Action of a cell wall proteinase (PI) from Streptococcus cremoris HP on bovine β-casein

Summary The specificity of a cell wall proteinase (P_I) from Streptococcus cremoris strain HP in its action on bovine -casein was determined. To this end an enzymic digest (pH 6.2; 15° C) of -casein was brought to pH 4.6 and the soluble fraction separated by semi-preparative reversed-phase HPLC. Purified peptides were analyzed by amino acid and end-group analysis. Ten chromatographic components were identified, which together accounted for at least seven cleavage sites all being located in the C-terminal fifty-residue part of -casein. In five cases it concerned a Gln-X or X-Gln peptide linkage. The specificity of this proteinase from S. cremoris HP shows similarity to that reported for a cell wall proteinase from S. lactis NCDO 763 in its action on -casein.Presented at the second FEMS Symposium on Lactic Acid Bacteria held in 1987 at Wageningen, Netherlands     

Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine αs1-, β-, and κ-Casein

Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine α_s1-, β-, and κ-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM₁, was concluded. The action of the HP-type proteinase P₁ (also detectable in strains Wg₂, C₁₃, and TR) was established by electrophoretic methods to be directed preferentially towards β-casein. The AM₁-type proteinase P_III (also detectable in strain SK₁₁) was also able to degrade β-casein, but at the same time split α_s1- and κ-casein more extensively than did P_I. Strain FD₂₇ exhibited mainly P_I activity but also detectable P_III degradation characteristics. The cell wall proteinase preparation of strain E₈ showed low P_I as well as low P_III activity. All proteinase preparations produced from κ-casein positively charged degradation products with electrophoretic mobilities similar to those of degradation products released by the action of the milk-clotting enzyme chymosin. The differences between P_I and P_III in mode of action, as detected by gel electrophoresis and thin-layer chromatography, were reflected by the courses of the initial degradation of methyl-¹⁴C-labeled β-casein and by the effect of α_s1- plus κ-casein on these degradations. The results are discussed in the light of previous comparative studies of cell wall proteinases in strains of S. cremoris and with respect to the growth of this organism in milk.     

Effects of ethanol,octanoic and decanoic acids on fermentation and the passive influx of protons through the plasma membrane of Saccharomyces cerevisiae

Ethanol, octanoic and decanoic acids are known toxic products of alcoholic fermentation and inhibit yeast functions such as growth and fermentation. pH-stat measurements showed that, in a concentration range up to 20 mg/l, octanoic and decanoic acids increase the rate of passive H⁺ influx across the plasma membrane of Saccharomyces cerevisiae IGC 3507. Decanoic acid was more active than octanoic acid, which agrees with its higher liposolubility. The fatty acids probably act as H⁺ carriers, since the magnitude of the effect depended on pH and correlated with the concentration of protonated fatty acids. Esterification of the fatty acids partially abolished the enhancing effect on passive H⁺ influx. Passive H⁺ influx showed saturation kinetics with half-maximal activity at 6.6 M H⁺ (pH 5.2). Contrary to previous findings, ethanol inhibited H⁺ influx exponentially up to a concentration of 8% (v/v). At higher concentrations, ethanol reactivated H⁺ influx; the original rate of H⁺ uptake was reached at 14% (v/v) ethanol. In the same concentration ranges that affected passive H⁺ influx, ethanol, octanoic and decanoic acids inhibited the fermentation rate. This inhibitory effect of the fatty acids on fermentation rate depended on liposolubility, pH, and esterification in the same way as that found for their effect on passive H⁺ influx. Inhibition of fermentation by octanoic and decanoic acids could therefore result from their effect on the rate of passive H⁺ influx. Correspondence to: S. Stevens     

Detection of Anorectal and Cervicovaginal Chlamydia Trachomatis Infections following Azithromycin Treatment: Prospective Cohort Study with Multiple Time-Sequential Measures of rRNA,DNA, Quantitative Load and Symptoms

Background

Determination of Chlamydia trachomatis (Ct) treatment success is hampered by current assessment methods, which involve a single post-treatment measurement only. Therefore, we evaluated Ct detection by applying multiple laboratory measures on time-sequential post-treatment samples.

