Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins

Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.     

The lipid composition and permeability to the triazole antifungal antibiotic ICI 153066 of serum-grown mycelial cultures of Candida albicans

The total lipid content of Candida albicans (serotype A: NCPF 3153) exponential-phase mycelial cultures grown in tissue-culture medium 199 (containing 10%, v/v, foetal calf serum) was 29.8 +/- 8 mg (g dry weight)-1 (mean +/- SD). The weight ratios of phospholipid to neutral lipid and phospholipid to non-esterified sterol were 2.6 +/- 0.4 and 24.9 +/- 0.5, respectively. The major phospholipid was phosphatidylcholine with smaller amounts of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and diphosphatidylglycerol; the most abundant fatty acids were palmitic, palmitoleic, oleic and linoleic acids. The major neutral lipids comprised esterified sterol, triacylglycerol and non-esterified fatty acid with a smaller amount of non-esterified sterol. The fatty acid compositions of the three fatty-acid-containing neutral lipids were distinct from each other and the phospholipids. Comparison with previous data on yeast cultures of C. albicans A grown in glucose broth shows that mycelial cultures have a larger lipid content, lower phospholipid to neutral lipid ratio and higher phospholipid to non-esterified sterol ratio. We now show that mycelial cultures were more permeable to a [14C]triazole antifungal antibiotic compared with exponentially growing yeast cultures of several azole-sensitive strains. Taken together these data are consistent with there being a relationship between the phospholipid/non-esterified sterol ratio of a culture and its ability to accumulate a triazole.     

T cell recognition of HIV synthetic peptides in a natural infection

Because T cell responses are critical for defense against viral infections, a series of synthetic peptides derived from the predicted sequence for HIV-1 proteins gp41, pg120, gag, and viral polymerase were used to test the T cell proliferative response of HIV-1 seropositive individuals. Of HIV-1-infected donors from various clinical categories 90% (27/30) had sensitized cells that proliferated in response to at least one of 21 HIV peptides tested. Cells from HIV seronegative controls did not proliferate (0/9) in response to these HIV peptides. Individuals with fewer clinical manifestations of HIV-1 disease responded to a greater number of peptides (average for asymptomatic seropositives = 8.1 peptides; AIDS patients averaged 2.0). The number of peptides recognized also correlated with absolute number of CD4+ cells, but not with delayed cutaneous hypersensitivity to a (non-HIV) battery of Ag. However, clinical stage at no time correlated with the response to any particular peptide. Response patterns differed considerably among individuals, and some peptides stimulated proliferation in many (48%) HIV-infected donors (peptides gp41-2 and pol-3), whereas another peptide elicited no T cell response in any donor tested (peptide gp120-8). We have also begun to investigate the basis for individual heterogeneity of T lymphocyte proliferative responses of HIV-infected donors to the 21 HIV synthetic peptides. Peptide structure and HLA class II determinants both influenced patterns of lymphocyte responses. Reactivity correlated with peptide size, the presence of alpha and beta secondary structure and lack of reverse turn potential. Hydropathy and charge had no predictive value. Peptides derived from HIV sequences that vary highly among strains tended to be recognized less frequently. HIV-infected lymphocyte donors were HLA typed to examine the influence of the MHC on T lymphocyte proliferation. Analysis of the frequencies of individuals reacting to specific peptides, when compared to the allele frequencies in the population at large, indicated association of some responses to DR alleles. More DR association was observed with peptides that showed "moderate" reactivity than with those that were "highly" reactive. We suggest that highly reactive peptides are capable of forming a structure closer to an "ideal" T cell epitope that can associate with many DR alleles. In contrast, "moderately" reactive determinants have less favorable structures for interaction, are more limited in their ability to interact and therefore show more restriction to specific class II alleles.     

Low-affinity, high-capacity system of glucose transport in the ruminal bacterium Streptococcus bovis: evidence for a mechanism of facilitated diffusion

The glucose phosphotransferase system (PTS) of Streptococcus bovis could not account for the glucose consumption of exponential cultures, and the kinetics of glucose transport were biphasic. A PTS-deficient mutant lost the high-affinity, low-capacity system but retained its ability to take up glucose at high substrate concentrations. The low-affinity, high-capacity system did not require a proton motive force or ATP and could not be driven by an artificial membrane potential in the presence or absence of sodium. Since low-affinity transport was directly proportional to the external substrate concentration and exhibited counterflow kinetics, it appeared that a facilitated-diffusion mechanism was responsible for glucose transport at high substrate concentrations.     

Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.     

Metabolism of halogenated bisphosphonates by the cellular slime mould Dictyostelium discoideum.

Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonates was taken up by the amoebae and incorporated into the corresponding beta, gamma-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.     

