RAPD analysis of long term micropropagated rootstock plants of Apple Malling 7.

Apple is the most dominating fruit crop of Himachal Pradesh, constituting about 40 per cent of total area under fruit crops and about 90 per cent of the total fruit production. Plant tissue culture has encouraged production of quality planting material which is disease free and true to type. Due to current trends in favour of high density apple plantations and to fulfill the requirement of planting material from the farmers, commercially viable in vitro propagation method of semidwarf clonal apple rootstock Malling 7 (M7) was developed by axillary branching, initially developed from shoot tip explants. A few thousand plants were transferred to field conditions. In order to check any tissue culture induced variation in these long term micropropagated plants, the genetic fidelity was assessed using Random Amplified Polymorphic DNA (RAPD) markers. Out of the 20 primers screened, 12 generated a total of 44 scorable and distinct bands with an average of 3.7 bands per primer. All the banding patterns for each primer were highly uniform and monomorphic across in vitro multiple shoot clusters and field transplanted micropropagated plants as well as comparable to the field grown mother clone from which the cultures had been established. Our results have shown that the micropropagation protocol developed by this study is appropriate and applicable for clonal propagation of apple rootstock M7 on large scale over a long period without any risk of genetic instability.

A cohort study of vulvar cancer over a period of 10 years and review of literature.

OBJECTIVE: The objective of this study was to study the risk factors, management protocols, and the outcome of vulvar cancer cases over a period of 10 years in a tertiary care hospital. METHODOLOGY: It is a retrospective cohort study. The hospital records of 41 patients with histologically proven vulvar cancer were studied from the Department of Obstetrics and Gynaecology and the Department of Radiotherapy (RT). The presence of risk factors, stage of disease, treatment modalities used, and disease outcomes in terms of survival were studied. The data collected were analyzed and compared with the published literature. RESULTS: The mean age for the diagnosis of vulvar cancer was 52 years and the peak incidence was seen in the age group of 50-70 years. Incidence was significantly more in multiparous (P = 0.001) and postmenopausal women (P = 0.007). An average of 4.1 cases were seen per year. Nearly, 97.56% of the cases were squamous cell carcinomas. Twenty cases belonged to the early stage of the disease (Stage I and II) whereas 21 cases had advanced disease (Stage III and IV). Nearly, 48.78% of the cases were primarily treated with surgery, 26.83% with RT, 7.3% with chemotherapy, and 17.07% with combined chemoradiation. Seventy-eight percent of the surgically treated cases had a mean survival of 5 years. Mean survival of 1 year was recorded in advanced disease cases. Limitation of the study was poor follow-up after treatment. CONCLUSION: Incidence of vulvar cancer is significantly high in multiparous and postmenopausal women. Conservative surgical treatment is the best option in the early stage of the disease (Stage I and II) and gives high survival rates whereas advanced disease treated with chemoradiation has a poor survival.

Bone-Subtracted Spinal CT Angiography Using Nonrigid Registration for Better Visualization of Arterial Feeders in Spinal Arteriovenous Fistulas.

BACKGROUND AND PURPOSE: Pretreatment diagnosis for the location of shunts and arterial feeders of spinal arteriovenous fistulas is crucial. This study aimed to evaluate the utility of subtracted CT angiography imaging by using nonrigid registration (R-CTA) in patients with spinal arteriovenous fistulas compared with conventional CTA imaging. MATERIALS AND METHODS: The records of 15 consecutive subjects (mean age, 65 years; 2 women) who had undergone CTA and digital subtraction angiography for clinically suspected spinal arteriovenous fistula were reviewed. From CTA images obtained at the arterial and late arterial phases, warped images of the late arterial phase were obtained by using nonrigid registration that was adjusted to the arterial phase images. R-CTA images were then obtained by subtracting the warped images from the arterial phase images. The accuracies of using nonrigid registration and conventional spinal CTA and the time required for detecting arterial feeders in spinal arteriovenous fistulas were analyzed for each patient with DSA results as a standard reference. The difference between R-CTA and conventional spinal CTA was assessed by the Welch test and the McNemar χ(2) test. RESULTS: R-CTA had a higher accuracy compared with conventional spinal CTA (80% versus 47%, P = .025). The time for interpretation was reduced in R-CTA compared with conventional spinal CTA (45.1 versus 97.1 seconds, P = .002). CONCLUSIONS: Our subtracted CTA imaging by using nonrigid registration detects feeders of spinal arteriovenous fistulas more accurately and quickly than conventional CTA.

Human fascioliasis with biliary complications.

