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Mohammed A Eldesouky,1 Molham A Elbakary,1 Saly Sabik,1 Mohamed M Shareef2 1Oculoplastic Unit, Ophthalmology Department, Faculty of Medicine, Tanta University, 2Pathology Department, Faculty of Medicine, Tanta University, Tanta, Egypt Purpose: The incidence and clinical and imaging criteria of different pathological forms of lacrimal fossa lesions in the Delta region of Egypt were studied.Methods: A retrospective study of patients with lacrimal fossa lesions for the past 10 years was conducted. A total of 146 cases were identified. Their medical records were reviewed for clinical and imaging data (computed tomography scan, magnetic resonance imaging scan, or both). A definitive diagnosis based on pathological examination of biopsies was also reviewed.Results: Among the patients reviewed, 43.15% had inflammatory lacrimal gland lesions, 26.71% had lymphoproliferative lesions, and 21.92% had epithelial lesions; 8.22% had rare lesions (5.48% were dacryops and 2.74% had hemangioma). The study included 71.92% benign lesions and 28.08% malignant lesions, which were distributed between 19.18% malignant lymphoma and 8.9% malignant epithelial tumors. According to the pathological origin of the lesions, they may be classified into 78.08% nonepithelial lesions and 21.92% epithelial lesions (16.44% epithelial tumors, and 5.48% dermoid cysts).Conclusion: Lacrimal fossa lesions show a wide pathological range. Inflammatory lesions are most frequent, followed by lymphoproliferative and epithelial lesions. Analysis of clinical and radiological criteria is helpful in the differential diagnosis of lacrimal gland lesions. Keywords: lacrimal gland, tumor, clinical and imaging criteria

Objective. The intend of the present study was to assess the diagnostic performance of strain elastography in investigating the thyroid nodule malignancy taking the surgical biopsy as a gold standard reference test. Methods. The study included 120 patients with 123 thyroid nodules, of which 67 had total thyroidectomy. The American College of Radiology Thyroid Imaging Reporting and Data Systems (ACR-TIRADS) were evaluated for all nodules. All suspicious nodules were referred for a fine needle aspiration cytology (FNAC) if they fulfilled the required size. Strain elastography was performed for each suspicious nodule. Ultrasound-guided FNAC was performed for all suspicious nodules. Total thyroidectomy was performed in those whom the suspicious nodules were proven by FNAC. Results. Strain ratio had a sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy of 84%, 81%, 95%, 85%, and 84%, respectively, with a cut point 1.96. Elasticity score had a sensitivity, specificity, PPV, NPV, and diagnostic accuracy of 100%, 80%, 95%, 85% and 87%, respectively, with a cut point 0.96. The elasticity score had a statistically significantly odds ratio for detecting the benignity 3.9 C. I (1.6-9.3). Conclusion. Strain elastography has a high diagnostic performance in detecting the malignant as well as benign nodules, thus it can limit the rate of unneeded FNAC or surgery especially among B3 and B4 groups with indeterminate cytology., (© 2024 Sarah Abd Elmageed Mahmoud et al., published by Sciendo.)

Objectives: To evaluate epidermal growth factor receptor and Ki-67 immunohistochemical expression in colorectal adenoma and carcinoma cases, and to relate their expression with the available clinicopathological data., Method: The retrospective study was conducted at the Faculty of Medicine, Kafrelsheikh University, Egypt, from September 2019 to October 2020, and comprised formalin-fixed and paraffin-embedded specimens related to cases of colorectal adenoma and those of colorectal carcinoma, who had no previous radiation or chemotherapeutic treatment. Immunohistochemical staining of all thecases was done using anti-epidermal growth factor receptor and anti-Ki-67 antibodies. Data was analysed using SPSS 20., Results: Of the 70 cases, 20(28.5%) were of colorectal adenoma; mean age 52.95±13.47 years, and male-to-female ratio 1:1. The remaining 50(71.5%) cases had colorectal carcinoma; mean age 51.08±13.49 years, and male-to-female ratio 1.17:1. Epidermal growth factor receptor and Ki-67 overexpression related significantly to villous histopathological type and high-grade dysplasia in colorectal adenoma cases(p<0.05). In colorectal carcinoma cases, epidermal growth factor receptor overexpression related significantly to tumour grade (p<0.05). Ki-67 overexpression related significantly to increased pathological stage (p<0.05)., Conclusions: Overexpression of epidermal growth factor receptor and Ki-67 was found to be an ominous sign of colorectal adenoma aggressiveness, and the risk of progression to colorectal carcinoma.

Lupus nephritis (LN) is a major risk factor for morbidity and mortality in systemic lupus erythematosus (SLE). Urinalysis has an invaluable role in the diagnosis of various renal and urological diseases. Examinations of the urinary sediment using phase contrast microscopy (PCM) may add more information to help earlier diagnoses of LN. This cross-sectional study aimed to assess the possible role of the components of urinary sediment examined using PCM in discriminating the proliferative classes of LN (III and IV ± V) from the non-proliferative classes (I, II, and V), and to detect the correlation between the components of urinary sediment and indices of both activity and chronicity found by the renal biopsy. The study was conducted on 40 SLE patients for whom a renal biopsy was indicated. Clinical, demographic, and laboratory data and the results of the histopathological renal biopsy were collected. The morning before the renal biopsy; urine samples were collected from every patient and examined by PCM. Receiver operating characteristic curves were used to detect the area under the curve to predict proliferative LN. The correlations of counts of leukocytes, erythrocytes, all dysmorphic erythrocytes, acanthocytes, and stomatocytes with the indices of activity and chronicity were significant (activity: P = 0.027, P = 0.015, P = 0.033, P = 0.040, and P <0.001*; chronicity: P = 0.035, P = 0.009, P = 0.027, P = 0.010, and P <0.001, respectively). For patients with SLE, urinary sediment examinations can suggest a renal biopsy for the histopathology of LN., (Copyright © 2022 Copyright: © 2022 Saudi Journal of Kidney Diseases and Transplantation.)

Urinary tract infection (UTI) is common in children. It is caused by bacteria and can lead to complications such as renal scarring in cases of late diagnosis and a lack of early treatment. Vesicoureteral reflux (VUR) occurs when urine flows from the bladder to the ureter and kidney, and is more common in children with UTI. VUR predisposes patients to pyelonephritis and can cause renal scarring. This retrospective study aimed to determine whether VUR was associated with the evolution to renal scarring in 132 infants aged 9 days to 24 months admitted to a tertiary care hospital in Jeddah, Saudi Arabia, with the first episode of febrile UTI (mean age = 4.48 months [standard deviation = 5.48]; 82 [62.1%] males; 50 [37.9%] females). Data from their medical records were investigated[INLINE:1]for VUR and renal scarring. The VUR was related to renal scarring (P = 0.001). C-reactive protein (CRP) levels were significantly higher in patients with VUR (P = 0.027) and renal scars (P = 0.05). There was a statistically significant difference between VUR and renal scarring with the first UTI (P = 0.001). In conclusion, our study revealed a significant association of CRP with renal scarring (P = 0.05) and VUR and (P = 0.027).

