Localization to Xq27 of a susceptibility gene for testicular germ-cell tumours.

Testicular germ-cell tumours (TGCT) affect 1 in 500 men and are the most common cancer in males aged 15-40 in Western European populations. The incidence of TGCT has risen dramatically over the last century. Known risk factors for TGCT include a history of undescended testis (UDT), testicular dysgenesis, infertility, previously diagnosed TGCT (ref. 7) and a family history of the disease. Brothers of men with TGCT have an 8-10-fold risk of developing TGCT (refs 8,9), whereas the relative risk to fathers and sons is fourfold (ref. 9). This familial relative risk is much higher than that for most other types of cancer. We have collected samples from 134 families with two or more cases of TGCT, 87 of which are affected sibpairs. A genome-wide linkage search yielded a heterogeneity lod (hlod) score of 2.01 on chromosome Xq27 using all families compatible with X inheritance. We obtained a hlod score of 4.7 from families with at least one bilateral case, corresponding to a genome-wide significance level of P=0.034. The proportion of families with UDT linked to this locus was 73% compared with 26% of families without UDT (P=0.03). Our results provide evidence for a TGCT susceptibility gene on chromosome Xq27 that may also predispose to UDT.

Muscle of dark and normal beef differs metabolically.

Reduction in muscle glycogen triggered by adverse antemortem handling events alters postmortem energy metabolism and results in a high ultimate pH and dark, firm and dry beef, often referred to as 'dark-cutting'. However, the relationship between atypical dark (AT) beef, postmortem energy metabolism and underlying tissue characteristics remains somewhat unclear. Cattle harvested in the US and Canada representing normal (pH < 5.6), AT dark (pH 5.6-5.8) and dark cutting (DC; pH > 5.8) beef were analyzed for tissue characteristics related to energy metabolism. Results show AT dark beef is more oxidative but similar to normal beef in glycolytic potential and nucleotide abundance. Mitochondria DNA content (P < 0.05, Canada; P < 0.005, US) and oxidative enzymes for DC and AT dark beef were greater (P < 0.01; Canada and US) compared to normal beef. Myoglobin tracked (P < 0.01) with color classification. These findings show both DC and AT beef are inherently more oxidative and raise the possibility that more oxidative muscle may be more prone to develop dark beef.

Time of dehairing alters pork quality development.

Studies investigating the effect of scald time on pork quality are confounded with time of dehairing. To understand better pork quality development and two-toning in hams, twenty-four carcasses were assigned to an 8- or 16-min dwell time prior to the dehairing, with or without scalding (n = 6 per trt). Semimembranosus (SM) muscles were collected following dehairing and at 24 h postmortem. Protracted time to dehair improved ultimate pH (pHu; P < 0.005) and reduced (P < 0.05) color variation. One hundred forty-two carcasses were then subjected to protracted (control, 10-min) dwell times (15-min, or 20-min) in an industrial setting. Lightness was improved with 15-min dwell times compared to control, however 20-min dwell decreased the pHu (P < 0.001), increased lightness (P < 0.05), and percent purge (P < 0.001) in the SM. Also, lightness of the longissimus muscle (LM) increased (P < 0.001) with dwell time. These data show time to dehairing impacts pork quality development and suggest dehairing may be critical to quality development in a muscle-dependent manner.

Reduced scald time does not influence ultimate pork quality.

Fresh pork color is a function of pigment, and the pH and temperature conditions in the carcass postmortem. To explore the role of scald on color development, carcasses (n = 16) were subjected to either a 4- or 8-min scald. Semimembranosus (SM) muscle samples were collected before and after scalding, and at 24 h postmortem. A 50% reduction in scald time resulted in lighter color (L*) across the muscle early postmortem (P < 0.001), yet the 8-min scald treatment was lighter (P = 0.001) at 24 h. An interaction between scald time and sampling time showed in an increase in L* values at 4-min immediately following scald (P < 0.001). Two-hundred carcasses were then subjected to a modified scald time (6.5 min, or 7.5 min) in an industrial setting. Lowering scald time failed to recapitulate results. In fact, darker meat (L* value; P = 0.0166) was noted in the SM across longer scalds. These data suggest modest changes in scald time may not be responsible for changes in pork quality development.

Inherent factors influence color variations in semimembranosus muscle of pigs.

