Wolbachia infection in D. bifasciata in Hokkaido island of Japan, in contrast to the presence of infection on the proximal island of Honshu, was associated with failure of the infection to function properly on the Hokkaido genetic background.Selfish genetic elements that distort the sex ratio are found widely. Notwithstanding the number of records of sex ratio distorters, their incidence is poorly understood. Two factors can prevent a sex ratio distorter from invading: inability of the sex ratio distorter to function (failure of mechanism or transmission), and lack of drive if they do function (inappropriate ecology for invasion). There has been no test to date on factors causing variation in the incidence of sex ratio distorting cytoplasmic bacteria. We therefore examined whether absence of the male-killing D. bifasciata may occur at different densities in Hokkaido and Honshu populations, giving some credence to the idea that ecological differentiation could be important.The male-killer both transmitted and functioned well following introgression to each of 24 independent isofemale inbred lines carrying Hokkaido genetic backgrounds. This was maintained even under stringent conditions of temperature. We therefore reject the hypothesis that absence of infection is due to its inability to kill males and transmit on the Hokkaido genetic background. Further trap data indicates that The absence of the infection from the Hokkaido population is not caused by failure of the male-killer to function on the Hokkaido genetic background. Selfish genetic elements that distort the sex ratio of their host are known widely in arthropods . DespiteDrosophila pseudoobscura, frequency declines at both high latitude and high altitude. This is not associated with resistance to the action of the driver (no resistance is known), and variation in rates of multiple mating is suspected as a cause [D. subobscura, X drive is present in North Africa and absent in Europe. Here, absence of X drive is associated with the less efficient function of X drive on the European genetic background [Factors causing variation in prevalence/incidence over space may be either ecological or associated with differences in host genetic constitution. For instance, in the case of X chromosome meiotic drive in  a cause . In contckground .Drosophila prosaltans, for instance, there is intra-population host genetic variation in refractoriness to male-killer action/transmission [et al. [This study pertains to the factors affecting the incidence of male-killing bacteria. These bacteria pass from a female to her progeny, and kill any males they enter. Male-killers are common in insects, but an appreciation of the factors underlying their incidence is lacking . In the smission . Thus, ismission . For ins [et al.  have demWolbachia in Drosophila bifasciata in Japan. Drosophila bifasciata feeds on sap fluxes in deciduous forests, and sampling across 10 populations within Honshu island in Japan revealed a relatively constant frequency of infection, with between 5 and 7% of females infected with the male-killer [Wolbachia still present on Honshu to this day [In this paper, we examine the factors that could cause incidence variation for the male-killing e-killer . The malthis day .D. bifasciata populations [In contrast to collections from Honshu, past surveys across Hokkaido, the North island of Japan, indicated flies in this area are not infected with the male-killer, despite the relative proximity of the sites to the infected populations in the Northern most sites in Honshu. In total, 559 flies from six locations within Hokkaido were tested, with no evidence of sex ratio distortion in any case . We can ulations . Second,D. bifasciata ecology between Hokkaido and Honshu islands. Our results indicated that the Hokkaido genetic background supported the transmission and action of the male-killer even under stringent conditions, ruling out genetic differentiation as a cause of the absence of the male-killer from Hokkaido. We did observe higher capture rates of D. bifasciata in the island of Hokkaido, and future work should therefore be focussed on the degree to which ecological differences affecting the drive of the infection dictates incidence in this species.We tested whether the absence of infection in Hokkaido was associated with an effect of host genotype on the efficiency of male-killer transmission or strength of male-killing ability. Beyond this, we examined whether trap collection data were consistent with difference in Twenty-eight female flies were collected from the field in Hokkaido. Of these, 4 failed to produce progeny. The remaining 24 all produced a normal sex ratio, consistent with continued absence of the male-killing trait in Hokkaido.Wolbachia from Honshu by introgression of the infection onto the Hokkaido genetic background. The progeny from the 24 Hokkaido females were maintained as isofemale inbred lines for three generations to capture genetic variation within them. The male-killing Wolbachia from Honshu was then placed onto each inbred background via crossing infected females from Honshu to males from each of the Hokkaido lines, with subsequent generations being maintained through further crossing to males from the appropriate Hokkaido line.We tested whether the Hokkaido genetic background supported the male-killing The sex ratio produced following introgression of the infection to the Hokkaido genetic background was female biased and penetrance of the male-killing phenotype was perfect in the first generation. A few males appeared sporadically in six of the 24 lines in one or more subsequent generations, with highest frequency in lines 15 and 25  than in the two samples from the Northern Honshu populations (Mimmaya, Morioka) and the population from Mid Honshu (Kiyosumi). Increased capture rate in Hokkaido was also reflected in an increase in the proportion of all drosophilids sampled that were bifasciata . This high efficiency was maintained even at the threshold for complete male-killing in Honshu, 23.5 C . Thus, the male-killer is proficient at being transmitted and killing males on the Hokkaido background even under relatively stressful environmental conditions. Further, we continue to maintain the infection on the Hokkaido background (we are now at generation 15) without any loss of the infection and without appearance of males. Thus, it is not tenable to argue that the hosts themselves are not genetically suitable for the function of male-killer, at least on an ecological timescale. This situation contrasts with the case of meiotic drive in the related fly D. subobscura, where absence of drive in Europe was associated with refractoriness to the action of the sex ratio distorting element.Previous study has shown that male-killing bifasciata captured at lower rate in the populations from Honshu than in Hokkaido. Thus, ecological heterogeneity is a possible source of the incidence pattern. There are three parameters in male-killer dynamics that may be environmentally influenced. First, the advantage to male-killing may not be as strong in Hokkaido populations. Second, the cost of infection to female flies may be higher in Hokkaido than in Honshu. Third, the transmission efficiency may alter, mediated via elevated temperature, or possibly by reduced overwinter temperature.In the absence of variation in the ability of the male-killer to function on the different genetic backgrounds, the question arises as to the features that cause infection to be absent from the Hokkaido population. Our study did reveal differences in trap collection rates between the populations of Hokkaido and those of Northern Honshu, with In our view, the latter factor is unlikely to be driving the observed pattern. It is notable that the survey of Ikeda revealed the infection to be present in