Proliferative diabetic retinopathy: Role of bevacizumab in decreasing the occurrence of vitreous hemorrhage after panretinal photocoagulation.

PURPOSE: To investigate the beneficial effect of bevacizumab injection one week prior to panretinal photocoagulation (PRP) on the occurrence of vitreous hemorrhage (VH) following PRP in high-risk proliferative diabetic retinopathy (PDR). METHODS: This was a case-control pilot study conducted on two groups: an anti-VEGF treatment group, treated with bevacizumab injection one week prior to the first PRP session, and a control group of treatment-naive PDR patients who underwent PRP treatment and were not given an intravitreal bevacizumab injection, consecutively recruited. In both groups, a complete ophthalmological examination was conducted prior to PRP and at 4, 9, and 16 weeks following treatment. The primary endpoint studied was the occurrence of VH. RESULTS: The control group included 69 patients (mean age 63±12.3 years) with high-risk PDR who received PRP treatment only, and the anti-VEGF treatment group included 67 patients (mean age 63.13±10.3 years). None of the demographic variables or comorbidities showed any significant difference between the two groups. The number of PRP sessions was not significantly correlated to the occurrence of VH in either of the groups (P=0.167). Vitreous hemorrhage within 16 weeks following laser treatment occurred in 10 patients (14.5%) in the control group and in only 3 patients (4.5%) in the anti-VEGF group (P=0.047). CONCLUSION: Our case-control pilot study demonstrates that a bevacizumab injection preceding the initial PRP session might be beneficial in reducing the occurrence of VH in the first 16 weeks following PRP.

FFA-induced hepatic insulin resistance in vivo is mediated by PKCδ, NADPH oxidase, and oxidative stress.

Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKCδ and oxidative stress play causal roles in this process and whether this occurs via IKKß- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased IκBα content, increased JNK phosphorylation (markers of IKKß and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKCδ activation. Furthermore, an antisense oligonucleotide against PKCδ prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKCδ, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA → PKCδ → NADPH oxidase and oxidative stress → IKKß/JNK → impaired hepatic insulin signaling.

Proteins kinase Cɛ is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes.

Protein kinase C (PKC)ɛ, a member of the novel PKC family, has key roles in mitogenesis and survival in normal and cancer cells. PKCɛ is frequently overexpressed in epithelial cancers, particularly in lung cancer. Using a short-hairpin RNA approach, here we established that PKCɛ is required for non-small cell lung carcinoma (NSCLC) growth in vitro as well as tumor growth when inoculated into athymic mice. Moreover, sustained delivery of a PKCɛ-selective inhibitor peptide, ɛV1-2, reduced xenograft growth in mice. Both RNA interference depletion and pharmacological inhibition of PKCɛ caused a marked elevation in the number of apoptotic cells in NSCLC tumors. PKCɛ-depleted NSCLC cells show elevated expression of pro-apoptotic proteins of the Bcl-2 family, caspase recruitment domain-containing proteins and tumor necrosis factor ligands/receptor superfamily members. Moreover, a Gene Set Enrichment Analysis revealed that a vast majority of the genes changed in PKCɛ-depleted cells were also deregulated in human NSCLC. Our results strongly suggest that PKCɛ is required for NSCLC cell survival and maintenance of NSCLC tumor growth. Therefore, PKCɛ may represent an attractive therapeutic target for NSCLC.

Effects of portal free fatty acid elevation on insulin clearance and hepatic glucose flux.

We tested the hypothesis that, due to greater hepatic free fatty acid (FFA) load, portal delivery of FFAs, as in visceral obesity, induces hyperinsulinemia and increases endogenous glucose production to a greater extent than peripheral FFA delivery. For 5 h, 10 microeq.kg(-1).min(-1) portal oleate (n = 6), equidose peripheral oleate (n = 5), or saline (n = 6) were given intravenously to conscious dogs infused with a combination of portal and peripheral insulin to enable calculation of hepatic insulin clearance during a pancreatic euglycemic clamp. Peripheral FFAs were similar with both oleate treatments and were threefold greater than in controls. Portal FFAs were 1.5- to 2-fold greater with portal than with peripheral oleate. Peripheral insulin concentrations were greatest with portal oleate, intermediate with peripheral oleate (P < 0.001 vs. portal oleate or controls), and lowest in controls, consistent with corresponding reductions in plasma insulin clearance and hepatic insulin clearance. Although endogenous glucose production did not differ between the two routes of oleate delivery, total glucose output (endogenous glucose production plus glucose cycling) was greater with portal than with peripheral oleate (P < 0.001) despite the higher insulin levels. In conclusion, during euglycemic clamps in dogs, the main effect of short-term elevation in portal FFA is to generate peripheral hyperinsulinemia. This may, in the long term, contribute to the metabolic and cardiovascular risk of visceral obesity.

