Expression of oncofoetal marker carcinoembryonic antigen in oral cancers in South India--a pilot study.

Expression of the oncofoetal glycoprotein, carcinoembryonic antigen (CEA), has been observed in a number of malignancies and is also being pursued as a target for anti-cancer therapy. This study explored the status of this biochemical entity in the oral squamous cell carcinoma (SCC) in South India caused by extensive chewing habits. Squamous cell carcinoma in the study belonged to grade I and grade II. Tumour staging of the patients recruited in the study ranged from T2N1M0 to T4N3M0. Of the grade II cases studied, 88% (7 out of 8) showed expression of CEA. The 2 cases of grade I SCC of buccal mucosa also showed positive anti-CEA staining. If the results from this pilot study can be validated with a larger sample size, a role can be attributed to this tumour marker in oral neoplasia, thereby opening up avenues for using CEA as an additional diagnostic marker in oral SCC in this population and as a possible target for anti-cancer therapy.

Telomerase from human leukemia cells: properties and its interaction with deoxynucleoside analogues.

Telomerase is a unique reverse transcriptase involved in the maintenance of genomic integrity. In an attempt to understand the properties of this enzyme and to study the effect of deoxynucleoside analogues, we have isolated and partially purified telomerase from the blast cells of a patient with acute myelogenous leukemia. During the course of purification of telomerase, three characteristic forms of this enzyme activity were separated. Two processive forms and one less processive form were noted. All forms of the enzyme activities could be abolished by RNase A and proteinase K treatments, implying that they are ribonucleoproteins. The major form of telomerase was characterized with respect to divalent ion requirements, effect of salt and nonionic detergents. The Km of deoxynucleoside triphosphates was determined with a modified telomerase repeat array protocol assay. Studies with deoxynucleoside analogues indicated that 3'-azido-3'deoxythymidine triphosphate is much more inhibitory than 2',3'-dideoxy 2',3'didehydrothymidine triphosphate, and the cytidine analogue ddCTP was not inhibitory. ddGTP was the most potent inhibitor among all dideoxynucleosides studied.

Microsurgical anatomy of the middle cerebral artery.

BACKGROUND: The microsurgical anatomy of the middle cerebral artery (MCA) is of particular interest to the cerebrovascular surgeon. The purpose of this study was to define the microsurgical anatomy of the MCA and its various branches in the Indian population. METHODS: Ten MCAs were studied from five cadaveric brain specimens. The authors studied the outer diameter, length, branches, perforators and site of these on the main trunk (M1), the division of the main trunk, the secondary trunks and their various cortical branches using the operating microscope under 5-20x magnification. RESULTS: The outer diameter of the MCA main trunk ranges from 2.5 to 4 mm with a mean of 3.35 mm. The superolateral branches consisted of polar temporal artery and anterior temporal artery that had a common origin and sometimes the uncal artery or the accessory uncal artery. Perforators or lenticulostriate arteries were seen in the inferomedial surface all along the length of M1. Eight bifurcations and two trifurcations were noted. Cortical branches and their origin are discussed. CONCLUSION: Although the microsurgical anatomy of the MCA in Indian population correlated with the findings in the western literature, some structural and statistical variations were noted.

Synthesis and biological evaluation of 2',3'-dideoxy-L-pyrimidine nucleosides as potential antiviral agents against human immunodeficiency virus (HIV) and hepatitis B virus (HBV).