Northern Honshu, but not in Southern Hokkaido. Given these two areas are geographically and climatically very close, temperature differences have poor explanatory power. Explanations based on temperature are also weak because this species exhibits a degree of homeostasis in temperature, moving to elevated altitudes to avoid excess temperature.bifasciata observed in Hokkaido translates into many females laying eggs in a single sap flux, male death will not greatly increase the survival of infected females over uninfected, and infection will decline in frequency. The second factor that may cause infection to decline in frequency is if costs of infection are higher in Hokkaido than in Honshu. This factor can be ecologically contingent. Ikeda demonstrated that the relative fitness of infected flies compared to uninfected flies was lower under high densities in the laboratory [This leaves us with two hypotheses to explain absence of infection in Hokkaido. The first is that there is a weaker advantage to male-killing in Hokkaido than on Honshu, such that there is insufficient drive to maintain the bacterium or permit its spread. The advantage of male-killing to the bacterium depends upon the number of females ovipositing in a single patch . If the boratory . Thus, iWolbachia from Hokkaido.Aside from these possibilities, which we consider most likely, other ecological discontinuities between Hokkaido and Honshu deserve investigation. Some symbionts, for instance, give the host protection against parasitoids , and thuWolbachia to function on different host genetic backgrounds that drives presence or absence of infection in the D. bifasciata- male-killing Wolbachia system. We have demonstrated that despite being absent from Hokkaido, Wolbachia can both be maintained and express male-killing on the Hokkaido host genetic background. We observe that the two populations show differences in trap capture rates, and argue that either ecologically contingent benefits or ecologically contingent costs of infection may explain presence and absence of infection in this species, and that future research be focussed at this level.It is not variation in the ability of D. bifasciata were collected on the campus of Hokkaido University, Sapporo (43°4'56"N, 141°20'21"E), Japan, in May 2003. These were then brought into the laboratory, where they were maintained individually in vials at 21°C on a modified cornmeal-agar diet . These female were checked for the presence of the male-killing trait through scoring of the sex ratio, and maintained by sib-sib inbreeding  for three generations to make inbred isofemale lines. In total, 24 isofemale inbred lines were created (4 lines went extinct), which were maintained thenceforth by simple tossing every three weeks.Twenty eight wild female Wolbachia infected virgin females extracted from a culture derived from Honshu island, Japan . This pished in ) with liTemperature is known to affect the stability of the male-killing trait, and previous study demonstrated that an upper threshold of 23.5 C existed for stable maintenance of the infection on the Honshu genetic background . We testDrosophila communities. Drosophila communities were sampled in 4 deciduous forests in Southern Hokkaido, 2 sites in Northern Honshu, and one site in Mid Honshu, in early-mid October over a number of years between 1973 and 1984 (Figure D. bifasciata is a typical forest-canopy dweller, sampling from the forest canopy is essential for estimating its population density. These traps were cleared 7 or 10 days after setting, and the capture rate of bifasciata per trap per day calculated at each locality to represent the density of this fly in this region. All drosophilid flies were identified, and the proportion of flies caught that were bifasciata recorded.Evidence of differences in fly density can be derived from sampling the ZV helped with the design of the crossing scheme, conducted the crosses involved, and performed the analysis of these crosses. MT designed and conducted the field sampling and scored trap collections. GH conceived the project, organised collection, helped with design of the crossing scheme, and wrote the paper. All authors read and commented on drafts of the manuscript, and approved the final manuscript.


Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT). HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. Immuno-precipitation and BIACore analysis revealed an interaction between HinT and the P80 domain of the membrane complex. As the membrane anchored P80 carries an N-terminal uncleaved signal peptide we have proposed that the N-terminus extends into the cytoplasm and interacts with the cytosolic HinT.Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. In E. coli and differing in the N-terminal region revealed that mutation of the +1 position of the mature protein (Asn to Pro) which is important for signal peptidase I recognition resulted in reduced P80 secretion. All other P80 variants were released into the supernatant, in general as a 74 kDa protein encompassing the helical part of P80. Incubation of M. hominis cells in phosphate buffered saline supplemented with divalent cations revealed that the release of mycoplasma proteins into the supernatant was inhibited by high concentrations of calciumions.Further characterization of P80 suggested that the 4.7 kDa signal peptide is protected from cleavage only in the membrane bound form. We found several proteins were released into the supernatant of a logarithmic phase mycoplasma culture, including P80, which was reduced in size by 10 kDa. Western blot analysis of recombinant P80 mutants expressed in M. hominis implies a two-step process. In general the P80 protein is transported across the membrane and remains complexed to P60, surface-exposed and membrane anchored via the uncleaved signal sequence. Loss of the 4.7 kDa signal peptide seems to be a pre-requisite for P80 secretion, which is followed by a proteolytic process leading to a helical 74 kDa product. We propose that this novel form of two-step secretion is one of the solutions to a life with a reduced gene set.Our model for secretion of the P80 protein of  Listeria, Yersinia and even some of the mycoplasmas are able to invade the host cells [The contact of a pathogenic bacterium with its eukaryotic host provokes a multitude of reactions. A prerequisite for successful infection with the host is the cytadhesion of the bacterium generally mediated by surface localized proteins . Besidesst cells -4. An inst cells -7.The large majority of exported proteins possess an N-terminal signal sequence . Most siMethanococcus jannaschii to 37% in the mollicute Mycoplasma pneumoniae [Bacillus subtilis found that only 50% of the secreted proteins were detected by genomic prediction, indicating that proteomic analyses of secreted proteins (the secretome) are necessary for a comprehensive definition of all secreted proteins [In the last few years, computer programs such as PSORT-II, PSORT-B, ExProt and SignalP have been developed to facilitate the identification of putative secreted proteins -18. Compeumoniae . Analysiproteins .M. hyopneumoniae. P102 is encoded as a precursor protein carrying a type I signal sequence and is found exclusively in the extra-cellular milieu suggesting cleavage by signal peptidase I. The expression of secreted P102 is coupled to that of the surface-exposed cilium adhesin P97, which seems to represent a new variant of processed surface antigens. P97 is derived from a 126 kDa precursor protein by cleavage at amino acid residue (aa) 195. The cleaved 22 kDa N-terminal fragment, which carries an uncleaved type I signal sequence is found embedded in the membrane, in the cytoplasm and in a soluble form in the supernatant, whereas the mature P97, proposed to be membrane bound, is the target of complex