Lysozyme: an endogenous antimicrobial protein with potent activity against extracellular, but not intracellular Mycobacterium tuberculosis.

Endogenous antimicrobial peptides (AMPs) play a key role in the host defense against pathogens. AMPs attack pathogens preferentially at the site of entry to prevent invasive infection. Mycobacterium tuberculosis (Mtb) enters its host via the airways. AMPs released into the airways are therefore likely candidates to contribute to the clearance of Mtb immediately after infection. Since lysozyme is detectable in airway secretions, we evaluated its antimicrobial activity against Mtb. We demonstrate that lysozyme inhibits the growth of extracellular Mtb, including isoniazid-resistant strains. Lysozyme also inhibited the growth of non-tuberculous mycobacteria. Even though lysozyme entered Mtb-infected human macrophages and co-localized with the pathogen we did not observe antimicrobial activity. This observation was unlikely related to the large size of lysozyme (14.74 kDa) because a smaller lysozyme-derived peptide also co-localized with Mtb without affecting the viability. To evaluate whether the activity of lysozyme against extracellular Mtb could be relevant in vivo, we incubated Mtb with fractions of human serum and screened for antimicrobial activity. After several rounds of sub-fractionation, we identified a highly active fraction-component as lysozyme by mass spectrometry. In summary, our results identify lysozyme as an antimycobacterial protein that is detectable as an active compound in human serum. Our results demonstrate that the activity of AMPs against extracellular bacilli does not predict efficacy against intracellular pathogens despite co-localization within the macrophage. Ongoing experiments are designed to unravel peptide modifications that occur in the intracellular space and interfere with the deleterious activity of lysozyme in the extracellular environment.

Chemotherapy for a secondary malignancy nearly restores complete chimerism in an SCID-patient after HSCT.

For patients with inborn errors of immunity (IEI) and other inborn diseases, mixed donor chimerism is a well-accepted outcome of hematopoietic stem cell transplantation (HSCT). Cytoreductive chemotherapy for a secondary malignancy is a potential challenge for the stability of the graft function after HSCT. We report on a boy with X-SCID who developed Ewing sarcoma ten years after HSCT which was successfully treated with cytoreductive chemotherapy, surgery and local radiation. Surprisingly, this treatment had a positive impact on mixed chimerism with an increase of donor-cell proportions from 40% for neutrophils and 75% for non-T-mononuclear cells (MNCs) to >90% for both. T-cell counts remained stable with 100% of donor origin. This is -to our knowledge- the first report on the impact of cytoreductive chemotherapy on post-HSCT mixed chimerism and provides an important first impression for future patients.

Analyzing and Engineering the Product Selectivity of a 2-Methylenebornane Synthase.

Terpenes constitute the largest class of natural products with more than 70 000 compounds. Many different terpenes find applications in the flavor and fragrance industry or can be used as fine chemicals or drugs. In some bacteria, noncanonical terpenes with 11 carbon atoms are synthesized via a GPP-C2-methyltransferase and the subsequent conversion of 2-methyl-GPP by certain terpene synthases into mainly 2-methylisoborneol and 2-methylenebornane. Many other C11-terpenes were reported as side products, but they are synthesized only in minor amounts by the bacterial C11-terpene biosynthesis pathway. To enable biotechnological synthesis of these largely unexplored natural products, we changed the product selectivity of the 2-methylenebornane synthase from Pseudomonas fluorescens by a semirational protein engineering approach. Active site amino acids with impact on the product selectivity were identified and variants with completely altered product spectra could be identified and characterized. The gathered data provide new insights into the structure-function relationship for C11-terpene synthases and demonstrate the production of formerly inaccessible noncanonical terpenes.

Off-axis astigmatism in the isolated chicken crystalline lens.

