Ulnar nerve morphology on magnetic resonance imaging predicts nerve recovery after surgery for cubital tunnel syndrome.

Magnetic resonance imaging (MRI) can evaluate nerve morphology in cubital tunnel syndrome (CuTS), but its value in predicting surgical outcome is unclear. The purpose of this study was to determine whether ulnar nerve morphology on MRI correlated with outcome after CuTS surgery. We reviewed 40 patients who had preoperative MRI and electrodiagnostic (EDX) examinations for CuTS and outcome evaluation 6 months and 2 years postoperatively. Using MRI, ulnar nerve cross-sectional area (UNCSA), changes in signal intensity, and any space-occupying lesion were evaluated. Other factors assessed were age, symptom duration and severity, type-2 diabetes and EDX parameters. Factors associated with unfavorable surgical outcome were identified. At 6 months postoperatively, 12 patients (30%) had excellent, 19 (47.5%) good, 8 (20%) fair and 1 (2.5%) poor results on modified Wilson-Krout criteria. On univariate analysis, unfavorable outcomes were associated with increased UNCSA, space-occupying lesion, and decreased motor nerve conduction velocity (mNCV), and on multivariate analysis with increased UNCSA 1 cm distal from the epicondyle only (model 1) or increased UNCSA 1 cm proximal from the epicondyle and decreased mNCV (model 2). At 2 years, 15 patients (37.5%) had excellent, 21 (52.5%) good, 3 (7.5%) fair and 1 (2.5%) poor results, and no factors correlated with unfavorable outcome. Increased UNCSA on MRI was associated with unfavorable outcome at 6 months but not at 2 years. This study suggests that morphologic ulnar nerve changes can predict delayed nerve recovery after surgery for CuTS.

SAPK2/p38-dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis.

In endothelial cells, H2O2 induces the rapid formation of focal adhesion complexes at the ventral face of the cells and a major reorganization of the actin cytoskeleton into dense transcytoplasmic stress fibers. This change in actin dynamics results from the activation of the mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38), which, via MAP kinase-activated protein (MAPKAP) kinase-2/3, leads to the phosphorylation of the actin polymerization modulator heat shock protein of 27 kD (HSP27). Here we show that the concomitant activation of the extracellular signal-regulated kinase (ERK) MAP kinase pathway by H2O2 accomplishes an essential survival function during this process. When the activation of ERK was blocked with PD098059, the focal adhesion complexes formed under the plasma membrane, and the actin polymerization activity led to a rapid and intense membrane blebbing. The blebs were delimited by a thin F-actin ring and contained enhanced levels of HSP27. Later, the cells displayed hallmarks of apoptosis, such as DEVD protease activities and internucleosomal DNA fragmentation. Bleb formation but not apoptosis was blocked by extremely low concentrations of the actin polymerization inhibitor cytochalasin D or by the SAPK2 inhibitor SB203580, indicating that the two processes are not in the same linear cascade. The role of HSP27 in mediating membrane blebbing was assessed in fibroblastic cells. In control fibroblasts expressing a low level of endogenous HSP27 or in fibroblasts expressing a high level of a nonphosphorylatable HSP27, H2O2 did not induce F-actin accumulation, nor did it generate membrane blebbing activity in the presence or absence of PD098059. In contrast, in fibroblasts that expressed wild-type HSP27 to a level similar to that found in endothelial cells, H2O2 induced accumulation of F-actin and caused bleb formation when the ERK pathway was inhibited. Cis-platinum, which activated SAPK2 but induced little ERK activity, also induced membrane blebbing that was dependent on the expression of HSP27. In these cells, membrane blebbing was not followed by caspase activation or DNA fragmentation. We conclude that the HSP27-dependent actin polymerization-generating activity of SAPK2 associated with a misassembly of the focal adhesions is responsible for induction of membrane blebbing by stressing agents.

Mode of action of poly(ADP-ribose) glycohydrolase.