proteolytic cleavage, which leads to the subsequent release of some fragments into the supernatant [M. fermentans is a further example of a surface-localized protein undergoing proteolysis after reaching its target. Site-specific cleavage leads to the generation of the membrane bound immune-stimulatory lipopeptide MALP-2 and the release of the remainder (the RF fragment) in a soluble form into the supernatant [Only a handful of mollicute genomes have been decoded, but no analyses of their secretomes have been conducted. A secreted protein, probably processed by the classical mechanism described above, has been characterized in the swine pathogen ernatant ,21. The M. hominis, which is mainly found as a commensal in the urogenital tract, but has also been associated with human urogenital tract infections [In fections ,24, two fections . Recentlfections . We havefections .The data in this paper suggest that the signal peptidase I recognition site is protected in the membrane bound form of P80, but is accessible as the first step in the release of the helical part of P80 into the cell culture supernatant, a process which is accompanied by a decrease in size of 10 kDa.E. coli, the TGA codons were mutated to TGG to allow expression. However, even though we mutated all TGA codons to TGG in the hitA gene, which encodes P80, we were not able to express and purify P80 peptides with an intact N-terminus. While P80-2, the C-terminal region of P80 from AA 320 to AA 713, was stably expressed in E. coli, P80-NT, the initial 21AA of the N-terminus, and P80-1, the N-terminal portion of P80 from AA 1 to AA 326, were degraded at the N-terminus during purification  and as type I signal peptidases share the unusual feature of being resistant to general inhibitors of the four other peptidase classes  we specuE. coli signal peptidase I, we characterized different mutants of the recombinant P80 protein according to their processing in E. coli. Using PCR, we constructed clones expressing the mature P80 protein without the 44 AA signal peptide (ΔSP) and an 89 AA N-terminal truncated P80 protein, which corresponds to the proposed helical, surface-localized part of P80 (Helic.). As the absence of proline at the +1 position of the mature protein is consistent with the observation that the SPase I of E.coli was inhibited by recombinant precursor proteins having a proline residue at this position [N/P) at the (+1) position in the mature protein expecting a reduced or complete inhibition of signal peptide cleavage  mutant, while the sizes of secreted P80 variants corresponded to that of the helical variant  and P80-(Helic.), the two variants lacking signal sequences as translocation signals, this transport may be mediated by an alternative, Sec-independent export mechanism.In order to test the hypothesis that the degradation of rP80 is mediated by position , we genee Figure . Westernt Figure . Small aE. coli is signal peptidase I-mediated and therefore we investigated the protection of the signal peptidase I recognition site of P80 in Mycoplasma hominis [The data suggest that the recombinant P80 processing in  hominis . TherefoAs might be expected the stationary phase culture contained several proteins that might be released from lysed cells, including P50, P60, OppA and cytoplasmic proteins such as P55 Figure . HoweverInterestingly, the P80 protein was the only one of the proteins analyzed that had a decreased size in the supernatant. To exclude the possibility that the reduction in size was simply a result of the large amount of albumin in the culture medium, we purified P80 from the supernatant of a culture in mid logarithmic growth using affinity chromatography. In addition we analyzed P60 as a control. As shown in Figure As the activity of proteases often depends on the presence of divalent cations, we examined the secretion of mycoplasma proteins in the presence or absence of divalent cations. Mycoplasma cells were incubated for 1 h at 37°C in phosphate buffered saline (PBS) supplemented with up to 5 mM magnesium or calcium chloride, or chelating agents as EDTA and EGTA. The addition of divalent cations did not increase the processing of P80 (data not shown). As the presence of 5 mM calcium ions resulted in a complete disappearence of P80 from the supernatant we examined the release of mycoplasma proteins from cells following stepwise addition of calcium ions at concentrations of 0 to 5 mM Figure . Silver Bacillus subtilis [The cytoplasmic proteins that were detected in the supernatant were not released from lysed cells as the titres of the cultures remained the same throughout the experiment (data not shown). These findings are in accordance with those of Antelmann and coworkers, who found several cytoplasmic proteins, such as elongation factor G (EF-G) and arginase (RocF) as to be components of the secretome of subtilis .2inhibited a release of most mycoplasma proteins from the cells  anchored in the membrane and exposed on the surface, and is also released into the extra-cellular environment. As P80 and P60 are concomitantly expressed and both complexed in the membrane fraction it is likely that the soluble P80 variant is derived from the membrane bound precursor. Interestingly, the findings from the heterologous expression of different P80 mutants in E. coli suggest that release of P80 is initiated by cleavage of the N-terminal type I signal sequence, followed by amino-terminal proteolysis by an unknown mechanism, leading to a stable P80 peptide with a predicted helical structure in the supernatant.The data presented here indicate that the membrane protein P80 of M. genitalium, the smallest known self-sufficient organism, lacks a gene for the type I signal peptidase [Mollicutes, despite the fact that several proteins with type I-signal sequences had been identified [Mycoplasma gallisepticum (MGA_1091) and M. pulmonis (MYPU_6300) [As the genome of eptidase  it was ientified ,25. HowePU_6300) ,32.M. hyopneumoniae in the extra-cellular matrix suggests that the intrinsic N-terminal type I signal peptide is immediately cleaved after translocation of the precursor through the membrane, as would be expected from current knowledge of the classical protein secretion pathway, the precursor proteins P80 and P120' of M. hominis are attached to the membrane without being processed or released into the supernatant [The release of mycoplasmal proteins into the extra-cellular milieu may follow different pathways to those described previously for other bacteria. While the exclusive detection of P102 of ernatant ,25,26.M. fermentans that leads to the generation of the immune-stimulatory lipopeptide MALP-2 and showed that the residual RF peptide was soluble and was released into the supernatant [M. hyopneumoniae appears to be quite complex. The precursor protein P126, which carries a type I signal sequence with a cleavage site between AA 31 and AA 32, is predominantly cleaved between AA 194 and AA 195, suggesting that the SP-I signature may be essential only for the translocation of the whole precursor protein across the membrane. The cleavage products P22 and P97 both remain closely associated with the membrane. While P97 is generally subjected to further site-directed proteolysis, leading to the release of peptides into the extra-cellular milieu, further processing of P22 occurs to be strain dependent [The later release of a surface-localized peptide is described here for the first time. Davis and Wise described site-specific proteolysis of the lipid-anchored MALP-404 of ernatant . Processependent .Mycoplasma hominis, and are considered to be required for the establishment of an infection at these sites. Recently in Legionella pneumophila a type II protein secretion system was characterized as a virulence factor linked to an intracellular infection [Release of proteins