MKP-1 expression and stabilization and cGK Ialpha prevent diabetes- associated abnormalities in VSMC migration

Diabetes mellitus is a major risk factor in the development of atherosclerosis and cardiovascular disease conditions, involving intimal injury and enhanced vascular smooth muscle cell (VSMC) migration. We report a mechanistic basis for divergences between insulin's inhibitory effects on migration of aortic VSMC from control Wistar Kyoto (WKY) rats versus Goto-Kakizaki (GK) diabetic rats. In normal WKY VSMC, insulin increased MAPK phosphatase-1 (MKP-1) expression as well as MKP-1 phosphorylation, which stabilizes it, and inhibited PDGF-mediated MAPK phosphorylation and cell migration. In contrast, basal migration was elevated in GK diabetic VSMCs, and all of insulin's effects on MKP-1 expression and phosphorylation, MAPK phosphorylation, and PDGF-stimulated migration were markedly inhibited. The critical importance of MKP-1 in insulin inhibition of VSMC migration was evident from several observations. MKP-1 small interfering RNA inhibited MKP-1 expression and abolished insulin inhibition of PDGF-induced VSMC migration. Conversely, adenoviral expression of MKP-1 decreased MAPK phosphorylation and basal migration rate and restored insulin's ability to inhibit PDGF-directed migration in GK diabetic VSMCs. Also, the proteasomal inhibitors lactacystin and MG132 partially restored MKP-1 protein levels in GK diabetic VSMCs and inhibited their migration. Furthermore, GK diabetic aortic VSMCs had reduced cGMP-dependent protein kinase Ialpha (cGK Ialpha) levels as well as insulin-dependent, but not sodium nitroprusside-dependent, stimulation of cGMP. Adenoviral expression of cGK Ialpha enhanced MKP-1 inhibition of MAPK phosphorylation and VSMC migration. We conclude that enhanced VSMC migration in GK diabetic rats is due at least in part to a failure of insulin-stimulated cGMP/cGK Ialpha signaling, MKP-1 expression, and stabilization and thus MAPK inactivation.     

Daily increments of light hours near vernal equinox synchronize circannual testicular cycle of tropical spotted munia

In some long-lived organisms, particularly in tropical birds and migrants that spend part of the year close to the equator, endogenous circannual rhythms have been demonstrated in seasonal events like reproduction, molt, and migration. These, like the circadian rhythms, are expressed only in constant conditions of illumination with a periodicity deviating from 1 yr. If birds followed this periodicity, they would soon be out of phase with the annual calendar and perish and, therefore, they would need to be synchronized. However, almost nothing is known as to how synchronization is achieved in birds. Herein, with the help of a suitable model, viz., the tropical spotted munia and long-term experiments conducted in series over a 5-yr period, we provide direct evidence for the first time indicating that the segment of annual photocycle with maximal rate of increase prior to vernal equinox (approximately between mid-February and mid-March) synchronizes the circannual reproductive cycle with the monsoon period of ample food supply through a phase delay. Data also indicate, contrary to the prevalent view, that birds in the tropics can perceive minor changes in day-length, that birds respond to progressive changes in day-length as distinct from responding to fixed photoperiods of particular durations, and that birds can actually distinguish the quality of the environmental signal, viz., vernal equinox from early spring, or increasing days of spring from decreasing days of autumn. The underlying mechanisms, although yet to be identified, appear to involve a gonado-inhibitory rather than the conventional gonado-stimulatory response to increasing day-length. The photoperiodic responses of spotted munia are distinctly different from that of any "long-day" birds described thus far and do not conform to the prevalent "circadian coincidence" hypothesis of photoperiodism.     

The C-terminus of CaMKII is truncated when expressed in E. coli

The neuronal enzyme Calcium/calmodulin dependent protein kinase type II (CaMKII) is a key molecule in biochemical events necessary for learning and memory. The alpha-subunit of CaMKII expressed in E. coli as well as in insect cells shows similar catalytic behavior [Praseeda, M., Pradeep, K. K., Krupa, A., Sri Krishna, S., Leena, S., Rajeev Kumar, R., John Cheriyan, Mayadevi, M., Srinivasan, N., and Omkumar, R. V. (2003) Biochem. J. In Press]. The association domain of the enzyme has been crystallized in its native multimeric form after expression in E. coli [Hoelz, A., Nairn, A. C. and Kuriyan, J. (2003) Molecular Cell 11, 1241]. However a major truncation product accompanies the full-length protein when expressed in E. coli. We show by epitope labeling and immunoblotting that the truncation occurs at the C-terminal half of the protein so that the N-terminal catalytic domain is complete in the truncated product. This supports the use of the preparation of alpha-CaMKII expressed in E. coli for studies on functions of the catalytic site. Our data will also be helpful in designing modified prokaryotic expression systems for CaMKII devoid of the trun-cation product, which are easier to use compared to the insect cell system.     