The turnover of the homopolymer of ADP-ribose, which is known to be involved in many DNA-related functions, is controlled by 2 principal enzymes. Poly(ADP-ribose) polymerase (EC 2.4.2.30) synthesizes the polymer from NAD, and poly(ADP-ribose) glycohydrolase (PARG) is the major enzyme responsible for its catabolism (Thomassin et al. (1992) Biochim. Biophys. Acta 1137, 171-181). In vivo, poly(ADP-ribose) polymers constitute a heterogeneous population of branched polymers attaining sizes of 200-400 residues. They are rapidly degraded by PARG, displaying variable kinetic parameters as a function of polymer size. Several studies have suggested that PARG acts exoglycosidically on its substrate but others observed that it could act endo/exo-glycosidically. We analysed the mode of action of PARG under conditions most suitable for expression of all the activities of PARG, using HPLC purified long free polymer and very pure PARG. We conclusively show that on large free polymers, PARG exhibits endoglycosidic activity along with exoglycosidic activity. This endoglycosidic activity could have a significant role during cellular response to DNA damage.

Characterization of anti-peptide antibodies directed towards the automodification domain and apoptotic fragment of poly (ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a highly conserved nuclear enzyme present in higher eukaryotes. PARP is activated following DNA damage, is implicated in DNA repair, and its proteolysis has been shown to be an early marker of programmed cell death or apoptosis. In order to better understand the role of PARP in apoptosis and DNA repair and also to study PARP automodification, we have developed anti-peptide sera directed against four peptides from the conserved automodification domain of PARP. Four peptides were synthesized according to the four branched Multiple Antigenic Peptide (MAP) system and injected into rabbits. Immune sera were titrated by ELISA and analysed in Western blotting experiments on cell lines. The sera were also analysed for their capacity to inhibit PARP activity in an in vitro assay. Of the eight sera developed (two for each peptide), a serum directed against a peptide localized at the C-terminal part of the automodification domain of PARP (#422) appeared to be the best antibody to detect PARP from different species. All antipeptide antibodies were efficient in detecting the apoptotic fragment of PARP during programmed cell death in HL-60 apoptotic cells. None of the serum alone was able to completely inhibit PARP activity but combinations of the sera could significantly reduce automodification of PARP consistent with the localization of half of the automodification sites on bovine PARP. Sera were also used to map proteolysed purified PARP and to immunoprecipitate purified bovine PARP.

Complete inhibition of poly(ADP-ribose) polymerase activity prevents the recovery of C3H10T1/2 cells from oxidative stress.

Activation of the poly(ADP-ribose) polymerase after oxidative damage is implicated in different responses of the cells, for example, cell recovery after sublethal damage or cell death after lethal damage. However, the extent and mechanism of involvement of the enzyme in these two processes appear to be different. Inhibitors of this polymerase, such as benzamides, which do not completely inhibit PARP have been shown to protect the cells from killing by massive oxidant damage, could neither reduce the cellular recovery after mild oxidant damage nor completely inhibit DNA repair in vitro. We report here that 1,5-dihydroxyisoquinoline, which was earlier shown to be a strong inhibitor of this polymerase in vitro, is also its potent inhibitor in vivo. Using sensitive techniques for measuring low levels of cellular poly(ADP-ribose) polymer, we show that this inhibitor can completely abolish oxidant-induced activation of the polymerase in C3H10T1/2 cells. We show that only a minor fraction of the poly(ADP-ribose) polymerase activity is sufficient in cellular recovery after sublethal oxidant damage. We also demonstrate that cells are unable to recover from oxidant damage in the complete absence of polymerase activity.

6-Aminonicotinamide sensitizes human tumor cell lines to cisplatin.