Variations in color, though a quality frustration, are common across the face of fresh and processed hams. Herein, we measured objective color across the semimembranosus (SM) muscle early postmortem and at 1440 min, then compared these differences against biochemical and metabolic characteristics responsible for pork quality development. Color (L*, a*) differed (P < 0.001) by zone and time but no interaction was evident. Lactate content and pH were highly correlated (R2 = 0.92) at 30 min, but weakened (R2 = 0.161412) by 1440 min. Lactate anaplerosis was not responsible for this lack of relationship. Glycolytic potential also differed across zone (P < 0.001) and time (P < 0.005). Differences in myoglobin expression and abundance, as well as mitochondrial DNA were notable (P < 0.05) across zone. These data suggest inherent differences in SM muscle are key determinants of ham color variation, while postmortem metabolism may play a lesser role in driving this quality attribute.

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family designated APRIL (for a proliferation-inducing ligand). Although transcripts of APRIL are of low abundance in normal tissues, high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. These findings suggest that APRIL may be implicated in the regulation of tumor cell growth.

BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center-like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function.

Genetic characteristics of the HeLa cell.

The genotype of the patient Henrietta Lacks from whose cervical carcinoma the HeLa cell was derived was deduced from the phenotypes of her husband and children, and from studies of the HeLa cell. Hemizygous expression of glucose-6-phosphate dehydrogenase in HeLa, together with the deduced heterozygosity of Mrs. Lacks, is consistent with clonal origin of her neoplasm.

Identification of CARDIAK, a RIP-like kinase that associates with caspase-1.

Members of the tumor necrosis factor receptor (TNFR) superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation and death. TNFR-1 recruits and assembles a signaling complex containing a number of death domain (DD)-containing proteins, including the adaptor protein TRADD and the serine/threonine kinase RIP, which mediates TNF-induced NF-kappa B activation. RIP also recruits caspase-2 to the TNFR-1 signaling complex via the adaptor protein RAIDD, which contains a DD and a caspase-recruiting domain (CARD). Here, we have identified a RIP-like kinase, termed CARDIAK (for CARD-containing interleukin (IL)-1 beta converting enzyme (ICE) associated kinase), which contains a serine/threonine kinase domain and a carboxy-terminal CARD. Overexpression of CARDIAK induced the activation of both NF-kappa B and Jun N-terminal kinase (JNK). CARDIAK interacted with the TNFR-associated factors TRAF-1 and TRAF-2, and a dominant-negative form of TRAF-2 inhibited CARDIAK-induced NF-kappa B activation. Interestingly, CARDIAK specifically interacted with the CARD of caspase-1 (previously known as ICE), and this interaction correlated with the processing of pro-caspase-1 and the formation of the active p20 subunit of caspase-1. Together, these data suggest that CARDIAK may be involved in NF-kappa B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta through activation of caspase-1.

Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces.

IMGT/HLA Database--a sequence database for the human major histocompatibility complex.

The IMGT/HLA Database (www.ebi.ac.uk/imgt/hla/) specialises in sequences of polymorphic genes of the HLA system, the human major histocompatibility complex (MHC). The HLA complex is located within the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes of diverse function. Many of the genes encode proteins of the immune system and these include the 21 highly polymorphic HLA genes, which influence the outcome of clinical transplantation and confer susceptibility to a wide range of non-infectious diseases. The database contains sequences for all HLA alleles officially recognised by the WHO Nomenclature Committee for Factors of the HLA System and provides users with online tools and facilities for their retrieval and analysis. These include allele reports, alignment tools and detailed descriptions of the source cells. The online IMGT/HLA submission tool allows both new and confirmatory sequences to be submitted directly to the WHO Nomenclature Committee. The latest version (release 1.7.0 July 2000) contains 1220 HLA alleles derived from over 2700 component sequences from the EMBL/GenBank/DDBJ databases. The HLA database provides a model which will be extended to provide specialist databases for polymorphic MHC genes of other species.

A clinical and epidemiological study of human leukocyte antigen-DPB alleles in Hodgkin's disease.

