Measurement of the Leakage of CO2 from Bundle-Sheath Cells of Leaves during C4 Photosynthesis

During C4 photosynthesis, CO2 is released in bundle-sheath cells by decarboxylation of C4 acids and then refixed via ribulose-1,5-bisphosphate carboxylase. In this study we examined the efficiency of this process by determining the proportion of the released CO2 that diffuses back to mesophyll cells instead of being refixed. This leak of CO2 was assessed by determining the amount of 14CO2 released from leaves during a chase in high [12CO2] following a 70-s pulse in 14CO2. A computer-based analysis of the time-course curve for 14CO2 release indicated a first-order process and provided an estimate of the initial velocity of 14CO2 release from leaves. From this value and the net rate of photosynthesis determined from the 14CO2 fixed in the pulse, the CO2 leak rate from bundle-sheath cells (expressed as a percentage of the rate of CO2 production from C4 acids) could be deduced. For nine species of Gramineae representing the different subgroups of C4 plants and two NAD-malic enzyme-type dicotyledonous species, the CO2 leak ranged between 8 and 14%. However, very high CO2 leak rates (averaging about 27%) were recorded for two NADP-malic enzyme-type dicotyledonous species of Flaveria. The results are discussed in terms of the efficiency of C4 photosynthesis and observed quantum yields.     

The influence of amylose on starch granule structure

Starch granules are principally composed of the two glucose polymers amylose and amylopectin. Native starch granules typically contain around 20% amylose and 80% amylopectin. However, it is possible to breed plants that produce starch with very different amylose and amylopectin contents. At present, the precise structural roles played by these two polymers are incompletely understood. In this study, small-angle X-ray scattering techniques have been applied to investigate the effect of varying amylose content on the internal structure of maize, barley and pea starch species. The results suggest that amylose disrupts the structural order within the amylopectin crystallites.     

A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors.

Cytokines manifest their function through alteration of gene expression. However, target genes for signals from cytokine receptors are largely unknown. We therefore searched for immediate-early cytokine-responsive genes and isolated a novel gene, CIS (cytokine inducible SH2-containing protein) which is induced in hematopoietic cells by a subset of cytokines including interleukin 2 (IL2), IL3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), but not by stem cell factor, granulocyte colony-stimulating factor and IL6. The CIS message encodes a polypeptide of 257 amino acids that contains an SH2 domain of 96 amino acids in the middle. To clarify the function of CIS in cytokine signal transduction, we expressed CIS in IL3-dependent hematopoietic cell lines under the control of a steroid-inducible promoter. The CIS product stably associated with the tyrosine-phosphorylated beta chain of the IL3 receptor as well as the tyrosine-phosphorylated EPO receptor. Forced expression of CIS by steroid reduced the growth rate of these transformants, suggesting a negative role of CIS in signal transduction. CIS induction requires the membrane-proximal region of the cytoplasmic domain of the EPO receptor as well as that of the common beta chain of the IL3, IL5 and GM-CSF receptor, whereas CIS binds to the receptor that is tyrosine phosphorylated by cytokine stimulation. Thus CIS appears to be a unique regulatory molecule for cytokine signal transduction.     

N-glycans of recombinant human interferon-gamma change during batch culture of chinese hamster ovary cells

A recombinant Chinese hamster ovary (CHO) cell line making human interfron-gamma (IFN-gamma) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition to cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-gamma was achieved using matrix-assisted laser desorption mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal(2)GlcNAc(4)Man(3) which was core alephl-6 fucosylated at Asn(25) but not at Asng(97)) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures was also observed. The proportion of the dominant core glycan structure (Gal(2)GlcNAc(4)Man(3) +/- Fuc(1)) decreased by 15-26% during batch culture, with increases in the proportion of oligomannose and truncated glycans over the same time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached to Asng(97) by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recombinant therapeutic products in vivo are discussed. (c) 1995 John Wiley & Sons, Inc.     

Molecular Genetic Characterization of Six Recessive Viable Alleles of the Mouse Agouti Locus

The agouti locus on mouse chromosome 2 encodes a secreted cysteine-rich protein of 131 amino acids that acts as a molecular switch to instruct the melanocyte to make either yellow pigment (phaeomelanin) or black pigment (eumelanin). Mutations that up-regulate agouti expression are dominant to those causing decreased expression and result in yellow coat color. Other associated effects are obesity, diabetes, and increased susceptibility to tumors. To try to define important functional domains of the agouti protein, we have analyzed the molecular defects present in a series of recessive viable agouti mutations. In total, six alleles (a(mJ), a(u), a(da), a(16H), a(18H), a(e)) were examined at both the RNA and DNA level. Two of the alleles, a(16H) and a(e), result from mutations in the agouti coding region. Four alleles (a(mJ), a(u), a(18H), and a(da)) appear to represent regulatory mutations that down-regulate agouti expression. Interestingly, one of these mutations, a(18H), also appears to cause an immunological defect in the homozygous condition. This immunological defect is somewhat analogous to that observed in motheaten (me) mutant mice. Short and long-range restriction enzyme analyses of homozygous a(18H) DNA are consistent with the hypothesis that a(18H) results from a paracentric inversion where one end of the inversion maps in the 5' regulatory region of agouti and the other end in or near a gene that is required for normal immunological function. Cloning the breakpoints of this putative inversion should allow us to identify the gene that confers this interesting immunological disorder.     