The nicotinamide analogue 6-aminonicotinamide (6AN) is presently undergoing evaluation as a potential modulator of the action of various antineoplastic treatments. Most previous studies of this agent have focused on a three-drug regimen of chemical modulators that includes 6AN. In the present study, the effect of single-agent 6AN on the efficacy of selected antineoplastic drugs was assessed in vitro. Colony-forming assays using human tumor cell lines demonstrated that pretreatment with 30-250 microM 6AN for 18 h resulted in increased sensitivity to the DNA cross-linking agent cisplatin, with 6-, 11-, and 17-fold decreases in the cisplatin dose that diminishes colony formation by 90% being observed in K562 leukemia cells, A549 non-small cell lung cancer cells, and T98G glioblastoma cells, respectively. Morphological examination revealed increased numbers of apoptotic cells after treatment with 6AN and cisplatin compared to cisplatin alone. 6AN also sensitized cells to melphalan and nitrogen mustard but not to chlorambucil, 4-hydroperoxycyclophosphamide, etoposide, or daunorubicin. In additional studies undertaken to elucidate the mechanism underlying the sensitization to cisplatin, atomic absorption spectroscopy revealed that 6AN had no effect on the rate of removal of platinum (Pt) adducts from DNA. Instead, 6AN treatment was accompanied by an increase in Pt-DNA adducts that paralleled the degree of sensitization. This effect was not attributable to 6AN-induced decreases in glutathione or NAD+, because other agents that depleted these detoxification cofactors (buthionine sulfoximine and 3-acetylpyridine, respectively) did not increase Pt-DNA adducts. On the contrary, 6AN treatment increased cellular accumulation of cisplatin. Further experiments revealed that 6AN was metabolized to 6-aminonicotinamide adenine dinucleotide (6ANAD+). Concurrent administration of nicotinamide and 6AN had minimal effect on cellular 6AN accumulation but abolished the formation of 6ANAD+, the increase in Pt-DNA adducts, and the sensitizing effect of 6AN in clonogenic assays. These observations identify 6AN as a potential modulator of cisplatin sensitivity and suggest that the 6AN metabolite 6ANAD+ exerts this effect by increasing cisplatin accumulation and subsequent formation of Pt-DNA adducts.

Biochemical properties and function of poly(ADP-ribose) glycohydrolase.

We describe here the latest observations on poly(ADP-ribose) glycohydrolase. There is now extensive evidence that this nuclear enzyme is an endo-exoglycosidase which has a key role to perform in the removal of polymers which interact with proteins through covalent and non-covalent interactions. Also, we have developed a zymogram which will permit the isolation of the various isoforms of the glycohydrolase and the eventual cloning of this enzyme. Finally, we have evidence that very short oligomers and even monomers of ADP-ribose covalently bound to proteins can be removed by poly(ADP-ribose) glycohydrolase.

Modulation of transcription in rat liver by benzo[a]pyrene.

Single intraperitoneal administration of benzo[a]pyrene (B[a]P) at 20 mg/kg body weight to male Wistar rats caused an early (2 h) inhibition in liver of gross transcription as measured by incorporation of [14C] orotic acid into nuclear RNA. This inhibition was reversed gradually into a partial stimulation at 2-4 days after administration of B[a]P, followed by another period of inhibition at 7 days which persisted up to 14 days. The early reversible inhibition at 2 h was probed further by studies on in vitro transcription using isolated nuclei from treated rats. It was observed that total expression of RNA polymerase activity was only marginally inhibited because, while expression of RNA polymerases II and III showed remarkable inhibition, RNA polymerase I showed stimulation in activity.

Coagulation cascade, fibrinolytic system, antithrombin III, protein C and protein S in patients maintained on continuous ambulatory peritoneal dialysis.

We studied the coagulation cascade, fibrinolytic system and naturally occurring anticoagulants in a group of 14 patients with end-stage renal disease maintained on continuous ambulatory peritoneal dialysis (CAPD). The results were compared with those obtained in a group of ten normal volunteers. Plasma procoagulant activities of factors XII, XI, IX, VIII, VII, X and II were significantly greater in the CAPD group as compared to the normal control group. Likewise plasma concentrations of total and free protein S were increased in the CAPD group. Although the mean value for plasma factor V activity in the CAPD group was higher than that found in the control group the difference did not attain statistical significance. In addition plasma fibrinogen concentration and factor VIII-related antigen level were significantly increased in CAPD patients. No significant difference was found between the CAPD patients and the control group with respect to plasma levels of protein C, antithrombin III, plasminogen or alpha 2-antiplasmin. In summary, the results demonstrate a tendency for increased levels of various coagulation factors and protein S in CAPD patients with no significant alterations in the levels of various fibrinolytic and endogenous anticoagulant agents, i.e. antithrombin III and protein C. The clinical significance and the mechanism responsible for the observed changes require further investigation.