into the surroundings of a mycoplasma cell should lead to an immediate alteration of the cell surface architecture and, as most membrane proteins are targets for the host immune response ,34, may nfection .M. pulmonis  the gene of which is followed by P60 (MYPU_0070) and HinT (MYPU_0080) gene homologues, and a rhoptry protein of Plasmodium yoelii yoelii (gi23481286). Rhoptry proteins are released during host cell invasion by apicomplexans [P80 has significant similarity with two protein sequences, a hypothetical protein of mplexans .2+-binding calmodulin [2+ ions – may activate the signal peptidase induced processing of the membrane bound P80 protein. This would lead to a soluble P80-helix and the membrane anchored signal peptide. After loss of the transmembrane spanning C-terminal amino acids or due to a leakage of the whole signal sequence (as described above) the signal peptide could reach the cytoplasm where an interaction with HinT may be a further step in modulating or promoting cellular processes such as growth or activation of gene expression, a process we are currently investigating.As P80 interacts with HinT, a cytoplasmic protein found in all kingdoms, which in eukaryotes hydrolyses AMP derivatives and in yeasts functions as a regulator of an RNA polymerase II domain , a furthlmodulin , or are lmodulin , where tlmodulin . Our resMycoplasma hominis. This is, to our knowledge, the first description of secreted protein with a type I signal sequence that is stably embedded in the membrane as precursor protein and, as we propose as a model of secretion, is subsequently released from the membrane into the supernatant after signal peptidase I cleavage. As in other mycoplasmas several proteins have been shown to either possess an uncleaved signal sequence or to be further processed after anchoring in the membrane, this may be a common phenomenon in mycoplasmas. Because of their minimal coding capacity, mycoplasmas may have evolved a strategy to use secretory proteins in a dual role, as surface localized proteins in the membrane for cell surface architecture, and, in response to a change in the environment, as a soluble protein released from the cell surface.The data presented here clearly demonstrate that P80 is a secreted antigen of Mycoplasma hominis strain FBG was amplified by PCR [Helic., AA 90–713), amplifying a fragment encoding the mature P80 polypeptide (ΔSP, AA 45–713), and mutating the asparagine codon at position +1 of the mature protein to a proline codon (N/P).The P80 protein encoding region (ACC Z29068) of d by PCR  using old by PCR . To facid by PCR , restricRcaI/BglII restricted amplicon in frame into the NcoI/BglII restricted plasmid pQE60 and inserted the BamHI/BglII restricted DHFR-fragment of pQE40 in the BglII site of the plasmid pQE60-P80 NT. Plasmids were propagated in DH5αF' for constitutive expression [Amplicons were cut at the restriction sites within the primers and ligated in-frame into the expression vector pQE30 (Qiagen, Hilden, Germany). To express the P80 N-terminus (AA 1–21) as a fusion protein with dihydrofolate reductase and a poly His-tag, we ligated the respective pression .g, 30 min, 4°C). In the secretion assay, the pelleted cells and supernatant, which was concentrated by lyophilizing, were subjected to Western blot analysis. The recombinant peptides P80-NT and P80-2 were purified by Ni-NTA chelation according to the manufacturer's protocol, while rP80 and P80-1 were purified with the Sepharose-coated P80-specific antibodies LF8 and NB12 as described by Henrich et al. [Cells from a mid-logarithmic phase culture in LB broth (Gibco BRL, Life Technologies Inc., Gaithersburg, Md.) containing ampicillin (100 μg/ml) were harvested by centrifugation . The integrity of the different plasmid sequences was confirmed by analysis on an ABI sequencer using the method of Sanger .Mycoplasma hominis strain FBG was cultivated in PPLO broth supplemented with arginine from frozen stocks of a mid-logarithmic phase broth culture as described previously [g, 10 min, 4°C), and the cell pellets and supernatants were subjected to Western blot analysis. Alternatively, cell pellets derived by low speed centrifugation (10,000 × g, 10 min, 4°C) were re-suspended in phosphate-buffered saline  containing 5.0, 2.5, 1.25, 0.63, 0.31 or 0.00 mM CaCl2 and incubated at 37°C for 60 min. Soluble proteins were then separated from insoluble material by centrifugation (15,000 × g, 15 min, 4°C), and all samples analyzed by Silver staining [eviously . The titeviously . For prostaining  and WestProteins were separated in 9.5% polyacrylamide gels , transfeM. hominis culture as part of her thesis. MH carried out the immunoassays and completed the secretion assays. BH carried out the molecular genetic studies, participated in the design of the study and drafted the manuscript. All authors have read and approved of the final manuscript.RH began the characterization of antigens released into the supernatant of a


Cancer of unknown primary remains a mallignancy of elusive biology and grim prognosis that lacks effective therapeutic options. We investigated angiogenesis in cancer of unknown primary to expand our knowledge on the biology of these tumors and identify potential therapeutic targets.Paraffin embedded archival material from 81 patients diagnosed with CUP was used. Tumor histology was adenocarcinoma (77%), undifferentiated carcinoma (18%) and squamous cell carcinoma (5%). The tissue expression of CD34, VEGF and TSP-1 was assessed immunohistochemically by use of specific monoclonal antibodies and was analyzed against clinicopathological data.VEGF expression was detected in all cases and was strong in 83%. Stromal expression of TSP-1 was seen in 80% of cases and was strong in 20%. The expression of both proteins was not associated with any clinical or pathological parameters. Tumor MVD was higher in tumors classified as unfavorable compared to more favorable and was positively associated with VEGF and negatively with TSP-1.Angiogenesis is very active and expression of VEGF is almost universal in cancers of unknown primary. These findings support the clinical investigation of VEGF targeted therapy in this clinical setting. Cancer of unknown primary (CUP) is a unique clinical entity that accounts for an approximately 3% of human cancers. PatientAngiogenesis, the formation of new vessels, is essential for tumor growth and the development of metastases. It evolves though a complex multifactor process that involves interaction of pro-angiogenic and anti-angiogenic signals from tumor, endothelial and stromal cells. The angiogenic activity is reflected in the development of novel microvessels in tumor tissue that is quantified by the intratumoral microvessel density (MVD). Among several molecules implicated, Vascular Endothelial Growth Factor (VEGF) and Thrombospondin-1 (TSP-1) appear to be most relevant. Much evidence indicates that VEGF is a key activator of angiogenesis,5 and TSWe were prompted to investigate angiogenesis in unknown primary cancer in an attempt to enrich our understanding of the biology of these tumors. We studied by immunohistochemistry the tissue expression of VEGF and TSP-1 in CUP and correlated with MVD and clinicopathological parameters. In a recently published study vascular endothelial growth factor, and CD34, factors were not found to be of prognostic value in adenocarcinoma of unknown primary.A total of 81 patients diagnosed with CUP and treated in three University Medical Oncology Settings  between January 1997 and December 2002 were selected on the basis of availability of archival tumor tissues and accessibility to medical notes. Pathology diagnosis was reviewed by two pathologists blinded to written pathology report and representative paraffin blocks were selected for immunohistochemistry.Eligible cases