Methods

A prospective cohort study was established with azithromycin-treated (1000 mg) Ct patients (44 cervicovaginal and 15 anorectal cases). Each patient provided 18 self-taken samples pre-treatment and for 8 weeks post-treatment (response: 96%; 1,016 samples). Samples were tested for 16S rRNA (TMA), bacterial load (quantitative PCR; Chlamydia plasmid DNA) and type (serovar and multilocus sequence typing). Covariates (including behavior, pre-treatment load, anatomic site, symptoms, age, and menstruation) were tested for their potential association with positivity and load at 3–8 weeks using regression analyses controlling for repeated measures.

Findings

By day 9, Ct positivity decreased to 20% and the median load to 0.3 inclusion-forming units (IFU) per ml (pre-treatment: 170 IFU/ml). Of the 35 cases who reported no sex, sex with a treated partner or safe sex with a new partner, 40% had detection, i.e. one or more positive samples from 3–8 weeks (same Ct type over time), indicating possible antimicrobial treatment failure. Cases showed intermittent positive detection and the number of positive samples was higher in anorectal cases than in cervicovaginal cases. The highest observed bacterial load between 3–8 weeks post-treatment was 313 IFU/ml, yet the majority (65%) of positive samples showed a load of ≤2 IFU/ml. Pre-treatment load was found to be associated with later load in anorectal cases.

Conclusions

A single test at 3–8 weeks post-treatment frequently misses Ct. Detection reveals intermittent low loads, with an unknown risk of later complications or transmission. These findings warrant critical re-evaluation of the clinical management of single dose azithromycin-treated Ct patients and fuel the debate on defining treatment failure. Clinicaltrials.gov Identifier: NCT01448876.     

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory,GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.     

Early and Middle Holocene Hunter-Gatherer Occupations in Western Amazonia: The Hidden Shell Middens

We report on previously unknown early archaeological sites in the Bolivian lowlands, demonstrating for the first time early and middle Holocene human presence in western Amazonia. Multidisciplinary research in forest islands situated in seasonally-inundated savannahs has revealed stratified shell middens produced by human foragers as early as 10,000 years ago, making them the oldest archaeological sites in the region. The absence of stone resources and partial burial by recent alluvial sediments has meant that these kinds of deposits have, until now, remained unidentified. We conducted core sampling, archaeological excavations and an interdisciplinary study of the stratigraphy and recovered materials from three shell midden mounds. Based on multiple lines of evidence, including radiocarbon dating, sedimentary proxies (elements, steroids and black carbon), micromorphology and faunal analysis, we demonstrate the anthropogenic origin and antiquity of these sites. In a tropical and geomorphologically active landscape often considered challenging both for early human occupation and for the preservation of hunter-gatherer sites, the newly discovered shell middens provide evidence for early to middle Holocene occupation and illustrate the potential for identifying and interpreting early open-air archaeological sites in western Amazonia. The existence of early hunter-gatherer sites in the Bolivian lowlands sheds new light on the region’s past and offers a new context within which the late Holocene “Earthmovers” of the Llanos de Moxos could have emerged.     

THE NEST OF THE KNYSNA SCRUB WARBLER (BRADYPTERUS SYLVATICUS) AND SOME NOTES ON PARENTAL BEHAVIOUR

Von Maltitz, F., Schmitt, M. B., &; Biggs, H. C. 1984. Measurements, moult and abundance of the Lizard Buzzard in the Transvaal. Ostrich 55: 177–181.

During a 12-year study 51 Lizard Buzzards Kaupifalco monogrammicus were captured in the Transvaal. South Africa. They occurred sporadically, with peaks in the years 1972, 1975, 1979 and 1983 and were significantly more common in winter. Moult was completed between January and June. Mass is given and recaptures documented.