7B2 Is a Specific Intracellular Binding Protein of the Prohormone Convertase PC2

Abstract: Biosynthetic pulse-chase analyses have previously demonstrated that the prohormone convertase PC2 is first synthesized as a precursor pro-PC2 and that zymogen activation to PC2 occurs following the slow exit of pro-PC2 from the endoplasmic reticulum (ER) and its concentration within the trans-Golgi network (TGN). The endocrine and neural protein 7B2 is first synthesized as a nonglycosylated precursor (pro-7B2), which is cleaved within the TGN by a furin-like ubiquitous convertase at the RRKRR¹⁵⁵S site to generate 7B2. In this report, we demonstrate that within the ER, pro-7B2 binds pro-PC2 but not any of the other convertases furin, PC1, PACE4, or PC5. This specific binding is Ca²⁺ dependent and does not require an N-glycosylated pro-PC2. Mutagenesis of the RRKRRS sequence demonstrated that the intact hexapeptide is critical for this binding, because the latter was abolished by mutations of the RR¹⁵² and greatly diminished by mutations of either the R¹⁵¹ or S¹⁵⁶ residues of pro-7B2. Once the complex is formed in the ER, it is then transported to the TGN where furin or a furin-like convertase cleaves both precursors, even when present as a complex. We also provide evidence that following zymogen cleavage, 7B2 remains bound to PC2, suggesting the presence of at least one other Ca²⁺-dependent binding site within the 7B2 sequence. Coexpression of 7B2 and PC2, although resulting in an elevation of the level of pro-PC2, did not eliminate the processing of pro-PC2 to PC2. Accordingly, cellular coexpression of 7B2 together with PC2 and proopiomelanocortin only marginally diminished the ability of PC2 to cleave proopiomelanocortin into β-endorphin in constitutive cells and had no effect in regulated cells. These results suggest that in vivo pro-7B2 is a specific PC2-binding protein that only transiently inhibits the processing of pro-PC2 until it reaches the TGN.     

Steroid hormone responsiveness of a family of closely related mouse proviral elements

Regulation of the mouse sex-limited protein (Slp) gene is unusual in that hormone response is conferred by the 5′ LTR of an upstream inserted provirus, dubbed the imposon (imp1). In a search for additional genes whose regulation has been affected by retrotransposition events, we isolated two partial proviral elements by stringent screening of a mouse genomic library. One clone (imp2) contained a portion of the envelope gene and a 3′ LTR that was nearly identical to the 3′ LTR of imp1; this similarity extended to insertion into a B1 repetitive element. The second proviral clone (imp3) contained a 5′ LTR and associated coding sequences, but lacked its 3′ LTR; the LTR of imp3 differed by 12% from the imp1 sequence. To assess potential hormone response, proviral enhancer regions cloned into reporter vectors were tested in transfection. The imp2 enhancer was similar in behavior to imp1, conferring both androgen and glucocorticoid induction in one fragment context and an androgen-specific response in another. In contrast, the imp3 enhancer allowed high expression in the absence of hormone and was less responsive to steroids in general and androgen in particular. These three proviral elements define a small family of steroid responsive proviruses in the mouse genome, and at least one member has had a lasting impact on an endogenous gene's regulation. Received: 29 April 1997 / Accepted: 14 July 1997     

Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation

Carotenoids are dietary antioxidants transported with plasma lipoproteins, primarily low-density lipoprotein (LDL). In this study in vitro methods were used to increase the amounts of specific, individual carotenoids in LDL. By addition of carotenoid to isolated LDL or to serum, followed by (re)isolation of the lipoproteins, samples of LDL were enriched 4- to 150-fold with lutein, 2- to 15-fold with lycopene, or 3- to 25-fold with β-carotene. Enrichment with specific carotenoids was achieved without affecting the electrophoretic mobility of the lipoprotein, its cholesterol to protein ratio, or the levels of other cartenoids or -tocopherol. The distributions among lipoproteins of carotenoid added to serum were similar, but not identical, to the distributions of the endogenous carotenoids. In particular, for added lutein, a greater proportion was found in HDL, and for added β-carotene, more was found in very low-density lipoprotein (VLDL). We then studied the effect of enriching LDL with specific carotenoids on its susceptibility to oxidation by copper ions. Lutein, β-cryptoxanthin, lycopene, and β-carotene, the four major plasma carotenoids, and -tocopherol were destroyed before the formation of lipid peroxidation products. The rates of destruction of the individual carotenoids differed; lycopene was destroyed most rapidly and lutein most slowly. Upon oxidation of β-carotene-enriched LDL, the rates of destruction of β-carotene, lycopene, and lutein were slowed and the lag times before the initiation of lipid peroxidation increased from 19 to 65 min. Neither effect was observed in LDL enriched with lutein or lycopene. Thus, β-carotene was unique among the carotenoids studied in having a small, but significant effect on LDL oxidation in vitro.     