categorized into unfavorable and more favorable subgroups tables  and 2. Pp = 0.04) and a longer survival, 11.5 vs 8.5 months (p = 0.01).Systemic chemotherapy was given in 64 patients (78%); four patients with cerebral metastases received whole brain irradiation. Chemotherapy was consisted of a platinum based combination. Objective response to chemotherapy was observed in 34 patients (53%) while one patient with brain metastases responded to radiotherapy. Median survival for all patients was 10.5 months  method. In brief, tissue sections were deparaffinised in xylene and dehydrated. They were immersed in citrate buffer (0,1 m, pH 0,6) and subjected to microwave twice for 15 min. Subsequently, all sections were treated for 30 min with 0,3% hydrogen peroxide in methanol to quench endogenous peroxidase activity. Mouse monoclonal antibodies directed against human CD34 antigen (M 7165, Dako) in dilution 1/50 β) VEGF Ab-3 (isoform 121, clone Jh121, Neomarkers) in dilution 1/50 and c) thrombospondin (Mob 315, DBS) in dilution1/50 were used. Positive control slides were included in all cases. All dilutions were made in TBS-1% BSA solution and were followed by overnight incubation.The assessment of immunostaining was made by two experienced pathologists using light microscope. Tumor specimens too small to provide sufficient sections for all the immunoassaying procedures were disregarded from the study.Staining of endothelial cells for CD34 was used to evaluate the MVD. Any CD34 positive endothelial cell clusters clearly separated from each other were considered as single countable microvessels. A lumen was not required to identify a vessel. Larger vessels with muscular walls were excluded from counting. In each sample three areas of most prominent vascular density (hot spots) were identified at ×40 power field and microvessel counting was done under ×400 magnification. Counting was performed by two independent observers blinded to clinical information. The median count was used to make distinction between low and high MVD.Immunoreactivity for VEGF was observed in stromal and epithelial cells. Only staining of tumor cells was considered for analysis. To evaluate the expression of VEGF protein, we devised a combined score that corresponds to the sum of staining intensity (0 = negative, 1 = weak, 2 = intermediate, 3 = strong staining) and percentile quadrants of positive cells (0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = >50%). The maximum score was 6. Score 2 was regarded to represent weak expression, score 3 intermediate and score 4–6 strong expression.Staining for TSP-1 was only considered in regard to extracellular matrix. The expression of TSP-1 was characterized according to the extent and the intensity of staining classified as negative, +1: mild, focal, +2: intermediate, multifocal, +3: strong, diffuse reactivity.Staining results were analyzed against clinical subgroup, histological differentiation, response to treatment and survival. The association between MVD and clinical subgroup, histological differentiation and response to treatment was assessed by an unpaired t test. A Fisher's exact test was used to determine associations between VEGF and TSP-1 and the clinical subgroup, histological differentiation and response to treatment. Spearman non parametric correlation test was used for associations between MVD, VEGF and TSP-1.Survival was calculated by Kaplan-Meier method and comparison of survival curves was performed by the log-rank test. For statistical significance a two-tailed p value was considered. The Graphpad Instat version 3.05 (Graphpad Software, Inc, San Diego, CA) and Prism version 4 (Graphpad Software, Inc, San Diego, CA) software programs were used for statistical analysis and graphing.Demographics of studied cases are depicted in Tables 2 (range, 16 to 300 microvessels/mm2)   declare that they have no competing interests.NP conceived, coordinated and designed the study, interpreted the data and drafted the manuscript; VK, EB, designed and carried out the study, performed the statistical analysis, interpreted the data and drafted the manuscript; VMM carried out the pathological and immunohistochemical study, interpreted data and drafted the manuscript; ET, EK carried out the pathological and immunohistochemical study, interpreted data and drafted the manuscript; HK, GF, TF participated in designing the study, acquisition and interpretation of data and revising critically the manuscript. All of the authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


There are three pathways of L-threonine catabolism. The enzyme L-threonine aldolase (TA) has been shown to catalyse the conversion of L-threonine to yield glycine and acetaldehyde in bacteria, fungi and plants. Low levels of TA enzymatic activity have been found in vertebrates. It has been suggested that any detectable activity is due to serine hydroxymethyltransferase and that mammals lack a genuine threonine aldolase.Thermotoga maritima TA. Almost all known functional residues are conserved between the two proteins including Lys242 that forms a Schiff-base with the cofactor, pyridoxal-5'-phosphate. The human TA gene is located at 17q25. It contains two single nucleotide deletions, in exons 4 and 7, which cause frame-shifts and a premature in-frame stop codon towards the carboxy-terminal. Expression of human TA mRNA was undetectable by RT-PCR. In mice, TA mRNA was found at low levels in a range of adult tissues, being highest in prostate, heart and liver. In contrast, serine/threonine dehydratase, another enzyme that catabolises L-threonine, is expressed very highly only in the liver. Serine dehydratase-like 1, also was most abundant in the liver. In whole mouse embryos TA mRNA expression was low prior to E-15 increasing more than four-fold by E-17.The 7-exon murine L-threonine aldolase gene (GLY1) is located on chromosome 11, spanning 5.6 kb. The cDNA encodes a 400-residue protein. The protein has 81% similarity with the bacterium Mice, the western-clawed frog and the zebrafish have transcribed threonine aldolase/GLY1 genes, but the human homolog is a non-transcribed pseudogene. Serine dehydratase-like 1 is a putative L-threonine catabolising enzyme. Elucidating the factors involved in threonine homoeostasis is important for the development of nutritional strategies in human clinical diets for treating patients suffering from wasting diseases. In farmed animals the regulation of livestock feed is required to ensure optimal growth and to reduce nitrogen excretion which poses environmental disposal problems. Threonine is required for protein synthesis and the removal of excess threonine by oxidation is needed to prevent its accumulation both intracellularly and in the circulation. The rate of catabolism of many amino acids, including threonine, increases when dietary protein exceeds the body's requirements. Gluconeogenesis occurs mainly in the liver where it helps maintain blood glucose homeostasis in mammals. During starvation amino acid catabolism increases to support gluconeogenesis. Glucocorticoids and glucagon hormones are known to up regulate and insulin down regulate the gene expression of many amino acid-catabolising enzymes .4+ and 2-ketobutyrate and TDH to yield 2-amino-3-ketobutyrate. The subsequent reaction between 2-amino-3-ketobutyrate and coenzyme A to form glycine and acetyl-CoA is catalysed by 2-amino-3-ketobutyrate coenzyme A ligase (KBL)(EC 2.3.1.29), also called glycine acetyltransferase (gene abbreviation GCAT).There are three L-threonine  degradation pathways in living organisms; via L-threonine aldolase (L-TA)EC 4.1.2.5)(gene abbreviation GLY1), via L-serine/threonine dehydratase (SDH)(EC 4.2.1.16)(gene abbreviation SDS) and via L-threonine 3-dehydrogenase (EC 1.1.1.103)(TDH) [.5, SDH uses threonine and serine as substrates to