Vitamin B-6 deficiency in rats reduces hepatic serine hydroxymethyltransferase and cystathionine beta-synthase activities and rates of in vivo protein turnover, homocysteine remethylation and transsulfuration

Vitamin B-6 deficiency causes mild elevation in plasma homocysteine, but the mechanism has not been clearly established. Serine is a substrate in one-carbon metabolism and in the transsulfuration pathway of homocysteine catabolism, and pyridoxal phosphate (PLP) plays a key role as coenzyme for serine hydroxymethyltransferase (SHMT) and enzymes of transsulfuration. In this study we used [H-2(3)]serine as a primary tracer to examine the remethylation pathway in adequately nourished and vitamin B-6-deficient rats pi and 0.1 mg pyridoxine (PN)/kg diet]. [H-2(3)]Leucine and [1-C-13]methionine were also used to examine turnover of protein and methionine pools, respectively, All tracers were injected intraperitoneally as a bolus dose, and then rats were killed (n = 4/time point) after 30, 60 and 120 min. Rats fed the low-PN diet had significantly lower growth and plasma and liver PLP concentrations, reduced liver SHMT activity, greater plasma and liver total homocysteine concentration, and reduced liver S-adenosylmethionine concentration. Hepatic and whole body protein turnover were reduced in vitamin B-6-deficient rats as evidenced by greater isotopic enrichment of [H-2(3)]leucine. Hepatic [H-2(2)]methionine production from [H-2(3)]serine via cytosolic SHMT and the remethylation pathway was reduced by 80.6% in vitamin B-6 deficiency. The deficiency did not significantly reduce hepatic cystathionine-beta-synthase activity, and in vivo hepatic transsulfuration flux shown by production of [H-2(3)]cysteine from the [H-2(3)]serine increased over twofold. In contrast, plasma appearance of [H-2(3)]cysteine was decreased by 89% in vitamin B-6 deficiency. The rate of hepatic homocysteine production shown by the ratio of [1-C-13]homocysteine/[1-C-13]methionine areas under enrichment vs. time curves was not affected by vitamin B-6 deficiency. Overall, these results indicate that vitamin B-6 deficiency substantially affects one-carbon metabolism by impairing both methyl group production for homocysteine remethylation and flux through whole-body transsulfuration.

The role of lipids and protein kinase Cs in the pathogenesis of diabetic retinopathy

Diabetic retinopathy is one of the most common complications of diabetes and is a major cause of new blindness in the working-age population of developed countries. While the exact pathogenic basis of this condition remains ill defined, it is clear that hyperglycaemia is a critical factor in its aetiology. Protein kinase C (PKC) activation is one of the sequelae of hyperglycaemia and it is thought to play an important role in the development of diabetic complications. This review questions the currently held dogma that PKC stimulation in diabetes is solely mediated through the overproduction of palmitate and oleate enriched diacylglycerols. Blood glucose concentrations are closely tracked by changes in the levels of free fatty acids and these, in addition to oxidative stress, may account for the aberrant activation of PKCs in diabetes. Little is known about why PKCs fail to downregulate in diabetes and efforts should be directed towards acquiring such information. Considerable evidence implicates the PKCbeta isoform in the pathogenesis of diabetic retinopathy, but other isoforms may also be of relevance. In addition to PKCs, it is evident that novel diacyglycerol-activated non-kinase receptors could also play a role in the development of diabetic complications. Therapeutic agents have been developed to inhibit specific PKC isoforms and PKCbeta antagonists are currently undergoing clinical trials to test their toxicity and efficacy in suppressing diabetic complications. The likely impact of these drugs in the treatment of diabetic patients is considered.

Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27(Kip-1)

Connective tissue growth factor (CTGF/CCN2) is a 38-kDa secreted protein, a prototypic member of the CCN family, which is up-regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down-regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27(Kip-1). CTGF stimulated the phosphorylation and cytoplasmic translocation of p27(Kip-1) on serine 10. Addition of the PI-3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate prevented p27(Kip-1) phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27(Kip-1) colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27(Kip-1), revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27(Kip-1) cytoplasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27(Kip-1). Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27(Kip-1) activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF-stimulated actin depolymerization only in wild-type mouse embryonic fibroblasts (MEFs) compared to Akt-1/3 (PKB alpha/gamma) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27(Kip-1) as a mediator of actin rearrangement.

MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival

Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Janus kinase inhibition and its effect upon the therapeutic landscape for myelofibrosis:from palliation to cure?

Following the discovery of the Janus kinase (JAK) 2 V617F mutation in 2005 the explosion of research and drug development activity has not only advanced our understanding of the pathogenesis of myeloproliferative neoplasms (MPNs) but also triggered debate about classification, allowed revised diagnostic and response criteria, provided a target for treatment and a mode of monitoring its success. These changes and the resultant clinical research are discussed in this article where we argue that discovery of the JAK2 V617F mutation has signalled the much delayed change in therapeutic paradigm for myelofibrosis and possibly other MPNs from palliation and allowing us to move closer to, but not yet attain, a cure.

Abnormal changes in voltage-gated sodium channels NaV1.1, NaV1.2, NaV1.3, NaV1.6 and in Calmodulin/calmodulin-dependent protein kinase II, within the brains of spontaneously epileptic rats and tremor rats

Voltage-gated sodium channels (VGSCs) play a crucial role in epilepsy. The expressions of different VGSCs subtypes are varied in diverse animal models of epilepsy that may reflect their multiple phenotypes or the complexity of the mechanisms of epilepsy. In a previous study, we reported that NaV1.1 and NaV1.3 were up-regulated in the hippocampus of the spontaneously epileptic rat (SER). In this study, we further analyzed both the expression and distribution of the typical VGSC subtypes NaV1.1, NaV1.2, NaV1.3 and NaV1.6 in the hippocampus and in the cortex of the temporal lobe of two genetic epileptic animal models: the SER and the tremor rat (TRM). The expressions of calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII) were also analyzed with the purpose of assessing the effect of the CaM/CaMKII pathway in these two models of epilepsy. Increased expression of the four VGSC subtypes and CaM, accompanied by a decrease in CaMKII was observed in the hippocampus of both the SERs and the TRM rats. However, the changes observed in the expression of VGSC subtypes and CaM were decreased with an elevated CaMKII in the cortex of their temporal lobes. Double-labeled immunofluorescence data suggested that in SERs and TRM rats, the four subtypes of the VGSC proteins were present throughout the CA1, CA3 and dentate gyrus regions of the hippocampus and temporal lobe cortex and these were co-localized in neurons with CaM. These data represent the first evidence of abnormal changes in expression of four VGSC subtypes (NaV1.1, NaV1.2, NaV1.3 and NaV1.6) and CaM/CaMKII in the hippocampus and temporal lobe cortex of SERs and TRM rats. These changes may be involved in the generation of epileptiform activity and underlie the observed seizure phenotype in these rat models of genetic epilepsy.

Autologous limbal transplantation in patients with unilateral corneal stem cell deficiency

Aim - To describe a surgical technique for autologous limbal stem cell transplantation and the outcome of a series of patients with unilateral stem cell deficiency. Methods - A report of six consecutive patients who underwent autologous limbal stem cell transplantation is presented. The primary diagnosis included alkali burn (n = 3), conjunctival intraepithelial neoplasia (CIN) (n = 1), recurrent pterygium (n = 1), and contact lens induced keratopathy (n = 1). The autologous transplanted tissue consisted of peripheral cornea, limbus, and conjunctiva obtained from the contralateral eye. Three of the above patients underwent penetrating keratoplasty in association with autolimbal transplantation. A significant modification to established techniques was the close monitoring of conjunctival epithelial migration in the immediate postoperative period. If conjunctival epithelium threatened to migrate on to the corneal surface, it was mechanically removed at the slit lamp and prevented from crossing the limbus. This was required in three patients. Results - The mean follow up was 18.8 months. The outcome was satisfactory in all cases: a stable corneal surface was restored and there was a substantial improvement in vision and symptoms. One patient had a primary failure of the corneal allograft associated with glaucoma, and 6 months later developed a retinal detachment. No complications were noted in the donor eye with the exception of one patient who developed filamentary keratitis along the edge of the donor site. Conclusion - Autologous limbal transplantation with corneal, limbal, and conjunctival carriers was found to be useful for ocular surface reconstruction, over a mid-term follow up, in patients with unilateral stem cell deficiency. Close monitoring of the migration of conjunctival epithelium in the immediate postoperative period, and preventing it from crossing the limbus, ensured that the corneal surface was re-epithelialised exclusively from epithelial cells derived from the transplanted limbal tissue. This approach should improve the success of this procedure.