Antigen expression in biofilm cells of Aeromonas hydrophila employed in oral vaccination of fish

Total protein, S-layer protein and lipopolysaccharides (LPS) of biofilm cells of Aeromonas hydrophila were analysed by SDS-PAGE and compared with that of planktonic cells. In the whole cell lysate of biofilm cells, about 15 proteins were repressed while three new proteins were expressed compared to that in planktonic cells. Interestingly, in biofilm cells the S-layer proteins were lost and LPS showed an additional high molecular weight band compared to that in planktonic cells. We propose that the change in LPS profile must have contributed to the loss of S-layer. Also, the high molecular weight band of LPS might play a role in the better performance of biofilm oral vaccine by eliciting a protective immune response.     

Protein kinase inhibitors block the stimulation of the AMP-activated protein kinase by 5-amino-4-imidazolecarboxamide riboside

The AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that plays a major role in energy sensing. AMPK is activated pharmacologically by 5-amino-4-imidazolecarboxamide (AICA) riboside monophosphate (ZMP), which mimics the effects of AMP on the AMPK cascade. Here we show that uptake of AICA riboside into cells, mediated by the adenosine transport system, is blocked by a number of protein kinase inhibitors. Under these conditions, ZMP does not accumulate to sufficient levels to stimulate AMPK. Our results demonstrate that careful interpretation is required when using AICA riboside in conjunction with protein kinase inhibitors to investigate the physiological role of AMPK.     

A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I,V, L,N, A,and K

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.2 kcal/mol per dimer more stable than the A-A interaction and 4.0 kcal/mol per dimer more stable than either the L-L or V-V interaction, and 7.0 kcal/mol per dimer more stable than the N-N interaction. Only lysine was less stable than alanine. An alanine-based double-mutant thermodynamic cycle calculated coupling energies between the a and a' positions in the heterodimer. The aliphatic amino acids L, V, and I prefer to form homotypic interactions with coupling energies of -0.6 to -0.9 kcal/mol per dimer, but the heterotypic aliphatic interactions have positive coupling energies of <1.0 kcal/mol per dimer. The asparagine homotypic interaction has a coupling energy of -0.5 kcal/mol per dimer, while heterotypic interactions with the aliphatic amino acids produce coupling energies ranging from 2.6 to 4.9 kcal/mol per dimer. The homotypic K-K interaction is 2.9 kcal/mol per dimer less stable than the A-A interaction, but the coupling energy is only 0.3 kcal/mol per dimer. Heterotypic interactions with lysine and either asparagine or aliphatic amino acids produce similar coupling energies ranging from -0.2 to -0.7 kcal/mol per dimer. Thus, of the amino acids that were examined, asparagine contributes the most to dimerization specificity because of the large positive coupling energies in heterotypic interactions with the aliphatic amino acids which results in the N-N homotypic interaction.     

Birth rights and rituals in rural south India: care seeking in the intrapartum period