Introduction:  We aimed to assess the accuracy of ultrasound elastography in detecting pediatric malignant cervical lymph nodes, and if this modality can obviate the need for surgical biopsies., Material and Methods:  A prospective study from September 2017 to September 2020 included 64 children with persistent cervical lymphadenopathy. Patients were evaluated by meticulous history and physical assessment. B-mode ultrasound, color Doppler, and sonoelastography were conducted thereafter. Elastography scans were classified into five patterns, and patterns from 3 to 5 were considered as malignancies. All children underwent open biopsies followed by pathological examination. Results of tissue diagnosis were compared with patterns of elastography to determine its accuracy., Results:  Twenty-eight patients (43.8%) had malignant nodes and the remaining 36 (56.2%) were due to benign causes. Elastography patterns of 1 and 2 were documented in 30 patients, and all of them were diagnosed as benign lesions. Patterns of 3 to 5 were demonstrated in 34 patients. Out of them, 28 were confirmed as malignancies, while 6 children were of benign nature (false positive). Ultrasound elastography achieved sensitivity and specificity of 100 and 85.7%, respectively, and an overall accuracy of 90.6% in the differentiation between malignant and benign entities. The overall accuracy of B-mode and color Doppler were 75 and 82.2%, respectively., Conclusion:  Elastography is a useful tool that should be added to ultrasound modalities during the diagnosis of pediatric cervical lymphadenopathy. Surgical biopsy in eligible patients is imperative to commence proper therapy or to discharge the child. Despite favorable results of elastography, it cannot replace surgical biopsy or change its indications., Competing Interests: None declared., (Thieme. All rights reserved.)

Background and Objectives: To compare the efficacy of autologous platelet-rich plasma (PRP), ablative fractional carbon dioxide (FCO 2 ) laser, and their combination in the treatment of atrophic acne scars, both clinically and immuno-histopathologically., Study Design/materials and Methods: Sixty patients were randomly divided into three equal groups. Group 1 received intradermal PRP injection sessions. Group 2 received FCO 2 laser sessions. Group 3 received FCO 2 laser followed by intradermal PRP injection sessions. Each group received three sessions at monthly intervals. The final assessment took place 3 months after the last session. Skin biopsies were obtained before and 1 month after treatment sessions with pathological evaluation., Results: Combined PRP and FCO 2 laser-treated patients had a better clinical response, fewer side effects, and shorter downtime than FCO 2 laser alone. PRP-treated patients had some improvement but significantly lower than the other two groups., Conclusion: The current study concluded that a combination of PRP and FCO 2 laser is an effective and safe modality in the treatment of atrophic acne scars with better results than PRP or FCO 2 laser alone. Lasers Surg. Med. © 2020 Wiley Periodicals LLC., (© 2020 Wiley Periodicals LLC.)

Background: Pilonidal sinus is a common chronic disease affecting young adults. Many surgical procedures have been developed for its treatment, but an optimal one has still not been achieved. This study presents a modification to the Karydakis operation., Methods: A total of 265 patients with chronic pilonidal sinus between May 2008 and May 2015 were included in this study and submitted to fascio-adipo-cutaneous lateral advancement flap as 1-day case surgery. Any Septic complication was treated firstly. Follow-up examinations were conducted during the first three postoperative months at the outpatient clinic at regular intervals and thereafter by phone calls or direct examination to check for recurrences., Results: Males constituted 93.6% with a mean age of 21.6 ± 7.7 year. The median disease duration was 15 months. Twenty-eight patients had recurrent disease. The mean operative time was 41.4 ± 12.7 min. The mean time out of work was 11.6 ± 4.6 days. Complications occurred in 19 patients (7.1%) in the form of seroma in six patients (2.2%), superficial wound infection occurred in nine patients (3.3%), cuticular wound disruption occurred in four patients (1.5%). All patients completed 3-months follow-up, but 48 patients were lost at variable intervals with the remaining 217 patients completed follow-up throughout the period of the study with a median follow-up of 43 months. No reported recurrence and 96.7% of the patients were satisfied about their wound., Conclusion: This modification is simple with low complication rate, no recurrence, and excellent patient satisfaction.

Edelfosine is a synthetic alkyl-lysophospholipid that possesses significant antitumor activity in several human tumor models. Here, we investigated the effects of edelfosine combined with androgen deprivation (AD) in LNCaP and VCaP human prostate cancer cells. This treatment regimen greatly decreased cell proliferation compared with single agent or AD alone, resulting in higher levels of apoptosis in LNCaP compared with VCaP cells. Edelfosine caused a dose-dependent decrease in AKT activity, but did not affect the expression of total AKT in either cell line. Furthermore, edelfosine treatment inhibited the expression of androgen receptor (AR) and was associated with an increase in activating transcription factor 3 (ATF3) expression levels, a stress response gene and a negative regulator of AR transactivation. ATF3 binds to AR after edelfosine + AD and represses the transcriptional activation of AR as demonstrated by PSA promoter studies. Knockdown of ATF3 using siRNA-ATF3 reversed the inhibition of PSA promoter activity, suggesting that the growth inhibition effect of edelfosine was ATF3 dependent. Moreover, expression of AR variant 7 (ARv7) and TMPRSS2-ERG fusion gene were greatly inhibited after combined treatment with AD and edelfosine in VCaP cells. In vivo experiments using an orthotopic LNCaP model confirmed the antitumor effects of edelfosine + AD over the individual treatments. A significant decrease in tumor volume and PSA levels was observed when edelfosine and AD were combined, compared with edelfosine alone. Edelfosine shows promise in combination with AD for the treatment of prostate cancer patients. Mol Cancer Ther; 15(6); 1353-63. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background and Study Aims: Gastric cancer is the second most common cause of cancer-related death worldwide. Claudins are a family of tight junction proteins that are biologically relevant in many cancer progression steps. This study aimed to investigate the expression of the intestinal claudin (claudin 4) in gastric carcinoma and to evaluate its relation to the different clinicopathologic prognostic parameters, especially lymphangiogenesis (production of new lymphatic vessels, measured by lymphovascular density (LVD)) and lymphovascular invasion (LVI)., Patients and Methods: Fifty-five gastric carcinoma specimens were immunohistochemically stained for claudin 4 and D2-40 (for detection of lymphatic vessel endothelium)., Results: High expression of claudin 4 was detected in 26 of 55 (47.3%) cases. Low expression of claudin 4 was related to poorly differentiated type (p=0.001), non-intestinal (diffuse) type (p=0.001), deeper tumour invasion (p<0.001), lymph node metastasis (p=0.001), and higher stage (p=0.001). In addition, higher LVD was related to poorly differentiated types (p=0.001), non-intestinal type (p=0.001), lymph node metastasis (p=0.015), and higher tumour, node, metastasis (TNM) stage (p=0.001). LVI was related to lymph node metastasis (p=0.025), higher TNM stage (p=0.001), and LVD (p=0.001). Claudin 4 significantly correlated with both LVD (p=0.009) and LVI (p=0.009)., Conclusions: High expression of claudin 4 was associated with the more differentiated intestinal-type gastric carcinoma and lost in poorly differentiated diffuse type. So, claudin 4 may be used as one of the differentiating markers between the two major types of gastric carcinoma (intestinal vs. diffuse). LVD and LVI were related to higher incidence of lymph node metastasis and therefore could be used as predictive markers for lymph node metastasis in limited specimens during early gastric carcinoma to determine the need for more invasive surgery. Low expression of claudin 4 was related to lymphangiogenesis. This may shed light on the relation of tight junction protein expression and lymphangiogenesis., (Copyright © 2015 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.)

