Establishment of effective and safe recipient preparation for germ-cell transplantation with intra-testicular busulfan treatment in pre-pubertal Barbari goats.

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and ß-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

The RelA NF-kappaB subunit and the aryl hydrocarbon receptor (AhR) cooperate to transactivate the c-myc promoter in mammary cells.

NF-kappaB/Rel transcription factors regulate many genes involved in control of cellular proliferation, neoplastic transformation, and apoptosis, including the c-myc oncogene. Recently, we have observed that levels of NF-kappaB and aryl hydrocarbon receptor (AhR), which mediates malignant transformation by environmental carcinogens, are highly elevated and appear constitutively active in breast cancer cells. Rel factors have been found to functionally interact with other transcription factors. Here we demonstrate a physical and functional association between the RelA subunit of NF-kappaB and AhR resulting in the activation of c-myc gene transcription in breast cancer cells. RelA and AhR proteins were co-immunoprecipitated from cytoplasmic and nuclear extracts of non-malignant MCF-10F breast epithelial and malignant Hs578T breast cancer cells. In transient co-transfection, RelA and AhR gene products demonstrated cooperation in transactivation of the c-myc promoter, which was dependent on the NF-kappaB elements, and in induction of endogenous c-Myc protein levels. A novel AhR/RelA-containing NF-kappaB element binding complex was identified by electrophoretic mobility shift analysis of nuclear extracts from RelA and AhR co-transfected Hs578T cells. Thus, the RelA and AhR proteins functionally cooperate to bind to NF-kappaB elements and induce c-myc gene expression. These findings suggest a novel signaling mechanism whereby the Ah receptor can stimulate proliferation and tumorigenesis of mammary cells.

Peptide transport in human lymphoblastoid and tumor cells: effect of transporter associated with antigen presentation (TAP) polymorphism.

CD8+ T-cells recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. These peptides bind to MHC class I molecules in the endoplasmic reticulum (ER) lumen. Antigenic peptides are translocated from the cytosol to the lumen of ER by transporter associated with antigen presentation (TAP) proteins. In this study, it is shown that TAP1 polymorphism influences the peptide substrate specificity in human B-lymphoblastoid and tumor cell lines. TAP1A and 1C alleles specifically enhance translocation of model peptides containing basic C-terminal amino acid residue. However, TAP1B allele does not show specificity for the peptide C-terminus. Human basophilic leukemia (Ku812), and hepatocellular carcinoma (PLC/PRF/5) cells express TAP1 molecules and exhibit TAP-mediated allele-specific peptide uptake after gamma-interferon (gamma-IFN) treatment. Ku812 cells express TAP1A and preferentially take up antigenic peptides with a basic C-terminus, however, PLC/PRF/5 cells with the TAP1B allele take up low but equivalent levels of peptides regardless of basic, acidic, or hydrophobic C-termini. Moreover, TAP2 polymorphisms have no influence on the peptide translocation in normal or tumor cell lines. In addition, Daudi, a beta2-microglobulin (beta2m) deficient human Burkitt lymphoma, cell line also showed TAP-dependent peptide uptake. Taken together, these results suggest that human TAP1 but not TAP2 polymorphisms influence the antigenic peptide transport and that this transport is independent of beta2m in this system.

HSP70-1 promoter region alleles and susceptibility to rheumatoid arthritis.

The distribution of HSP70-1 promoter alleles was studied in 90 adult Caucasian RA patients (65 European and 25 Asian Indian) and 113 normal control (60 European and 53 Asian Indian). The HSP70-1 promoter alleles were defined by oligonucleotide typing of polymerase chain (PCR)-amplified genomic DNA. The prevalence of HSP70-1 promoter allele "B" was significantly (p < 0.0004, pc < 0.0012; RR = 5.1) higher in RA patients (22.2%) compared to normal controls (5.3%). It is likely therefore that HSP70-1 promoter allele B is associated with susceptibility to RA.

Tension arachnoid cyst causing uncal herniation in a 60 year old: a rare presentation.