generate glycine which is used in gluconeogenesis. Serine dehydratase-like 1 gene (SDH1) is a second SDH gene found in vertebrates, but has yet to be characterised. I suggest that it is also a putative L-threonine catabolising enzyme.6-dependant enzymes can be grouped according to their fold type. L-TA belongs to fold type I. L-TA enzymes are unrelated to D-TA enzymes which possess type III folds [Vitamin BII folds -14. ThreII folds .Saccharomyces cerevisiae, the glycine synthase-1 gene, GLY1 was identified as threonine aldolase [Candida albicans [Escherichia coli, Aeromonas jandaei, Pseudomonas and Thermatoga maritima the GLY1 gene has been cloned and their enzymatic activity characterised [Arabidopsis thaliana, there are two threonine aldolase genes (THA1 and THA2). THA1 has been shown to play a role in seed nutritional quality [Thermotoga maritima have been determined as the apo-enzyme, bound to L-allo-threonine and to glycine [L-TAs from a number of species of bacteria and fungi have been isolated and characterized (reviewed in ). In thealdolase ,18. Prevaldolase . But it albicans . In a nucterised -24. In t quality . Putativ quality . Recentl glycine .E. coli and the yogurt bacterium, Streptococcus thermophilus, have TA activity [These GLY1/threonine aldolases are distinct from the serine hydroxymethyltransferases (EC 2.1.2.1)(SHMT). However, some SHMT also possess some threonine aldolase enzymatic activity. SHMT from activity ,27. SHMTactivity . Consequactivity . Here I E. coli TA protein [I conducted a search of the GenBank database for a putative mouse L-threonine aldolase gene using the sequence of the  protein . PCR pri protein . The preT. maritima [T. maritima. However, the mouse protein has an additional putative amino-terminal mitochondrial import leader peptide. Given long evolutionary distance between mouse and bacteria this high degree of homology strongly suggests that this murine protein is also a threonine aldolase. Most functional residues are conserved between the two proteins. By homology with the T. maritima protein, Lys242 is expected to form a Schiff-base with the cofactor, pyridoxal-5'-phosphate (PLP), with Asp211 and Arg214 expected to interact with PLP. Those residues that contact the ligands L-allo-threonine and glycine, Ser45, His123, Tyr127, Arg214 and Arg372, are conserved. T. maritima His125 from the second subunit is predicted to bind the hydroxyl group of L-threonine. This residue is homologous to murine Tyr168, a conservative substitution since both residues are polar and aromatic. In other TA proteins from diverse phyla this residue is mainly histidine, but in rice it is a tyrosine also. At the catalytic dimer interface electrostatic interactions occur among the side chains of Arg44-Glu71, Thr47-Asp66 and Arg274-Ser241. These residues are conserved. Residues involved in ion coordination are also conserved with Ala246, Thr49 and Ser241 contacting Ca2+ with Arg112 contacting a chloride ion. In the Arabidopsis THA1 enzyme a Gly114 to Arg mutation, located between two beta-sheets, results in loss of enzymatic activity [A comparison of the predicted secondary structure of the murine TA protein with the known secondary structure of maritima  is shownTakifugu rubripes)(accession No. BK005561). The exon/exon boundaries on the proteins are highly conserved between mouse and Japanese puffer fish with only one being displaced slightly. The Japanese puffer fish gene encodes a 421-residue protein that has 46% identity and 74% similarity to the murine protein. Similar L-threonine aldolase cDNAs for the western-clawed frog and the zebrafish were identified (accession Nos. BK005562 and AAH72718 respectively). The proteins are of similar lengths with the functional residues identified in T. maritima being well conserved. Homology extends throughout their lengths, apart from the amino-terminal regions. Despite their low sequence identity in the amino-terminal region all four proteins contain putative mitochondrial import leader peptides, being positively charged. They possess also a predicted cleavage site that would be utilised during their import into mitochondria. After cleavage of the mitochondrial import sequence the mature murine TA enzyme would have a mass 39,778 Da and pI 6.11. Recently, two other vertebrate GLY1 genes have been sequenced. In the dog there is a complete gene (GenBank accession number NW_140385) that would encode a 391-residue protein with 82% identity and 94% similarity to murine TA. Three overlapping unassigned genomic DNA sequences from the freshwater puffer fish, Tetraodon nigroviridis, would encode a gene encoding a 431-residue protein with 44% identity and 73% similarity to murine TA. Additionally, homologous coding ESTs from mammals (rat, pig and cow), birds (chicken), amphibians (African clawed frog) and fish  were identified, indicating that L-threonine aldolase expression in vertebrates is widespread.Database searches revealed the presence of other L-threonine aldolase genes in other vertebrates Fig. . There iA database search identified 17q25 as the location for the human GLY1 gene. All exon/intron boundaries found in the murine threonine aldolase gene are conserved in man. The mouse to human conserved synteny map shows that both GLY1 genes have the synaptogyrin 2 (SYNGR2), baculoviral IAP repeat-containing 5 (BIRC5) and soluble thymidine kinase 1 (TK1) genes as near neighbours. Starting with human liver RNA, I was neither able to amplify any threonine aldolase transcripts using a variety of 5' and 3' RACE methods nor to detect any transcripts in a wide range of tissue and cell line cDNAs by RT-PCR. A search of the human EST database identified five potential EST transcripts scattered throughout the threonine aldolase gene, but they lack supporting evidence that they are truly transcribed sequences, being unspliced singletons. There is a potential polyadenylation signal site in the human threonine aldolase gene with good homology to the murine site. However, corresponding 3'UTR ESTs and SAGE tags are conspicuously absent from the databases leading to the conclusion that the GLY1 threonine aldolase gene is not transcribed in man.T. maritima, and vertebrates. One residue that would be expected to interact with the PLP ligand, murine Arg214, would be mutated to Gln in man. Murine residue Arg372 that would be expected to interact with threonine is mutated to Ala. The side chains of two residues that form electrostatic interactions at the catalytic dimer interface are also mutated, murine Thr47 to Lys, and murine Arg274 to His. If the frame-shifts were not present, the mouse and human proteins would have 66% identity and 85% similarity. Likewise, the chimpanzee threonine aldolase gene is a pseudogene possessing the same frame-shifts as the human gene. Additionally, it has lost the splice donor site in exon 1 and, by comparison with the mouse gene, has a 64 bp deletion in exon 7 . In mammals, these two genes are adjacent, being arranged in a 5' to 5' orientation. Database searches identified both the SDH and SDH1 genes in man, rat, freshwater puffer fish and the Western and African clawed frogs. But in the chicken only the SDH1 gene is present since SDH is absent from the draft genome and all expressed sequences. A comparison of vertebrate SDH and SDH1 proteins with the crystal structure of rat SDH  suggestsTo identify those tissues which are likely to contribute to TA activity in the mouse, the expression of TA mRNA in adult tissues was examined by RT-real time PCR normalised to the expression of the housekeeping genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Low levels of TA mRNA were detected in all tissues examined. They varied 20-fold between tissues, being highest in prostate, heart and liver Fig. . In contThe mRNA expression of threonine catabolic enzymes was examined