Various 2',3'-dideoxy-L-cytidine,2',3'-dideoxy-L-uridine, and 3'-deoxy-L-thymidine analogues have been synthesized and evaluated in vitro as potential anti-HIV and anti-HBV agents. Coupling of 1-O-acetyl-5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-L-ribofuranose (1) with silylated derivatives of 5-fluorocytosine, cytosine, 5-fluorouracil, uracil, and thymine in the presence of ethylaluminum dichloride gave the corresponding nucleosides 2, 3, 4, 5, 10, 11, 12, 16, 17, and 18 as a mixture of alpha- and beta-anomers, which were then deblocked to yield the corresponding 2',3'-dideoxy-L-5-fluorocytidine derivatives, 6 and 7, 2',3'-dideoxy-L-cytidine derivatives, 8 and 9, 2',3'-dideoxy-beta-L-fluorouridine (13), 2',3'-dideoxy-beta-L-uridine (14), and 3'-deoxy-L-thymidine derivatives, 15 and 19. Among these 2',3'-dideoxy-L-nucleoside analogues, 2',3'-dideoxy-beta-L-5-fluorocytidine (6, beta-L-FddC) was found to be the most active against HIV-1, which is approximately 3 and 4 times more active against HIV-1 in vitro than 2',3'-dideoxy-beta-D-cytidine (ddC) and 2',3'-dideoxy-beta-D-5-fluorocytidine (beta-D-FddC) with ED50 values of 0.5, 1.5, and 2 microM, respectively. The dose-limiting toxicity of ddC is severe neuropathy which may be caused by the inhibition of the synthesis of mitochondrial DNA. ddC has an IC50 value of 0.022 microM against host mitochondrial DNA synthesis. Conversely, the IC50 values for beta-L-FddC and beta-L-ddC are > 100 microM; therefore, neuropathy may not present itself to be a problem with beta-L-FddC and beta-L-ddC as chemotherapeutic agents. In addition, beta-L-FddC and 2',3'-dideoxy-beta-L-cytidine (8, beta-L-ddC) demonstrated equally potent activity against HBV in vitro by having the same ED50 value of 0.01 microM. Both beta-L-FddC and beta-L-ddC, which have an "unnatural" L-configuration in the sugar moiety, are approximately 1000 and 280 times more potent, respectively, against HBV than the D-configuration beta-D-FddC and ddC which have an ED50 values of 10 and 2.8 microM. In view of the potent antiviral activity of beta-L-FddC against both HIV-1 and HBV and potent antiviral activity of beta-L-ddC against HBV in vitro, their low cytotoxicity, and especially the negligible inhibitory effect on host mitochondrial DNA synthesis, beta-L-FddC and beta-L-ddC merit further development as potential anti-HIV and anti-HBV agents.

Structure-activity relationships of cytotoxic cholesterol-modified DNA duplexes.

Short DNA duplexes with cholesterol linked at the 3'-terminus of each strand have unique, selective cytotoxic properties. The structural requirements for biological activity were explored through chemical synthesis of analogs and testing in cultured hepatoma cells. Effects of modifications to the sequence, backbone, 3'-sterol, 3'-linker, and 5'-terminus were evaluated. Self-complementary 3'-modified oligodeoxynucleotide (ODN) 10-mers were prepared from solid supports bearing the modification and linker of interest. Any changes to the normal phosphodiester backbone were poorly tolerated. The presence of cholesterol or a closely related sterol was an absolute requirement for activity. The length and position of attachment of the linker to cholesterol was important, with longer linkers showing reduced activity. Large, lipophilic groups at the 5'-terminus gave reduced cytotoxicity and poor solubility properties. The short length and unique structure of these ODNs allowed efficient automated synthesis on a 400 mumol scale and simplified purification.

Structure--activity relationships of 1-(2-Deoxy-2-fluoro-beta-L-arabinofuranosyl)pyrimidine nucleosides as anti-hepatitis B virus agents.

Since 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU) has been shown to be a potent anti-HBV agent in vitro, it was of interest to study the structure-activity relationships of related nucleosides. Thus, a series of 1-(2-deoxy-2-fluoro-beta-L-arabinofuranosyl)pyrimidine nucleosides have been synthesized and evaluated for antiviral activity against HBV in 2.2.15 cells. For this study, L-ribose was initially used as the starting material. Due to the commercial cost of L-ribose, we have developed an efficient procedure for the preparation of L-ribose derivative 6. Starting from L-xylose, 6 was obtained in an excellent total yield (70%) through the pyridinium dichromate oxidation of the 3-OH group followed by stereoselective reduction with NaBH4. It was further converted to the 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-alpha-L-arabinofuranose (10), which was then condensed with various 5-substituted pyrimidine bases to give the nucleosides. Among the compounds synthesized, the lead compound, L-FMAU (13), exhibited the most potent anti-HBV activity (EC50 0.1 microM). None of the other uracil derivatives showed significant anti-HBV activity up to 10 microM. Among the cytosine analogues, the cytosine (27) and 5-iodocytosine (35) derivatives showed moderately potent anti-HBV activity (EC50 1.4 and 5 microM, respectively). The cytotoxicity of these nucleoside analogues has also been assessed in 2.2.15 cells as well as CEM cells. None of these compounds displayed any toxicity up to 200 microM in 2.2.15 cells. Thus, compound 13 (L-FMAU), 27, and 35 showed a selectivity of over 2000, 140, and 40, respectively.

Antiviral activity of 2',3'-dideoxy-beta-L-5-fluorocytidine (beta-L-FddC) and 2',3'-dideoxy-beta-L-cytidine (beta-L-ddC) against hepatitis B virus and human immunodeficiency virus type 1 in vitro.

