U2AF homology motifs: protein recognition in the RRM world

Recent structures of the heterodimeric splicing factor U2 snRNP auxiliary factor (U2AF) have revealed two unexpected examples of RNA recognition motif (RRM)-like domains with specialized features for protein recognition. These unusual RRMs, called U2AF homology motifs (UHMs), represent a novel class of protein recognition motifs. Defining a set of rules to distinguish traditional RRMs from UHMs is key to identifying novel UHM family members. Here we review the critical sequence features necessary to mediate protein-UHM interactions, and perform comprehensive database searches to identify new members of the UHM family. The resulting implications for the functional and evolutionary relationships among candidate UHM family members are discussed.     

Promotion of U937 cell adhesion on polypropylene surfaces bearing phosphorylcholine functionalities

Phosphorylcholine (PC) groups were grafted onto ammonia plasma-treated biaxially-oriented polypropylene (BOPP) surfaces, via (a) reductive amination of phosphorylcholine glyceraldehyde and (b) a two-step procedure involving the chemical amplification of surface amine groups with tris(2-aminoethyl amine) and subsequent reductive amination of phosphorylcholine glyceraldehyde. The occurrence of grafting was ascertained by X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance Fourier-transform infrared (ATR-FT-IR) spectroscopy. The wettability of PC-modified surfaces was assessed by dynamic contact-angle measurements using the Wilhelmy plate method. Human U937 macrophages adhered and proliferated to a significantly larger extent on PC-modified surfaces, compared to unmodified or ammonia plasma-modified BOPP.     

Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens (n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.     

Effects of concurrent inspiratory and expiratory muscle training on respiratory and exercise performance in competitive swimmers

The efficiency of the respiratory system presents significant limitations on the bodys ability to perform exercise due to the effects of the increased work of breathing, respiratory muscle fatigue, and dyspnoea. Respiratory muscle training is an intervention that may be able to address these limitations, but the impact of respiratory muscle training on exercise performance remains controversial. Therefore, in this study we evaluated the effects of a 12-week (10 sessions week^–1) concurrent inspiratory and expiratory muscle training (CRMT) program in 34 adolescent competitive swimmers. The CRMT program consisted of 6 weeks during which the experimental group (E, n=17) performed CRMT and the sham group (S, n=17) performed sham CRMT, followed by 6 weeks when the E and S groups performed CRMT of differing intensities. CRMT training resulted in a significant improvement in forced inspiratory volume in 1 s (FIV_1.0) (P=0.050) and forced expiratory volume in 1 s (FEV_1.0) (P=0.045) in the E group, which exceeded the S groups results. Significant improvements in pulmonary function, breathing power, and chemoreflex ventilation threshold were observed in both groups, and there was a trend toward an improvement in swimming critical speed after 12 weeks of training (P=0.08). We concluded that although swim training results in attenuation of the ventilatory response to hypercapnia and in improvements in pulmonary function and sustainable breathing power, supplemental respiratory muscle training has no additional effect except on dynamic pulmonary function variables.     

The surface-associated protein of Staphylococcus saprophyticus is a lipase

Staphylococcus saprophyticus surface-associated protein (Ssp) was the first surface protein described for this organism. Ssp-positive strains display a fuzzy layer of surface-associated material in electron micrographs, whereas Ssp-negative strains appear to be smooth. The physiologic function of Ssp, however, has remained elusive. To clone the associated gene, we determined the N-terminal sequence, as well as an internal amino acid sequence, of the purified protein. We derived two degenerate primers from these peptide sequences, which we used to identify the ssp gene from genomic DNA of S. saprophyticus 7108. The gene was cloned by PCR techniques and was found to be homologous to genes encoding staphylococcal lipases. In keeping with this finding, strains 7108 and 9325, which are Ssp positive, showed lipase activity on tributyrylglycerol agar plates, whereas the Ssp-negative strain CCM883 did not. Association of enzyme activity with the cloned DNA was proven by introducing the gene into Staphylococcus carnosus TM300. When wild-type strain 7108 and an isogenic mutant were analyzed by transmission electron microscopy, strain 7108 exhibited the fuzzy surface layer, whereas the mutant appeared to be smooth. Lipase activity and the surface appendages could be restored by reintroduction of the cloned gene into the mutant. Experiments using immobilized collagen type I did not provide evidence for the involvement of Ssp in adherence to this matrix protein. Our experiments thus provided evidence that Ssp is a surface-associated lipase of S. saprophyticus.     

Three novel human VMD2-like genes are members of the evolutionary highly conserved RFP-TM family

The RFP-TM protein family was first described in Caenorhabditis elegans as hypothetical transmembrane proteins containing a conserved 350-400 amino acid domain including the invariant peptide motif RFP. The VMD2 gene underlying Best disease was shown to represent the first human member of the RFP-TM protein family. More than 97% of the disease-causing mutations are located in the N-terminal RFP-TM domain implying important functional properties. Here, we have identified three novel VMD2-related human genes (VMD2L1, VMD2L2 and VMD2L3) demonstrating a high degree of conservation in their respective RFP-TM domains. Each of the VMD2-like proteins has a unique C-terminus that lack similarity to other proteins or motifs. By FISH analysis, VMD2L1 was localised to chromosome 19p13.2-p13.12, VMD2L2 to 1p32.3-p33 and VMD2L3 to 12q14.2-q15. RT-PCR analyses revealed tissue-restricted expression of the three genes with both VMD2L1 and VMD2L2 abundantly transcribed in colon. VMD2L1 is present in the retinal pigment epithelium while VMD2L3 shows predominant expression in skeletal muscle.     

Neurological Manifestations of COVID-19 Feature T Cell Exhaustion and Dedifferentiated Monocytes in Cerebrospinal Fluid

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Comparison of GB virus C, HIV, and HCV infection markers in hemophiliacs exposed to non-inactivated or inactivated factor concentrates.

BACKGROUND: Until the mandatory introduction of viral inactivation techniques of blood plasma products in the early 1980s many recipients of these products were infected with various viral pathogens. OBJECTIVES: To determine the rate of transmission of GB virus C/hepatitis G virus (GBV-C/HGV) HCV, and HIV through non-virus-inactivated clotting factor concentrates in hemophiliacs, as well as the relation between amount of administered clotting factor and risk for GBV-C/HGV infection. STUDY DESIGN: In this cross-sectional study, we determined retrospectively the rates of infection markers for GBV-C/HGV, HCV, and HIV in a German cohort of hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor concentrates (group A) and in a second group of hemophiliacs who were treated exclusively with virus-inactivated clotting factor (group B). The presence of anti-virus antibodies was determined by ELISA. Viral RNA was detected by RT-PCR. Markers for viral infections were compared to amounts of administered non-virus-inactivated clotting factor. RESULTS: Among hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor the prevalence for GBV-C/HGV, HCV, and HIV was 40.3%, 98.6%, and 56.3%, respectively. In contrast to HIV, the rate of GBV-C/HGV infections did not increase with increasing amounts of consumed non-inactivated clotting factor. Even in the subgroup of heavily treated hemophiliacs the rate of GBV-C/HGV infection markers did not exceed 45%. CONCLUSIONS: The amount of non-virus-inactivated clotting factor is not predictive for the risk of GBV-C/HGV infection in hemophiliacs. Despite repeated parenteral exposure more than 55% of hemophiliacs were not infected with GBV-C/HGV. Our findings indicate a high frequency of host factors preventing parenteral transmission of GBV-C/HGV.