The Mouse Spam1 maps to proximal Chromosome 6 and is a candidate for the sperm dysfunction in Rb(6.16)24Lub and Rb(6.15)lAld heterozygotes

We have determined the chromosomal localization of the murine gene encoding the 68-kDa sperm adhesion molecule 1, Spam1 or Ph-20. Using two independent approaches, fluorescence in situ hybridization (FISH) and interspecific backcross analysis, we show that Spam1 maps to proximal mouse Chromosome (Chr) 6. This map position is within the conserved linkage group corresponding to human Chr 7q, where the human homolog, SPAM 1, has been shown to map previously. Genetic mapping shows the gene to be very closely linked to Met, one of the most proximal loci on MMU 6. It thus places the gene near the centromere and the junction of the Rb(6.16)24Lub and Rb(6.15)1Ald translocations. The essential role of the Spam1 sperm antigen in mouse sperm-egg interactions and its gene location provide strong support for its candidacy as the gene involved in the dysfunction of mouse sperm bearing the Rb(6.16)24Lub or Rb(6.15)1Ald translocation. Received: 16 July 1996 / Accepted: 23 September 1996     

Randomised trial of octreotide for long term management of cirrhosis after variceal haemorrhage.

OBJECTIVE: To assess the efficacy of long term octreotide as adjuvant treatment to programmed endoscopic sclerotherapy after acute variceal haemorrhage in cirrhotic portal hypertension. DESIGN: Randomised clinical trial. SETTING: University hospital. SUBJECTS: 32 patients with cirrhotic portal hypertension. INTERVENTIONS: Programmed injection sclerotherapy with subcutaneous octreotide 50 micrograms twice daily for 6 months, or programmed injection sclerotherapy alone. MAIN OUTCOME MEASURES: Episodes of recurrent variceal bleeding and survival. RESULTS: Significantly fewer patients receiving combined octreotide and sclerotherapy had episodes of recurrent variceal bleeding compared with patients given sclerotherapy alone (1/16 v 7/16; P = 0.037, Fisher''s exact test), and their survival was significantly improved (P < 0.02, log rank test); this improvement was maintained for 12 months after the end of the study. Combined treatment also resulted in a sustained decrease in portal pressure (median decrease -6.0 mm Hg, interquartile range -10 to -4.75 mm Hg, P = 0.0002) compared with sclerotherapy alone (median increase 1.5 mm Hg, interquartile range 0.25 to 3.25 mm Hg), as well as a significant improvement in liver function as assessed by plasma concentrations of bilirubin, albumin, and alanine aminotransferase and by hepatocyte metabolism of aminopyrine labelled with carbon-14. CONCLUSION: Long term octreotide may be a valuable adjuvant to endoscopic sclerotherapy for acute variceal haemorrhage in cirrhotic portal hypertension.     

The genes encoding the peripheral cannabinoid receptor and alpha-L-fucosidase are located near a newly identified common virus integration site, Evi11.

A new common region of virus integration, Evi11, has been identified in two retrovirally induced murine myeloid leukemia cell lines, NFS107 and NFS78. By interspecific backcross analysis, it was shown that Evi11 is located at the distal end of mouse chromosome 4, in a region that shows homology with human 1p36. The genes encoding the peripheral cannabinoid receptor (Cnr2) and alpha-L-fucosidase (Fuca1) were identified near the integration site by using a novel exon trapping system. Cnr2 is suggested to be the target gene for viral interference in Evi11, since proviruses are integrated in the first intron of Cnr2 and retroviral integrations alter mRNA expression of Cnr2 in NFS107 and NFS78. In addition, proviral integrations were demonstrated within the 3' untranslated region of Cnr2 in five independent newly derived CasBrM-MuLV (mouse murine leukemia virus) tumors, CSL13, CSL14, CSL16, CSL27, and CSL97. The Cnr2 gene encodes a seven-transmembrane G-protein-coupled receptor which is normally expressed in hematopoietic tissues. Our data suggest that the peripheral cannabinoid receptor gene might be involved in leukemogenesis as a result of aberrant expression of Cnr2 due to retroviral integration in Evi11.     

An intragenic deletion of the P gene is the common mutation causing tyrosinase-positive oculocutaneous albinism in southern African Negroids.

Tyrosinase-positive oculocutaneous albinism (OCA2), an autosomal recessive disorder of the melanin biosynthetic pathway, is the most common recessive disorder occurring in southern African Bantu-speaking Negroids, with an overall prevalence of 1/3,900. The OCA2 gene, P, has been mapped to chromosome 15q11-q13, and recently alterations in the P gene have been identified in OCA2 individuals. An intragenic deletion has been described and proposed to be of African origin because of its occurrence in four unrelated African American OCA2 individuals and in two individuals, one from Zaire and the other from Cameroon. This study shows that the intragenic deletion is a common cause of OCA2 in southern African Negroids (114/146 [.78]; OCA2 chromosomes) and is associated with one common haplotype (43/55 [.78]; OCA2 chromosomes), confirming the African origin of this allele. On the basis of haplotype data, it would appear that at least seven additional, less frequent OCA2 mutations occur in this population.