We report a case of human fascioliasis adding to the few of the previously reported cases in India. A young boy from rural background in Bihar presented with diarrhea, vomiting, hepatic tenderness, jaundice and fever along with peripheral eosinophilia. Examination of stool revealed yellow-brown eggs of Fasciola hepatica. Human fascioliasis should be kept in mind in patients with cholangitis and eosinophilia especially in areas of sporadic occurrence.

Analysis and comparison of clinicohematological parameters and molecular and cytogenetic response of two Bcr/Abl fusion transcripts.

Different forms of p210 are produced by alternative splicing, namely b2a2 and b3a2. There have been many contrasting data establishing a relationship between the two Bcr/Abl transcripts and platelet counts and also response to treatment. However, the data published to date have been on a small group of patients. The aim of the present study was to determine whether there was any difference between clinical and hematological parameters at diagnosis between the two Bcr/Abl fusion transcripts in our population, and whether the two transcripts responded differently or similarly to imatinib treatment. RT-PCR was performed in 202 cases for detection of Bcr/Abl transcripts in newly diagnosed chronic myelogenous leukemia cases in one year. The two transcripts were compared and correlated with clinical, hematological and FISH data and with response to treatment. A total of 138 cases were of b3a2 and 64 were of b2a2 transcript. There was no correlation between the hematological parameters and the type of transcript. There was a significant association of blast crisis with b2a2, especially with myeloid blast crisis. When compared to FISH results, 10% of b3a2 were found to have a significant association with 5'Abl deletion as compared to 3% of b2a2. On analyzing the therapeutic response, we did not find any difference between the two transcripts. In conclusion, our findings confirm that the b3a2 type transcript is not significantly associated with thrombocytosis, that the short transcript, b2a2, occurs with acute phase, i.e., blast crisis, and that there is no difference in treatment response between the two transcripts. However, further studies are required to understand the molecular pathways involved in the Bcr/Abl mechanism.

Arbitrary-primed PCR for genomic fingerprinting and identification of differentially regulated genes in Indian isolates of Leishmania donovani.

The arbitrary-primed PCR (AP-PCR) technique was employed with the twin goals of identifying genetic polymorphisms within the Indian isolates and to identify differentially expressed gene sequences. The parasite isolates from Indian Kala-azar patients could be differentiated from Leishmania donovani isolates from distinct geographic regions. Moreover, differences within the Indian isolates could also be identified. A majority (17/19) of the Indian isolates gave identical AP-PCR pattern, while two isolates gave consistently divergent pattern. The distinctive AP-PCR fragments obtained with Indian isolates were used as probes in Northern blot analysis. Three such fragments were found to represent transcribed sequences that were differentially expressed in the two stages of the parasite. These sequences led to cloning and characterization of Leishmania Centrin gene and a novel gene termed A-1 that is over-expressed in amastigote stage of the parasite. The study demonstrates the utility of random genome sampling methods in genomic fingerprinting and in identifying differentially transcribed sequences that could potentially contribute to parasite virulence.

Diagnosis of visceral leishmaniasis: comparative potential of amastigote antigen, recombinant antigen and PCR.

Development of simple, economical and non-invasive tests for the early diagnosis of visceral leishmaniasis (VL) or kala-azar (KA) remains a challenge, and serological studies based on antigen prepared from the amastigote stage of Leishmania donovani, the stage that causes infection, are lacking. In the present study, circulating antibodies to total antigen isolated from the promastigote and amastigote stages of the parasite, as well as to recombinant K39 (rK39) antigen, are measured by enzyme-linked immunosorbent assay (ELISA) and the results compared with a polymerase chain reaction (PCR) test for KA diagnosis. In 116 samples of KA examined, the amastigote antigen gave significantly higher mean absorbance values in ELISA than did the promastigote antigen. The sensitivity for KA detection was significantly higher using the amastigote antigen (94%) than the promastigote antigen (90.5%). Analysis in 91 controls showed that specificity was higher with amastigote antigen (92.3%) than with promastigote antigen (86.8-89.0%). Reliability of ELISA diagnosis with amastigote antigen was only marginally lower than that with rK39 ELISA or with the PCR test. Easy availability and low cost of indigenous amastigote antigen, together with the simplicity of ELISA compared with PCR, make ELISA based on amastigote antigen a promising choice for the diagnosis of KA.

Diagnosis of visceral leishmaniasis: comparative potential of amastigote antigen, recombinant antigen and PCR.