by real time PCR in cDNAs derived from whole mouse embryos from days 7, 11, 15 and 17 Fig. . OverallT. maritima with an expect value of 2e-73, being clearly distinct from glycine dehydrogenase, the second most closely related protein, with an expect value of 0.002. This remarkable conservation, over billions of years of evolution since the last common ancestor, shows the general importance of these metabolic pathways. However, the presence of some abnormal TA mRNA splicing in mouse, the low levels of mRNA found in mouse tissues, together with the low levels of TA enzymatic activity found in rat liver [In vertebrates, L-threonine is one of the indispensable amino acids. It is obtained from protein in the diet, typically being the second or third limiting amino acid in herbivorous diets. Some of it is utilized in synthesising new protein, but the rest is converted to other amino acids by oxidative catabolism by three different enzymes that are found in most organisms; TDH, TA and SDH. Both the TDH and TA pathways produce glycine. However, the TDH pathway occurs in two steps, requiring KBL as the second step. Using protein homology searches of the mouse genome with the bacterial enzymes has allowed me to identify and clone TDH and KBL cDNAs ,37. GLY1at liver ,15 plus allo-threonine. These can be divided into three types based on the stereospecificity towards the β-carbon of threonine. Low-specificity L-TA can use both L-threonine and L-allo-threonine as substrates. L-TA only acts on L-threonine and L-allo-TA is specific to L-allo-threonine [allo isomer in a manner similar to the T. maritima enzyme, because Tyr127 (Tyr87 in T. maritima) in the TA active site is conserved, a residue which appears to be involved in discriminating L-threonine from L-allo-threonine [The L-TA enzymes can act on the stereoisomers, L-threonine and L-hreonine . Murine hreonine .S. cerevisiae, revealed a cytosolic localisation for TA [All the vertebrate TA proteins have putative amino-terminal mitochondrial import sequences, suggesting that the mitochondrion is its intracellular localisation. In contrast, fractionation studies in the yeast, n for TA . Addition for TA ,37,39. HHumans have lost two of the three enzymes of threonine catabolism with both GLY1 and TDH  genes beWith the loss of TA and TDH genes in humans this leaves serine/threonine dehydratase as our only major threonine catabolic enzyme. However, vertebrates also have a second SDH gene, called SDH-like-1 which, by homology, is likely to function also as a serine/threonine dehydratase since all the residues that bind the PLP co-factor are conserved between the two proteins. Only the SDH1 gene is present in the chicken, therefore the serine dehydratase activity found in chick livers  must be The SHMT enzymes are members of the α-class of pyridoxal phosphate enzymes, catalyzing the reversible interconversion of serine and glycine. Mammals have two SHMT genes. One encodes a cytosolic and the other a mitochondrial enzyme. Purified SHMT enzyme from rat liver possesses some threonine aldolase activity  and bothin vitro and relative contribution to threonine catabolism under different physiological states in vivo. Changes in TA and SDH-1 mRNA expression in response to diet have yet to be examined, but rats fed a high protein diet or fasted showed an increase in TA enzymatic activity [With the identification of murine TA and SDH-1 mRNA the way is open to study their enzymatic activity activity . In contactivity .I have shown that GLY1/TA genes are present in vertebrates. TA genes and enzymatic activities have been previously isolated from bacteria, fungi and plants. These enzymes are distinct from the serine hydroxymethyltransferases. The mouse GLY1 gene is located on chromosome 11, band E2 and the 1855 bp cDNA from this gene encodes a 400-residue threonine aldolase. The presence of a positively-charged amino-terminal import leader peptide sequence in mammalian, amphibian and fish TA proteins, that are not present in bacterial proteins, suggests that the vertebrate TA enzymes are mitochondrial. Man and chimpanzees have lost a functional GLY1 gene. Vertebrates also have a second SDH gene, SDH1, that by homology to the crystal structure of SDH may function as a threonine dehydratase and contribute to threonine catabolism.Total RNA was extracted from mouse liver using guanidine thiocyanate and treated with DNase-I to remove any contaminating genomic DNA . Total RNA was reversed transcribed with AMV RNase H- reverse transcriptase (ThermoScript, Life Technologies, UK) at 50°C using an oligo-dT primer. The cDNA was amplified by touchdown PCR using the Advantage cDNA polymerise mix (Clontech, UK) on a Perkin-Elmer 2400 thermocycler. Amplification conditions for the first 10 cycles were 94°C for 5 sec, 72°C less 0.4°C per cycle for 3 min and for the next 20 cycles 94°C for 5 sec, 68°C for 10 sec, 72°C for 3 min per cycle using primers (100 nM) derived from the sequence of the mouse genomic DNA from clone RP23-268N22 (accession number AL591433 forward 5'-ATAGTGCCCCGGGCTTGC-3' and, first reverse 5'-TTTTTTTTTTTTTTTGTGCCTTCAGTATTT-3' and (Amersham-Pharmacia Biotech, UK). PCR amplicons were electrophoresised in a low-melting point agarose gel stained with ethidium bromide. They were excised from the gel. The agarose digested with agarase (Promega, U.K.). These PCR amplicons were cloned into pCR-II-TOPO, a T-A vector (Invitrogen, The Netherlands) and sequenced in both directions using the big dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase FS on an ABI 373XL Stretch Sequencer (both from PE Applied Biosystems, UK). SDH and SDH-1 cDNAs were cloned in a similar manner and were used as positive controls in RT-PCR assays.+ selected RNA by reverse transcriptase using an oligo-dT primer (BD Clontech, UK). Approximately 8.0 ng of cDNA were used for each PCR. Tissue master mixes were divided into gene specific mixes and primers were added to a final concentration of 200 μM. The primers were: TA, CCCAGAGATTCGCTAAAGGACTC (exon 6/7) and CACGGCCAGTCTGAGCCAC (exon 7), which produced a 171 bp amplicon; SDH, TTTTACGAACACCCCATTTTCTC (exon 7) and AGAATCTTCTCATCGTCCACGAA (exon 8/7), which produced an 89 bp amplicon; SDH1, CCTGCCAGACATCACCAGTGT (exon 6/7) and GCGCTCATCGTCCAGGAA (exon 8/7), which produced a 154 bp amplicon; G3PDH, TCCCACTCTTCCACCTTCGA and GTCCACCACCCTGTTGCTGTA, which produced a 111 bp amplicon. Primers for beta-actin, TDH and KBL have been described previously [t]) was used as a measure of abundance. A comparative Ct method was used to determine gene expression. Expression levels in each tissue cDNA sample were normalised to the average expression levels of the housekeeping genes beta-actin and G3PDH (ΔCt). Ratios of gene of interest mRNA/housekeeping mRNA from each tissue were standardised to that of the highest expressing tissue for that gene which was taken as 100% (ΔΔCt). Formula E-ΔΔCt was used to calculate relative expression levels where E is the efficiency of the PCR per cycle. Amplification specificity was confirmed by melting curve analysis and agarose gel electrophoresis.Quantitative PCR was carried out on a GeneAmp 5700 Sequence Detection System (AB Applied Biosystems) and a Rotor Gene 3000 utilising a CAS-1200 robotic precision liquid handling system  using a SYBR Green I double-stranded DNA binding dye assay (Applied Biosystems, UK). For the determination of TA, SDH, SDH1, TDH and KBL mRNA expression in adult mouse tissues and whole mouse embryos, cDNAs were generated from polyAeviously . AmplifiT. maritima [The predicted start site of transcription of murine TA mRNA was determined using the program Eponine . The premaritima  using 3Dmaritima . Mitochomaritima . Cleavagmaritima .A.J.E. initiated and carried out the molecular genetic studies, drafted the manuscript and approved the final manuscript.


