Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase C�� to Associate with Microtubules and to Recruit Dynein

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Cι (aPKCι) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCι in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCι were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin, we characterized the distribution of tyrosinated/detyrosinated α-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated α-tubulin; however, aPKCι was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCι-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway and associates with vesicular tubular clusters (VTCs)² located between the endoplasmic reticulum (ER) and the cis-Golgi compartment (1, 2). VTCs are pleomorphic structures that sort anterograde-directed cargo from recycling proteins and trafficking machinery retrieved to the ER (3-6). Rab2 bound to a VTC microdomain stimulates recruitment of soluble factors that results in the release of vesicles containing the recycling protein p53/p58 (7). In that regard, we have previously reported that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical PKC ι (aPKCι) are Rab2 effectors that interact directly with the Rab2 amino terminus and with each other (8, 9). Their interaction requires Src-dependent tyrosine phosphorylation of GAPDH and aPKCι (10). Moreover, GAPDH is a substrate for aPKCι (11). GAPDH catalytic activity is not required for ER to Golgi transport indicating that GAPDH provides a specific function essential for membrane trafficking from VTCs independent of glycolytic function (9). Indeed, phospho-GAPDH influences MT dynamics in the early secretory pathway (11).GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs) (12) and subsequently was shown to interact with the carboxyl terminus of α-tubulin (13). The binding of GAPDH to MTs promotes formation of cross-linked parallel MT arrays or bundles (14, 15). GAPDH has also been reported to possess membrane fusogenic activity, which is inhibited by tubulin (16). Similarly, aPKC associates directly with tubulin and promotes MT stability and MT remodeling at specific intracellular sites (17-21). It may not be coincidental that these two Rab2 effectors influence MT dynamics because recent studies indicate that the cytoskeleton plays a central role in the organization and operation of the secretory pathway (22).MTs are dynamic structures that grow or shrink by the addition or loss of α- and β-tubulin heterodimers from the ends of protofilaments (23). Their assembly and stability is regulated by a variety of proteins traditionally referred to as microtubule-associated proteins (MAPs). In addition to the multiple α/β isoforms that are present in eukaryotes, MTs undergo an assortment of post-translational modifications, including acetylation, glycylation, glutamylation, phosphorylation, palmitoylation, and detyrosination, which further contribute to their biochemical heterogeneity (24, 25). It has been proposed that these tubulin modifications regulate intracellular events by facilitating interaction with MAPs and with other specific effector proteins (24). For example, the reversible addition of tyrosine to the carboxyl terminus of α-tubulin regulates MT interaction with plus-end tracking proteins (+TIPs) containing the cytoskeleton-associated protein glycine-rich (CAP-Gly) motif and with dynein-dynactin (27-29). Additionally, MT motility and cargo transport rely on the cooperation of the motor proteins kinesin and dynein (30). Kinesin is a plus-end directed MT motor, whereas cytoplasmic dynein is a minus-end MT-based motor, and therefore the motors transport vesicular cargo toward the opposite end of a MT track (31).Although MT assembly does not appear to be directly regulated by small GTPases, Rab proteins provide a molecular link for vesicle movement along MTs to the appropriate target (22, 32-34). In this study, the potential interaction of Rab2 with MTs and motor proteins was characterized. We found that Rab2 does not bind directly to preassembled MTs but does associate when both GAPDH and aPKCι are present and bound to MTs. Moreover, the MTs predominantly contained tyrosinated α-tubulin (Tyr-tubulin) suggesting that a dynamic pool of MTs that differentially binds MAPs/effector proteins/motors associates with VTCs in response to Rab2. To that end, we determined that Rab2-promoted dynein/dynactin binding to membranes and that the recruitment required aPKCι.     

Evaluation of cause of deaths' validity using outcome measures from a prospective, population based cohort study in Tehran, Iran

Objective

The aim of this study was to evaluate the validity of cause of death stated in death certificates in Tehran using outcome measures of the Tehran Lipid and Glucose Study (TLGS), an ongoing prospective cohort study.

Methods

The cohort was established in 1999 in a population of 15005 people, 3 years old and over, living in Tehran; 3551 individuals were added to this population three years later. As part of cohort''s outcome measures, deaths occurring in the cohort are investigated by a panel of medical specialists (Cohort Outcome Panel-COP) and underlying cause of death is determined for each death. The cause of death assigned in a deceased''s original death certificate was evaluated against the cause of death determined by COP and sensitivity and positive predictive values (PPV) were determined. In addition, determinants of assigning accurate underlying cause of death were determined using logistic regression model.