Arachnoid cysts are congenital benign cysts accounting for approximately 1% of all intracranial mass lesions. Uncal herniation due to arachnoid cyst is a rare mode of presentation. It is hypothesized that only tension arachnoid cyst could cause the life-threatening condition that results from a progressive deterioration and worsening of a simple and usually congenital arachnoid cyst, associated with the formation of a "ball valve" at the point of an opening on the cyst wall. To-date only one case of an arachnoid cyst causing Uncal herniation has been reported to the best of our knowledge. We present a rare case of uncal herniation in a 60-year-old lady caused by a giant left temporal arachnoid cyst. She presented to us in emergency room after experiencing headaches since last one week followed by vomiting, seizures, and altered state of consciousness. She was operated immediately and marsupialization of the arachnoid cyst was performed. She showed good recovery. Although arachnoid cyst is a benign slowly growing pathology, it can lead to Uncal herniation as a "tension" arachnoid cyst, possible due to "ball-valve" mechanism. Elective treatment should be considered to prevent progressive significant enlargement of cyst.

Establishment of a comprehensive epilepsy center in pakistan: initial experiences, results, and reflections.

Background. Developing countries, home to 80% of epilepsy patients, do not have comprehensive epilepsy surgery programs. Considering these needs we set up first epilepsy surgery center in Pakistan. Methods. Seventeen teleconferences focused on setting up an epilepsy center at the Aga Khan University (AKU), Karachi, Pakistan were arranged with experts from the University of Alberta Hospital, Alberta, Canada and the University of West Virginia, USA over a two-year period. Subsequently, the experts visited the proposed center to provide hands on training. During this period several interactive teaching sessions, a nationwide workshop, and various public awareness events were organized. Results. Sixteen patients underwent surgery, functional hemispherectomy (HS) was done in six, anterior temporal lobectomy (ATL) in six, and neuronavigation-guided selective amygdalohippocampectomy (SAH) using keyhole technique in four patients. Minimal morbidity was observed in ATL and, SAH groups. All patients in SAH group (100%) had Grade 1 control, while only 5 patients (83%) in ATL group, and 4 patients (66%) in HS group had Grade 1 control according to Engel's classification, in average followups of 12 months, 24 months and 48 months for SAH, ATL, and HS, respectively. Conclusion. As we share our experience we hope to set a practical example for economically constrained countries that successful epilepsy surgery centers can be managed with limited resources.

Traumatic retroclival epidural hematoma in pediatric patient-Case report and review of literature.

BACKGROUND: Retroclival epidural hematoma (REDH) is a very rare entity in the practical field of neurosurgery. Only a few cases have been reported in literature. The authors present to you case of a 12-year-old female, a victim of road traffic accident (RTA), who had presented to us with loss of consciousness and seizures. CASE DESCRIPTION: Magnetic resonance imaging revealed retroclival hematoma. She was managed on conservative grounds and discharged with assurance of multiple follow-up visits. CONCLUSION: Very few cases of REDH have been reported in pediatric population to date. It should be suspected in children with head and neck injuries who have been a victim of RTAs. Most likely underdiagnosed due to its rarity; therefore, MRI should be considered when the suspicion is high. Atlanto-occipital dislocation should always be kept under consideration in all cases, and therefore should be managed and monitored very cautiously. In this report, the authors also present concise review of the literature pertaining to the pathogenesis and management of this rare clinical entity which has a high likelihood to be encountered and underdiagnosed by neurosurgeons in Emergency Room.

Major histocompatibility-encoded human proteasome LMP2. Genomic organization and a new form of mRNA.

