Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum

DNA sequencing of the region downstream of the cellulose synthase catalytic subunit gene of Acetobacter xylinum led to the identification of an open reading frame coding for a polypeptide of 86 kDa. The deduced amino acid sequence of this polypeptide matches from position 27 to 40 with the N-terminal amino acid sequence determined for a 93 kDa polypeptide that copurifies with the cellulose synthase catalytic subunit during purification of cellulose synthase. The cellulose synthase catalytic subunit gene and the gene encoding the 93 kDa polypeptide, along with other genes probably, are organized as an operon for cellulose biosynthesis in which the first gene is the catalytic subunit gene and the second gene codes for the 93 kDa polypeptide. The function of the 93 kDa polypeptide is not clear at present, however it appears to be tightly associated with the cellulose synthase catalytic subunit. Sequence analysis of the polypeptide shows that it is a membrane protein with a signal sequence at the N-terminal end and a transmembrane helix in the C-terminal region for anchoring it into the membrane.     

Oestrogen receptor gene structure and function in breast cancer

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoRI, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.     

The acetycholinesterase gene ofAnopheles stephensi

1. The acetylcholinesterase (AChE) gene from the important malaria vector Anopheles stephensi has been isolated by homology to the Drosophila acetylcholinesterase gene. 2. The complete sequence and intron-exon organization has been determined. The encoded protein has 69% identity to Drosophila AChE and 38 and 36% identity to Torpedo AChE and human butyrylcholinesterase, respectively.     

In situ activation of mouse macrophages and therapy of spontaneous renal cell cancer metastasis by liposomes containing the lipopeptide CGP 31362

Summary We determined whether the intravenous administration of multilamellar vesicle liposomes (MLV) containing a lipopeptide analogue of a fragment from the cell wall of gram-negative bacteria (CGP 31 362) can render BALB/c mouse alveolar macrophages tumoricidal in situ and reduce the incidence of spontaneous lung metastasis of syngeneic renal carcinoma (RENCA) cells. Alveolar macrophages (a) incubated in vitro with MLV containing CGP 31 362 (MLV-31 362) and (b) harvested from mice injected i.v. with MLV-31 362 were rendered cytotoxic against the RENCA cells. Maximum cytotoxic activity of the macrophages was induced by injecting 5 µmol MLV consisting of 250 mg phospholipids and 0.5 mg CGP 31 362. The single i.v. injection of 5 µmol MLV-31 362 produced activation of macrophages that lasted for up to 4 days. Repeated i. v. injections of MLV-31 362 produced a continuous antitumor activity in alveolar macrophages. To study the lipopeptide's effects on metastasis, we injected the left kidneys of BALB/c mice with RENCA cells. The kidney with growing tumor was resected 10 days later and, after a further 2 days, groups of mice were injected i.v. with MLV-31 362 or with MLV-HBSS (twice weekly for 3 weeks). Treatment with MLV-31 362 significantly decreased the median number of spontaneous lung metastases. These data demonstrate that the systemic administration of MLV-31 362 can activate murine lung macrophages in situ and reduce the incidence of spontaneous RENCA lung metastases.     

Differences between the catabolism and tumour distribution of intact monoclonal antibody (791T/36) and its Fab/c fragment in mice with tumour xenografts revealed by the use of a residualizing radiolabel (dilactitol-125I-tyramine) and autoradiography

Summary Radioiodine-labelled 791T/36 monoclonal antibody (mAb) and its Fab/c fragment, consisting of one Fab arm and the Fc portion, have identical whole-body survival curves in BALB/c mice (t1/2 = 3.75 days). Therefore, these two forms of this antibody provide a suitable model for studying the role of valency in the targeting efficiency of antibodies to tumours in vivo. 791/T36 antibody and its Fab/c fragment were labelled either by direct iodination using the iodogen method (¹²⁵I) or by dilactitol-¹²⁵I-tyramine (¹²⁵I-DLT), a residualizing label, which accumulates in the cells involved in degradation of the carrier protein. In tumour-bearing nude mice, the percentage of injected dose of mAb or Fab/c fragment reaching the specific 791T tumour was similar, and these proteins appeared to be catabolized at a similar rate in this tissue. mAb, but not the Fab/c fragment, was found to be very actively catabolized by the liver and spleen of tumour-bearing mice compared to control nude mice, this probably resulting from clearance of immune complexes. This effect was most pronounced when the mAb was labelled with¹²⁵I-DLT, the percentage of injected dose of mAb reaching the spleen and liver being higher than the percentage of injected dose reaching the tumour. This effect was not seen with the Fab/c fragment. Autoradiographic studies on tumour sections, which exhibit antigenic sites throughout the tumour mass, showed that the Fab/c fragment was already homogeneously distributed in the tumour 12 h after injection whereas the whole antibody was mainly localized at the periphery of the tumour. Those results suggest a binding site barrier effect. Overall, these results indicate that the highest valency and affinity may not be the optimal choice for mAb to be used for therapeutic purposes.     

