Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner

During membrane traffic, transport carriers are first tethered to the target membrane prior to undergoing fusion. Mechanisms exist to connect tethering with fusion, but in most cases, the details remain poorly understood. GM130 is a member of the golgin family of coiled-coil proteins tat is involved in membrane tethering at the endoplasmic reticulum (ER) to Golgi intermediate compartment and cis-Golgi. Here, we demonstrate that GM130 interacts with syntaxin 5, a t-SNARE also localized to the early secretory pathway. Binding to syntaxin 5 is specific, direct, and mediated by the membrane-proximal region of GM130. Interestingly, interaction with syntaxin 5 is inhibited by the binding of the vesicle docking protein p115 to a distal binding site in GM130. The interaction between GM130 and the small GTPase Rab1 is also inhibited by p115 binding. Our findings suggest a mechanism for coupling membrane tethering and fusion at the ER to Golgi intermediate compartment and cis-Golgi, with GM130 playing a central role in linking these processes. Consistent with this hypothesis, we find that depletion of GM130 by RNA interference slows the rate of ER to Golgi trafficking in vivo. The interactions of GM130 with syntaxin 5 and Rab1 are also regulated by mitotic phosphorylation, which is likely to contribute to the inhibition of ER to Golgi trafficking that occurs when mammalian cells enter mitosis.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

CCL3L1-CCR5 genotype influences durability of immune recovery during antiretroviral therapy of HIV-1-infected individuals

The basis for the extensive variability seen in the reconstitution of CD4(+) T cell counts in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) is not fully known. Here, we show that variations in CCL3L1 gene dose and CCR5 genotype, but not major histocompatibility complex HLA alleles, influence immune reconstitution, especially when HAART is initiated at <350 CD4(+) T cells/mm(3). The CCL3L1-CCR5 genotypes favoring CD4(+) T cell recovery are similar to those that blunted CD4(+) T cell depletion during the time before HAART became available (pre-HAART era), suggesting that a common CCL3L1-CCR5 genetic pathway regulates the balance between pathogenic and reparative processes from early in the disease course. Hence, CCL3L1-CCR5 variations influence HIV pathogenesis even in the presence of HAART and, therefore, may prospectively identify subjects in whom earlier initiation of therapy is more likely to mitigate immunologic failure despite viral suppression by HAART. Furthermore, as reconstitution of CD4(+) cells during HAART is more sensitive to CCL3L1 dose than to CCR5 genotypes, CCL3L1 analogs might be efficacious in supporting immunological reconstitution.     

Developmental changes in BDNF protein levels in the hamster retina and superior colliculus

Quantitative studies of ontogenetic changes in the levels of brain‐derived neurotrophic factor (BDNF) mRNA and its effector, BDNF protein, are not available for the retinal projection system. We used an electrochemiluminescence immunoassay to measure developmental changes in the tissue concentration of BDNF within the hamster retina and superior colliculus (SC). In the SC, we first detected BDNF (about 9 pg/mg tissue) on embryonic day 14 (E14). BDNF protein concentration in the SC rises about fourfold between (E14) and postnatal day 4 (P4), remains at a plateau through P15, then declines by about one‐third to attain its adult level by P18. By contrast, BDNF protein concentration in the retina remains low (about 1 pg/mg tissue) through P12, then increases 4.5‐fold to attain its adult level on P18. The developmental changes in retinal and collicular BDNF protein concentrations are temporally correlated with multiple events in the structural and functional maturation of the hamster retinal projection system. Our data suggest roles for BDNF in the cellular mechanisms underlying some of these events and are crucial to the design of experiments to examine those roles. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 173–187, 2001     

Acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA without increasing proteolysis in skeletal muscle