Result

A total of 231 death certificates were evaluated. The original death certificates over reported deaths due to neoplasms and underreported death due to circulatory system and transport accidents. Neoplasms with sensitivity of 0.91 and PPV of 0.71 were the most valid category. The disease of circulatory system showed moderate degree of validity with sensitivity of 0.67 and PPV of 0.78. The result of logistic regression indicated if the death certificate is issued by a general practitioner, there is 2.3 (95% CI 1.1, 5.1) times chance of being misclassified compared with when it is issued by a specialist. If the deceased is more than 60 years, the chance of misclassification would be 2.5 times (95% CI of 1.1, 5.9) compared with when the deceased is less than 60 years.     

Orexin receptor type-1 antagonist SB-334867 inhibits the development of morphine analgesic tolerance in rats

Herein the effect of orexin receptor type-1 antagonist SB-334867 on the development of tolerance to analgesic effects of morphine was studied in rats. To incite tolerance, morphine sulfate was injected intraperitoneally (i.p., 10mg/kg) once a day for 7 days. The tail flick test was used to evaluate antinociceptive effects of the morphine. A selective OxR1 receptor antagonist, SB-334867, was microinjected (i.c.v.) into the right cerebral ventricle (10 μg/10 μl) immediately before each morphine injection. Repeated morphine application resulted in tolerance to morphine analgesic effects as a decreasing trend during 7 days. Also, repeated administration of SB-334867 (i.c.v.) alone was without significant effect on the nociception as compared to control. Microinjection of SB-334867 prior to each morphine injection inhibited the development of tolerance, so that the analgesic effects of morphine were significantly higher in SB-334867 plus morphine treated rats than that of vehicle plus morphine treated ones on days 4-7. It is concluded that orexin receptor type-1 might be involved in the development of tolerance to morphine analgesic effects.     

Reference Values for Serum Zinc Concentration and Prevalence of Zinc Deficiency in Adult Iranian Subjects

Zinc, as an essential trace element for health, plays various biological roles in human body functions. Serum zinc reference values are essential for assessing zinc-associated abnormalities and the prevalence of zinc deficiency. This study aims at determining age- and sex-specific reference values for serum zinc concentrations in adult Iranian subjects. Serum zinc concentration was measured by flame atomic absorption spectrometry in 4,698 adult subjects, aged 20?C94?years, randomly selected from the population of the Tehran, Lipid, and Glucose Study. After application of exclusion criteria, reference values for serum zinc were determined in 2,632 apparently healthy subjects according to guidelines of the International Federation of Clinical Chemistry (nonparametric method). Dietary zinc was assessed in 2,906 individuals, of which 1,685 were healthy subjects, using a validated semiquantitative food frequency questionnaire. Reference values for serum zinc concentrations ranged between 9.6 and 31.6, 8.9 and 29.9, and 9.3 and 30.8???mol/L in men, women, and the total population, respectively. Prevalence of serum zinc deficiency was 3.0 and 2.4?% in men and women, respectively (p?=?0.267); in men, but not in women, the prevalence increased significantly with age (p for trend <0.001). Of the total participants, 10.3?% (6.5 men and 3.8?% women, p?<?0.01) had lower zinc intake compared to dietary reference intakes. The zinc density of the population was 6.3?mg/1,000?kcal. In conclusion, this study presents reference values for serum zinc concentration in adult Iranian subjects for both sexes and different age groups. Prevalence of serum zinc deficiency and dietary zinc inadequacy seems to be lower in Iranians, compared to some other populations.     

Haplotype frequency distribution for 7 microsatellites in chromosome 8 and 11 in relation to the metabolic syndrome in four ethnic groups: Tehran Lipid and Glucose Study

Introduction

Different variants of haplotype frequencies may lead to various frequencies of the same variants in individuals with drug resistance and disease susceptibility at the population level.

Materials and methods

In this study, the haplotype frequencies of 4 STR loci including the D8S1132, D8S1779, D8S514 and D8S1743, and 3 STR loci including D11S1304, D11S1998 and D11S934 were investigated in 563 individuals of four Iranian ethnic groups in the capital city of Iran, Tehran. One hundred thirty subjects had the metabolic syndrome. Haplotype frequencies of all markers were calculated.

Results

There were significant differences in the haplotype frequencies in short and long alleles between the metabolic affected subjects and controls. In addition, haplotype frequencies were significant in the four ethnic groups in both chromosomes 8 and 11.

Conclusion

Our findings show a relation between the short allele of D8S1743 in all related haplotype frequencies of subjects with metabolic syndrome. These findings may require more studies of some candidate genes, including the lipoprotein lipase gene, in this chromosomal region.     

