The UBIAD1 Prenyltransferase Links Menaquione‐4 Synthesis to Cholesterol Metabolic Enzymes

Schnyder corneal dystrophy (SCD) is an autosomal dominant disease characterized by germline variants in UBIAD1 introducing missense alterations leading to deposition of cholesterol in the cornea, progressive opacification, and loss of visual acuity. UBIAD1 was recently shown to synthesize menaquinone‐4 (MK‐4, vitamin K₂), but causal mechanisms of SCD are unknown. We report a novel c.864G>A UBIAD1 mutation altering glycine 177 to glutamic acid (p.G177E) in six SCD families, including four families from Finland who share a likely founder mutation. We observed reduced MK‐4 synthesis by UBIAD1 altered by SCD mutations p.N102S, p.G177R/E, and p.D112N, and molecular models showed p.G177‐mutant UBIAD1 disrupted transmembrane helices and active site residues. We show UBIAD1 interacts with HMGCR and SOAT1, enzymes catalyzing cholesterol synthesis and storage, respectively, using yeast two‐hybrid screening and immunoprecipitation. Docking simulations indicate cholesterol binds to UBIAD1 in the substrate‐binding cleft and substrate‐binding overlaps with GGPP binding, an MK‐4 substrate, suggesting potential competition between these metabolites. Impaired MK‐4 synthesis is a biochemical defect identified in SCD suggesting UBIAD1 links vitamin K and cholesterol metabolism through physical contact between enzymes and metabolites. Our data suggest a role for endogenous MK‐4 in maintaining cornea health and visual acuity.     

Somatic Alterations Contributing to Metastasis of a Castration‐Resistant Prostate Cancer

Metastatic castration‐resistant prostate cancer (mCRPC) is a lethal disease, and molecular markers that differentiate indolent from aggressive subtypes are needed. We sequenced the exomes of five metastatic tumors and healthy kidney tissue from an index case with mCRPC to identify lesions associated with disease progression and metastasis. An Ashkenazi Jewish (AJ) germline founder mutation, del185AG in BRCA1, was observed and AJ ancestry was confirmed. Sixty‐two somatic variants altered proteins in tumors, including cancer‐associated genes, TMPRSS2‐ERG, PBRM1, and TET2. The majority (n = 53) of somatic variants were present in all metastases and only a subset (n = 31) was observed in the primary tumor. Integrating tumor next‐generation sequencing and DNA copy number showed somatic loss of BRCA1 and TMPRSS2‐ERG. We sequenced 19 genes with deleterious mutations in the index case in additional mCRPC samples and detected a frameshift, two somatic missense alterations, tumor loss of heterozygosity, and combinations of germline missense SNPs in TET2. In summary, genetic analysis of metastases from an index case permitted us to infer a chronology for the clonal spread of disease based on sequential accrual of somatic lesions. The role of TET2 in mCRPC deserves additional analysis and may define a subset of metastatic disease.     

Cyclooxygenase-2 expression in uveal melanoma: novel classification of mixed-cell-type tumours

BACKGROUND: The expression of cyclooxygenase-2 (COX-2), an inducible prostaglandin (PG) synthase, has been investigated in various human malignant diseases, such as cutaneous melanoma. We investigated the expression of COX-2 in uveal melanoma and related the findings to prognostic factors. METHODS: In 40 cases of uveal melanoma, immunostaining for COX-2 was done. COX-2 expression was related to histopathological prognostic markers, such as cell type, the presence of lymphocytic infiltration and vascular closed loops in the tumour, and cytomorphometry results. RESULTS: COX-2 expression was found in 58% of the cases, and it correlated with markers of poor prognosis, such as epithelioid cell type and the presence of lymphocytic infiltration and vascular closed loops. The uveal melanomas expressing COX-2 had larger nuclei, as determined by cytomorphometry. INTERPRETATION: Whereas epithelioid tumours carry a worse prognosis than spindle cell tumours, until now it has not been possible to give a strong indication of prognosis in mixed-cell tumours. This study showed that mixed-cell tumours, representing the majority of uveal melanomas, may be further subclassified according to COX-2 expression, which serves as a marker of poor prognosis. The role of COX-2 in uveal melanoma should be further elucidated, and the use of COX-2 inhibitors warrants investigation as adjuvant treatment for this life-threatening malignant disease.     

The E5 Protein of HPV-6, but Not HPV-16, Associates Efficiently with Cellular Growth Factor Receptors

The transforming activity of the prototype E5 protein of bovine papillomavirus type 1 (BPV-1) is associated with its binding to, and activation of, both the platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. The E5 proteins of human papillomavirus types 6 and 16 (HPV-6, HPV-16) also transform rodent cells in the presence of the EGF receptor. In this study we examined whether epitope-tagged HPV E5 proteins could associate with three different tyrosine kinase-containing growth factor receptors: the EGF receptor, the erbB2 receptor, and the PDGF receptor. The HPV-6 E5 protein was found to associate efficiently with all three of these growth factor receptors, while the HPV-16 E5 protein did not. These findings suggest either that the in vitro transforming activities of HPV-6 and HPV-16 E5 proteins involve a similar mechanism unrelated to receptor binding (e.g., binding to the 16-kDa membrane pore protein) or that they proceed along distinct pathways, with receptor binding being important for HPV-6. Regardless of the ultimate mechanisms, the differences between the HPV-6 and HPV-16 E5 proteins in binding to growth factor receptors may potentially contribute to the distinctive morphologies of their respective neoplastic lesions.     