Development of simple, economical and non-invasive tests for the early diagnosis of visceral leishmaniasis (VL) or kala-azar (KA) remains a challenge, and serological studies based on antigen prepared from the amastigote stage of Leishmania donovani, the stage that causes infection, are lacking. In the present study, circulating antibodies to total antigen isolated from the promastigote and amastigote stages of the parasite, as well as to recombinant K39 (rK39) antigen, are measured by enzyme-linked immunosorbent assay (ELISA) and the results compared with a polymerase chain reaction (PCR) test for KA diagnosis. In 116 samples of KA examined, the amastigote antigen gave significantly higher mean absorbance values in ELISA than did the promastigote antigen. The sensitivity for KA detection was significantly higher using the amastigote antigen (94%) than the promastigote antigen (90.5%). Analysis in 91 controls showed that specificity was higher with amastigote antigen (92.3%) than with promastigote antigen (86.8-89.0%). Reliability of ELISA diagnosis with amastigote antigen was only marginally lower than that with rK39 ELISA or with the PCR test. Easy availability and low cost of indigenous amastigote antigen, together with the simplicity of ELISA compared with PCR, make ELISA based on amastigote antigen a promising choice for the diagnosis of KA.

Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite.

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca(2+) binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca(2+) binding site suggest that Ca(2+) binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.

Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis.

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.

Immunoblot analysis of the antibody response to antigens of Leishmania donovani in Indian kala-azar.

When infected with Leishmania donovani, patients develop specific antibodies that constitute the basis of serodiagnosis. Using immunoblot analysis, we examined the antibody response to antigens of L. donovani in 35 kala-azar (KA) patients and 67 controls. Sera from KA patients recognised numerous antigens with molecular weights ranging from 14-110 kDa. Antigens of 40 kDa, 55 kDa, 65 kDa, 70 kDa and 82 kDa were recognised most frequently. All KA patients produce an antibody response to one or more of these antigens. The majority (83%) of KA cases recognised at least four of these five parasite antigens. The 70 kDa antigen showed the greatest sensitivity for Indian KA, and produced a positive reaction in 94% of patients. This antigen gave 10% false-positive reactions in controls comprising patients with related diseases (i.e. tuberculosis, leprosy and malaria) and in healthy controls. Data indicated that the 70 kDa antigen may include a member of the heat shock protein 70 family. Studies with four clinical isolates of L. donovani showed that the 70 kDa component was expressed in all the strains examined. Immunoblot assay (Western blotting) provided a sensitive diagnostic test for KA patients, and identified the 70 kDa parasite antigen that is promising as a potential target antigen for the development of less complex serodiagnostic assays for KA.

Prognostic significance of pre discharge one hour electrocardiographic monitoring in patients recovering from acute myocardial infarction.

During a 15 months period, 97 patients with AMI were monitored continuously at hospital discharge for one hour. The VPCs or no VPC noted during this period of continuous monitoring were correlated with subsequent cardiac events during one year follow up. Patients with complicated VPCs had a significantly higher incidence of cardiac deaths and non-fatal re-infarctions during one year follow up (54.5%) as compared to patients with no VPCs (6.5%) (p less than .001). Patients with higher grades of VPCs had more complicated course (30.3% to 66.7% for Lown Grade II to IV) as compared to 6.5% and 20.8% for grade Lown O and I. Though, the results of this 1 hour continuous monitoring at hospital discharge are not as sensitive as with ambulatory Holter monitoring as is evident from this study where complicated VPCs were detected in 11.4% patients vs. 33% with Holter monitoring, it could be a useful tool in institutions where the facility of Holter monitoring does not exist.

Effect of trifluralin on growth, morphology, and nucleic Acid synthesis.

Roots and shoots of corn seedlings (Zea mays L. var. Dixie 18) germinated in trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) solutions are characterized by radial enlargement of the cortical cells and by multinucleate cells in the meristematic regions. Trifluralin inhibits elongation of Avena coleoptile sections at concentrations of 0.1 mum to 10 mum. Synthesis of DNA, RNA, and protein is suppressed in the root tips while no significant effect is noticeable in the shoots of corn germinated in trifluralin. A (32)P time-course study of 48, 72, and 96 hours utilizing phenol extraction and MAK column separation of corn root and shoot nucleic acids showed suppression of (32)P incorporation in the treated roots; however, the 72 and 96 hour treated shoots incorporated a much greater amount than the control with most of the increased incorporation found in the sRNA and DNA fractions. The increased activity in the DNA may be due to a high G-C type DNA. No selective suppression or enhancement of any particular RNA species was noticed in the treated plants.