Limbal stem cell deficiency:Concept, aetiology, clinical presentation, diagnosis and management

Defects in renewal and repair of ocular surface as a result of limbal stem cell deficiency are now known to cause varying ocular, surface morbidity including persistent photophobia, repeated and persistent surface breakdown and overt conjunctivalisation of the cornea. Ocular conditions with abnormalities of ocular surface repair include pterygium, limbal tumours, aniridia, severe scarring following burns, cicatricial pemphigoid and Stevens-Johnson Syndrome, sequelae of mustard gas exposure and Herpes simplex epithelial disease, radiation keratopathy, contact lens induced keratopathy, neuroparalytic keratitis and drug toxicity. Restoring ocular health in these eyes has traditionally been frustrating. An understanding of these intricate cell renewal and maintenance processes has spurred the evolution in recent years of new treatment methods for several blinding diseases of the anterior segment; many more exciting modalities are in the offing. However, there is inadequate awareness among ophthalmologists about the current principles of management of ocular surface disorders. The purpose of this article is to help elucidate the important principles and current treatment methods relevant to ocular surface disorders.

Allo-limbal transplantation in patients with limbal stem cell deficiency

Aim - To report the outcome of a series of patients with stem cell deficiency who underwent allo-limbal transplantation and to describe a technique for this procedure. Methods - Six consecutive patients underwent allo-limbal stem cell transplantation. The primary diagnosis included alkali burn (n = 2), trachoma (n = 1), chronic rosacea blepharitis and keratoconjunctivitis (n = 1), aniridia (n = 1), and Stevens-Johnson syndrome (n = 1). The limbal rim consisted of peripheral cornea and perilimbal sclera, FK-506 was used postoperatively for immunosuppression. Results - The length of follow up ranged from 3 to 24 months (mean follow up 11.8 (SD 9.3) months). The outcome was considered satisfactory in five of six cases. The corneal surface was completely epithelialised within 2 weeks, and there was a substantial improvement in vision and symptoms. One patient had recurrent epithelial defects related to eyelid abnormalities. No side effects associated with systemic immunosuppression were noted. Conclusion - Allo-limbal transplantation, with systemic immunosuppression with FK-506 is useful in reconstruction of the ocular surface with improvement in vision in patients with severe stem cell deficiency.

A systematic literature review of surgical interventions for limbal stem cell deficiency in humans

PURPOSE: To evaluate the relative benefits and to identify any adverse effects of surgical interventions for limbal stem cell deficiency (LSCD).

DESIGN: Systematic literature review.

METHODS: We searched the following electronic databases from January 1, 1989 through September 30, 2006: MEDLINE, EMBASE, Science citation index, BIOSIS, and the Cochrane Library. In addition, reference lists were scanned to identify any additional reports. The quality of published reports was assessed using standard methods. The main outcome measure was improvement in vision of at least two Snellen lines of best-corrected visual acuity (BCVA). Data on adverse outcomes also were collected.

RESULTS: Twenty-six studies met the inclusion criteria. There were no randomized controlled studies. All 26 studies were either prospective or retrospective case series. For bilateral severe LSCD, keratolimbal allograft was the most common intervention with systemic immunosuppression. Other interventions included eccentric penetrating keratolimbal allografts and cultivated autologous oral mucosal epithelial grafts. An improvement in BCVA of two lines or more was reported in 31% to 67% of eyes. For unilateral severe LSCD, the most common surgical intervention was contralateral conjunctival limbal autograft, with 35% to 88% of eyes gaining an improvement in BCVA of two lines or more. The only study evaluating partial LSCD showed an improvement in BCVA of two lines or more in 39% of eyes.

CONCLUSIONS: Studies to date have not provided strong evidence to guide clinical practice on which surgery is most beneficial to treat various types of LSCD. Standardized data collection in a multicenter LSCD register is suggested.