Studies on the metabolism of oestrone and oestradiol-17 beta in the kidneys of minipigs of different ages and sexes (author's transl)]

After incubation of [4-14C]oestrone (E1) with kidney cortex slices of minipigs, [4-14C]oestradiol-17 beta (E2) and small amounts of a polar metabolite were detected in the ether-soluble fraction. E1, E2 and polar metabolites were found in the protein-bound fraction. The water-soluble fraction contained E1-3-glucuronide (80% of total glucuronides), E2-3-glucuronide and trace amounts of the 3-monoglucuronide of oestriol (E3). When E2 was used as substrate, the main product formed was E1; it was detected in both the ether-soluble and protein-bound fractions. E1-3-Glucuronide was the main metabolite in the water-soluble fraction, which also contained some E2-3-Glucuronide. In male minipigs, the mast, the rate of conversion of E1 and E2 as well as the formation of glucuronides were significantly greater in fertile females than in infertile females. Whereas no sex difference was observed in the metabolism of oestrogens in kidneys of infertile minipigs, the rate of oxidoreduction and glucuronidation was more pronounced in fertile female animals than in the corresponding males. The present results suggest that, in the kidneys of minipigs, the ratio of E1 to E2 is shifted towards the former; furthermore, by a comparatively rapid metabolism of the oestrogenic hormone, the renal tissue contributes to the maintenance of hormonal equilibrium.     

Pyridoxal phosphate. An anionic probe for protein amino groups exposed on the outer and inner surfaces of intact human red blood cells.

Pyridoxal phosphate is a potent probe for exploring the "sidedness" of proteins in the membrane of the intact red blood cell. It reacts with amino groups with a high degree of specificity, forming a Schiff's base that can be fixed as an irreversible bond upon reduction with NaBH4; its binding site can be identified by use of [3-H]pyridoxal phosphate or NaB3-H4; it can be used as a surface probe under conditions of minimal penetration, or it can be used as a probe for proteins on the inside of the membrane under conditions of substantial uptake. Pyridoxal phosphate uptake involves a rapid and a slow component. The former represents the binding to the outer surface of the membrane; it is not substantially affected by pH and temperature, but is reduced considerably by pretreatment of cells by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid, a specific inhibitor of anion transport. The slow component represents penetration into the cell; it is blocked by high pH, low temperature, or pretreatment with the disulfonic stilbene. Pyridoxal phosphate itself is also an effective and specific inhibitor of the permeation of other anions. Under conditions of minimal uptake, the only labeled proteins are three glycoproteins and a protein of apparent molecular weight 95,000. Under conditions of substantial uptake into the cell, the other major protein bands seen by staining on acrylamide gels after electrophoresis are labeled. It is concluded that virtually all of the major membrane proteins interact with pyridoxal phosphate from one side of the membrane or the other. The differences in labeling under conditions of minimal or maximal uptake can, therefore, be attributed to the sidedness in the distribution of the membrane proteins rather than to differences in their reactivity.     

An improved breeding method for solitarious locusts

In order to unravel the physiological, endocrine, and behavioral differences between gregarious and solitarious forms of the desert locust, Schistocerca gregaria (Forsk.) (Orthoptera, Acrididae), a constant supply of rather large numbers of solitary individuals has to be guaranteed. This represents a bottleneck, mainly because of the intensity of the labor involved and limited laboratory accommodation. The method we describe here substantially reduces the space and manpower needed. The survival rate we obtained in the solitarised population was relatively high, reaching about 55%. The optimal rearing temperature proved to be 32–36 °C. Cabbage leaves and oat flakes sufficed for feeding all year round. Special racks have been designed that enable high density stacking and easy handling. The solitarisation process was monitored over ten consecutive generations. Changes in morphometrics, eye stripes, color, and behavior were recorded, of which only morphometrics, temperature related development, and mortality are discussed. A shift towards the solitarious phase was recorded, with clear differences between gregarious, 1^st generation and 7^th to 10^th generation solitarious locusts.     

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein‐like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat‐inducible and appears to play a major role in Lpl1 interaction. Pre‐incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1‐Hsp90 interaction induces F‐actin formation, thus, triggering an endocytosis‐like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl‐Hsp90 interaction.     

Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting

Ubiquitin accumulation in amyloid plaques is a pathological marker observed in the vast majority of neurodegenerative diseases, yet ubiquitin function in these inclusions is controversial. It has been suggested that ubiquitylated proteins are directed to inclusion bodies under stress conditions, when both chaperone-mediated refolding and proteasomal degradation are compromised or overwhelmed. Alternatively, ubiquitin and chaperones may be recruited to preformed inclusions to promote their elimination. We address this issue using a yeast model system, based on expression of several mildly misfolded degradation substrates in cells with altered chaperone content. We find that the heat shock protein 70 (Hsp70) chaperone pair Ssa1/Ssa2 and the Hsp40 cochaperone Sis1 are essential for degradation. Substrate ubiquitylation is strictly dependent on Sis1, whereas Ssa1 and Ssa2 are dispensable. Remarkably, in Ssa1/Ssa2-depleted cells, ubiquitylated substrates are sequestered into detergent-insoluble, Hsp42-positive inclusion bodies. Unexpectedly, sequestration is abolished by preventing substrate ubiquitylation. We conclude that Hsp40 is required for the targeting of misfolded proteins to the ubiquitylation machinery, whereas the decision to degrade or sequester ubiquitylated proteins is mediated by the Hsp70s. Accordingly, diminished Hsp70 levels, as observed in aging or certain pathological conditions, might be sufficient to trigger ubiquitin-dependent sequestration of partially misfolded proteins into inclusion bodies.     

Lipid Droplet-Binding Protein TIP47 Regulates Hepatitis C Virus RNA Replication through Interaction with the Viral NS5A Protein

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.