Overexpression of proteins containing tyrosine- or leucine-based sorting signals affects transferrin receptor trafficking.

Targeting of many transmembrane proteins to post-Golgi compartments is dependent on cytoplasmically exposed sorting signals. The most widely used signals conform to the tyrosine- or the leucine-based motifs. Both types of signals have been implicated in protein localization to the same intracellular compartments, but previous results from both cell-free experiments and studies of transfected cell lines have indicated that the two types of signals interact with separate components of the sorting machinery. We have overexpressed several transmembrane proteins in stably transfected Madin-Darby canine kidney cells using an inducible promoter system. Overexpression of proteins containing tyrosine- or leucine-based sorting signals resulted in reduced internalization of the transferrin receptor, whereas recycling and polarized distribution was not influenced. Our results indicate that proteins with tyrosine- and leucine-based sorting signals can be transported along common saturable pathways.     

The Occurrence of Mycoplasmas and Bacteria in Lungs from Sheep in Southern Norway

In order to study the occurrence of mycoplasmas among Norwegian sheep, lungs from a great number of different herds were collected at 3 abattoirs in Southern Norway. The presence of fermenting mycoplasmas and bacteria was examined in both normal and pneumonic lungs to determine whether recovery of these agents could be related to pneumonic changes. Pneumonic lungs demonstrated lesions typical of the condition described as subacute or chronic pneumonia. Mycoplasma ovipneumoniae was found in 87 % of the 126 pneumonic lungs and in 6 % of the 83 normal lungs. Bacteria, mostly Pasteurella haemolytica, were less frequently encountered in the pneumonic lungs, and usually in combination with M. ovipneumoniae. It is concluded that M. ovipneumoniae is widespread among sheep in Southern Norway and can be considered to have etiological significance in subacute or chronic pneumonia, whereas bacteria probably occur mainly as secondary invaders. Changes resulting from moderate invasion by lungworm were found in about half of the lungs, but just as frequently in normal as in pneumonic lungs, and accordingly did not appear to contribute to the pneumonia investigated.     

Drivers of Vaginal Drug Delivery System Acceptability from Internet-Based Conjoint Analysis

Vaginal microbicides potentially empower women to protect themselves from HIV and other sexually transmitted infections (STIs), especially when culture, religion, or social status may prevent them from negotiating condom use. The open literature contains minimal information on factors that drive user acceptability of women’s health products or vaginal drug delivery systems. By understanding what women find to be most important with regard to sensory properties and product functionality, developers can iteratively formulate a more desirable product. Conjoint analysis is a technique widely used in market research to determine what combination of elements influence a consumer’s willingness to try or use a product. We applied conjoint analysis here to better understand what sexually-active woman want in a microbicide, toward our goal of formulating a product that is highly acceptable to women. Both sensory and non-sensory attributes were tested, including shape, color, wait time, partner awareness, messiness/leakage, duration of protection, and functionality. Heterosexually active women between 18 and 35 years of age in the United States (n = 302) completed an anonymous online conjoint survey using IdeaMap software. Attributes (product elements) were systematically presented in various combinations; women rated these combinations of a 9-point willingness-to-try scale. By coupling systematic combinations and regression modeling, we can estimate the unique appeal of each element. In this population, a multifunctional product (i.e., broad spectrum STI protection, coupled with conception) is far more desirable than a microbicide targeted solely for HIV protection; we also found partner awareness and leakage are potentially strong barriers to use.     

The susceptibility of Salvelinus fontinalis (Mitchill) to Gyrodactylus salaris Malmberg (Platyhelminthes; Monogenea) under experimental conditions

The host specificity of Gyrodaclylus Solaris is examined experimentally with respect to its ability to infect the brook trout, Salvelinus fontinalis . The parasite readily attached to and reproduced on parr of this host and infections grew for c. 20 days from first monitoring (c. 30 days from first infection) before declining. Parasites could persist on this host for up to 70 days before finally disappearing. The pattern of infection resembled that seen in many other gyrodactylid species on their normal hosts, and suggested the action of a host response, In this respect infections of G. salaris on parr of S. fontinalis , anadromous Salvelinus alpinus, Oncorhynchus mykiss, Thymallus thymallus and Baltic Salmo salar follow a normal pattern, while infections of Norwegian S. salar are unusual in a continued unchecked growth, until the host dies, under pooled laboratory conditions.     

Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by 15N NMR using magnetization transfer and indirect detection via protons

NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, 15N labeling being used to identify specific backbone 15N and 1H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence of hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neurophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of 15N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

1,25-Dihydroxyvitamin D3 suppresses human T helper/inducer lymphocyte activity in vitro

The active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), suppresses in vitro immunoglobulin (Ig) production by activated peripheral blood mononuclear cells (PBM) from normal human subjects by inhibiting T helper/inducer TH cell activity. Normal PBM were fractionated into B, TH and T suppressor/cytotoxic (Ts) cells by fluorescence-activated cell sorting techniques. The resultant subsets were activated with mitogens and were cultured in the presence or absence of a receptor-saturating concentration of 1,25-(OH)2-D3. The sterol reduced [3H]thymidine incorporation in TH cells by 56%, with no effect on Ts or B cells. When 1,25-(OH)2-D3-treated TH cells were co-cultured with untreated B cells and culture supernatants assayed for Ig production, 1,25-(OH)2-D3 abrogated the inducing effect of TH cells on Ig synthesis by B cells. There was no inhibitory effect of the sterol on Ts or B cell activity. In addition, 1,25-(OH)2-D3 produced a dramatic inhibition of interleukin 2 (IL 2) production by activated PBM, but did not inhibit IL 2 receptor generation by these cells. Other vitamin D metabolites tested did not produce this effect. These results suggest that the TH lymphocyte is the specific cellular target for the immunoinhibitory effects of 1,25-(OH)2-D3.     

Differences in the host resistance of Atlantic salmon, Salmo salar L., stocks to the monogenean Gyrodactylussalaris Malmberg, 1957

The susceptibility and resistance of hatchery-reared salmon parr, native to the rivers Neva (U.S.S.R. Baltic Sea), Alta (northern Norway) and Lone (western Norway) (both eastern Atlantic Ocean), to Gyrodactylus salaris from Norway, was examined. The level of resistance to the parasite was assessed from counts, made on anaesthetized salmon, ofthe numbers of G. salaris after an initial experimental exposure (2 weeks) to G. salaris-infected salmon. Three experiments, all in water at c. 12° C, were carried out: (1) 50 Alta and 50 Neva salmon, initial mean parasite intensity c. 12; (2) 50 Lone and 50 Neva salmon, initial mean parasite intensity c. 60; (3) 10 Lone and 10 Neva salmon individually isolated, initial intensity one gravid G. salaris . In both the Norwegian salmon stocks, the G. salaris infrapopulations steadily increased during the experimental period of 5 weeks, in contrast to a prominent decline in the Neva salmon stock, after, respectively: (Exp. 1) week 3, average peak intensity 32.6; (Exp. 2) week 2, average peak intensity 58.7; and (Exp. 3) week 3, average peak intensity 6.3. The hatchery-reared Baltic Neva stock demonstrated both an innate and an acquired resistance towards G. salaris , in contrast to the highly susceptible, Norwegian Alta and Lone salmon stocks.