N-acetylcysteine and taurine prevent hyperglycemia-induced insulin resistance in vivo: possible role of oxidative stress.

Exposure to high concentrations of glucose and insulin results in insulin resistance of metabolic target tissues, a characteristic feature of type 2 diabetes. High glucose has also been associated with oxidative stress, and increased levels of reactive oxygen species have been proposed to cause insulin resistance. To determine whether oxidative stress contributes to insulin resistance induced by hyperglycemia in vivo, nondiabetic rats were infused with glucose for 6 h to maintain a circulating glucose concentration of 15 mM with and without coinfusion of the antioxidant N-acetylcysteine (NAC), followed by a 2-h hyperinsulinemic-euglycemic clamp. High glucose (HG) induced a significant decrease in insulin-stimulated glucose uptake [tracer-determined disappearance rate (Rd), control 41.2 +/- 1.7 vs. HG 32.4 +/- 1.9 mg. kg-1. min-1, P < 0.05], which was prevented by NAC (HG + NAC 45.9 +/- 3.5 mg. kg-1. min-1). Similar results were obtained with the antioxidant taurine. Neither NAC nor taurine alone altered Rd. HG caused a significant (5-fold) increase in soleus muscle protein carbonyl content, a marker of oxidative stress that was blocked by NAC, as well as elevated levels of malondialdehyde and 4-hydroxynonenal, markers of lipid peroxidation, which were reduced by taurine. In contrast to findings after long-term hyperglycemia, there was no membrane translocation of novel isoforms of protein kinase C in skeletal muscle after 6 h. These data support the concept that oxidative stress contributes to the pathogenesis of hyperglycemia-induced insulin resistance.

Insulin inhibits glucose production by a direct effect in diabetic depancreatized dogs during euglycemia.

In our previous studies in nondiabetic dogs and humans, insulin suppressed glucose production (GP) by both an indirect extrahepatic and a direct hepatic effect. However, insulin had no direct effect on GP in diabetic depancreatized dogs under conditions of moderate hyperglycemia. The present study was designed to investigate whether insulin can inhibit GP by a direct effect in this model under conditions of euglycemia. Depancreatized dogs were made euglycemic (approximately 6 mmol/l), rather than moderately hyperglycemic (approximately 10 mmol/l) as in our previous studies, by basal portal insulin infusion. After approximately 100 min of euglycemia, a hyperinsulinemic euglycemic clamp was performed by giving an additional infusion of insulin either portally (POR) or peripherally at about one-half the rate (1/2 PER) to match the peripheral venous insulin concentrations. The greater hepatic insulin load in POR resulted in greater suppression of GP (from 16.5 +/- 1.8 to 12.2 +/- 1.6 micromol. kg(-1). min(-1)) than 1/2 PER (from 17.8 +/- 1.9 to 15.6 +/- 2.0 micromol. kg(-1). min(-1), P < 0.001 vs. POR), consistent with insulin having a direct hepatic effect in suppressing GP. We conclude that the direct effect of insulin to inhibit GP is present in diabetic depancreatized dogs under conditions of acutely induced euglycemia. These results suggest that, in diabetes, the prevailing glycemic level is a determinant of the balance between insulin's direct and indirect effects on GP.

Free fatty acid-induced hepatic insulin resistance: a potential role for protein kinase C-delta.