An international study to investigate the role of human leukocyte antigen (HLA)-DPB alleles in Hodgkin's disease was conducted with 17 participating centers in 12 countries. A total of 741 patients and 686 controls were typed using polymerase chain reaction amplification of HLA-DPB alleles and subsequent sequence specific oligonucleotide hybridization. The frequency of HLA-DPB1*0301 was found to be significantly increased in white patients, compared with ethnically matched controls. In this population group, the DPB1*0301 allele is associated with a relative risk of 1.95 (P < 0.01). There was also a significant reduction in the frequency of HLA-DPB1*0401 in patients from Japan and Taiwan (relative risk, 0.15; P < 0.01). Clinical analysis from data on 551 patients demonstrated a significantly inferior remission duration in patients with HLA-DPB1*0901, overall (P < 0.05), and in the Japanese and Taiwanese populations (P = 0.02), where this allele is most prevalent. This analysis suggests an epidemiological as well as a possible prognostic association between HLA-DPB alleles and Hodgkin's disease.

Loss of caspase-8 expression in highly malignant human neuroblastoma cells correlates with resistance to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis.

Human neuroblastoma (NB) is a highly heterogeneous childhood cancer that is aggressively malignant or can undergo spontaneous regression that may involve apoptosis. NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Noninvasive S-type cell lines (NB cell lines of substrate adherent phenotype) are highly sensitive to TRAIL, whereas invasive N-type cell lines (NB cell lines of neuronal phenotype) are resistant. Whereas both S- and N-type cell lines express TRAIL-R2, FADD, and caspase-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a malignant stage IV NB tumor when compared with a benign ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage NB tumors. Caspase-8 expression can be induced by demethylation with 5-aza-2'deoxycytidine, which enhances sensitivity to TRAIL. Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.

The t(14;18) chromosomal translocation and Bcl-2 protein expression in Hodgkin's disease.

A polymerase chain reaction analysis of biopsy specimens from a total of 52 patients with Hodgkin's disease has revealed the presence of the t(14;18) chromosomal translocation in 14 cases (28%). Twelve involved the major breakpoint region and two included the minor cluster region of the bcl-2 gene. Direct sequencing of the amplified 14q+ junctions from the initial four positive cases (from 21 biopsies) has been previously published and demonstrated the similarity in nature of the break-points to those described in follicular lymphoma and lymphoid hyperplasia. The 52 biopsies have also been studied with a monoclonal antibody to immunolocalize the Bcl-2 protein. In all cases Bcl-2 positivity was observed in the majority of surrounding lymphocytes. However, in 11 cases, positive immunostaining in the Sternberg-Reed cells was also observed. Three of these cases contained the t(14;18) translocation, but 11 cases which were positive for the t(14;18) by PCR did not show Bcl-2 protein staining in the Sternberg-Reed cells. This data confirms the presence of t(14;18) in 28% of biopsies from Hodgkin's disease and demonstrates Bcl-2 protein staining in a variable proportion of Sternberg-Reed cells of some cases.

p53 protein expression in Reed-Sternberg cells of Hodgkin's disease.

Hodgkin's disease (HD) is perceived to be a malignant disease of the lymphoid system. One of the main obstacles into the investigation of the cell biology of Hodgkin's disease is the relative paucity of Reed-Sternberg cells (or variants), the presumed neoplastic component of this condition, which often make up less than 1% of the total cell number.

Nuclear lamin expression reveals a surprisingly high growth fraction in childhood acute lymphoblastic leukemia cells.

In an attempt to better characterize leukemic bone marrow cells of children with ALL in G0/G1, we studied the variation of the nuclear projection area (NPA) during the cell cycle. Approximately half of the increase of the nuclear volume during the cell cycle occurred before DNA synthesis. Next, we assessed by in situ hybridization (ISH) the expression of nuclear envelope type A/C and type B lamins in leukemic lymphoblast and unstimulated as well as stimulated normal peripheral blood mononuclear cells (PBMC). It was found that 82.0 +/- 16.0% of the ALL cells expressed B-type and 5.8 +/- 3.1% A/C-type lamins. The in vitro 3HdT pulse-labeling index (3HdT LI) of ALL cells varied from 1.3 to 16.8%. Of unstimulated PBMC 2.9 and 1.2% expressed lamin type B and A/C, respectively. The 3HdT LI was 0.8%. In conA-stimulated PBMC, the corresponding values were 95.3 and 74.8% and 31.0%, respectively. In view of the current concepts regarding G1 events and regulation of cell proliferation, we considered B-type lamin expression an early marker for the commitment to proliferation and used it for growth fraction (GF) determinations. By this method, a surprisingly high GF was found in ALL cell populations; there was no correlation between GF and the 3HdT LI, as seen in normal cells.