LMP2 is one of the two proteasome subunits encoded by genes in the major histocompatibility complex class II region. Here we report the genomic organization of human LMP2 gene. Sequence analysis of polymerase chain reaction-amplified cDNA from a number of lymphoblastoid cell lines demonstrated two forms of LMP2 mRNA, one (LMP2.1) complete and homologous to the published LMP2 genomic sequence from cosmid clones, and the other (LMP2.s) a smaller transcript resulting from splicing of a 30-base pair fragment from the first exon. Antibodies to recombinant LMP2.s protein (22.3 kDa) were raised in rabbits. This anti-LMP2.s serum recognized both recombinant proteins (LMP2.1 = 23.3 kDa and LMP2.s = 22.3 kDa) and a single protein of 21.5 kDa molecular mass in lysates from human lymphoblastoid cell lines. Pulse-chase experiments demonstrated that LMP2 polypeptide also undergoes processing from 22.3- to 21.5-kDa protein when incorporated into proteasomes. These data suggest that the processing of human LMP2 subunit takes place both at the transcription and post-translational levels. Northern blot analysis showed that the LMP2 mRNA is expressed in lymphoblastoid cell lines and in fibroblasts following gamma-interferon induction, but not in brain, smooth muscle, fibroblasts (uninduced), and colon epithelial cells.

Aziridinyl steroid-induced lesions in DNA and apoptosis in promyelocytic leukemia cells.

Variety of synthetic steroids are reported to be mutagenic as well as carcinogenic. The mutagenic and carcinogenic nature of these compounds have been related to their potential of being reactive to genetic material and production of reactive oxygen species. Here we have analyzed the action of aziridinyl steroid on calf thymus DNA and human promyelocytic leukemia cell line HL-60 under in vitro conditions. Calf thymus DNA when treated with various doses of aziridinyl steroid induced a high degree of stand separation and sensitivity/susceptibility to S1 nuclease hydrolysis. The treatment also induced an increasing number of strand breaks per molecule of DNA as determined by alkaline unwinding assay. Relatively higher doses of steroid, however, displayed a reduced susceptibility to S1 nuclease hydrolysis and did not increase the number of strand breaks in DNA. Moreover, the high dose treatments result increased melting temperature and an enhanced rate of reanealing after thermal denaturation, indicating that interstrand crosslinks are induced at higher doses of steroid treatment. Moreover, steroid treatment caused cell death in human promyelocytic leukemia cell line HL-60 and induced DNA degradation, characteristic of apoptosis. The test steroid has the ability to produce reactive oxygen intermediates (ROI) as determined by chemical methods. Incorporation of oxygen radical scavengers into the system blocked the damaging effect of steroid in calf thymus DNA and HL-60 cells. These observations strongly suggest that aziridinyl steroid, a pharmaceutical, damages mammalian DNA and induces apoptosis by the production of ROI in the test system.

The bioflavonoid galangin blocks aryl hydrocarbon receptor activation and polycyclic aromatic hydrocarbon-induced pre-B cell apoptosis.

Bioflavonoids are plant compounds touted for their potential to treat or prevent several diseases including cancers induced by common environmental chemicals. Much of the biologic activity of one such class of pollutants, polycyclic aromatic hydrocarbons (PAH), is mediated by the aryl hydrocarbon receptor/transcription factor (AhR). For example, the AhR regulates PAH immunotoxicity that manifests as pre-B cell apoptosis in models of B cell development. Because bioflavonoids block PAH-induced cell transformation and are structurally similar to AhR ligands, it was postulated that some of them would suppress PAH-induced, AhR-dependent immunotoxicity, possibly through a direct AhR blockade. This hypothesis was tested using a model of B cell development in which pre-B cells are cultured with and are dependent on bone marrow stromal or hepatic parenchymal cell monolayers. Of seven bioflavonoids screened, galangin (3,5,7-trihydroxyflavone) blocked PAH-induced but not C(2)-ceramide- or H(2)O(2)-induced pre-B cell apoptosis. Because galangin blocked AhR-dependent reporter gene expression, AhR complex-DNA binding, and AhR nuclear translocation, inhibition of a relatively early step in AhR signaling was implicated. This hypothesis was supported by the ability of galangin to bind the AhR and stabilize AhR-90-kDa heat shock protein complexes in the presence of AhR agonists. These studies demonstrate the utility of pre-B cell culture systems in identifying compounds capable of blocking PAH immunotoxicity, define at least one mechanism of galangin activity (i.e., repression of AhR activation), and motivate the use of this and similar dietary bioflavonoids as relatively nontoxic inhibitors of AhR agonist activity and as pharmacologic agents with which to dissect AhR signaling pathways.