The mechanisms of the impairment in hepatic glucose metabolism induced by free fatty acids (FFAs) and the importance of FFA oxidation in these mechanisms remain unclear. FFA-induced peripheral insulin resistance has been linked to membrane translocation of novel protein kinase C (PKC) isoforms, but the role of PKC in hepatic insulin resistance has not been assessed. To investigate the biochemical pathways that are induced by FFA in the liver and their relation to glucose metabolism in vivo, we determined endogenous glucose production (EGP), the hepatic content of citrate (product of acetyl-CoA derived from FFA oxidation and oxaloacetate), and hepatic PKC isoform translocation after 2 and 7 h Intralipid + heparin (IH) or SAL in rats. Experiments were performed in the basal state and during hyperinsulinemic clamps (insulin infusion rate, 5 mU. kg(-1). min(-1)). IH increased EGP in the basal state (P < 0.001) and during hyperinsulinemia (P < 0.001) at 2 and 7 h. Also, 7-h infusion of IH induced resistance to the suppressive effect of insulin on EGP (P < 0.05). Glycerol infusion (resulting in plasma glycerol levels similar to IH infusion) did not have any effect on EGP. IH increased hepatic citrate content by twofold, independent of the insulin levels and the duration of IH infusion. IH induced hepatic PKC-delta translocation from the cytosolic to membrane fraction in all groups. PKC-delta translocation was greater at 7 compared with 2 h (P < 0.05). In conclusion, 1) increased FFA oxidation may contribute to the FFA-induced increase in EGP in the basal state and during hyperinsulinemia but is not associated with FFA-induced hepatic insulin resistance, and 2) the progressive insulin resistance induced by FFA in the liver is associated with a progressive increase in hepatic PKC-delta translocation.

Dopamine D(3) receptor antagonists. 1. Synthesis and structure-activity relationships of 5,6-dimethoxy-N-alkyl- and N-alkylaryl-substituted 2-aminoindans.

5,6-Dimethoxy-2-(N-dipropyl)-aminoindan (3, PNU-99194A) was found to be a selective dopamine D(3) receptor antagonist with potential antipsychotic properties in animal models. To investigate the effects of nitrogen substitution on structure-activity relationships, a series of 5,6-dimethoxy-N-alkyl- and N-alkylaryl-substituted 2-aminoindans were synthesized and evaluated in vitro for binding affinity and metabolic stability. The results indicate that substitution at the amine nitrogen of the 2-aminoindans is fairly limited to the di-N-propyl group in order to achieve selective D(3) antagonists. Thus, combinations of various alkyl groups were generally inactive at the D(3) receptor. Although substitution with an N-alkylaryl or N-alkylheteroaryl group yields compounds with potent D(3) binding affinity, the D(2) affinity is also enhanced, resulting in a less than 4-fold preference for the D(3) receptor site, and no improvements in metabolic stability were noted. A large-scale synthesis of the D(3) antagonist 3 has been developed that has proven to be reproducible with few purification steps. The improvements include the use of 3,4-dimethoxybenzaldehyde as a low-cost starting material to provide the desired 5,6-dimethoxy-1-indanone 5c in good overall yield (65%) and the formation of a soluble silyl oxime 17 that was reduced efficiently with BH(3).Me(2)S. The resulting amino alcohol was alkylated and then deoxygenated using a Lewis acid and Et(3)SiH to give the desired product 3 in good overall yield of ( approximately 65%) from the indanone 5c.

Shear-induced assembly of lambda-phage DNA.

Recombinant DNA technology, which is based on the assembly of DNA fragments, forms the backbone of biological and biomedical research. Here we demonstrate that a uniform shear flow can induce and control the assembly of lambda-phage DNA molecules: increasing shear rates form integral DNA multimers of increasing molecular weight. Spontaneous assembly and grouping of end-blunted lambda-phage DNA molecules are negligible. It is suggested that shear-induced DNA assembly is caused by increasing the probability of contact between molecules and by stretching the molecules, which exposes the cohesive ends of the otherwise undeformed lambda-phage DNA molecules. We apply this principle to enhance the kinetics and extent of DNA concatenation in the presence of ligase. This novel approach to controlled DNA assembly could form the basis for improved approaches to gene-chip and recombinant DNA technologies and provide new insight into the rheology of associating polymers.

Shape anisotropy of a single random-walk polymer.

Random walks have been used to describe a wide variety of systems ranging from cell colonies to polymers. Sixty-five years ago, Kuhn [Kuhn, W. (1934) Kolloid-Z. 68, 2-11] made the prediction, backed later by computer simulations, that the overall shape of a random-walk polymer is aspherical, yet no experimental work has directly tested Kuhn's general idea and subsequent computer simulations. By using fluorescence microscopy, we monitored the conformation of individual, long, random-walk polymers (fluorescently labeled DNA molecules) at equilibrium. We found that a polymer most frequently adopts highly extended, nonfractal structures with a strongly anisotropic shape. The ensemble-average ratio of the lengths of the long and short axes of the best-fit ellipse of the polymer was much larger than unity.

Dynamics of individual flexible polymers in a shear flow.