2',3'-Dideoxy-beta-L-5-fluorocytidine (beta-L-FddC) and 2',3'-dideoxy-beta-L-cytidine (beta-L-ddC), two nucleosides with "unnatural L-configuration," have been synthesized and found to have potent antiviral activity against hepatitis B virus (HBV) and human immunodeficiency virus type 1 (HIV-1) in vitro with very little toxicity. At 1 microM, both beta-L-ddC and beta-L-FddC inhibited the growth of HBV by more than 90%, while at the same concentration the D-configuration counterparts, 2',3'-dideoxy-beta-D-cytidine (ddC) and 2',3'-dideoxy-beta-D-5-fluorocytidine (beta-D-FddC), did not show antiviral activity against HBV. The order of anti-HIV-1 activity was beta-L-FddC > ddC; beta-D-FddC > beta-L-ddC. The dose-limiting toxicity of ddC is neuropathy which is believed to be caused by the inhibition of the synthesis of mitochondrial DNA. ddC severely inhibited the mitochondrial DNA synthesis of CEM cells yielding an IC50 value of 0.022 microM. Conversely, both beta-L-FddC and beta-L-ddC did not demonstrate any inhibition against mitochondrial DNA synthesis up to 100 microM concentration.

Tumor promotion studies in rat tracheal epithelium.

The tracheal epithelium of the Fischer 344 rat is histologically very similar to that of the human bronchus. Also, carcinomas of tracheal origin in F-344 rats are similar in morphology to human bronchogenic carcinomas. Tumor promotion in rat tracheal epithelium was studied by using two model systems. The first is a heterotopic transplant system in which rat tracheas are implanted subcutaneously on the backs of isogenic recipents. In the first system, the epithelium was topically exposed to pellets containing 7,12-dimethylbenz(a)anthracene (DMBA), used as the initiating agent, followed by pellets containing the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the promoting agent. After 98 weeks, a three- to fourfold increase in the percentage of tracheas having malignant tumors was seen in tracheal transplants receiving both DMBA and TPA compared to DMBA alone. Exposure of the tracheal grafts to TPA alone resulted in epithelial hyperplasia and inflammation, but no dysplastic lesions. The second system is an organ culture-cell culture system in which small pieces of trachea are grown in organ culture, then epithelial cells are grown from these pieces as primary cell cultures. The organ cultures were exposed to the direct alkylating agent, N-methyl-N'-nitro-N -nitrosoguanidine (MNNG) used as the initiator, then multiple short exposures to TPA were used to promote. Primary cell cultures and cell lines were then established from these explants. After 52 weeks, a five-fold increase in the percentage of explants producing tumorigenic cell lines was observed when MNNG + TPA-exposed explants were compared to MNNG-exposed explants. Tracheal explants exposed to TPA alone produced many cell lines but none tested were tumorigenic. These two systems provide a means to study tumor promotion in respiratory epithelium. The evidence more importantly suggests that airborne promoting substances may play a key role in the development of bronchogenic carcinoma.

Treatment modalities for hepatitis B viral infection and resistance to antiviral therapy.

The worldwide problem of hepatitis B viral infection has been the focus of a large number of investigations. In the last decade advances have been made in the understanding of the viral function as well as strategies to combat the virus. Lamivudine, Hepsera, and interferon-alpha therapies that are currently used in the clinic are not optimal because of the emergence of resistance in the former modality and/or adverse effects. There are several new nucleosides that are under development for treatment of HBV. With the combination of a number of treatment protocols, it should be possible to bring down the viral load to undetectable levels and minimize the risk of generating mutations that confer resistance to the therapeutic agent.

Evaluation of mutagenic effect of the fungicide fenaminosulf in Drosophila melanogaster.

Fenaminosulf (p-dimethylaminobenzenediazo sodium sulfonate, CAS registry No. 140-56-7) which is an active ingredient in several commercial fungicides was reported to be mutagenic in Salmonella typhimurium (McCann et al., 1975), Bacillus subtilis (Kada et al., 1974) and shown to cause chromosome aberrations in plants (Zutshi and Kaul, 1975). Since fenaminosulf has structural similarity to the potent carcinogen, butter yellow (p-dimethylaminoazobenzene, CAS registry No. 60-11-7), the present studies were undertaken to evaluate the mutagenic potential of this fungicide in Drosophila melanogaster. Fenaminosulf administered at 10 mg/100 ml food medium failed to induce sex-linked recessive mutations in Drosophila. Since Drosophila has drug-metabolizing enzymes similar to those of mammals (Vogel, 1975), it is suggested that the lack of mutagenic activity of fenaminosulf could be due to the conversion of fenaminosulf to non-mutagenic derivatives in Drosophila.

