT-diaphorase were performed spectrophotometrically. Human fibroblasts were exposed to 4 microm DNR for 2 h, and the cells placed in drug-free medium for 6, 12, 24, 48, and 72 h. Cellular levels of GSH and total glutathione decreased following exposure to DNR. However, the ratio of GSH to total glutathione returned to control levels only in trisomic cells. These changes were concomitant with increasing glutathione-S-transferase and glutathione reductase activities. DNR also significantly increased the activity of Se-independent peroxidase and DT-diaphorase in trisomic fibroblasts. Marked increases in the activity of Se-dependent peroxidase and DT-diaphorase alone were seen in normal cells. The results provide the first evidence that DNR can induce alterations in the level of glutathione and glutathione-dependent enzymes in trisomic fibroblasts as compared to normal cells, which may provide additional protection against daunorubicin-induced oxidative stress in trisomic fibroblasts.     

Pyrimidine auxotrophy and the complementation map of the Rudimentary locus of Drosophila melanogaster

Summary The complementation pattern of twelve rudimentary mutations has been analyzed at two different levels. When analyzed on the basis of complementation for a wing abnormality the mutations can be divided into three groups, each of which is believed to affect the activity of one of the first three enzymes of pyrimidine synthesis (Norby, 1973; Jarry and Falk, 1974; Rawls and Fristrom, 1975). However, when the mutants are analyzed for complementation on the basis of a second phenotype, pyrimidine auxotrophy, the distinction between two of these three groups is not evident. The disparity in the two patterns probably reflects a different threshold of gene activity required for the detection of an auxotrophic phenotype as compared to that at which a wing abnormality is detectable.The biochemical basis of these results is interpreted in light of recent data suggesting that at least the first two enzymes of pyrimidine synthesis are contained within a single multifunctional protein complex (Soderholm et al., 1975).     

Denaturation map of polyoma DNA.

A denaturation map of polyoma DNA cleaved by Eco R1 to form linear molecules was established by electron microscopy. Partial denaturation, under the same conditions, of fragments obtained by Haemophilus influenzae restriction enzymes allowed us to align the denaturation map with the already established physical map of polyoma DNA (Griffin et al., 1974).     

Epstein-Barr virus DNA XII. A variable region of the Epstein-Barr virus genome is included in the P3HR-1 deletion.

The P3HR-1 subclone of Jijoye differs from Jijoye and from other Epstein-Barr virus (EBV)-infected cell lines in that the virus produced by P3HR-1 cultures lacks the ability to growth-transform normal B lymphocytes (Heston et al., Nature (London) 295:160-163, 1982; Miller et al., J. Virol. 18:1071-1080, 1976; Miller et al., Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974; Ragona et al., Virology 101:553-557, 1980). The P3HR-1 virus was known to be deleted for a region which encodes RNA in latently infected, growth-transformed cells (Bornkamm et al., J. Virol. 35:603-618, 1980; Heller et al., J. Virol. 38:632-648, 1981; King et al., J. Virol. 36:506-518, 1980; Raab-Traub et al., J. Virol. 27:388-398, 1978; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934, 1980). This deletion is now more precisely defined. The P3HR-1 genome contains less than 170 base pairs (and possibly none) of the 3,300-base pair U2 region of EBV DNA and is also lacking IR2 (a 123-base pair repeat which is the right boundary of U2). A surprising finding is that EBV isolates vary in part of the U2 region. Two transforming EB viruses, AG876 and Jijoye, are deleted for part of the U2 region including most or all of a fragment, HinfI-c, which encodes part of one of the three more abundant cytoplasmic polyadenylated RNAs of growth-transformed cells (King et al., J. Virol. 36:506-518, 1980; King et al., J. Virol. 38:649-660, 1981; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934).     

New vistas for P-aminobenzoate participation in the biosynthesis of dihydro folate: a tentative model of the tetrahydro folate multienzyme complex.

Figures 6a and 6b and Table 2 show the united pattern of possible pathways of THFA biosynthesis with the substrates and the enzymes involved. With substrate selection ten different individual enzyme activities can be distinguished, but A2′ is identical with A2, and their apparent molecular weight is 28,000 daltons ±7%, and similarly c1–4 are the same enzymes with an apparent molecular weight of 40,000 daltons ±5% (Tóth-Martinez et al., 1974a). The identity of these enzymes has preliminary been shown, and construction of the THFA-MEC model was partly based on these findings.So, no distinction can be made among functioning MEC-es. The different pathways, mentioned in the introductory part of this paper, can be a product of the separated study of the individual enzymic steps of DHFA (THFA) biosynthesis. By all means it is an important approach to understand the dynamics of the integrated process what we tentatively suggest in this paper for further elucidation.     

Glycogen synthesizing function of hepatocytes in rats with liver cirrhosis after treatment with chorionic gonadotropin]

Using rat liver hepatocytes, methods of cytofluorimetry (Kudryavtseva et al., 1974) and biochemistry were applied to comparative studies of the total glycogen content, including its labile (LF) and stable (SF) fractions, and activities of glucose-6-phosphatase, glycogen phosphorylase and glycogensynthetase in these. The liver hepatocytes were examined in norm, and under conditions of CCl4 poisoning of rats, both 6 months after a chronic poisoning, and 1, 3 and 6 months following poisoning cessation. All the experimentally poisoned rats were divided into two conventional groups: rats of one group received, apart from poisoning, a complex treatment with chorionic gonadotropin (CG); the other group rats received, no treatment. The material used for examination was obtained from serial functional biopsies of each experimental animal. It has been shown that under cirrhosis the content of the total glycogen in hepatocytes increased by 3 times, and that of its SF even by 9.7 times. The treatment with CG for 1 month resulted in its reducing to the norm, and 3 to 6 months treatments normalized contents of both the glycogen fractions. In the group of non-treated rats no similar changes were registered. Besides, in the cirrotic rats the activity of glucose-6-phosphatase was shown to increase by 4 times. After CG treatment it was seen to decrease by 3 times. Thus, CG may be regarded as an optimum and more effective agent for restoring abnormalities in cirrotic liver, compared to some other stimulating factors, such as hepatectomy (Kudryavtseva et al., 1996) or rich-carbohydrate diet (Kudryavtseva et al., 1998).     

Characterization of some erythrocyte G6PD variants by isoelectric focusing

Summary The existence of a microheterogeneity of glucose-6-phosphate dehydrogenase (G6PD) in human erythrocyte lysates has been previously demonstrated using isoelectric focusing (Der Kaloustian et al., 1974; Turner et al., 1975). The application of this method, modified in some aspects, to the identification of various G6PD variants led to interesting conclusions. The results reported here have been obtained from a study of four distinct molecular types: Gd(-)Mediterranean, Gd(-) Kabyle, the African Gd(+) A, and a new almost undescribed G6PD variant with severe enzyme deficiency named Gd(-) Muret.     

Unusual behaviour of SPO1 DNA with respect to restriction and modification enzymes recognizing the sequence 5''-G-G-C-C

Summary SPO1 DNA contains only 5 cleavage sites for restriction enzymes which recognize and cleave the sequence 5-G-G-C-C (HaeIII or BsuR). Fragments of SPO1 DNA cloned in E. coli to substitute 5-hydroxymethyluracil (HMU) by thymine (T) remain resistant to HaeIII indicating that this unexpectedly small number of cleavages by HaeIII is not correlated with the presence of HMU in the normal phage DNA. It was previously shown that SPO1 is neither subject to B. subtilis R restriction (Trautner et al., 1974) nor modification in vivo (Günthert et al., 1975). We now show that SPO1 DNA can however be restricted and modified in vitro.