A novel metabolic form of the 32 kDa-D1 protein in the grana-localized reaction center of photosystem II

Two forms of the 32 kDa-D1 reaction center protein of photosystem II (PSII), having slightly different mobilities on denaturing polyacrylamide gels, have been resolved in Spirodela oligorrhiza, Glycine max L., Gossypium hirsutum L., Triticum aestivum L., and Zea mays L. The protein band with faster mobility is identified as the 32 kDa-D1 protein, and the less mobile band as a novel form, designated 32*. The two forms are structurally similar based on immunological and partial proteolytic tests. 32* is associated exclusively with the grana and is present in the PSII reaction center. Temporally, 32* appears several hours after the translocation of newly synthesized and processed 32 kDa-D1 protein from the stroma lamellae to the grana. Formation of the 32* is strictly light-dependent under physiological light intensities and correlates with a reciprocal loss of the 32-kDa form. Light induced formation of 32* is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is not coupled to linear electron transport.     

MMP-9 gene ablation mitigates hyperhomocystenemia-induced cognition and hearing dysfunction

Hyperhomocysteinemia (HHcy) is associated with cognitive decline and hearing loss due to vascular dysfunction. Although we have shown that HHcy-induced increased expression of matrix metalloproteinase-9 (MMP-9) is associated with cochlear pathology in cystathionine-β-synthase heterozygous (CBS^+/?) mice, it is still unclear whether MMP-9 contributes to functional deficit in cognition and hearing. Therefore, we hypothesize that HHcy-induced MMP-9 activation causes vascular, cerebral and cochlear remodeling resulting in diminished cognition and hearing. Wildtype (WT), CBS^+/?, MMP-9^?/? and CBS^+/?/MMP-9^?/? double knock-out (DKO) mice were genotyped and used. Doppler flowmetry of internal carotid artery (ICA) was performed for peak systolic velocity [PSV], pulsatility index [PI] and resistive index [RI]. Cognitive functions were assessed by Novel Object Recognition Test (NORT) and for cochlear function Auditory brainstem response (ABR) was elicited. Peak systolic velocity, pulsatility and resistive indices of ICA were decreased in CBS^+/? mice, indicating reduced perfusion. ABR threshold was increased and maximum ABR amplitude and NORT indices (recognition, discrimination) were decreased in CBS^+/? mice compared to WT and MMP-9^?/?. All these parameters were attenuated in DKO mice suggesting a significant role of MMP-9 in HHcy-induced vascular, neural and cochlear pathophysiology. Regression analysis of PSV with ABR and cognitive parameters revealed significant correlation (0.44–0.58). For the first time, MMP-9 has been correlated directly to functional deficits of brain and cochlea, and found to have a significant role. Our data suggests a dual pathology of HHcy occurring due to a decrease in blood supply (vasculo-neural and vasculo-cochlear) and direct tissue remodeling.     

Mediator structure and conformation change

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Exploring mechanisms of sex differences in longevity: lifetime ovary exposure and exceptional longevity in dogs

To move closer to understanding the mechanistic underpinnings of sex differences in human longevity, we studied pet dogs to determine whether lifetime duration of ovary exposure was associated with exceptional longevity. This hypothesis was tested by collecting and analyzing lifetime medical histories, age at death, and cause of death for a cohort of canine ‘centenarians’– exceptionally long‐lived Rottweiler dogs that lived more than 30% longer than average life expectancy for the breed. Sex and lifetime ovary exposure in the oldest‐old Rottweilers (age at death, ≥ 13 years) were compared to a cohort of Rottweilers that had usual longevity (age at death, 8.0–10.8 years). Like women, female dogs were more likely than males to achieve exceptional longevity (OR, 95% CI = 2.0, 1.2–3.3; P = 0.006). However, removal of ovaries during the first 4 years of life erased the female survival advantage. In females, a strong positive association between ovaries and longevity persisted in multivariate analysis that considered other factors, such as height, body weight, and mother with exceptional longevity. A beneficial effect of ovaries on longevity in females could not be attributed to resistance against a particular disease or major cause of death. Our results document in dogs a female sex advantage for achieving exceptional longevity and show that lifetime ovary exposure, a factor not previously evaluated in women, is associated with exceptional longevity. This work introduces a conceptual framework for designing additional studies in pet dogs to define the ovary‐sensitive biological processes that promote healthy human longevity.     

Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

Lignin confers recalcitrance to plant biomass used as feedstocks in agro‐processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3‐dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3‐dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild‐type plants, lines expressing QsuB contain higher amounts of protocatechuate, p‐coumarate, p‐coumaraldehyde and p‐coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D‐NMR spectroscopy and pyrolysis‐gas chromatography/mass spectrometry (pyro‐GC/MS) reveal an increase of p‐hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size‐exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain‐of‐function strategy for spatiotemporal reduction of lignin in plant biomass.     

How Well Does the World Health Organization Definition of Domestic Violence Work for India?

Domestic violence (DV) is reported by 40% of married women in India and associated with substantial morbidity. An operational research definition is therefore needed to enhance understanding of DV epidemiology in India and inform DV interventions and measures. To arrive at a culturally-tailored definition, we aimed to better understand how definitions provided by the World Health Organization and the 2005 India Protection of Women from Domestic Violence Act match the perceptions of behaviors constituting DV among the Indian community. Between September 2012 and January 2013, 16 key informant interviews with experts in DV and family counseling and 2 gender-concordant focus groups of lay community members were conducted in Pune, India to understand community perceptions of the definition of DV, perpetrators of DV, and examples of DV encountered by married women in Pune, India. Several key themes emerged regarding behaviors and acts constituting DV including 1) the exertion of control over a woman’s reproductive decision-making, mobility, socializing with family and friends, finances, and access to food and nutrition, 2) the widespread acceptance of sexual abuse and the influences of affluence on sexual DV manifestations, 3) the shaping of physical abuse experiences by readily-available tools and the presence of witnesses, 4) psychological abuse for infertility, dowry, and girl-children, and 5) the perpetration of DV by the husband and other members of his family. Findings support the need for a culturally-tailored operational definition that expands on the WHO surveillance definition to inform the development of more effective DV intervention strategies and measures.     

Cadherin Expression,Vectorial Active Transport,and Metallothionein Isoform 3 Mediated EMT/MET Responses in Cultured Primary and Immortalized Human Proximal Tubule Cells

Background

Cultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells.

Methods

Immunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells.

Results

It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells.

Conclusions

The results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.     

The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells

Glioblastoma multiforme (GBM) is the most common and most aggressive malignant brain tumor. Despite optimal treatment and evolving standard of care, the median survival of patients diagnosed with GBM is only 12–15 months. In this study, we combined progesterone (PROG) and temozolomide (TMZ), a standard chemotherapeutic agent for human GBM, to test whether PROG enhances the antitumor effects of TMZ and reduces its side effects. Two WHO grade IV human GBM cells lines (U87MG and U118MG) and primary human dermal fibroblasts (HDFs) were repeatedly exposed to PROG and TMZ either alone or in combination for 3 and 6 days. Cell death was measured by MTT reduction assay. PROG and TMZ individually induced tumor cell death in a dose-dependent manner. PROG at high doses produced more cell death than TMZ alone. When combined, PROG enhanced the cell death-inducing effect of TMZ. In HDFs, PROG did not reduce viability even at the same high cytotoxic doses, but TMZ did so in a dose-dependent manner. In combination, PROG reduced TMZ toxicity in HDFs. PROG alone and in combination with TMZ suppressed the EGFR/PI3K/Akt/mTOR signaling pathway and MGMT expression in U87MG cells, thus suppressing cell proliferation. PROG and TMZ individually reduced cell migration in U87MG cells but did so more effectively in combination. PROG enhances the cytotoxic effects of TMZ in GBM cells and reduces its toxic side effects in healthy primary cells.