Background: Psoriasis is an erythematosquamous dermatosis, which has strong expression of antimicrobial peptides (AMPs), imparting a high resistance to lesional skin infection. Granulysin is a proinflammatory AMP that has been found in some infection-resistant dermatoses., Aim: To assess granulysin expression in psoriasis., Methods: Immunohistochemical expression of granulysin was assessed in lesional skin biopsies taken from 30 patients with psoriasis and 10 normal skin specimens from healthy controls (HCs) matched for age, gender and skin site. Granulysin expression was found to be high in 11 patients (36.7%), moderate in 10 (33.3%) and low in 9 (30%). A highly significant (P = 0.001) difference in granulysin expression between patients with psoriasis and HCs was found. There were also significant differences in granulysin expression between patients with low Psoriasis Area and Severity Index (PASI) (≤ 10) and those with high PASI (> 10) (P = 0.001), and between patients with early-onset (< 40 years of age) and those with late-onset (> 40 years of age) disease (P < 0.04). There was a significant (P = 0.001) positive correlation between granulysin expression and PASI (r = 0.84) and a significant (P = 0.02) negative correlation with age of onset (r = -0.38). Patients with psoriasis hadhigh granulysin expression, which increased with increased clinical severity of the disease., Conclusions: These findings suggest a role for granulysin in psoriasis pathogenesis, and may explain the triggering effect of skin infection in psoriasis. If the pathogenic role of granulysin is substantiated by further studies, granulysin may be a potential target for immunomodulatory therapy for psoriasis., (© 2015 British Association of Dermatologists.)

Background: Multiple complex pathways are operable in the evolution of cutaneous T cell lymphomas (CTCLs). These pathways involve interaction between neoplastic T cells and cells of the immune system (especially dendritic cells and the non-malignant T cells). Granulysin is a proinflammatory antimicrobial peptide which has an immune alarmin function, activating dendritic cells, as well as an active role in tumor immunology and prognosis. FOXP3+ regulatory T cells Tregs are an important player in the immune system. Much controversy is found in the literature about the role of Tregs in CTCL., Aim: The present study aimed to investigate the expression of granulysin and FOXP3 in mycosis fungoides (MF), its precursor lesion large plaque parapsoriasis and its leukemic form Sezary syndrome (SS)., Materials and Methods: Immunohistochemical expression of granulysin and FOXP3 were assessed in lesional skin biopsies taken from 58 patients (4 large plaque parapsoriasis, 48 MF and 6 SS)., Results: Granulysin positivity was cytoplasmic and higher in MF than in parapsoriasis en plaque and higher in the more advanced stages of MF (p<0.001). All groups showed significant differences between each other except between MF tumor stage and SS. FOXP3 positivity was nuclear and higher in early stage MF (plaque and patch stages) than in tumor stages and SS (p<0.001). However the FOXP3 count was lower in parapsoriasis en plaque than in other stages of MF. All the groups showed significant differences between each other except between parapsoriasis and SS and between patch and plaque stages of MF., Conclusions: The present study supports a role for granulysin in MF progression and proposes a novel hypothesis about the effect of FOXP3 +veTregs in the suppression of the activity of the neoplastic cells in MF.

Atrazin is currently the most widely used herbicide in agriculture with lots of adverse effects on human health. Curcumin is a polyphenol known for its antioxidant, anti-inflammatory, and anticancer properties. In the present study, the protective effect of curcumin on atrazin-intoxicated rats is evaluated. Toxicity was induced by oral administration of atrazine (400 mg/kg/day) for 3 weeks. Curcumin at a dose of 400 mg/kg/day was given simultaneously by oral route. Redox status, mitochondrial function, 8-hydroxydeoxyguanosine (8-OHdG) level by immunoassay, and caspace-3 expression by immunohistochemistry were evaluated. Curcumin showed significant cardiac protection with improvement of redox status, mitochondrial function, 8-OHdG level, caspase-3 immunoreactivity, and cardiac muscle degeneration. From this current study, it can be concluded that administration of curcumin improved atrazine-induced cardiotoxicity through its modulatory effect on redox status, mitochondrial function, and caspase-3 expression., (© 2014 Wiley Periodicals, Inc.)

A series of 6 consecutive cases of orbital metastasis from hepatocellular carcinoma (HCC) were reported in the last 5 years. All patients were men. The age of patients ranged between 47 and 70 years. Four patients presented with recent onset of unilateral proptosis, 1 patient presented with inflammatory manifestations, and 1 patient presented with unilateral ptosis. Pain was present in 4 patients. Three patients had a previous history of HCC, while the orbital affection was the first manifestation of the disease in 3 cases. All patients had chronic hepatitis C. CT scan of 5 patients showed a mass in upper lateral orbital quadrant, and 1 patient had the mass in the upper central orbit. Bone changes (thinning, notching, or erosion) were detected in all patients. Diagnosis was confirmed by incision biopsy in all cases. Life span of 5 patients in the study had a mean of 10.2 (±2.3) months.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation afflicting any part of the bowel wall as a result of a deregulated and inappropriate immune response. In recent years, experimental and clinical evidence has demonstrated that infection with parasitic worms could protect hosts from IBD. The aims of this study were to determine if the underlying mechanism of the host immune regulation inherent to Trichinella spiralis infection involves Foxp3-expressing regulatory T cells, and to gain insight about time-related interactions between intestinal nematode infection and induced colitis using an experimental model for ulcerative colitis. Mice were experimentally subjected to acetic acid-induced colitis, which was either preceded or followed by T. spiralis infection. Assessment of colitis was done by histopathological examination of the colon and determination of pentraxin 3 levels. Immunohistochemistry was done for demonstration of Foxp3-expressing regulatory T cells in colonic tissues. It was evident that T. spiralis infection ameliorated the severe inflammation induced by acetic acid, evidenced by amelioration of histopathological changes and diminution of pentraxin 3 levels. The amelioration was more pronounced when T. spiralis infection preceded the induction of colitis. Regarding the immunohistochemical staining of regulatory T cells, T. spiralis infection induced recruitment of Foxp3-expressing regulatory T cells to areas of inflammation. In conclusion, T. spiralis regulatory mechanism can improve inflammation of the colon through the 'inflammatory-regulatory' axis. Finally, it would be of great importance to apply these results to the development of new therapeutic approaches for the treatment of ulcerative colitis.