The Interconnection between Sympathetics, Microcirculation, and Insulin Resistance in Hypertension

The pathophysiology of the frequent association of insulin resistance and hypertension has not been elucidated. The skeletal muscle is the major site of insulin resistance; when stimulated with insulin, the hypertensive skeletal muscles extract less glucose than the normotensive. We postulate that hypertension-related changes in the skeletal muscle microcirculation contribute to the impaired glucose uptake in hypertension. Vascular rarefaction in hypertension impairs the delivery of insulin and glucose to muscle cells. Insulin resistance has been described both in human and experimental hypertension and both conditions are associated with vascular rarefaction. Functional studies (response to whole body or forearm exercise) and anatomic investigations (conjunctival photography, mesenteric and muscle biopsies) show vascular rarefaction in human hypertension. In addition, patients with hypertension are known to have a larger proportion of insulin resistant, poorly vascularized fast twitch muscle fibers. A few interventions can increase or decrease insulin resistance and these effects can be explained on hemodynamic grounds. Beta adrenergic blocking agents aggravate insulin resistance, and their main hemodynamic effect is a decrease of cardiac output. Converting enzyme inhibitors, alpha adrenergic blocking agents and possibly calcium antagonists decrease the insulin resistance, and their major hemodynamic effect is vasodilation. Physical training decreases insulin resistance; a higher capillary density in skeletal muscles is the hallmark of physical training. A hypothesis ought to rest on sufficient supporting data and its validity ought to lend itself to experimental verification. We believe our hypothesis meets both criteria. After outlining the supporting evidence we propose a number of tests to prove or disprove the hypothesis. In addition to the testable hypothesis we also speculate on the possible cause of the frequent association between hypertension and insulin resistance. We propose that both insulin resistance and blood pressure elevation represent a facet of the “defense reaction” which might have offered an early survival advantage and may, over evolutionary times, have fostered natural selection of subjects with both conditions.     

Recreational Exercise and Cardiovascular Status in the Rural Community of Tecumseh, Michigan

Background. Because of population stability, Tecumseh, a rural community in Michigan, was the site of health surveys over a period of three decades.

Methods. In the recent survey anthropometrics, blood pressure (BP), blood chemistry, echo/Doppller cardiac exam, personality and exercise questionnaires were collected on site.

Results. In this rural community 70% of subjects (447 men, 410 women, average age 30 years) do not engage in recreational exercise. Sedentary subjects were heavier (4 kg), had higher BP (2.3/2 mm Hg), faster heart rate (4 beats/min), and lower stroke volume (2 ml/m²) than physically more active subjects (p = 0.02 to 0.00001). Measures of cardiac structure and function were more favorable in exercising subjects. Cholesterol (+ 8 mg/dl), triglycerides (+ mg/dl) and insulin (+ 2.4 μU/ml) were higher and HDL cholesterol was lower (- 2 mg/dl) in the sedentary group (p = 0.04 to 0.003). Being sedentary was associated with more anxiety, anger and feeling time pressure (p = 0.001 to 0.00001). Exercise demands at work had no effect while even once a week recreational exercise was associated with a more favorable cardiovascular risk status. A difference in cardiovascular status between the sedentary and exercising subjects was not apparent throughout childhood, adolescence or early adult life, suggesting that subjects who exercise presently were not a priori healthier than presently sedentary subjects.

Conclusions. Seventy percent of the residents studied in Tecumseh are physically inactive and have a less favorable cardiac risk profile. Enhancement of exercise habits may beneficially affect cardiovascular status and, presumably, the prognosis.     

Methodological Aspects of Flow Cytometry DNA Analysis in Endometrial Carcinoma, with Special Reference to Sampling and Reproducibility

Two adjacent paraffin-embedded sections manifested concordance in ploidy status in 96% of cases (45/47), and the standard deviation (SD) for SPF was 2.7%. Analysis of 'micro-heterogeneity', within a distance of ≤700μ, yielded results for concordant ploidy status in 94% of cases, and the SD for SPF was 1.9% (n = 17). Frozen and paraffin-embedded material yielded concordant results for ploidy status in 87% (39/45) of cases, and SPF values were significantly lower (mean difference 1.5%) in the frozen samples. Diagnostic and repeat curettage material yielded concordant results for DNA ploidy status in 85% (40/47) of cases, and no significant difference in mean SPF (12% vs. 11%) was found. Discordant DNA ploidy results were attributable to small differences in the DNA histograms influencing the interpretation of near-diploid, near-tetraploid and small non-diploid cell populations, and the influence of debris on SPF estimation. On the basis of our findings and the practical advantage we recommend paraffin-embedded material from diagnostic curettage for FCM DNA analysis; the results are available sooner and the handling and transportation of tumor samples is more convenient.