Incidence of Chronic Kidney Disease and Its Risk Factors,Results of Over 10 Year Follow Up in an Iranian Cohort

To examine, the predictors of incident chronic kidney disease (CKD) in a community-based cohort of Middle East population, during a mean follow-up of 9.9 years. In a sample of 3313 non-CKD Iranian adults ≥20 years the estimated glomerular filtration rate (eGFR) was calculated at baseline and at three year intervals during three consecutive phases. The eGFR <60 mL/min/1.73 m2 was defined as CKD. Multivariate Logistic regression analysis was used to determine the independent variables associated with incident CKD. The incidence density rates of CKD were 285.3 and 132.6 per 10,000 person-year, among women and men, respectively. Female gender per se was associated with higher risk of CKD, compared with males. Among women, age, eGFR, known diabetes, being single or divorced/widowed, hypertension (marginally significant) and current smoking were independent risk factors for CKD; however the intermediate degree of education and family history of diabetes decreased the risk by 40% (P<0.05). Among male subjects, independent predictors of developing CKD included aging and hypertension (with significantly higher risk than in women, P for interaction<0.05), eGFR, new diagnosed diabetes, high normal blood pressure; abdominal obesity decreased the risk of CKD about 30% which was marginally significant. In the Iranian population,>2% of individuals develops CKD each year. Our findings confirmed that sex- specific risk predictors should be considered in primary prevention for incident CKD.     

Oncogenic human papillomavirus genital infection in southern Iranian women: population-based study versus clinic-based data

ABSTRACT: BACKGROUND: Epidemiological studies on genital human papilloma viruses infection (HPVs) in general population are crucial for the implementation of health policy guidelines for developing the strategies to prevent the primary and secondary cervical cancer. In different parts of Iran, there is a lack of population-based studies to determine the prevalence of HPV in the general population. The aim of this population-based study is to compare the prevalence rate of genital HPV infection among reproductive women with our previous clinic-based data, which showed a prevalence rate of 5% in women in southern Iran. RESULTS: Using general primers for all genotypes of HPV, of 799 randomly selected women, five (0.63%, 95% CI 0.23-1.55%) tested positive for HPV DNA. Overall, seven different HPV genotypes were detected: six types (16, 18, 31, 33, 51 and 56) were carcinogenic, or "high risk genotypes" and one genotype (HPV-66) was "probably carcinogenic." CONCLUSIONS: In a population-based study, the prevalence of HPV infection among southern Iranian women was lower than that observed worldwide. However, our gynaecological clinic-based study on the prevalence of HPV infection showed results comparable with other studies in the Middle East and Persian Gulf countries. Since gynaecological clinic-based data may generally overestimate HPV prevalence, estimates of prevalence according to clinic-based data should be adjusted downward by the population-based survey estimates.     

Targeting high affinity and epitope-distinct oligoclonal nanobodies to HER2 over-expressing tumor cells

Modern anti-HER2 antibody therapy tends to exploit a panel of different antibodies against different epitopes on the antigen. For this aim, nanobodies are very striking targeting agents and can be easily produced against any cell-specific membrane antigen. The oligoclonal nanobodies can be used to block more than one functional epitope on a target antigen and inhibit the generation of escape variants associated with cancer therapy. In this study, 12 nanobody clones selected from an immune camel library were examined for their ability to differ between tumor markers. These oligoclonal nanobodies targeted breast cancer cells better than each individual nanobody. In epitope mapping, several nanobodies overlapped in the epitope recognized by trastuzumab and some of the non-overlapping nanobodies could affect the binding of trastuzumab to HER2. This study demonstrates that the oligoclonal nanobodies are potential therapeutic tools that can be used instead of, or in combination with trastuzumab to assess tumor viability during treatment.     

Patterns of axial and appendicular movements during aquatic walking in the salamander Siren lacertina

Most studies of salamander locomotion have focused either on swimming or terrestrial walking, but some salamanders also use limb-based locomotion while submerged under water (aquatic walking). In this study we used video motion analysis to describe the aquatic walking gait of Siren lacertina, an elongate salamander with reduced forelimbs and no hindlimbs. We found that S. lacertina uses a bipedal-undulatory gait, which combines alternating use of the forelimbs with a traveling undulatory wave. Each forelimb is in contact with the substrate for about 50% of the stride cycle and forelimbs have little temporal overlap in contact intervals. We quantified the relative timing and frequency of limb and tail movements and found that, unlike the terrestrial gaits of most salamanders, axial and appendicular movements are decoupled during aquatic walking. We found no significant relationship between stride frequency and aquatic walking velocity, but we did find a statistically significant relationship between tailbeat frequency and aquatic walking velocity, which suggests that aquatic walking speed is mainly modulated by axial movements. By comparing axial wavespeed and distance traveled per tailbeat during swimming (forelimbs not used) and aquatic walking (forelimbs used), we found lower wavespeed and greater distance traveled per tailbeat during aquatic walking. These findings suggest that the reduced forelimbs of S. lacertina contribute to forward propulsion during aquatic walking.