Polymer dynamics are of central importance in materials science, mechanical engineering, biology and medicine. The dynamics of macromolecular solutions and melts in shear flow are typically studied using bulk experimental methods such as light and neutron scattering and birefringence. But the effect of shear on the conformation and dynamics of individual polymers is still not well understood. Here we describe observations of the real-time dynamics of individual, flexible polymers (fluorescently labelled DNA molecules) under a shear flow. The sheared polymers exhibit many types of extended conformation with an overall orientation ranging from parallel to perpendicular with respect to the flow direction. For shear rates much smaller than the inverse of the relaxation time of the molecule, the relative populations of these two main types of conformation are controlled by the rate of the shear flow. These results question the adequacy of assumptions made in standard models of polymer dynamics.

Quantitative Analysis of Anions at ppb/ppt Levels with Capillary Electrophoresis and Conductivity Detection: Enhancement of System Linearity and Precision Using an Internal Standard.

Capillary electrophoresis with conductivity detection is evaluated for quantitative analysis of anions at low- to sub-ppb concentration levels in the presence and absence of a conductive sample matrix composed of 2 ppm ammonia and 50 ppb hydrazine. The low-level sensitivity is extended by a transient isotachophoretic stacking procedure. The linear range of the CE system and conductivity detector is graphically evaluated on the basis of absolute and corrected (normalized) chloride and sulfate peak profiles using an ASTM linearity criterion. The influence of random contamination bias from ubiquitous entities of nonsample chloride and sulfate levels introduced by liquid handling, laboratory atmosphere, and bulk chemical residues is quantitatively compared against an internal (contamination) reference ion.

Conductivity detection in capillary electrophoresis--a powerful tool in ion analysis.

Conductivity detection in CE has recently become available in a commercial CE instrument. The new conductivity cell is based on an end-capillary concept. The conductivity sensor and the detection end of the fused-silica capillary are permanently encapsulated in two individually modified coupling connectors (ConTip, ConCap [both from Orion Research, Boston, MA, U.S.A.]). This open-architecture cell permits interchangeability of sensors and capillaries, while maintaining a precisely defined detection volume between those two components when inserted into the detector cell block. The detector's performance is evaluated for sensitivity, linearity, and reproducibility using low-mobility electrolytes. Electropherograms comprising a variety of ionic class separations including inorganic and organic anions, organic surfactants, alkali metals, alkaline earths, transition metals, and organic amines are shown along with separations of actual samples.

Antioxidants attenuate endotoxin-gentamicin induced acute renal failure in rats.

The synergistic mechanism by which endotoxin enhances the nephrotoxic potential of gentamicin is unknown. In this study, we attempted to shed light on this mechanism by injecting rats with endotoxin plus gentamicin. Renal injury was assessed by measuring creatinine, inulin and PAH clearance, NADH levels and electrolyte reabsorption, for 24 hr following this injection. Gentamicin alone (20 mg/100 g) induced no renal injury, while endotoxin without gentamicin (0.075 mg/100 g) induced mild injury. However, endotoxin plus gentamicin resulted in acute renal failure. In an attempt to halt the progressive renal dysfunction, the antioxidants NAO (5 mg/100 g), Vitamin E (0.2 mg/100 g per day) and dimethylthiourea (DMTU-50 mg/100 g) were administered, or early endotoxin tolerance was induced before injecting the rats with endotoxin plus gentamicin. The reduction in renal function was markedly slower in rats administered with antioxidants compared with untreated rats. Similar results were obtained with endotoxin tolerance. These data suggest that NAO, vitamin E, DMTU and endotoxin tolerance are potentially beneficial in arresting progressive renal damage associated with endotoxin plus gentamicin.

Anthelmintic activity of dioxapyrrolomycin.

Dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 produced by UC 11065 were evaluated as anthelmintics. Assays used to examine these compounds included effects on the free-living nematode Caenorhabditis elegans, ability to clear target nematodes (Haemonchus contortus and Trichostrongylus colubriformis) from jirds, and clearance of Haemonchus contortus from lambs. A crude extract of UC 11065 containing dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 was active against C. elegans and against H. contortus in the jird. Purified and/or synthetic samples of dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 were tested in the jird model; only dioxapyrrolomycin exhibited appreciable activity against H. contortus (greater than or equal to 90.9% clearance at 0.33 mg/jird), while none of the compounds showed appreciable activity against T. colubriformis. Dioxapyrrolomycin cleared 99.9% of H. contortus from lambs at 12.5 mg/kg. An in vitro migration study using susceptible and closantel-resistant H. contortus showed there is cross-resistance between dioxapyrrolomycin and closantel. Dioxapyrrolomycin appears to be a narrow-spectrum anthelmintic which works through a closantel-like mode-of-action.