Regulation of expression of a wound-inducible tomato inhibitor I gene in transgenic nightshade plants

A wound-inducible proteinase Inhibitor I gene from tomato containing 725 bp of the 5 region and 2.5 kbp of the 3 region was stably incorporated into the genome of black nightshade plants (Solanum nigrum) using an Agrobacterium Ti plasmid-derived vector. Transgenic nightshade plants were selected that expressed the tomato Inhibitor I protein in leaf tissue. The leaves of the plants contained constitutive levels of the inhibitor protein of up to 60 g/g tissue. These levels increased by a factor of about two in response to severe wounding. Only leaves and petioles exhibited the presence of the inhibitor, indicating that the gene exhibited the same tissue specificity of expression found in situ in wounded tomato leaves. Inhibitor I was extracted from leaves of wounded transformed nightshade plants and was partially purified by affinity chromatography on a chymotrypsin-Sepharose column. The affinity-purified protein was identical to the native tomato Inhibitor I in its immunological reactivity and in its inhibitory activity against chymotrypsin. The protein exhibited the same M _r of 8 kDa as the native tomato Inhibitor I and its N-terminal amino acid sequence was identical to that of the native tomato inhibitor I, indicating that the protein was properly processed in nightshade plants. These expriments are the first report of the expression of a member of the wound-inducible tomato Inhibitor I gene family in transgenic plants. The results demonstrate that the gene contains elements that can be regulated in a wound-inducible, tissuespecific manner in nightshade plants.     

The use of 13C-n.m.r. spectroscopy to monitor alginate biosynthesis in mucoid Pseudomonas aeruginosa.

The biosynthesis of alginate by a mucoid strain of Pseudomonas aeruginosa, isolated from a cystic-fibrosis patient, was monitored by using 13C-n.m.r. spectroscopy of bacterial cultures incubated with 1-13C- or 2-13C-enriched fructose. When 1-13C- or 2-13C-enriched fructose was used as the precursor of alginate, enrichment with 13C in the constituent uronic acid monomers of the polysaccharide could only be detected in C-1 or C-2 respectively, indicating that alginate is synthesized in Ps. aeruginosa directly from fructose, with the hexose molecule being retained intact; this rules out the involvement of C3 intermediates, which occurs when glucose is the alginate precursor. The absence of detectable poly-L-gluluronate block sequences from the alginate of Ps. aeruginosa was confirmed, and it was shown that there is no modification of the arrangement of the constituent uronic acids between polymerization to form alginate and the appearance of the mature alginate in the extracellular medium. The 13C-n.m.r. data also provided independent evidence for acetylation on D-mannuronate residues and for the ratio of D-mannuronate to L-guluronate residues in newly synthesized alginate, which had previously been determined only for material secreted from bacteria into the extracellular medium.     

Sporicidal action of alkaline glutaraldehyde: factors influencing activity and a comparison with other aldehydes

The sporicidal efficacy of glutaraldehyde (2% w/v) was investigated under various conditions. Numerous factors influenced its activity: method of spore production, inherent spore resistance characteristics, alkalination, storage time and storage temperature. The sporicidal action of 2% alkaline glutaraldehyde at room temperature was compared with that of other aldehydes and commercially available formulations. Cidex (glutaraldehyde) and Sporicidin (glutaraldehyde + phenol full strength) were the most effective, followed by 8% (w/v) formaldehyde and 10% (v/v) Gigasept, a formaldehyde-containing product. Five per cent (v/v) Gigasept and 10% (w/v) glyoxal also had good sporicidal activity, though that of Sporicidin (1 : 16) was poor. No activity was observed with 10% (w/v) butyraldehyde.     

Feeding and growth of juvenile sea bass: the effect of ration and temperature on growth rate and efficiency

The effect of ration on the growth of pairs of juvenile sea bass Dicentrarchus labrax fed squid mantle was recorded at four temperatures: 6, 10, 14 and 18) C, covering the range typical of Welsh coastal waters. Initial weight of the fish ranged from 2.8 to 15.9 g. A predictive model for the maximum meal size (M_max) at temperatures between 10 and 18) C, accounted for 95% of the variance in lnM_max. Even when offered excess food, bass at 6) C had a low rate of food consumption [0.19% body weight (BW) day^?1] and lost weight (G=?0.04% day^?1). Predictive regression models for specific growth rate (G) accounted for 86% of the variance at reduced rations and 70% at maximum meals. The relationship between G (calculated for total biomass per tank) and ration was a decelerating curve. G at maximum meals increased with temperature, at lower rations G decreased with temperature. For a pair of bass with a combined weight of 15 g, predicted maintenance ration ranged between 0.7 and 2.3% BW day^?1 and increased with temperature. Maximum meal size was more sensitive to temperature than maintenance ration. At 18) C optimum ration was 7.4% BW day^?1. At lower temperatures, the optimum ration was the maximum meal. The maximum gross growth efficiency was 17.4% at 18) C. Mean absorption efficiency was 94.8%. Ration level had no significant effect on absorption efficiency, which was lowest at 6) C. Condition indices (Fulton condition factor, wet and dry liver—somatic indices and body depth index) increased with meal size at all temperatures except 6) C. An increase in temperature between 10 and 18) C generally resulted in a decrease in condition indices at a given ration. When comparisons were made at a given standard length, gut and carcass weight increased with ration. Visceral fat and gut weight decreased with increased temperature.