The effect of some new platinum (II) and palladium (II) coordination complexes on rat hepatic nuclear transcription in vitro.

Several new L-amino acid derivatives of 2,2'-bipyridine and 1,10-phenanthroline complexes of platinum (Pt) and palladium (Pd) and a few binuclear 2,2'-bipyridine complexes of these metals were tested for their potential to inhibit rat hepatic nuclear transcription in vitro. Pd complexes were generally more effective inhibitors of transcription than the corresponding Pt complexes. Among Pd-diimine chlorides, the 2,2'-bipyridine complex was nearly 10 times more active than the corresponding 1,10-phenanthroline complex. Both Pt-diimine chlorides, however, showed same level of inhibitory activity. Amino acid derivatives were less inhibitory with respect to the parent metal diimine chlorides except for 1,10-phenanthroline complexes of Pd. For binuclear 2,2'-bipyridine complexes of Pt, the increase in length of linking hydrocarcon chain increased the inhibitory potential of the complex. The mechanism of inhibition of transcription by these metal complexes was sought to be understood by use of actinomycin-D and poly[d(I-C)] to differentiate effect on the two major components of transcription machinery viz. the template and the enzyme. These studies along with studies on reconstituted system of transcription using either pretreated template or enzyme indicate that these metal complexes displayed dual effect on transcription by inhibiting both the template and the enzymes.

Modulation by plant flavonoids and related phenolics of microsome catalyzed adduct formation between benzo[a]pyrene and DNA.

Twenty-six flavonoids and related compounds were screened for their ability to modulate microsome mediated covalent adduct formation between [3H]benzo[a]pyrene ([3H]BP) and DNA in vitro. Some of these flavonoids, notably robinetin, quercetin, isorhamnetin and kaempferol were observed to inhibit the adduct formation significantly at very low levels. The unsubstituted flavone and some of the other flavonoids moderately inhibited this adduct formation, while some flavonoids were inactive, viz., most of the isoflavonoids and methylether derivatives of polyhydroxylated flavonoids. Structural features contributory towards the inhibitory activity of flavonoids appeared to be hydroxyl groups in 3 position of C ring, 5,7-positions of A ring and 3',4'- and 5'-positions of B ring. Methylation or glycosylation of hydroxyl group rendered the flavonoid less active or inactive. Flavanones, with saturated 2,3 double bond, were also inactive. Metabolic activation of BP to proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydro-BP (BP-7,8-dihydrodiol) was also measured in presence of some of these flavonoids. The extent of inhibition of metabolism by these flavonoids did not correlate with their ability to inhibit the adduct formation. Thus, suppression of metabolism did not appear to be a major contributory factor towards inhibition of adduct formation. The solvolysis in aqueous dioxane of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE I), the ultimate carcinogen of BP, was accelerated in presence of selected flavonoids. Inactivation of BPDE I, therefore, appeared to be the major mechanism by which some of these flavonoids inhibited the adduct formation between BP and DNA, and this could be the basis for the anti-carcinogenic nature of these flavonoids.

Alteration in hepatic nuclear RNA polymerase activity following benzo[a]pyrene administration in rat.

Benzo[a]pyrene was administered intraperitoneally to male Wistar rats as a single dose of 20 mg.kg-1 body weight. Two hours after its administration, livers were excised and the enzyme RNA polymerase was solubilized from purified nuclei. The enzyme was resolved into three forms, polymerase I, II and III by partial purification on DEAE-Sephadex A-25 column. All the three forms of nuclear RNA polymerase were inhibited in response to administration of benzo[a]pyrene. This was evident in terms of total yield as well as specific activity of each enzyme. The inhibition of nuclear transcription following administration of benzo[a]pyrene as observed previously (1) along with the present results on the enzyme inhibition clearly suggest that benzo[a]pyrene acts on both the major components of transcription machinery i.e. the template chromatin and the enzyme RNA polymerases.