Expression of carbonic anhydrase 9 (CA9) is associated with poor prognosis and increased tumor aggressiveness and does not always correlate with HIF-1α expression. Presently, we analyzed the regulation of CA9 expression during hypoxia by HIF-1α, Notch3, and the von Hippel-Lindau (VHL) in breast carcinoma cells. Both HIF-1α and Notch3 were absolutely required for the expression of CA9 mRNA, protein, and reporter. Reciprocal co-immunoprecipitation of HIF-1α, Notch3 intracellular domain (NICD3), and pVHL demonstrated their association. The presence of common consensus prolyl hydroxylation and pVHL binding motifs (L(XY)LAP);LLPLAP(2191) suggested an oxygen-dependent regulation for NICD3. However, unlike the HIF-1α protein, NICD3 protein levels were not modulated with hypoxia or hypoxia-mimetic agents. Surprisingly, mutations of the common prolyl hydroxylation and pVHL binding domain lead to the loss of CA9 mRNA, protein, and reporter activity. Chromatin immunoprecipitation assay demonstrated the association of NICD3, HIF-1α, and pVHL at the CA9 promoter. Further, the NICD3 mutant defective in prolyl hydroxylation and subsequent pVHL binding caused a reduction in cell proliferation of breast carcinoma cells. We show here for the first time that the interaction of HIF-1α with NICD3 is important for the regulation of CA9 expression. These findings suggest that although CA9 is a hypoxia-responsive gene, its expression is modulated by the interaction of HIF-1α, Notch3, and VHL proteins. Targeting the expression of CA9 by targeting upstream regulators could be useful in cancer/stem cell therapy.

The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the μm-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely α-helical fold into a predominantly β-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Eukaryotic nuclei contain regions of differentially staining chromatin (heterochromatin), which remain condensed throughout the cell cycle and are largely transcriptionally silent. RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability. Here we report a similar phenotype for the telomeric partner of HP1, HOAP, and roles for both proteins in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the masterswitch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, Sxl(Pe), is essential to females, as it provides SXL protein to initiate the productive female-specific splicing of later Sxl transcripts, which are transcribed from the maintenance promoter (Sxl(Pm)) in both sexes. HOAP mutants show inappropriate Sxl(Pe) firing in males and the concomitant inappropriate splicing of Sxl(Pm)-derived transcripts, while females show premature firing of Sxl(Pe). HP1 mutants, by contrast, display Sxl(Pm) splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with Sxl(Pe) sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to Sxl(Pe) are severely compromised. Our genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at Sxl(Pe)., Competing Interests: The authors have declared that no competing interests exist.

Both the p53- and E2F1-signaling pathways are defective in almost all types of tumors, suggesting very important roles for their signaling networks in regulating the process of tumorigenesis and therapy response. Studies on Radiation Therapy Oncology Group tissue samples have identified aberrant expression of p53, MDM2 (an E3 ubiquitin ligase that targets p53 for proteosomal degradation), and p16 (an upstream regulator of retinoblastoma and hence E2F1 in prostate cancer); abnormal expression of these biomarkers has been associated with clinical outcome after radiotherapy ± androgen deprivation therapy. Although the proapoptotic properties of p53 are well documented, a relatively new aspect of p53 function as an active mediator of prosurvival signaling pathways is now emerging. E2F1 is a transcription factor that possesses both proapoptotic and prosurvival properties. Thus, the role of E2F1 in the process of tumorigenesis versus apoptosis is a contested issue that needs to be resolved. Furthermore, the role of E2F1 in DNA repair is being increasingly recognized. Thus, novel approaches to curb the prosurvival and DNA repair capability of E2F1 while promoting apoptotic function are of interest. In this review, we discuss the challenges involved in targeting the p53/E2F1 pathways and the crosstalk networks, and further propose potential therapeutic strategies for prostate cancer management., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Recent studies have suggested that mycophenolate mofetil (MMF) may offer advantages over intravenous cyclophosphamide (IVC) for the treatment of lupus nephritis. The aim of this study was to evaluate the efficacy of MMF compared with IVC in the induction therapy of proliferative lupus nephritis., Methods: We randomly assigned 47 patients with newly diagnosed active proliferative lupus nephritis class III or IV to open-label oral MMF 2 g/day for 6 months or intravenous cyclophosphamide 0.5-1 g/m(2) monthly for 6 months in addition to corticosteroids., Results: In the intention-to-treat analysis, 14 of the 24 patients (58.33%) receiving MMF and 12 of the 23 patients receiving cyclophosphamide (52.17%) had remission (P = 0.48); complete remission occurred in 6 of the 24 patients (25%) and 5 of the 23 patients (21.74%), respectively (P = 0.53). Improvements in packed cell volume, the erythrocyte sedimentation rate, anti-double-stranded DNA antibodies titer (anti-dsDNA), serum complement, proteinuria, urinary activity, renal function, serum soluble interleukin-2 receptor alpha concentration and the systemic lupus activity measure score were similar in both groups. Two patients assigned to MMF and another patient assigned to IVC developed end-stage renal failure with commencement of dialysis. Adverse events were similar. Major infections occurred in two patients in each group. There was no difference in gastrointestinal side effects, but more diarrhea occurred in those receiving MMF., Conclusion: In this 24-week trial, MMF or IVC combined with corticosteroids demonstrated equal efficacy in inducing remission of proliferative lupus nephritis.