Distribution and fate of free and liposome-encapsulated [3H]nor-muramyl dipeptide and [3H]muramyl tripeptide phosphatidylethanolamine in mice

The pharmacokinetics and metabolism of i.v. administered free (unencapsulated) or liposome-encapsulated hydrophilic [3H]-labeled nor-muramyl dipeptide (nor-MDP) and lipophilic [3H]-labeled muramyl tripeptide phosphatidylethanolamine (MTP-PE) were evaluated. In addition we also examined the distribution and fate of these immunomodulators subsequent to intranasal (i.n.) administration. Unique patterns of circulatory clearance, organ distribution, metabolism, and excretion were observed for each of the four preparations. Nor-MDP in saline was rapidly cleared from the circulation and excreted in the urine as intact molecules. MTP-PE dissolved in saline was cleared from the circulation at a slow rate and found within various organs as intact MTP-PE, lyso-MTP-PE, and MDP. Following the i.v. administration of nor-MDP or MTP-PE in liposomes, patterns of clearance and organ distribution corresponded to that of liposome distribution, i.e., the reticuloendothelial system. Extensive dissociation of hydrophilic nor-MDP from the carrier liposomes occurred, and the immunomodulator was recovered in the urine. In contrast, MTP-PE entrapped in liposomes was retained in target organs for the duration of the study. The i.n. instillation of radiolabeled nor-MDP or MTP-PE was associated with the accumulation of these immunomodulators in the brain. Our results demonstrate the feasibility of targeting hydrophilic and lipophilic immunomodulators to cells of the macrophage-histiocyte series.     

Observations on 6-mercaptopurine activity in Saccharomyces cerevisiae

Abstract Hydrogen fluoride treatment of [¹⁴C-glycerol]lipoteichoic acid synthesized by growing Streptococcus faecium ATCC9790 in the presence of 1,3[¹⁴C]glycerol produced five radioactive, water-soluble products which were identified by chromatographic and analytical techniques to be tetraglucosyl glycerol, triglucosyl glycerol, diglucosyl glycerol, monoglucosyl glycerol and unsubstituted glycerol. The percent composition of each varied modestly from culture to culture and ranged between 7 and 8% for the tetra-, 20.5 and 31.2% for the tri-, 11.3 to 23.5% for the di-, 20.9 to 26.8% for the mono-, and 23.1 to 34.8% for the unsubstituted glycerol. The same glucosylated glycerol compounds could be obtained in an in vitro reaction in which a 30 000 × g particulate enzyme catalyzed the incorporation of [³H]glucose from UDP [³H]glucose into lipoteichoic acid.     

Sulphate reduction in oxic and sub-oxic North-East Atlantic sediments

Abstract Oxic and sub-oxic N.-E. Atlantic sediments were examined for sulphate-reducing activity. Oxygen and/or nitrate reduction are probably the dominant mineralisation processes in the abyssal plain sediment studied. A low rate of sulphate reduction (0.1 nmol SO²⁻₄/ml/day) was recorded in the surface 5 cm of the continental slope sediment, together with the presence of a range of sulphate-reducing bacteria (SRB). A higher activity of sulphate reduction (2.2 nmol SO²⁻₄/ml/day) occurred in the continental shelf sediment which led to a small decrease in pore water sulphate and an increase in titration alkalinity. This sediment contained approx. 10²–10³ acetate, lactate and propionate oxidising SRB/ml. No low- M _r organic acids were detected in these sediments. However, amendment with 75 μM acetate stimulated sulphate-reducing activity in the shelf sediment.     

Domes,arches and urchins: The skeletal architecture of echinoids (Echinodermata)

Summary A combination of simple membrane theory and statical analysis has been used to determine how stresses are carried in echinoid skeletons. Sutures oriented circumferentially are subject principally to compression. Those forming radial zig-zags are subject to compression near the apex and tension near the ambitus. Radial and circumferential sutures in Eucidaris are equally bound with collagen fibers but in Diadema, Tripneustes, Psammechinus, Arbacia and other regular echinoids, most radial sutures are more heavily bound, and thus stronger in tension. Psammechinus, Tripneustes and several other echinoids have radial sutures thickened by ribs which increase the area of interlocking trabeculae. Ribs also increase flexural stiffness and carry a greater proportion of the stress. Further, ribs effectively draw stress from weaker areas pierced by podial pores, and increase the total load which can be sustained.Allometry indicates that regular echinoids become relatively higher at the apex as size increases, thus reducing ambital stresses. Some spatangoids with very high domes (eg Agassizia) maintain isometry, but others (eg Meoma) become flatter with size. Both holectypoids (Echinoneus) and cassiduloids (Apatopygus) maintain a constant height to diameter relationship. Flattening, and consequently ambital tensile stress, is greatest in the clypeasteroids. In this group the formation of internal buttresses which preferentially carry stress, reaches maximum development. A notable exception, however, is the high domed Clypeaster rosaceus.In this analysis it was assumed that local buckling or bending does not occur. The test of some echinoids (e.g. Diadematoida) have relatively wide sutures swathed in collagen, which allows local deformation. Others (e.g. Arbacia) have rigid sutures with reduced collagen. In Psammechinus and other members of the Order Echinoida, in addition to rib formation, inner and outer surface trabeculae are thickened so that the individual plates are stiffened. Some spatangoids (Meoma, Paleopneustes) have extensive sutural collagen, but the cassiduloid Apatopygus has collagen confined to junctions of sutures, and elsewhere the joints are strengthened and stiffened by fusion of trabeculae. Fusion of surface trabeculae is almost complete in the holectypoid, Echinoneus, and the sutures are obscured.