Peritoneal catheters: a comparative study of column disc and Tenckhoff catheters.

A functioning peritoneal access is crucial to the success of peritoneal dialysis. We report retrospective analysis of our experience using 44 Tenckhoff and 23 column disc, double-cuff, catheters in 46 patients receiving peritoneal dialysis. Postoperative care was identical in both groups. Both catheter groups were comparable with regards to age, sex, obesity and prior abdominal surgery. Catheter removal due to drainage failure was significantly greater with the column disc than the Tenckhoff catheters (22% vs 5%, p = 0.04). In addition, 39% of column disc catheters compared to 11% Tenckhoff catheters were removed as a result of therapy resistant peritonitis (p = 0.011). Furthermore, there was a greater incidence of peritonitis with the column disc than with the Tenckhoff catheters at the end of the first year (71% vs 42%, p less than 0.01). There was no difference between the two groups with respect to other complications, such as pericatheter leak, catheter infections, catheter cuff-extrusion or hernia. Our experience indicates that the column disc catheter is associated with higher complication rates and does not offer any advantage over the Tenckhoff catheter.

Elevated plasma lipoprotein(a) levels and hypoalbuminemia in peritoneal dialysis patients.

Plasma lipoprotein(a), Lp(a), is strongly and independently associated with atherosclerosis, and levels are elevated in hemodialysis (HD) patients and in some studies of those on peritoneal dialysis (PD). We hypothesized that protein losses and hypoalbuminemia could stimulate hepatic Lp(a) synthesis, and this effect would be accentuated in PD patients with malnutrition. The PD subjects (n = 24) had higher plasma Lp(a) levels than those (n = 10) on HD (median 34.4 vs 21.0 mg/dl, p < 0.05), and values exceed normal in 62.5% vs 20% of the subjects (p < 0.03), respectively. The serum albumin levels inversely correlated with concentrations of Lp(a) and apolipoprotein B, as well as the apolipoprotein B/AI ratio. In conclusion, plasma Lp(a) concentrations are frequently elevated in PD as well as HD patients. Measuring Lp(a) levels is useful in identifying patients at increased atherogenic risk, which may not be reflected in routine lipid profiles. The negative correlation between plasma Lp(a) and albumin levels suggests that the latter may be linked pathophysiologically to hepatic Lp(a) production. The association of hypoalbuminemia with higher Lp(a) values is of particular concern because malnutrition frequently occurs in PD patients.

Peritoneal leucocyte response to bacterial peritonitis in patients receiving peritoneal dialysis.

We evaluated the quantitative peritoneal leucocyte response to antibiotic therapy in 25 CAPD patients with 57 episodes of bacterial peritonitis. Eighty-eight percent of the peritonitis episodes were initially treated with a first generation cephalosporin, but results of microbial sensitivity studies led to a change in the initial antibiotic regimen in 23 episodes. Overall, 47/57 (82%) episodes were cured by antibiotic therapy alone (responders), while 10/57 (18%) required removal of the peritoneal catheter as a curative procedure (nonresponder). Neither the duration of symptoms on initial presentation nor the status of being a nonresponder could be related to the baseline peritoneal leucocyte values, either the total (PLC) or polymorphonuclear counts (PMN). Since the baseline PLC and PMN showed a 500-fold variation, subsequent changes were expressed as a percent [PLC (%) and PMN-PLC (%)] of the baseline value. On day 3 of peritonitis, PLC (%) and PMN-PLC (%) were less in responders (26% and 10%) than nonresponders (251% and 254%) (p less than 0.001). Differentiation between responders and nonresponders based on PLC (%) and PMN-PLC (%) was associated with a high degree of sensitivity (90%) and specificity (90%). Similar results were obtained for day 4. These data suggest that the temporal pattern of PLC and PMN, when expressed as a percentage of the baseline value, may be useful in predicting those episodes of peritonitis which require removal of the peritoneal catheter.

A study of basal metabolic rate in adult Kashmiris.

