Unusual conformational preferences of β-alanine containing cyclic peptides. VII

In the present paper we describe the synthesis, purification, and single crystal x-ray analysis of the cyclic pentapeptide cyclo-(Pro-Phe-Phe-β-Ala-β-Ala). This compound crystallizes in the orthorhombic space group P2₁2₁2₁ from methanol and adopts in the solid state an unusual conformation characterized by a cis β-Ala⁵-Pro¹ peptide bond and by an intramolecular hydrogen bond stabilizing a C₁₁- and a C₁₂-ring structure. The C₁₁, structure contains the Phe³ and the β-Ala⁴ at the corner position of the turn; it is the first observation of a type II β-turn enlargement due to the insertion of an extra methylene group of the β-alanine residue. The rest of the molecule participates in a newly characterized C₁₂-ring structure, which incorporates a β-Ala residue at position i of the turn. © 1996 John Wiley & Sons, Inc.     

Normal rat intestinal cells IEC-18: characterization and transfection with immortalizing oncogenes

IEC-18 cells, a cell line derived from the ileum of rat intestine, have the characteristics of normal cells since they have a contact inhibited cell growth, do not form colonies in soft agar and are not tumorigenic when injected in nude mice. IEC-18 cells were transfected with nuclear oncogenes, c-myc, v-myc and SV40 T antigen in order to obtain immortal cell lines. Independent clones were isolated and characterized for the growth properties. Expression of v-myc altered the morphology of the cells and shortened the doubling time. A slow growth together with a low cloning efficiency was associated with the expression of SV40 T antigen. No changes either in growth or in morphology were observed in c-myc-expressing IEC-18 cells. Expression of these nuclear oncogenes did not result in the neoplastic transformation of the IEC-18 cells, since none of the clones lost the anchorage dependence or were able to form tumors in vivo. The c-myc-containing IEC-18 cells were unable to secrete in the growth medium TGF and exposure to TGF inhibited the growth rate by 30%. All these observations are consistent with the conclusion that the expression of nuclear oncogenes does not lead to the neoplastic transformation of these cells.     

Environmental vibrio phage–bacteria interaction networks reflect the genetic structure of host populations

Phages depend on their bacterial hosts to replicate. The habitat, density and genetic diversity of host populations are therefore key factors in phage ecology, but our ability to explore their biology depends on the isolation of a diverse and representative collection of phages from different sources. Here, we compared two populations of marine bacterial hosts and their phages collected during a time series sampling program in an oyster farm. The population of Vibrio crassostreae, a species associated specifically to oysters, was genetically structured into clades of near clonal strains, leading to the isolation of closely related phages forming large modules in phage–bacterial infection networks. For Vibrio chagasii, which blooms in the water column, a lower number of closely related hosts and a higher diversity of isolated phages resulted in small modules in the phage–bacterial infection network. Over time, phage load was correlated with V. chagasii abundance, indicating a role of host blooms in driving phage abundance. Genetic experiments further demonstrated that these phage blooms can generate epigenetic and genetic variability that can counteract host defence systems. These results highlight the importance of considering both the environmental dynamics and the genetic structure of the host when interpreting phage–bacteria networks.     

Combined effect of ambient temperature and solar radiation on maned sloths' behaviour and detectability

Changes in ambient temperature and solar radiation may affect sloths' metabolic rate and body temperature, with consequent changes in activities, postures and microhabitat selection. Although the separate effect of temperature and solar radiation on sloth's behaviour have been previously studied, the combined effect of these climatic factors on behavioural aspects of sloths has never been systematically evaluated in field conditions. Here we evaluated the influence of hourly ambient temperature variation on maned sloth (Bradypus torquatus) activities, postures and tree crown positions, under sunny and cloudy conditions; and tested if any of the animal posture and position increase their exposure to human detection. We performed 350 h of visual observation on eight maned sloths, equipped with radio-backpacks, in northern Bahia, Brazil, recording their activities, and their resting postures and positions on tree crowns. We also recorded the time taken to visualize the sloths on 58 days to analyse if sloths' detection is affected by posture and position. Higher ambient temperature, within a range of 21–33°C, increased the sloths' activity levels in cloudy conditions but reduced their activity in sunny conditions. Increasing ambient temperature also reduced the frequency of huddled posture and increased the frequency of extended posture and permanence in the inner tree crown. Lastly, the postures and positions did not influence sloths' detectability. Thus, the direction of the temperature–activity relationship depends on climatic conditions (sunny/cloudy), and individuals rely on resting postures and positions to thermoregulate. The warmer and drier future climate, expected to occur in the northern Atlantic Forest, may impose change in the diurnal activity levels and postural pattern for this threatened species, leading maned sloths to reduce its activity on sunny and warmer days and adopting an extended posture.     

