Association between testosterone, estradiol and sex hormone binding globulin levels in men with type 1 diabetes with nephropathy

Male sex is a risk factor for development and progression of diabetic nephropathy; however, the relationship between sex hormone levels and diabetic nephropathy in type 1 diabetic men is unknown. This was a prospective follow-up study as part of the nationwide Finnish Diabetic Nephropathy (FinnDiane) Study; 297 patients were followed for 5.9 ± 1.5 years. Serum total testosterone (Tt) and estradiol (Te), calculated free testosterone (cFt) and estradiol (cFe) and sex hormone binding globulin were measured at baseline and correlated with urinary albumin excretion rate, estimated glomerular filtration rate and markers of metabolic syndrome. Diabetes without renal disease was associated with decreased Tt (p < 0.001), Te (p < 0.001) and cFt (p = 0.001) levels compared with healthy non-diabetic men. With progression of renal disease from micro- to macroalbuminuria, this decrease in serum Tt was even more pronounced. Cox regression showed that cFt and cFe were independent predictors of the progression from macroalbuminuria to end-stage renal disease. Our study shows that men with type 1 diabetes exhibit dysregulated sex hormone levels, which is most pronounced in men with progressive renal disease, suggesting that sex hormones may play a role in the pathogenesis of diabetic nephropathy associated with type 1 diabetes.     

The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation

Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinson's disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2-3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.     

Up- and down-modulation of liver cytochrome P450 activities and associated events in two murine malaria models

Background

The mechanisms by which malaria up and down-regulates CYP activities are not understood yet. It is also unclear whether CYP activities are modulated during non-lethal malaria infections. This study was undertaken to evaluate the time course of CYP alterations in lethal (Plasmodium berghei ANKA) and non-lethal (Plasmodium chabaudi chabaudi) murine malaria. Additionally, hypotheses on the association of CYP depression with enhanced nitric oxide (NO) production, and of CYP2a5 induction with endoplasmic reticulum dysfunction, enhanced haem metabolism and oxidative stress were examined as well.

Methods

Female DBA-2 and C57BL/6 mice were infected with P.berghei ANKA or P. chabaudi and killed at different post-infection days. Infection was monitored by parasitaemia rates and clinical signs. NO levels were measured in the serum. Activities of CYP1a (ethoxyresorufin-O-deethylase), 2b (benzyloxyresorufin-O-debenzylase), 2a5 (coumarin-7-hydroxylase) and uridine-diphosphoglucuronyl-transferase (UGT) were determined in liver microsomes. Glutathione-S-transferase (GST) activity and concentrations of gluthatione (GSH) and thiobarbituric acid-reactive substances (TBARS) were determined in the liver. Levels of glucose-regulated protein 78 (GRP78) were evaluated by immunoblotting, while mRNAs of haemoxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) were determined by quantitative RT-PCR.

Results

Plasmodium berghei depressed CYP1a and 2b and induced 2a5 in DBA-2 mice. In P.berghei-infected C57BL/6 mice CYP activities remained unaltered. In both strains, GST and UGT were not affected by P.berghei. Plasmodium c. chabaudi depressed CYP1a and 2b and induced 2a5 activities on the day of peak parasitaemia or near this day. CYP2a5 induction was associated with over-expression of HO-1 and enhanced oxidative stress, but it was not associated with GRP78 induction, a marker of endoplasmic reticulum stress. Plasmodium chabaudi increased serum NO on days near the parasitaemia peak in both strains. Although not elevating serum NO, P.berghei enhanced iNOS mRNA expression in the liver.

Conclusion

Down-regulation of CYP1a and 2b and induction of 2a5 occurred in lethal and non-lethal infections when parasitaemia rates were high. A contribution of NO for depression of CYP2b cannot be ruled out. Results were consistent with the view that CYP2a5 and HO-1 are concurrently up-regulated and suggested that CYP2a5 induction may occur in the absence of enhanced endoplasmic reticulum stress.     

