A quantitative study of the recruitment potential of all intracellular tyrosine residues on EGFR, FGFR1 and IGF1R

Receptor tyrosine kinases transmit and process extracellular cues by recruiting intracellular signaling proteins to sites of tyrosine phosphorylation. Using protein microarrays comprising virtually every human SH2 and PTB domain, we generated quantitative protein interaction maps for three well-studied receptors--EGFR, FGFR1 and IGF1R--using phosphopeptides derived from every intracellular tyrosine residue on each receptor, regardless of whether or not they are phosphorylated in vivo. We found that, in general, peptides derived from physiological sites of tyrosine phosphorylation bind to substantially more SH2 or PTB domains than do peptides derived from nonphysiological sites, supporting the idea that kinases and interaction domains co-evolve and suggesting that new sites arise predominantly through selection favoring advantageous interactions, rather than through selection disfavoring unwanted interactions. We also found substantial qualitative overlap in the recruitment profiles of these three receptors, suggesting that their different biological effects arise, at least in part, from quantitative differences in their affinities for the proteins they recruit.     

A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.     

Systematic studies invalidate the neonatally androgenized rat as a model for polycystic ovary disease

As an adult, the neonatally androgenized (AZ) rat is anovulatory and exhibits follicular cysts. Thus, the AZ rat has been used as a model for polycystic ovary disease (PCO). However, its correlation with the human disease is not clear; so we have studied the AZ rat to determine its suitability as a PCO model. In Experiment I, reproductive hormones were measured at specific intervals between postnatal Days 15 and 90 in saline-treated and AZ rats. In Experiment II, AZ rats were treated with exogenous follicle-stimulating hormone (FSH) or subjected to unilateral ovariectomy (ULO) as analogies to therapies that have been used to treat human PCO. The results demonstrate that the luteinizing hormone (LH), FSH, testosterone (T), and estradiol (E2) concentrations of the AZ rat were not different from control values. Additionally, FSH therapy did not increase the E2 concentrations or the ovarian weight of the AZ rat. Furthermore, control and AZ rats exhibited similar post-ULO rises in FSH, but compensatory ovarian hypertrophy was not evident in the AZ rat. We conclude that 1) the hormonal and morphological patterns observed in the AZ rat do not correlate with those of PCO and 2) the androgenized rat does not provide an adequate model to study PCO.     

Anesthetic and nephrotoxic effects of Telazol in New Zealand white rabbits.

Telazol was evaluated as an anesthetic for rabbits. Two groups of five rabbits each were injected intramuscularly with 32 or 64 mg/kg of Telazol, and the depth and duration of anesthesia period monitored. At both doses, the righting reflex was lost within 2 minutes postinjection. Animals in both groups responded to noxious stimuli for the duration of the anesthesia. Hematology and urinalyses were performed daily for 7 days postinjection. Hematologic parameters remained unchanged in both groups. In the high-dose group, blood urea nitrogen and serum creatinine levels increased 1 day postinjection and continued steadily throughout the week. Elevations in urine protein and the presence of casts correlated with this increase. In the low-dose group, blood urea nitrogen and creatinine levels increased and protein was present in the urine of four of five rabbits beginning approximately 5 days postinjection. Histologically, severe renal tubular necrosis was evident 7 days postinjection in all high-dose rabbits and in three rabbits in the low-dose group. Our results indicate that Telazol does not produce analgesia in rabbits and is nephrotoxic at both 32 and 64 mg/kg. We conclude that Telazol is contraindicated for use in rabbits.     

Caloric Restriction Engages Hepatic RNA Processing Mechanisms in Rhesus Monkeys

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Protein A as a fusion partner for the expression of heterologous proteins in Lactobacillus

An expression system based on the Staphylococcus aureus protein A gene (spa) was developed to allow the production and export of proteins in Lactobacillus. Plasmid shuttle vectors were constructed that carried the eZZ gene, a synthetic gene based on the Protein A gene (spa) but lacking the carboxy-terminal membrane-anchoring region. A gene fusion was created between the eZZ gene and the VD4 region of a chlamydial major outer-membrane protein gene. Expression studies demonstrated the recognition of the spa regulatory signals by several Lactobacillus, with the recombinant protein being expressed (from 0.1 μg of EZZVD4 fusion protein per ml in L. plantarum up to 10 μg of EZZ protein per ml in L. fermentum) and exported (levels up to 20% in L. fermentum) in several Lactobacillus strains. Received: 27 August 1996 / Received revision: 27 November 1996 / Accepted: 29 November 1996     

Molecular and kinetic alterations of muscle AMP deaminase during chronic creatine depletion

We examined apossible mechanism to account for the maintenance of peak AMPdeamination rate in fast-twitch muscle of rats fed the creatine analog-guanidinopropionic acid (-GPA), in spite of reduced abundance ofthe enzyme AMP deaminase (AMPD). AMPD enzymatic capacity (determined atsaturating AMP concentration) and AMPD protein abundance (Western blot)were coordinately reduced ~80% in fast-twitch white gastrocnemiusmuscle by -GPA feeding over 7 wk. Kinetic analysis of AMPD in thesoluble cell fraction demonstrated a single Michaelis-Menten constant(K_m; ~1.5 mM) in control muscle extracts. An additional high-affinityK_m (~0.03 mM)was revealed at low AMP concentrations in extracts of -GPA-treated muscle. The kinetic alteration in AMPD reflects increased molecular activity at low AMP concentrations; this could account for high ratesof deamination in -GPA-treated muscle in situ, despite the loss ofAMPD enzyme protein. The elimination of this kinetic effect bytreatment of -GPA-treated muscle extracts with acid phosphatase invitro suggests that phosphorylation is involved in the kinetic controlof skeletal muscle AMPD in vivo.

     

Recruitment of TBK1 to cytosol‐invading Salmonella induces WIPI2‐dependent antibacterial autophagy

Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti‐bacterial autophagy relies on the core autophagy machinery, cargo receptors, and “eat‐me” signals such as galectin‐8 and ubiquitin that label bacteria as autophagy cargo. Anti‐bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti‐bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella‐associated “eat‐me” signals, including host‐derived glycans and K48‐ and K63‐linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host.     

Controlled Release of Food Lipids Using Monoglyceride Gel Phases Regulates Lipid and Insulin Metabolism in Humans

We describe a solid vegetable oil–water gel structure which is stabilized through the use of low concentrations of monoglycerides, containing no added trans fats or saturated fats. The resulting structure consists of oil droplets encapsulated in self-assembled crystalline monoglyceride multilayers, surrounded by a continuous water phase. Acute ingestion human feeding trials indicated that the serum triglyceride loading was significantly lower after ingestion of the structured gel rather than a simple oil–water mixture, resulting in an attenuated increase in serum insulin. This indicates the effectiveness of encapsulation in modulating blood lipid and insulin response in humans, and suggests a strategy that can be employed for the controlled release of food macronutrients. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.