Escherichia coli Heat Shock Protein DnaK: Production and Consequences in Terms of Monitoring Cooking

Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F₇₀¹⁰ of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55°C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50°C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55°C, F₇₀¹⁰ = 3 min) accompanied by a 12-h recovery (containing 76,786 ± 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 ± 6,056 molecules/cell) when later heated to 60°C for 50 min (F₇₀¹⁰ = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment.     

Evidence for two possible glnB-type genes in Herbaspirillum seropedicae.

Two glnB-like genes have been isolated from Herbaspirillum seropedicae by complementation of the Klebsiella pneumoniae glnB502 mutant for growth on nitrate. One of these glnB-like genes has been sequenced and shows strong identity with GlnB proteins derived from other organisms. A Tn5-20 mutation of this glnB was Nif negative.     

MK-801 Prevents 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism in Primates

In cynomologus monkeys, systemic administration of MK-801, a noncompetitive antagonist for the N-methyl-D-aspartate receptor, prevented the development of the parkinsonian syndrome induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MK-801 also attenuated dopamine depletion in the caudate and putamen and protected dopaminergic neurons in the substantia nigra from the degeneration induced by the neurotoxin. Nevertheless, 7 days after MPTP administration in the caudate and putamen of monkeys also receiving MK-801, the levels of toxic 1-methyl-4-phenylpyridinium were even higher than those measured in monkeys receiving MPTP alone. This indicates that the protective action of MK-801 is not related to MPTP metabolism and strongly suggests that, in primates, the excitatory amino acids could play a crucial role in the mechanism of the selective neuronal death induced by MPTP.     

Highly concentrated urine-purified Tac peptide fails to inhibit IL-2-dependent cell proliferation in vitro.

Tac peptide, i.e., the p55 chain of the human interleukin-2 receptor (IL-2R) complex, is detectable as a soluble from (sIL-2R) in normal sera and, at increased levels, in patients with different diseases. Since several immunological abnormalities are observed in most conditions associated with an increase in sIL-2R levels, a down-regulatory effect on IL-2-dependent functions has been postulated as a consequence of binding and functional block of IL-2 by the excess of sIL-2R. To test this hypothesis, we purified sIL-2R from the urine of a patient with hairy cell leukemia and investigated the possible inhibitory effect of this peptide on the in vitro IL-2-induced cell proliferation. The urine-purified molecule was detectable by the specific immunoassay utilized to measure the serum Tac peptide and was constructed by a single polypeptide of about 50 kDa which was able to bind IL-2. Experiments performed with the IL-2-dependent murine CTLL-2 cell line and with PHA-stimulated human peripheral blood mononuclear cells showed that the purified sIL-2R at concentrations up to about 300 nM was unable to block IL-2-dependent cell proliferation. According to these data, which can be explained by the low affinity for IL-2 of the p55 IL-2R chain, it seems unlikely that in vivo the soluble Tac peptide can exert a down regulatory effect on IL-2-induced phenomena through a functional block of IL-2.     

Seasonal commitment of HMGCoA reductase activity to vitellogenin production

In female frogs (Rana Esculenta) during gametogenesis the cholesterol synthesized in the liver by 3-hydroxy-3-methylglutaryl coenzyme A reductase is mostly exported into the blood and taken up by the oocytes.In order to understand the fate of the neosynthesized cholesterol, female and male frogs and estrogenized male controls were injected with the labelled precursor¹⁴C mevalonate.In females and in estrogenized controls, mevalonate-derived radioactivity is found in a plasmatic lipoprotein that has been identified as vitellogenin by immunological detection.The increased 3-hydroxy-3-methylglutaryl coenzyme A reductase activity present in females in Fall is likely to be committed to provide cholesterol for the lipidation of this cholesterol-rich protein.