Background and Study Aims: Despite the growing understanding of the involvement of protooncogenes and tumour suppressor genes in the oncogenesis of CRC, the exact biological and molecular mechanisms underpinning this process remain poorly understood. The signal transducer and activator of transcription (STAT3) has been implicated in the regulation of growth and malignant transformation. Accumulating evidences have come to indicate that abnormalities in the Janus kinase (JAK)/STAT pathway are involved in oncogenesis of several cancers. The aim of this study was to investigate the expression of JAK3 and STAT3 in both normal and activated forms by immunohistochemistry in adenomas of the colon, ulcerative colitis and CRC compared to normal colonic mucosa., Patients and Methods: Tissues from 30 cases with primary CRC and seven cases with ulcerative colitis (UC), removed by colectomy, were included. In addition, tissues from 10 colonic adenomas, 15 CRC and eight cases with UC, obtained by endoscopic biopsies, were examined histopathologically. Immunohistochemical evaluation of STAT3, p-STAT3, JAK3 and p-JAK3 expression in tissue sections was completed. Statistical analysis and correlation of data were then performed., Results: Normal colonic mucosa showed expression of STAT3 only. Immunoreactivity of p-JAK3 increased significantly (p<0.05) and correlated with the degree of dysplasia in colonic adenomas. Immunoreactivity of p-STAT3 increased significantly (p<0.05) and correlated with the degree of dysplasia in cases with UC. In CRC a significant positive correlation was found between p-STAT3 expression and grading, STAT3, JAK3 and p-JAK3 and TNM or Dukes' staging, and p-STAT3 and nodal status excluding distant metastasis (p<0.05)., Conclusion: JAK3 and STAT3, and particularly their activated forms, were found to correlate significantly with the degree of dysplasia in adenomas and UC, indicating their potential role in colorectal carcinogenesis. They also correlate with anaplasia and invasion, suggesting a definitive role in progression of CRC., (Copyright © 2009 Arab Journal of Gastroenterology. Published by Elsevier Ltd. All rights reserved.)

In this study, we investigated the functional role of early growth response-1 (Egr1 gene) in the regulation of radiation-induced clonogenic inhibition and apoptosis in p53 wild-type and mutant prostate cancer cells 22Rv1 and DU145, respectively. 22Rv1 cells were more sensitive to irradiation compared with DU145 cells, and the sensitivity was enhanced by overexpression of EGR-1 in both cells. Dominant-negative EGR-1 mutant (dnEGR-1) or repressor of EGR-1, NGFIA binding protein 1 (NAB1), increased radioresistance of these cells. Significant activation of caspases 3 and 9 and Bcl2-associated X (Bax) with increased poly(ADP-ribose) polymerase (PARP) cleavage and cytochrome c release was observed in radiation-exposed EGR-1 overexpressing cells. Gel shift analysis and chloramphenicol acetyl transferase (CAT) reporter assays indicate that EGR-1 transactivates the promoter of the Bax gene. Interaction of EGR-1 and Yes kinase-associated protein 1 (YAP-1) through the WW domain of YAP-1 enhances the transcriptional activity of EGR-1 on the Bax promoter as shown by chromatin immunoprecipitation and reporter assays. Irradiation of PC3 cell xenografts that were treated with adenoviral EGR-1 showed significant regression in tumor volume. These findings establish the radiation-induced pro-apoptotic action of EGR-1, in a p53-independent manner, by directly transactivating Bax, and prove that alters the B-cell CLL/lymphoma 2 (Bcl-2)/Bax ratio as one of the mechanisms resulting in significant activation of caspases, leading to cell death through the novel interaction of EGR-1 with YAP-1.

TGF-β signaling regulates several different biological processes involving cell-growth, differentiation, apoptosis, motility, angiogenesis, epithelial mesenchymal transition and extracellular matrix production that affects embryonic development and pathogenesis of various diseases, including cancer, its effects depending on the cellular context and physiological environment. Growth suppression mediated by TGF-β signaling often associated with inhibition of c-myc, cdks and induction of p15, p27, Bax and p21. Despite its growth inhibitory effect, in certain conditions TGF-β may act as a promoter of cell proliferation and invasion. Loss of responsiveness to growth suppression by TGF-β due to mutation or loss of TGF-beta type II receptor (TβRII) and Smad4 in several different cancer cells are reported. In addition, TGF-β binding to its receptor activates many non-canonical signaling pathways. Radiation induced TGF-β is primarily involved in normal tissue injury and fibrosis. Seminal studies from our group have used radio-adjuvant therapies, involving classical components of the pathway such as TβRII and SMAD4 to overcome the growth promoting effects of TGF-β. The main impediment in the radiation-induced TGF-β signaling is the induction of SMAD7 that blocks TGF-β signaling in a negative feedback manner. It is well demonstrated from our studies that the use of neutralizing antibodies against TGF- β can render a robust radio-resistant effect. Thus, understanding the functional interactions of TGF-β signaling components of the pathway with other molecules may help tailor appropriate adjuvant radio-therapeutic strategies for treatment of solid tumors.

Cryoablation is one of the established treatment modalities for prostate cancer management. Although, it is target specific, it may still lead to damage to the nerve fibers around the prostate tumor. In this study, by directly exposing the co-cultures of prostate cancer cells, PC-3 and Schwann cell-Dorsal Root Ganglion neuron (SC-DRG) to cryo-shock and by exposing SC-DRG to cryo-shock conditioned media (CSCM) obtained from PC-3 cells, robust neuro-protective effects were observed. Since this neuro-protective effect originated from cryotherapy-treated PC-3 cells, the presence of putative factors secreted by PC-3 cells in the medium following cryo-shock was analyzed. Using human cytokine antibody array analysis, differential release of cytokines in CSCM was observed with induced release of cytokines involved in neuro-protection like IL-1α, MIP-4, MIP-5, Leptin, IL-15 and ICAM-1 with simultaneous inhibition of TNFRI and TNFRII that are implicated in killing of nerve cells. Further, using Matrix Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) sequencing, two proteins were identified namely, CypA (cyclophilin A) and NM23 (nonmetastatic protein 23) in the CSCM. CypA functions as a mediator of intracellular as well as extracellular neuro-protective mechanisms and NM23 has been implicated as a potential suppressor protein of tumor metastasis. Thus, this study revealed the presence of factors in CSCM that has the potential to protect normal neuronal cells and suppress metastasis.

Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones.

Schistosoma mansoni (S. mansoni) eggs trapped in the host liver elicit a chain of oxidative processes that may be, at least in part, responsible for the pathology and progression of fibrosis associated with schistosomal hepatitis. This study was designed to assess the protective effect of the antioxidant coenzyme-Q10 (Co-Q10) against experimental S. mansoni-induced oxidative stress in the liver, and its potential role as an adjuvant to praziquantel (PZQ) therapy. The oxidative stress and overall liver function were improved under Co-Q10 therapy as evidenced by significant reduction in oxidative stress markers and preservation of antioxidant factors. Liver fibrosis was also reduced with a positive impact on liver function. Moreover, addition of Co-Q10 to PZQ therapy caused: significant reduction of liver egg load, significant improvement of the redox status, and lastly decreased liver fibrosis.