Acute alcohol intoxication decreases muscle protein synthesis, but there is a paucity of data on the ability of alcohol to regulate muscle protein degradation. Furthermore, various types of atrophic stimuli appear to regulate ubiquitin-proteasome-dependent proteolysis by increasing the muscle-specific E3 ligases atrogin-1 and MuRF1 (i.e., "atrogenes"). Therefore, the present study was designed to test the hypothesis that acute alcohol intoxication increases atrogene expression leading to an elevated rate of muscle protein breakdown. In male rats, the intraperitoneal injection of alcohol dose- and time-dependently increased atrogin-1 and MuRF1 mRNA in gastrocnemius, the latter of which was most pronounced. A comparable change was absent in the soleus and heart. The ability of in vivo-administered ethanol to increase atrogene expression was independent of the route of alcohol administration (intraperitoneal vs. oral), as well as of nutritional status (fed vs. fasted) and gender (male vs. female). The increase in atrogin-1 and MuRF1 was independent of alcohol metabolism, and the overproduction of endogenous glucocorticoids and could not be prevented by maintaining the circulating concentration of insulin-like growth factor-I. Despite marked changes in atrogene expression, acute alcohol in vivo did not alter the release of either 3-methylhistidine (MH) or tyrosine from the isolated perfused hindlimb, suggesting that the rate of muscle proteolysis remains unchanged. Moreover, alcohol did not increase the directly determined rate of protein degradation in isolated epitrochlearis muscles or cultured myocytes. Finally, no increase in atrogene expression or 3-MH release was detected in muscle from rats fed an alcohol-containing diet. Our results indicate that although acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA preferentially in fast-twitch skeletal muscle, this change was not associated with increased rates of muscle proteolysis. Therefore, the loss of muscle mass/protein in response to chronic alcohol abuse appears to result primarily from a decrement in muscle protein synthesis, not an increase in degradation.     

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.     

An enzyme-linked immunosorbent poly(ADP-ribose) polymerase biomarker assay for clinical trials of PARP inhibitors

Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. The DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is activated by DNA damage and has critical roles in DNA repair. Inhibition of PARP potentiates the activity of DNA-damaging agents such as temozolomide, topoisomerase inhibitors and radiation in both in vitro and in vivo preclinical models. Recently, several PARP inhibitors have entered clinical trials either as single agents or in combination with DNA-damaging chemotherapy. Because PARP inhibitors are not cytotoxic, a biomarker assay is useful to guide the selection of an optimal biological dose. We set out to develop an assay that enables us to detect 50% PAR reduction in human tumors with 80% power in a single-plate assay while assuring no more than a 10% false-positive rate. We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) to measure PARP activity that meets the above-mentioned criterion. This robust assay is able to detect PAR levels of 30-2000 pg/ml in both tumor and peripheral blood monocyte samples. In a B16F10 mouse syngeneic tumor model, PARP inhibitor ABT-888 potentiates the effect of temozolomide in suppressing tumor growth, and PARP activity is greatly reduced by ABT-888 at efficacious doses. In summary, the ELISA assay described here is suitable for biomarker studies in clinical trials of PARP inhibitors.     

Diagnosis of pulmonary hamartoma by fine needle biopsy

OBJECTIVE: To review the features of pulmonary hamartoma (PH) on fine needle biopsy (FNB), with emphasis on features that allow specific diagnosis. STUDY DESIGN: Fourteen cases of PH diagnosed on FNB were reviewed. The presence and volume of aspirate components were recorded. Attention was paid to features that might lead to false positive diagnosis of malignancy. Immunohistochemical staining for S100 was performed on cell block material. RESULTS: Fibromyxoid stroma and chondroid material were seen in 93% and 79% of cases, respectively; 71% demonstrated both components. Fibromyxoid stroma was prominent in the majority of cases; chondroid material was less abundant, being scanty in over half of cases. There were no cases in which epithelial cells represented the sole prominent component, and significant epithelial atypia was not identified. S100 staining was demonstrable in all cases in which stromal material was present in the cell block. CONCLUSION: A correct specific diagnosis of pH requires identification and correct interpretation of either fibromyxoid stroma or cartilaginous material. These components may show variable appearance on smears, with a range of potential mimics and pitfalls, but specific features are recognizable. Immunohistochemical staining of stromal material with S100 may lend support to the diagnosis.