The chicken eye was previously found to have little off-axis astigmatism which is not explained by its special corneal shape but rather by the optical properties of the crystalline lens. To learn more about lens design, we studied off-axis astigmatism in the chicken lens in situ and compared it to a glass lens of similar power but with homogenous refractive index. After euthanasia, enucleated eye balls were cut in the equatorial plane right behind the scleral ossicles. The anterior segment was placed in a water-filled chamber. Several thin laser beams were projected in two perpendicular meridians through the lens under various eccentricities and the focal lengths were determined. Off-axis astigmatism across the horizontal visual field was determined as the differences in power in the two meridians. The same procedure was used for the glass lens. On-axis, the chicken crystalline lens had slightly more power in the vertical than in the horizontal meridian (-2.8±0.7D (SEM)). Astigmatism flipped sign and increased with eccentricity to reach +6.1±2.1D (SEM) at 33.5deg off-axis, as expected from off-axis astigmatism. Even though this value appears high, it was still 2.5 times lower than in the glass lens. A ZEMAX model of a lens with a homogeneous index and with surface profiles taken of the natural chicken lens revealed even higher levels of off-axis astigmatism. Obviously, the natural chicken lens displays much less off-axis astigmatism than a glass lens with similar power. Since its shape does not explain the low off-axis astigmatism, it must be due to a refined internal refractive index structure.

Lack of oblique astigmatism in the chicken eye.

Primate eyes display considerable oblique off-axis astigmatism which could provide information on the sign of defocus that is needed for emmetropization. The pattern of peripheral astigmatism is not known in the chicken eye, a common model of myopia. Peripheral astigmatism was mapped out over the horizontal visual field in three chickens, 43 days old, and in three near emmetropic human subjects, average age 34.7years, using infrared photoretinoscopy. There were no differences in astigmatism between humans and chickens in the central visual field (chicks -0.35D, humans -0.65D, n.s.) but large differences in the periphery (i.e. astigmatism at 40° in the temporal visual field: humans -4.21D, chicks -0.63D, p<0.001, unpaired t-test). The lack of peripheral astigmatism in chicks was not due to differences in corneal shape. Perhaps related to their superior peripheral optics, we found that chickens had excellent visual performance also in the far periphery. Using an automated optokinetic nystagmus paradigm, no difference was observed in spatial visual performance with vision restricted to either the central 67° of the visual field or to the periphery beyond 67°. Accommodation was elicited by stimuli presented far out in the visual field. Transscleral images of single infrared LEDs showed no sign of peripheral astigmatism. The chick may be the first terrestrial vertebrate described to lack oblique astigmatism. Since corneal shape cannot account for the difference in astigmatism in humans and chicks, it must trace back to the design of the crystalline lens. The lack of peripheral astigmatism in chicks also excludes a role in emmetropization.

Is adaptation to perceived interocular differences in height explained by vertical fusional eye movements?

PURPOSE: To find out whether adaptation to a vertical prism involves more than fusional vertical eye movements. METHODS: Adaptation to a vertical base-up 3 prism diopter prism was measured in a custom-programmed Maddox test in nine visually normal emmetropic subjects (mean age 27.0 ± 2.8 years). Vertical eye movements were binocularly measured in six of the subjects with a custom-programmed binocular video eye tracker. RESULTS: In the Maddox test, some subjects adjusted the perceived height as expected from the power of the prism while others appeared to ignore the prism. After 15 minutes of adaptation, the interocular difference in perceived height was reduced by on average 51% (from 0.86°-0.44°). The larger the initially perceived difference in height in a subject, the larger the amplitude of adaptation was. Eye tracking showed that the prism generated divergent vertical eye movements of 1.2° on average, which was less than expected from its power. Differences in eye elevation were maintained as long as the prism was in place. Small angles of lateral head tilt generated large interocular differences in eye elevation, much larger than the effects introduced by the prism. CONCLUSIONS: Vertical differences in retinal image height were compensated by vertical fusional eye movements but some subjects responded poorly to a vertical prism in both experiments; fusional eye movements were generally too small to realign both foveae with the fixation target; and the prism adaptation in the Maddox test was fully explained by the changes in vertical eye position, suggesting that no further adaptational mechanism may be involved.