Multidrug resistance (MDR) is associated with the overproduction of the 170-kDa transmembrane protein P-glycoprotein (MDR1) caused by transcriptional activation. However, the activity of the MDR1 promoter in response to different doses of ionizing radiation has not been investigated. In this study, two squamous cell carcinoma oral cavity cell lines, T-167 and T-409, were exposed to either a standard clinical dose of 2 Gy or low-dose fractionated radiation therapy (LDFRT), delivered as 0.5 Gy in four fractions. MDR1 gene expression and degree of cell death were assessed. Clinically relevant 2-Gy dose of radiation resulted in increased expression of MDR1 by reverse transcription-PCR and luciferase reporter assays in both cell lines (T-167 and T-409), whereas LDFRT did not. LDFRT caused enhanced apoptosis when compared with the 2-Gy dose in T-167 and T-409 cells as assessed by terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assays. Transcription of the MDR1 gene is regulated by numerous transcription factors, which include nuclear factor-kappaB (NF-kappaB), NF-Y, SP1, YB1, MEF1 (MDR1 promoter-enhancing factor 1), p53, and NF-R1. Interestingly, 2 Gy robustly induced both NF-kappaB and NF-Y in T-167 and T-409 cells, but did not show induction when exposed to LDFRT. Silencing the expression of the DNA binding subunit of NF-kappaB, p50, by small interfering RNA vector resulted in a decrease of MDR1 function by rhodamine 123 efflux assay in T167 cells exposed to 2 Gy. Together, these results provide evidence for the lack of induction of P-glycoprotein expression by LDFRT, which has important implications in combinatorial cancer therapy, including the use of LDFRT as an adjuvant for chemotherapy.

In the present study, ionizing radiation (IR)-induced bystander effects were investigated in two lung cancer cell lines. A549 cells were found to be more resistant to radiation-conditioned medium (RCM) obtained from A549 cells when compared with the H460 exposed to RCM procured from H460 cells. Significant release of tumor necrosis factor-alpha (TNF-alpha) was observed in A549 cells after IR/RCM exposure, and the survival was reversed with neutralizing antibody against TNF-alpha. In H460 cells, significant release of TNF-related apoptosis-inducing ligand (TRAIL), but not TNF-alpha, was observed in response to IR, RCM exposure, or RCM + 2Gy, and neutralizing antibody against TRAIL diminished clonogenic inhibition. Mechanistically, TNF-alpha present in RCM of A549 was found to mediate nuclear factor-kappaB (NF-kappaB) translocation to nucleus, whereas the soluble TRAIL present in RCM of H460 cells mobilized the nuclear translocation of PAR-4 (a proapoptotic protein). Analysis of IR-inducible early growth response-1 (EGR-1) function showed that EGR-1 was functional in A549 cells but not in H460 cells. A significant decrease in RCM-mediated apoptosis was observed in both A549 cells stably expressing small interfering RNA EGR-1 and EGR-1(-/-) mouse embryonic fibroblast cells. Thus, the high-dose IR-induced bystander responses in A549 may be dependent on the EGR-1 function and its target gene TNF-alpha. These findings show that the reduced bystander response in A549 cells is due to activation of NF-kappaB signaling by TNF-alpha, whereas enhanced response to IR-induced bystander signaling in H460 cells was due to release of TRAIL associated with nuclear translocation of PAR-4.

Quinsy (peritonsillar abscess) is a common emergency seen in otolaryngology practice. These patients are often screened for glandular fever in addition to routine haematological tests. In our unit, we have screened 66 patients with quinsy for glandular fever over a period of 12 months. All these patients were screened for glandular fever by rapid immunoassay. Only one out of 66 patients was tested positive for glandular fever. Due to the extremely low incidence of glandular fever in quinsy patients, we do not see any relevance in screening for glandular fever in quinsy patients. Hence we recommend that routine screening for glandular fever in quinsy patients is an unnecessary invasive investigation for the patients and not cost effective for the hospital.

Purpose: To address the functional role of radiation-induced transforming growth factor-beta (TGF-beta) signaling in a normal epithelial background, we selected a spontaneously immortalized lung epithelial cell line derived from the normal lung tissue of a dominant-negative mutant of the TGF-beta RII (DeltaRII) transgenic mouse that conditionally expressed DeltaRII under the control of the metallothionein promoter (MT-1), and assessed this cell line's response to radiation., Methods and Materials: A spontaneously immortalized lung epithelial cell culture (SILECC) was established and all analyses were performed within 50 passages. Colony-forming and terminal transferase dUPT nick end labeling (TUNEL) assays were used to assess clonogenic inhibition and apoptosis, respectively. Western-blot analysis was performed to assess the kinetics of p21, bax, and RII proteins. Transforming growth factor-beta-responsive promoter activity was measured using dual-luciferase reporter assay., Results: Exposure to ZnSO(4) inhibited TGF-beta signaling induced either by recombinant TGF-beta1 or ionizing radiation. The SILECC, treated with either ZnSO(4) or neutralizing antibody against TGF-beta, showed a significant increase in radio-resistance compared to untreated cells. Furthermore, the expression of DeltaRII inhibited the radiation-induced up-regulation of the TGF-beta effector gene p21(waf1/cip1)., Conclusions: Our findings imply that inhibition of radiation-induced TGF-beta signaling via abrogation of the RII function enhances the radio-resistance of normal lung epithelial cells, and this can be directly attributed to the loss of TGF-beta signaling function.

A 32-year-old woman presented with bilateral lower eyelid margin erosion and erythema of several years' duration. She reported no improvement with the use of topical corticosteroid therapy. A full-thickness excisional biopsy was performed, and the histopathology of the specimens was consistent with lichen planus.

Establishing diagnosis of a granulomatous lesion of the nose is often difficult. Here we report a case of granulomatous lesion of the nose caused by Leishmania--an unlikely cause in the UK. The diagnosis and management of the case is discussed here.

Objective: To examine the relationship between obesity and cardiovascular risk factors among men aged 20 years and above., Methods: The study involved a cross-sectional survey of 2,250 Saudi male soldiers aged between 20 and 60 years residing in a military city in northern Saudi Arabia conducted in 2004. Anthropometric measurements, blood pressure, and a brief medical history were obtained in a pre-set questionnaire. Serum lipid profile and fasting plasma sugar were requested for all the subjects. A total of 1,079 subjects responded with a response rate of 47.9%. A multivariate analysis was performed to assess the relationship between general obesity, abdominal obesity, and cardiovascular risk factors., Results: Over 82% of the subjects were either overweight or obese. Abdominal obesity was found in one third, and approximately half were either current or ex-smokers. The means of anthropometric and laboratory measured risk factors for cardiovascular disease showed a progressive rise with increase in age, abdominal, and general obesity., Conclusion: This study has shown a high prevalence of overweight and obesity positively correlated with the prevalence of cardiovascular risk factors among Saudi adult male soldiers. There is a need for concerted efforts aimed at achieving ideal body-weight together with a reduction in the co-existent risk factors for cardiovascular disease.