Mycoplasma pneumoniae Pneumonia—Experience at a Referral Center

Mycoplasma pneumoniae pneumonia is usually a benign illness, and respiratory complications and extrapulmonary manifestations occur rarely. In this series, patients admitted to a referral hospital with this disorder had unusual symptoms, signs and findings on chest roentgenograms and laboratory studies. Pneumonia was often severe and extrapulmonary manifestations were frequent, resulting in prolonged hospital stays and illnesses. Although this extreme end of the spectrum of disease caused by M pneumoniae is not representative of this type of pneumonia as seen in outpatients, it is important to realize that patients admitted to hospital with severe, complicated pneumonia frequently have unusual manifestations of a common disease.     

Relationship of alpha MSH-specific neurons to the arcuate opiocortin neuronal system as determined by dual antigen immunocytochemical procedures

The cross-immunoreactivity, topography, and fiber projections of the alpha MSH-immunoreactive specific neurons in the forebrain of the rat appear to be distinctly different from that of the neurons in the hypothalamic arcuate opiocortin system. The cell bodies, immunoreactive only to -MSH, have a specific pattern of distribution in the dorsal and lateral hypothalamic regions from the level of the retrochiasmatic region to the premammillary area of the posterior hypothalamus. Immunoreactive fibers of these cells appear to extend into regions of the cerebral cortex and hippocampus. An antomical relationship between the immunostained fibers and/or terminals of the arcuate opiocortin pool of neurons and the -MSH-immunoreactive perikarya is described utilizing the ABC (Avidin-Biotin-Peroxidase Complex) and ABC-GO (Glucose Oxidase) or glucose oxidase-antiglucose oxidase complex methods of immunocytochemistry in which two tissue antigens with contrasting colors are demonstrated in the same tissue section.     

Substrate Degradation and Pressure Tolerance of Free-Living and Attached Bacterial Populations in the Intestines of Shallow-Water Fish

This report compares recovery of non-O1 Vibrio cholerae strains from seven California coastal sites during the winter and summer of 1983. A total of 41 identified and 27 presumptive nn-O1 V. cholerae strains were recovered from six of seven coastal sites in the summer. A 5-to 56-fold increase in the numbers of organisms isolated from different sites occurred in the summer months, when water temperatures were 1.9 to 5.1 degrees C higher. At the three sites where the highest levels of non-O1 V. cholerae were found, pollution, as measured by the total number of coliforms, exceeded the legal limit (less than 1,000 coliforms per 100 ml.).     

Blood chemistry of the common marmoset (Callithrix jacchus) maintained in an indoor-outdoor environment: Primate comparisons

Blood chemistry values were collected over a three-year period from at least 10 colony-born and 24 wild-born apparently normal common marmosets. BUN, SGOT, creatinine, calcium, phosphorus, alkaline phosphatase, protein, albumin, cholesterol, triglycerides, uric and glucose values were determined. A statistical comparison of baseline values was made between wild-born, colony-born, male and female marmosets. Also the same comparison was made between common marmosets and cotton-top tamarins, white lipped tamarins and human subjects.     

The effect of cycloheximide on Euglena gracilis phenylalanyl-tRNA synthetases

The effect of cycloheximide on the chloroplastic, cytoplasmic and mitochondrial phenylalanyltransferRNA synthetases of Euglena gracilis was studied by growing both logarithmic and stationary phase cultures in the presence of the antibiotic. Enzyme activity was measured relative to untreated control cultures. At very low concentrations of cycloheximide (1 g/ml), all three log phase enzymes showed an increase in activity of 40–50%. At slightly higher concentrations (2.5 g/ml), the phenylalanyl-tRNA synthetase activities were comparable to those of the control cultures. At a cycloheximide concentration of 5g/ml the enzyme activities from stationary phase cultures showed only very slight decreases (5–20%). The cytoplasmic and mitochondrial enzymes behaved similarly in log phase cultures at this concentration. However, the chloroplastic phenylalanyl-tRNA synthetase from log phase cultures treated with 5g/ml cycloheximide showed a marked decrease in activity (70%). A further increase in antibiotic concentration to 10g/ml resulted in significant losses of activity of all three enzymes, from both growth stages. The implications of the data with regard to identification of the site(s) of chloroplast enzyme synthesis are discussed.