The Arabidopsis NIMIN proteins affect NPR1 differentially.

NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is the central regulator of the pathogen defense reaction systemic acquired resistance (SAR). NPR1 acts by sensing the SAR signal molecule salicylic acid (SA) to induce expression of PATHOGENESIS-RELATED (PR) genes. Mechanistically, NPR1 is the core of a transcription complex interacting with TGA transcription factors and NIM1-INTERACTING (NIMIN) proteins. Arabidopsis NIMIN1 has been shown to suppress NPR1 activity in transgenic plants. The Arabidopsis NIMIN family comprises four structurally related, yet distinct members. Here, we show that NIMIN1, NIMIN2, and NIMIN3 are expressed differentially, and that the encoded proteins affect expression of the SAR marker PR-1 differentially. NIMIN3 is expressed constitutively at a low level, but NIMIN2 and NIMIN1 are both responsive to SA. While NIMIN2 is an immediate early SA-induced and NPR1-independent gene, NIMIN1 is activated after NIMIN2, but clearly before PR-1. Notably, NIMIN1, like PR-1, depends on NPR1. In a transient assay system, NIMIN3 suppresses SA-induced PR-1 expression, albeit to a lesser extent than NIMIN1, whereas NIMIN2 does not negatively affect PR-1 gene activation. Furthermore, although binding to the same domain in the C-terminus, NIMIN1 and NIMIN2 interact differentially with NPR1, thus providing a molecular basis for their opposing effects on NPR1. Together, our data suggest that the Arabidopsis NIMIN proteins are regulators of the SAR response. We propose that NIMINs act in a strictly consecutive and SA-regulated manner on the SA sensor protein NPR1, enabling NPR1 to monitor progressing threat by pathogens and to promote appropriate defense gene activation at distinct stages of SAR. In this scenario, the defense gene PR-1 is repressed at the onset of SAR by SA-induced, yet instable NIMIN1.

NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) and some NPR1-related proteins are sensitive to salicylic acid.

NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1; also known as NIM1) is a master regulator of systemic acquired resistance (SAR). SAR is induced by salicylic acid (SA), leading to the expression of PATHOGENESIS-RELATED (PR) genes. Current evidence suggests that NPR1 is part of a transcription complex tethered to activation sequence-1 (as-1)-like cis-acting elements in PR-1 gene promoters through TGA transcription factors, and that SA-dependent PR-1 gene expression is regulated by NIM1-INTERACTING (NIMIN) proteins. In Arabidopsis, NPR1 is active only after SA induction. Regulation of Arabidopsis NPR1 activity has been proposed to comprise cysteine-156 (Cys-156), mediating SA-induced cytoplasmic oligomer-nuclear monomer exchange, and Cys-521 and Cys-529, mediating SA-dependent transcriptional activation. Tobacco NPR1 does not harbour these residues. To understand the function of tobacco NPR1, we analysed its biochemical capabilities in a heterologous system: yeast. Tobacco NPR1 differs from Arabidopsis NPR1 in its subcellular localization and its transactivation potential. Yet, both tobacco and Arabidopsis NPR1, as well as tobacco NIM1-like1, alter some of their biochemical activities in response to SA. Whereas the addition of SA to yeast growth medium induces transcriptional activity in tobacco NPR1, its interaction with NIMIN2-type proteins is suppressed. The effects of SA are specific, sensitive and occur coordinately. They are abolished completely by mutation of the arginine residue within the invariable penta-amino acid motif LENRV, as present in the nonfunctional Arabidopsis nim1-4 allele. Furthermore, NPR1 proteins with the LENRV domain coincidently harbour a broad and strongly conserved NIMIN1/NIMIN2 binding site. Our data suggest that NPR1 and some NPR1-like proteins are sensitive to the plant hormone SA, altering some of their biochemical capabilities to enable stimulus-dependent gene expression. The sensitivity of NPR1 proteins to SA, together with their differential interaction with diverse NIMIN proteins, seems a plausible molecular basis for the timely and coordinated activation of PR genes during SAR.