Munc18-1, also referred to as p67, co-purifies with Cdk5 and has an important role in neurotransmitter release. The role of Munc18-1 for functional connectivity of the nervous system was demonstrated by gene knockout experiments in mice, wherein accumulation of neurotransmitter and silencing of synaptic activity was observed. Our earlier studies have shown that both Munc18-1 and Cdk5 co-purify and co-localize with cytoskeletal components, implying that apart from having a regulatory role in vesicle docking and fusion, Munc18-1 could also affect the dynamics of neuronal cytoskeleton. In the present study we have shown the presence of Munc18-1 in nuclear rich fraction from rat brain and confirmed the nuclear localization of this protein in PC12 cells and adult rat brain neurons by immunofluorescence and immunoelectron microscopy. We also demonstrate the binding of Munc18-1 to double stranded (ds) DNA. The ability of Munc18-1 to bind dsDNA, albeit the lack of DNA binding domains, suggests that the binding may be mediated through protein-protein interaction through some other DNA-binding proteins. The presence of both nuclear import and export signals in Munc18-1 primary structure corroborates its nuclear localization and makes it a putative shuttle protein between nuclear and cytoplasmic compartments, the precise physiological relevance of which needs to be elucidated.

Glial progenitors from the brain of normal adult Sprague-Dawley rats were compared to their initiated and malignant counterparts that were isolated from apparently normal brains of animals exposed to methylnitrosourea (MNU). Fibroblast growth factor-2 (FGF-2) or platelet-derived growth factor (PDGF)-A or -B induced differentiation of normal progenitors to a pro-astrocytic or oligodendrocytic morphology, respectively, whereas the combination of these factors resulted in their terminal differentiation to oligodendrocytes and senescence. In contrast, initiated progenitors did not exit the cell cycle when stimulated with PDGF and/or FGF-2. cDNA oligoarray analysis and RT-PCR verification showed an early upregulation/ induction of growth factor/receptors, PDGF-A, PDGFR-beta, IGFR-1, IGF-1 and -2, IL-6, MEGF-5, FRAG-1, IRS-2, HSPG, and FGFR-1, followed by a late increase in the expression IGFBP-6, PDGF-alpha, FGFR-4A, c/ERB-A, and FGFR-4, 2, and 1 during the tumorigenic progression. Western blot analyses demonstrated that MNU exposure caused progressive reduction of p21 protein levels, an increase of Rb phosphorylation, activation of AKT and CDK2, and upregulation of FGF receptors. Double immunofluorescence labeling showed progressive increase in nuclear colocalization of FGFR1, 2, and 4, which peaked in malignant lines. It is postulated that transition of normal rat glial progenitors to an initiated state is driven by IGF-1 and 2, IL-6, and the upregulation of the receptors PDGFR-beta and FGFR-1, 2, and 4. Deregulation of the cell cycle in this state involves reduction of p21 protein, concomitant upregulation of CDC2, and an increase in Rb phosphorylation that favors expression and nuclear translocation of FGFR-4 and FRAG-1 and 2. These events are associated with progressive activation of AKT and RAS. Malignant transformation is enhanced by near elimination of p21 and PC3, induction of AP-1 (upregulation of JUN-B, c-JUN, FRA-1), activation of the NF-kB pro-survival pathway, and inhibition of the TGF-beta pro-apoptotic pathway possibly in response to changes in the expression of nerve growth factor (NGF) I-A and NGFI-B. These data demonstrate that the events leading to malignancy in the rat brain in response to MNU treatment are to a great extent similar to those described for secondary glial malignancies in humans.

Methionine deprivation imposes a metabolic stress, termed methionine stress, that inhibits mitosis and induces cell cycle arrest and apoptosis. The methionine-dependent central nervous system tumor cell lines DAOY (medulloblastoma), SWB61 (anaplastic oligodendroglioma), SWB40 (anaplastic astrocytoma), and SWB39 (glioblastoma multiforme) were compared with methionine-stress resistant SWB77 (glioblastoma multiforme). The cDNA-oligoarray analysis and reverse transcription-PCR verification indicated common changes in gene expression in methionine-dependent cell lines to include up-regulation/induction of cyclin D1, mitotic arrest deficient (MAD)1, p21, growth arrest and DNA-damage-inducible (GADD)45 alpha, GADD45 gamma, GADD34, breast cancer (BRCA)1, 14-3-3sigma, B-cell CLL/lymphoma (BCL)1, transforming growth factor (TGF)-beta, TGF-beta-induced early response (TIEG), SMAD5, SMAD7, SMAD2, insulin-like growth factor binding protein (IGFBP7), IGF-R2, vascular endothelial growth factor (VEGF), TNF-related apoptosis-inducing ligand (TRAIL), TNF-alpha converting enzyme (TACE), TRAIL receptor (TRAIL-R)2, TNFR-related death receptor (DR)6, TRAF interacting protein (I-TRAF), IL-6, MDA7, IL-1B convertase (ICE)-gamma, delta and epsilon, IRF1, IRF5, IRF7, interferon (IFN)-gamma and receptor components, ISG15, p65-NF-kappaB, JUN-B, positive cofactor (PC)4, C/ERB-beta, inositol triphosphate receptor I, and methionine adenosyltransferase II. On the other hand, cyclins A1, A2, B1 and B2, cell division cycle (CDC)2 and its kinase, CDC25 A and B, budding uninhibited by benzimidazoles (BUB)1 and 3, MAD2, CDC28 protein kinase (CKS)1 and 2, neuroepithelial cell transforming gene (NET)1, activator of S-phase kinase (ASK), CDC14B phosphatase, BCL2, TGF-beta activated kinase (TAK)1, TAB1, c-FOS, DNA topoisomerase II, DNA polymerase alpha, dihydrofolate reductase, thymidine kinase, stathmin, and MAP4 were down-regulated. In the methionine stress-resistant SWB77, only 20% of the above genes were affected, and then only to a lesser extent. In addition, some of the changes observed in SWB77 were opposite to those seen in methionine-dependent tumors, including expression of p21, TRAIL-R2, and TIEG. Despite similarities, differences between methionine-dependent tumors were substantial, especially in regard to regulation of cytokine expression. Western blot analysis confirmed that methionine stress caused the following: (a) a marked increase of GADD45alpha and gamma in the wt-p53 cell lines SWB61 and 40; (b) an increase in GADD34 and p21 protein in all of the methionine-dependent lines; and (c) the induction of MDA7 and phospho-p38 in DAOY and SWB39, consistent with marked transcriptional activation of the former under methionine stress. It was additionally shown that methionine stress down-regulated the highly active phosphatidylinositol 3'-kinase pathway by reducing AKT phosphorylation, especially in DAOY and SWB77, and also reduced the levels of retinoblastoma (Rb) and pRb (P-ser780, P-ser795, and P-ser807/811), resulting in a shift in favor of unphosphorylated species in all of the methionine-dependent lines. Immunohistochemical analysis showed marked inhibition of nuclear translocation of nuclear factor kappaB under methionine stress in methionine-dependent lines. In this study we show for the first time that methionine stress mobilizes several defined cell cycle checkpoints and proapoptotic pathways while coordinately inhibiting prosurvival mechanisms in central nervous system tumors. It is clear that methionine stress-induced cytotoxicity is not restricted by the p53 mutational status.