Replicational organization of three weakly expressed loci in Physarum polycephalum

We previously mapped early-activated replication origins in the promoter regions of five abundantly transcribed genes in the slime mold Physarum polycephalum. This physical linkage between origins and genes is congruent with the preferential early replication of the active genes in mammalian cells. To determine how general this replicational organization is in the synchronous plasmodium of Physarum, we analyzed the replication of three weakly expressed genes. Bromodeoxyuridine (BrdUrd) density-shift and gene dosage experiments indicated that the redB (regulated in development) and redE genes replicate early, whereas redA replicates in mid-S phase. Bi-dimensional gel electrophoresis revealed that redA coincides with an origin that appears to be activated within a large temporal window in S phase so that the replication of the gene is not well defined temporally. The early replication of the redB and redE genes is due to the simultaneous activation of flanking origins at the onset of S phase. As a result, these two genes correspond to termination sites of DNA replication. Our data demonstrate that not all the Physarum promoters are preferred sites of initiation but, so far, all the expressed genes analyzed in detail either coincide with a replication origin or are embedded into a cluster of early firing replicons.     

Buoyant density gradient fractionation and flow cytometric analysis of embryonic rat cortical neurons and progenitor cells

We have used the property of natural cell buoyant density to selectively fractionate embryonic rat neocortical cells into 20 subpopulations ranging in phenotype from proliferatively active progenitors to terminally postmitotic neurons. Immunocytochemical and cell cycle analysis of the cellular fractions with flow cytometry revealed an inverse relationship between cell buoyant density and neuronal differentiation. The most buoyant fractions contained predominantly terminally postmitotic, tubulin betaIII-positive, tetanus toxin-positive, and nestin-negative differentiating neurons, while immature, bromodeoxyuridine-positive and nestin-positive proliferating cells were more prevalent in less buoyant fractions. Double loading of isolated cells with voltage- and Ca2+-sensitive fluorescent indicator dyes followed by simultaneous recordings of membrane potential and cytoplasmic [Ca2+] ([Ca2+]c]) using flow cytometry revealed that >50% of the least buoyant cells produced functional responses to veratridine, a Na+ channel agonist, and muscimol, a GABA(A) receptor agonist, but <10% responded to kainic acid, an agonist of a subset of glutamate receptors. As cells became more buoyant the percentage of cells that depolarized and produced a rise in [Ca2+]c to each ligand increased, particularly in response to kainic acid. Short-term culture of select fractions revealed a marked enrichment for cells with morphologies and epitopes characteristic of neuronal and progenitor cell subpopulations. The results show that embryonic cortical cells exhibit a range of naturally occurring buoyant densities that can be used to expeditiously fractionate cortical cells according to their pre- or postmitotic status, thus providing ready access for cellular and molecular studies of proliferation and differentiation.     

Remodeling of chromatin loops does not account for specification of replication origins during Xenopus development

We have investigated the possible relationship between replicons and chromatin loops during Xenopus development. In early embryos, replication of the ribosomal RNA genes (rDNA) can initiate at apparently any sequence. Nevertheless, the need for a regular spacing of replication origins suggests that some periodic chromatin folding might dictate which sites are actually used for initiation. After the midblastula transition, replication initiation is restricted to the rDNA intergenic spacers. A remodeling of chromatin folding could account for this change in origin usage. Here, it is reported that nuclear matrix anchorage of the Xenopus rDNA occurs at multiple, apparently random sequences, throughout embryonic development as well as in adult cells. In vitro matrix rebinding assays confirmed the lack of specific anchoring sequences in the rDNA, before as well as after specific replication origins are established. Thus, no change in loop attachment sites could explain the change in origin usage at this locus. Nonspecific loop anchorage was a special feature of the rDNA locus, since the same nuclear matrices were able selectively to bind the scaffold attachment region (SAR) of the Drosophila histone gene cluster in vitro. Blastula and gastrula nuclear matrices bound a higher amount of SAR sequences than matrices from later stages or adult cells. This developmental change in SAR binding might explain the increase in size of the bulk of genomic DNA loops that occurs after the gastrula stage. However, no change in chromatin loop organization that could explain the midblastula stage transition from small to large replicons was observed. Received: 15 January 1998; in revised form: 4 March 1998 / Accepted: 9 March 1998