Sixty two adults, including forty one males (16-36 yrs.) and twenty one females (16-25 yrs.), were studied by closed circuit method as regards their B.M.R. Males showed a greater lowering of metabolism than the females when compared to MF and AD standards. The females actually showed a higher B.M.R. than the RR standards. Both the sexes had a higher basal metabolism than the value reported from other parts of the country. The difference, however, was significant only in the case of females. Winter metabolism was not sigificantly higher than the summer value in either sex, though females showed some what greater variation.

Incidence of P.T.C. taste sensitivity and threshold in Kashmiri population.

Taste sensitivity and threshold of Phenylthiourea (PTC) was determined in 800 individuals of Kashmir who form a distinct ethnic group. Correlation, if any, with age, sex, blood group, pH of Saliva, or smoking habits was investigated. The taste sensitivity of P.T.C. increased with advancing age. The percentage of non-tasters was more in blood group 'B' & 'O'. However, no correlation was observed as far as sex, pH of saliva and smoking habits were concerned.

Serum sialic acid levels in normal pregnant and non-pregnant women.

Serum sialic acid estimation was done in 97 pregnant and 61 non-pregnant healthy women. A progressive rise in the level was observed as the pregnancy advanced. The rise was higher in subjects above 30 years of age. Non-pregnant women weighing more than 50 kg a significantly higher serum sialic acid level, but the contrast was reversed during pregnancy and puerperium.

Environmental conditions, anthropometric parameters and arterial blood pressure.

The influence of environmental conditions and anthropometric parameters on arterial blood pressure level was studied in 280 healthy Kashmiri subjects, aged 18-50 years (140 males, 140 females) Men in the age group of 18-20 years had higher blood pressure than women and their systolic blood pressure showed significant correlation with height, weight and skinfold thickness. Females (18-20 years) showed association of diastolic blood pressure with weight, skinfold thickness. Females (18-20) years) showed association of diastolic blood pressure with weight, skinfold thickness and mid-arm circumference. After the age of 20 years females had higher blood pressure than males. Height and weight decreased with age in both the sexes. Skinfold thickness and midarm circumference increased with age in females. A comparison between the present study and those reported from Haryana and Delhi revealed a higher diastolic pressure in Kashmiris.

Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) activity in PARP-1 silenced tumour cells increases chemosensitivity to temozolomide and to a N3-adenine selective methylating agent.

We recently demonstrated that poly(ADP-ribose) polymerase (PARP)-1 is involved in angiogenesis and tumour aggressiveness. In this study we have compared the influence of abrogation of PARP-1 expression by stable gene silencing to that of the pharmacological inhibition of cellular PARP activity using PARP-1/-2 inhibitors on the chemosensitivity of tumour cells to the wide spectrum methylating agent temozolomide (TMZ) and to the N3-adenine selective methylating agent {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2-carboxamido}propane (Me-Lex). Silencing of PARP-1 in melanoma or cervical carcinoma lines enhanced in vitro sensitivity to TMZ and Me- Lex, and induced a higher level of cell accumulation at the G2/M phase of cell cycle with respect to controls. GPI 15427, which inhibits both PARP-1 and PARP-2, increased sensitivity to TMZ and Me-Lex both in PARP-1-proficient and - deficient cells. However, it induced different cell cycle modulations depending on PARP-1 expression, provoking a G2/M arrest only in PARP-1 silenced cells. Treatment of PARP-1 silenced cells with TMZ or Me-Lex resulted in a more extensive phosphorylation of Chk-1 and p53 as compared to PARP-1 proficient cells. The combination of the methylating agents with GPI 15427 increased Chk-1 and p53 phosphorylation both in PARP-1 proficient or deficient cells. When mice challenged with PARP-1 silenced melanoma cells were treated with the TMZ and PARP inhibitor combination there was an additional reduction in tumour growth with respect to treatment with TMZ alone. These results suggest the involvement of PARP-2 or other PARPs, in the repair of DNA damage provoked by methylating agents, highlighting the importance of targeting both PARP-1 and PARP-2 for cancer therapy.