Munc18-1 (p67, nSec1, rbSec1), a neuron-specific 67kDa protein was independently identified as a syntaxin-binding protein, and as a component that co-purifies with, and regulates the kinase activity of cyclin dependent kinase (Cdk5). Gene knockout studies have demonstrated a role for Munc18-1 in synaptic vesicle docking and neurotransmitter release. Mice lacking Munc18-1 gene were synaptically silent, but the gene deletion did not prevent normal brain assembly, including the formation of layered structures, fiber pathways and morphologically defined synapses. Previous study has shown that Munc18-1 facilitates Cdk5 mediated phosphorylation of KSPXK domains of the neuronal cytoskeletal elements, suggesting that Munc18-1 may function in the regulation of cytoskeletal dynamics. Present study demonstrates the co-purification and co-localization of Munc18 with cytoskeletal elements and forms first step towards understanding the role for Munc18-1 in cytoskeletal dynamics. Conversely, the cytoskeletal proteins and Cdk5 co-purifies with Munc18-1 in a Munc18-1 immuno-affinity chromatography, suggesting a strong protein-protein interaction. Findings from immunofluorescence studies in PC12 cells have shown co-localization of Munc18-1 and Cdk5 with neurofilaments and microtubules. Further, immunohistochemical and immuno-electron microscopic studies of rat olfactory bulb also demonstrated co-localization of Munc18-1 and Cdk5 with cytoskeletal elements. Thus, the biochemical evidence of strong interaction between Munc18-1 with cytoskeletal proteins and morphological evidence of their (Munc18 and cytoskeletal elements) identical sub-cellular localization is suggestive of the possible role for Munc18-1 in cytoskeletal dynamics.

Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Here we show direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain) (47). We find heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin.

We have used the highly conserved heterochromatin component, heterochromatin protein 1 (HP1), as a molecular tag for purifying other protein components of Drosophila heterochromatin. A complex of HP1 associated with the origin recognition complex (ORC) and an HP1/ORC-associated protein (HOAP) was purified from the maternally loaded cytoplasm of early Drosophila embryo. We propose that the DNA-binding activities of ORC and HOAP function to recruit underphosphorylatedisoforms of HP1 to sites of heterochromatin nucleation. The roles of highly phosphorylated HP1, other DNA-binding proteins known to interact with HP1, and histone modifying activities in heterochromatin assembly are also addressed.

A spectrophotometric method based on dinitrophenol (DNP) derivatization of proteolytic products was developed for monitoring the increase in NH(2)-groups as a function of protease activity. DNP derivatization of amino acids and proteolytic products was carried out at an alkaline pH of 8.8, in presence of 2,4-dinitrofluorobenzene (DNFB), followed by the stabilization of products by adjusting the pH to approximately 2.5. Using casein as substrate, under the defined assay conditions for proteases, trichloroacetic acid soluble proteolytic products were derivatized with DNFB reagent. Though alkaline pH favored the DNP derivatization of primary amino compounds, the products formed were found to be unstable. However, upon adjusting the pH to 2.5+/-0.1, DNP derivatives of amino acids and proteolytic products were found to be stable with identical lambda(max) of 395 nm. The utility of the method was evaluated by assaying the proteolytic activities of trypsin and calcium activated neutral protease (CANP). Proteolytic activity was quantified by employing the molar extinction coefficient of DNP derivatives of an equimolar concentration of glutamate and glycine. By employing this method, CANP activity in different regions of rat brain was determined. The proposed method to monitor the increase in NH(2)-end groups as a function of proteolytic activity could be employed to assay the activity of proteases., (Copyright 2002 Elsevier Science B.V.)

Quantification of total and individual amino acids in biological fluids such as plasma, urine and cerebrospinal fluid has an important diagnostic implication in laboratory medicine. The present paper describes protocols for the assay of total amino acids by modified method based on dinitrophenyl and HPLC profile involving pre-column derivatization with o-pthalaldehyde (OPA) derivatization, respectively. The method, based on the alkylation of-SH groups prior to OPA derivatization of amino acids followed by reverse phase high performance liquid chromatography, provide a comprehensive profile of more than twenty amino acids (including-SH group containing) in a single run lasting about 45 minutes. The present study, apart from establishing the normal profile of amino acids in plasma of Indian sub population, also presents HPLC profile for some of the rare amino acidopathies.

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

p67 (Munc-18), is a neuron-specific protein of 67 kDa, known for its ability to bind with syntaxin and also to copurify with neuronal cdc2-like kinase. Earlier, in situ hybridization and immunocytochemical analysis of rat trigeminal ganglion and hippocampal cells demonstrated the specific localization of p67 in nerve cells and its rich distribution in axons. In the present study, we have looked for p67 expression in normal human brain and various neuroectodermal tumors. Immunohistochemical and Western immunoblot analysis of normal human brain tissue using antibodies against the N- and C-termini of p67 demonstrated the specific localization of this protein in postmitotic neurons but not in glia. Among neuroectodermal tumors, expression of p67 was observed in 100% of the tumors of neuronal origin studied, especially in the mature neuronal cell population of these tumors. Western immunoblot analysis of non-neuronal neuroectodermal tumors failed to reveal the expression of this protein in majority of cases. However, in gliomas and meningiomas, mild cytoplasmic immunohistochemical staining of neoplastic cells was noted in 64.7% and 25% of cases, respectively. Observed mild immunohistochemical staining of these tumors could be due to immunoreactivity to low molecular weight degraded products of p67, as seen on Western blot. The findings suggest that p67, by virtue of its ability to be expressed in postmitotic neurons of adult human brain and in tumors of neuronal origin, may serve as a molecular tool to understand the growth and differentiation of the nervous system in general.