Maternal morbidity and mortality are high in the Indian context, but the majority of maternal deaths could be avoided by prompt and effective access to intrapartum care (WHO, 1999). Understanding the care seeking responses to intrapartum morbidities is crucial if maternal health is to be effectively improved, and maternal mortality reduced. This paper presents the results of a prospective study of 388 women followed through delivery and traditional postpartum in rural Karnataka in southern India. In this setting, few women use the existing health facilities and most deliveries occur at home. The analysis uses quantitative data, collected via questionnaires administered to women both during pregnancy and immediately after delivery. By virtue of its prospective design, the study gives a unique insight into intentions for intrapartum care during pregnancy as well as events following morbidities during labour. Routine care in the intrapartum period, both within institutions and at home, and impediments to appropriate care are also examined. The study was designed to collect information about health seeking decisions made by women and their families as pregnancies unfolded, rather than trying to capture women's experience from a retrospective instrument. The data set is therefore a rich source of quantitative information, which incorporates details of event sequences and health service utilization not previously collected in a Safe Motherhood study. Additional qualitative information was also available from concurrent in-depth interviews with pregnant women, their families, health care providers and other key informants in the area. The level of unplanned institutional care seeking during the intrapartum period within the study area was very high, increasing from 11% planning deliveries at a facility to an eventual 35% actually delivering in hospitals. In addition there was a significant move away from planned deliveries with the auxiliary nurse midwive (ANM), to births with a lay attendant or dai. The proportion of women who planned for an ANM to assist was 49%, as compared with the actual occurrence, which was less than half of this proportion. Perceived quality of care was found to be an important factor in health seeking behaviour, as was wealth, caste, education and experience of previous problems in pregnancy. Actual care given by a range of practitioners was found to contain both beneficial and undesirable elements. As a response to serious morbidities experienced within the study period, many women were able to seek care although sometimes after a long delay. Those women who experienced inadequate progression of labour pains were most likely to proceed unexpectedly to a hospital delivery.     

Neuromedin U has a physiological role in the regulation of food intake and partially mediates the effects of leptin

Intracerebroventricular (ICV) administration of Neuromedin U (NMU), a hypothalamic neuropeptide, or leptin, an adipostat hormone released from adipose tissue, reduces food intake and increases energy expenditure. Leptin stimulates the release of NMU in vitro, and NMU expression is reduced in models of low or absent leptin. We investigated the role of NMU in mediating leptin-induced satiety. ICV administration of anti-NMU immunoglobulin G (IgG) (5 nmol) to satiated rats significantly increased food intake 4 h after injection, an effect seen for 相似文献   

Repeated ICV administration of oxyntomodulin causes a greater reduction in body weight gain than in pair-fed rats

Oxyntomodulin (OXM) is a product of proglucagon processing in the intestine and the central nervous system. We reported that intracerebroventricular (ICV) and intranuclear administration of OXM caused an inhibition of food intake in rats (Dakin CL, Gunn I, Small CJ, Edwards CM, Hay DL, Smith DM, Ghatei MA, and Bloom SR. Endocrinology 142: 4244-4250, 2001). In this study, we investigated the effect of twice-daily ICV administration of OXM, 1 nmol, for 7 days. A pair-fed control was included. These animals were restricted to the food intake of the OXM group but injected twice daily with saline. OXM-treated animals gained significantly less weight than either control group (day 8: OXM, 12.2 +/- 1.9 g vs. pair fed, 21.0 +/- 2.1 g; P < 0.005). OXM treatment caused a reduction in epididymal white adipose tissue (OXM, 1.13 +/- 0.03 g vs. pair fed, 1.29 +/- 0.04 g; P < 0.05) and interscapular brown adipose tissue (OXM, 0.15 +/- 0.01 g vs. pair fed, 0.18 +/- 0.01 g; P < 0.05) and increased core temperature compared with saline control, suggestive of enhanced energy expenditure. The food restriction-induced suppression in plasma TSH, seen in the pair-fed group, was prevented by OXM, potentially via increased release of hypothalamic TRH. In summary, ICV OXM causes reduced body weight gain and body adiposity following chronic administration.     

Neurons are the Primary Target Cell for the Brain-Tropic Intracellular Parasite Toxoplasma gondii

Toxoplasma gondii, a common brain-tropic parasite, is capable of infecting most nucleated cells, including astrocytes and neurons, in vitro. Yet, in vivo, Toxoplasma is primarily found in neurons. In vitro data showing that interferon-γ-stimulated astrocytes, but not neurons, clear intracellular parasites suggest that neurons alone are persistently infected in vivo because they lack the ability to clear intracellular parasites. Here we test this theory by using a novel Toxoplasma-mouse model capable of marking and tracking host cells that directly interact with parasites, even if the interaction is transient. Remarkably, we find that Toxoplasma shows a strong predilection for interacting with neurons throughout CNS infection. This predilection remains in the setting of IFN-γ depletion; infection with parasites resistant to the major mechanism by which murine astrocytes clear parasites; or when directly injecting parasites into the brain. These findings, in combination with prior work, strongly suggest that neurons are not incidentally infected, but rather they are Toxoplasma’s primary in vivo target.