Secondary holocord syringomyelia with spinal hemangioblastoma: a report of two cases.

Intramedullary spinal hemangioblastoma is well known to be accompanied by syringomyelia. However, holocord secondary syringomyelia is uncommon. We present 2 cases of spinal hemangioblastoma, one in the conus medullaris and the other in midthoracic region, accompanied by holocord syrinx. In both the cases the secondary syrinx resolved following successful total tumor excision with good neurological recovery.

Foramen magnum metastatic malignant melanoma.

The foramen magnum as a site for brain metastasis is extremely rare. We report the case of a 24-year-old male who presented with features of increased intracranial pressure and lower cranial nerve palsies. Imaging revealed a foramen magnum tumor with extension up to the cerebellar vermis superiorly and into the spinal canal inferiorly (craniospinal mass) with mild obstructive hydrocephalus. A malignant melanoma was completely excised. The patient continued to be tumor-free 24 months later.

Ventral screw fixation of odontoid fracture, using single fluoroscope.

This paper is a detailed description of an improvised operative technique used by us, for ventral screw fixation in a case of type II odontoid fracture.

A fatal case of erythema necroticans.

Erythema nodosum leprosum (ENL) classically presents as tender, erythematous nodules over the face, arms and legs. Severe ENL can become vesicular or bullous and break-down and is termed erythema necroticans (Jopling & McDougall, 1996) and is treated with corticosteroids. The causes of death in a majority of leprosy patients are the same as in the general population, with the exception of renal damage in lepromatous leprosy. There is possible increased mortality from side-effects of antileprosy drugs, steroids, or other drugs used in reactions, from toxaemia in severe reactions, and from asphyxia due to glottic oedema (Jopling & McDougall, 1996). We report here a case of erythema necroticans, the cause of death being septicaemia, secondary to skin ulcers and urinary tract infection, precipitated by corticosteroids.

Development of sulforaphane-encapsulated microspheres for cancer epigenetic therapy.

Even though conventional chemotherapeutic management of cancer has reduced morbidity and mortality to a great extent, virtually all chemotherapeutic agents cause damage to healthy cells, necessitating exploration of novel anticancer agents that exert their effects through an alternate mode of action. Objectives of our research were twofold. First, we explored the promising potential of histone deacetylase inhibitor sulforaphane for epigenetic therapy for cancer as this therapeutic approach aims to reverse aberrant epigenetic modifications that affect gene expression. In vitro cell culture studies performed using B16 and S91 melanoma cells showed that sulforaphane inhibited growth and proliferation of cancer cells by downregulating deacetylation enzymes. The second part of our research investigated polymeric drug delivery systems to increase therapeutic efficacy and to minimize potential side effects of R,S-sulforaphane. Albumin microspheres encapsulating sulforaphane were developed by spray drying. Microspheres were characterized for their morphology, size and zeta potential. Cell culture studies using melanoma cells and in vivo studies in melanoma tumor-bearing C57BL/6 mice demonstrated that albumin based polymeric delivery system was efficacious and has the potential to enhance the therapeutic effect and anticancer activity of sulforaphane.

Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate.

AIM: The present study was directed on the assessment of the response of treatment-naive oral cancer cells to methotrexate (MTX) in vitro: and clinical response to MTX therapy. METHODS: A pilot study of in vitro: evaluation of MTX response of oral cancer cells from 10 patients was conducted using a cell viability assay to determine the sensitivity/resistance to MTX. Quantitative in vitro: data were correlated to the clinical outcome to MTX therapy. RESULTS: A positive correlation was observed between the effect of MTX on tumor cells in vitro: and clinical response for 7 out of 10 patients. CONCLUSIONS: Observations from the proof-of-principle pilot study suggests that oral cancer cells have intrinsically variable response to MTX. Confirmation of these findings with a larger cohort of patients could aid in the development of individualized therapies for this class of malignancy.

Purification of a bacterial organophosphate-hydrolysing phosphatase by Cibacron 3GA-Sepharose affinity chromatography.

A phosphatase catalysing the hydrolysis of organophosphorus pesticides was purified to homogeneity using Cibacron 3GA-Sepharose CL 6B affinity chromatography. The enzyme which is localized in the periplasm of the bacterium Alcaligenes NC5 was extracted by treating with 0.2M MgCl2, pH 8.4. The enzyme was adsorbed to the Cibacron-Sepharose at pH 7.0 and eluted with Tris-HCl buffer at pH 8.0, with 47 per cent recovery. The enzyme thus obtained was electrophoretically homogeneous. This simple affinity purification procedure enhances the potential for its use in large scale detoxification systems.