Development of a mechanism of action-based screen for anthelmintic microbial metabolites with avermectinlike activity and isolation of milbemycin-producing Streptomyces strains.

A high-volume screen for anthelmintic microbial metabolites with an avermectinlike mode of action was developed. The primary screen used the free-living nematode Caenorhabditis elegans in a whole-organism assay. The specificity for avermectinlike compounds resides in the secondary screen, which takes advantage of the chloride channel-opening properties of the avermectins. By using standard microelectrode techniques, membrane conductance changes following exposure to extracts of microbial cultures were measured in the walking leg stretcher muscle fibers of the lined shore crab Pachygrapsus crassipes. The avermectins and related milbemycins give a characteristic response of rapid loss of membrane resistance coupled with a slight hyperpolarization of the membrane. This is partially (near 50%) reversible with the chloride channel blocker picrotoxinin. Four morphologically similar cultures that produced avermectinlike activities were identified by this screen. Isolation of the active components from one of these cultures (strain UC 8984) followed by nuclear magnetic resonance spectroscopy resulted in the identification of milbemycins alpha 1 and alpha 3. These metabolites are members of a large family of milbemycins produced by Streptomyces hygroscopicus subsp. aureolacrimosus NRRL 5739. Systematic studies revealed that strain UC 8984 is also a S. hygroscopicus strain, but which is taxonomically distinct from NRRL 5739.

The psychoanalytic treatment of a preschool boy with a gender identity disorder.

A three-year, nine-month-old boy began analysis by wishing he was a girl and pretending he was a superheroine. Over the course of almost five years, the cross-gender defense against fear of loss of the object, anal loss, and castration by the object reorganized in all libidinal phases through early latency. Developmental arrests seemed to occur during the anal rapprochement and oedipal phases that led to observable cross-gender strivings by two and a half years of age. The role of early childhood illness, narcissistic vulnerability, mother's childhood wish for a sister, the mother's adult wish for a daughter, a shared fantasy between mother and child, identification with the perceived power and beauty of mother and grandmother, pathological sibling rivalry that influenced identification with his sister, were demonstrated in his play during sessions. Interwoven in the background was the impact of an emotionally absent father, a dying grandfather, and an accident-prone uncle. This paternal matrix seemed to discourage budding masculinity and encourage feminine identifications. The analyst's approach and the child's responses to interpretation of the transference manifestations, cross-gender behavior, fantasies, and play are presented. Finally, the gradual resolution of the conflicted wish to be a girl was supplanted by the emergence of appropriate gender identification. A two-year followup appeared to confirm his postanalytic gender stance and continuing consolidation of stable gender development.

Nature and course of pancreatitis caused by 6-mercaptopurine in the treatment of inflammatory bowel disease.

6-Mercaptopurine (6-MP) has an important role in the treatment of inflammatory bowel disease. Its most frequent short-term complication has proven to be pancreatitis, which we have now seen in 13 of 400 (3.25%) patients (12 Crohn's disease, 1 ulcerative colitis) and which we here describe. The timing of the pancreatitis was such that it could not be attributed to sulfasalazine, which was also being taken by 9 patients, or corticosteroids, which were being taken by 7 patients. The dosage of 6-MP ranged from 50 to 100 mg daily, and the pancreatitis, which was uncomplicated in all cases, occurred within 8-32 days with one exception (6.5 mo). Symptoms included epigastric pain, back pain, fever, and nausea. The serum amylase was elevated in 12 patients. The average elevation was 5.9 times normal. In all cases, the 6-MP was discontinued and symptoms and signs returned to normal over a period of 1-11 days. No other complications of 6-MP occurred; there was no leukopenia. Of 7 patients rechallenged with 6-MP, all developed recurrent pancreatitis, including 4 in less than 24 h. In 3 patients, desensitization attempted by a gradual increase in dose from 1/8 tablet (approximately 6 mg) daily also led to recurrence. The timing of the initial pancreatitis and the recurrence at rechallenge are best explained by an allergic reaction. 6-Mercaptopurine should not be reinstituted once it has caused pancreatitis.