Proper management of patients with Kallmann syndrome (KS) allows them to attain a normal reproductive health. The purpose of this study is to demonstrate the presentation modalities, phenotypes and the modes of inheritance among 32 patients with Kallmann syndrome in Jordan. Recognition of the syndrome allows for prompt proper management and provision of genetic counselling.Over a period of five years (1999–2004), the clinical and inheritance profiles of 26 male and 6 female patients with Kallmann syndrome from 12 families were evaluated at the National Center for Diabetes, Endocrinology and Genetics in Jordan.The patients belonged to twelve Jordanian and Palestinian families and their age at presentation ranged from 4 – 46 years. Nine boys aged 4–14 years presented with cryptorchidism and microphallus, all other males presented with delayed puberty, hypogonadism and/or infertility. The main presentation among six female patients was primary amenorrhea. Intrafamilial variability in clinical phenotype was specifically evident for renal abnormalities and sensorineural hearing impairment. Familial KS was diagnosed in 27 patients belonging to five families with the X-linked mode of inheritance and two families with the autosomal recessive mode of inheritance.(1) the majority of cases in this study represented the X-linked form of KS, which might point to a high prevalence of Kal 1 gene in the population. (2) Genetic counselling helps these families to reach a diagnosis at an early age and to decide about their reproductive options. (3) Children presenting with cryptorchidism and microphallus in our population should be investigated for KS. One of the most common causes of hypogonadotropic hypogonadism is Kallmann syndrome (KS). KS is a genetically heterogeneous condition that affects approximately one in 8000 males and one in 40,000–70,000 females -3. This To our knowledge, no data is available on the incidence of this syndrome in Jordan or in the Arab world.In addition to the sporadic form which is the most common , KS has Over a period of five years, thirty-two male and female patients from twelve Jordanian and Palestinian families were referred to the National Center for Diabetes, Endocrinology and Genetics (NCDEG) in Amman, Jordan for evaluation of hypogonadism among adults, or microphallus among children.Prospective evaluation was performed including pedigree construction fig , and comOlfactory testing was performed through a smell identification test designed by our department using odours that can be easily identified among all social classes in the local population. These odours included cinnamon, coffee, camomile, thyme, soap, oriental perfume and tobacco. The specific odoriferous substance was approximated to the nostrils while the patient had his eyes closed. The patient is then asked to name the olfactory stimulus. According to the number of substances identified by smelling, patients were divided into anosmic and hyposmic. Those who failed to identify all odours were categorized as anosmic, while those who identified three or less odours were categorized as hyposmic. Those who identified four to all seven odours were considered normosmic. First-degree relatives of the probands were also questioned and tested for the smell sensation in a similar fashion. The same investigator carried out olfactory testing for all patients and family members.In addition to the assessment of smell sensation among relatives of probands, their answers to specific questions were recorded. These questions addressed the issues of surgery to correct cryptorchidism, administration of testosterone and whether there were any complaints of delayed puberty or infertility. The specific questions and the olfactory testing led to the identification of further cases of KS in the family.Patients were asked to perform a screwdriver motion in one hand while the examiner watches for any similar non-voluntary movement in the other hand to assess mirror image movements. Subjects were also asked to perform alternate supination and pronation of one forearm while the examiner watches for a similar movement in the other arm . PhallusThe criteria for diagnosis of KS among adults included the presence of anosmia or hyposmia with clinical signs and symptoms of hypogonadism and a testosterone level <100 ng/dl among males 16 years and older, and estradiol level <20 pg/dl among adult females, together with basal low gonadotrophin level. In family II, loss of contact of three brothers hindered testing their hormonal levels. They were, however, included among our patients because clinical examination revealed hypogonadism, anosmia and micropenis. Among prepubertal males, the criteria for diagnosis of KS included presence of microphallus with anosmia/hyposmia, and/or absent olfactory bulbs on MRI.Pedigree analysis was used to establish the modes of inheritance of KS in the familial cases. Inheritance in a family was classified as X-linked if only males were affected in more than one sibship connected by females, or if two or more males were affected in the sibship with a negative family history and with associated synkinesia. Inheritance was classified as autosomal recessive if all affected individuals were members of the same generation and included at least one female.Over a period of five years (1999–2004), thirty-two patients were prospectively diagnosed with Kallmann syndrome at the National Center for Diabetes, Endocrinology and Genetics (NCDEG) in Amman, Jordan. The patients belonged to twelve Jordanian and Palestinian families and their ages at presentation ranged between 4 – 46 years. They included 26 males and 6 females with a male/female ratio of 4.34/1. Nine male patients were aged 14 years and younger.The clinical features among male patients are presented in table Twenty seven patients were anosmic and five patients were hyposmic table . In famiCryptorchidism was found or previously operated on in 19/26 (73%) and microphallus in 17/26 (65%) male patients respectively. All patients included in this study manifested high-arched palate. Renal abnormalities including unilateral renal agenesis, malrotated kidney, and horseshoe kidney were detected in 11/19 (58%)cases with the X-linked form of KS. Two sporadic cases showed renal anomalies. A variable degree of sensorineural hearing impairment was found in 4/19 patients with X-linked KS, and in none of the other mode of inheritance or the sporadic cases. Olfactory MRI revealed olfactory tract agenesis among 19/24 cases for which the investigation was done in the series.Among females diagnosed as KS in this series primary amenorrhea was the main presenting feature.Pedigrees were constructed for all families figure . Five caThis study points to the higher proportion of the X-linked form of Kallmann syndrome among all KS cases seen at an endocrine/genetic clinic in Jordan over a period of 5 years. Among 7 families with inherited KS, the X-linked form was the mode of inheritance in 5 families . None of the pedigrees was consistent with autosomal dominant inheritance in this series. In the two families with autosomal recessive inheritance, the probability of a non-penetrant autosomal dominant gene in either parent was considered remote because of absence of any relevant family history. Autosomal dominant inheritance was also considered a very remote possibility in the X-linked families because of absence of affected individuals in earlier generations and the pattern of inheritance as revealed by pedigree construction , and was not related to a specific mode of inheritance or etiology. Microphallus was present among 17/26 (65%) patients in this study, with several other patients reporting a history of treatment with testosterone; the exact number of treated patients or the treatment profile could not be precisely determined. Seminal fluid analysis was done for all patients of 16 years and above. The test showed azoospermia for all tested males except patient 1 in family IX, where oligospermia was reported with a sperm count of 10,000,000 per ml. None of the patients with the X-linked form manifested ichthyosis, mental retardation, short stature or ocular albinism, pointing to the underlying etiology being a mutation in the Kal 1 gene rather than a contiguous-gene deletion syndrome .Olfactory MRI revealed olfactory tract agenesis in 80% of cases for which the investigation was done in the series (19/24). Quinton et al, 1996, indicate that KS may be present with no pathology detected in the olfactory tract on MRI, and that phenotypic characterization of KS was effectively achieved by accurate estimation of olfactory sensation . KallmanThe majority of Kallmann syndrome cases in our study showed the X-linked mode of inheritance, which might indicate a high prevalence of Kal1 gene in the population. However, molecular studies for the Kal1 gene were not performed in this study. Patients in our series manifested a wide range of phenotypic heterogeneity with intrafamilial variability of clinical manifestations. We recommend an evaluation for Kallmann syndrome in our population in any child presenting with microphallus and cryptorchidism. Further studies are needed to establish the prevalence rate of Kallmann syndrome in Jordan and to define the causative mutations.The authors declare that they have no competing interests.MAJ and KA were the main researchers. HH and MAJ drafted the manuscript. All authors were part of the team that evaluated the patients


By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors.The JAMM  motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EX Protein structure studies suggest that deubiquitination in the proteasome is carried out by a protein with features of a zinc metalloprotease Many cellular proteins are degraded by the proteasome after they become covalently modified with a multiubiquitin chain. The 26S proteasome is a massive protein composed of a 20S core and two 19S regulatory particles . The 20SArabidopsis thaliana from genetic studies of constitutively photomorphogenic mutant plants (nHS/THX7SXXD), referred to as the JAMM motif, that is embedded within the larger JAB1/MPN/Mov34 domain (hereafter referred to as the JAMM domain) and is critical for Csn5 function in vivo  regulatory modification from the Cullin 1 (Cul1) subunit of the SCF (Skp1/Cdc53/Cullin/F-box receptor) Ub ligase is catalyzed by the COP9 signalosome (CSN) . The CSNt plants . It latet plants . Csn5 of in vivo . Removal in vivo . The JAM in vivo , and pro in vivo . Taken t in vivo , indicatEscherichia coli a variety of JAMM motif-containing proteins to find a suitable candidate for crystallographic analysis. The expression of all candidates except for AfJAMM led to insoluble aggregates. Unlike many JAMM proteins that contain an additional domain, the AfJAMM protein consists entirely of the JAMM domain. We were able to purify and crystallize native and selenomethionine-substituted AfJAMM; the latter was used for phasing by employing the multiwavelength anomalous diffraction (MAD) technique , flanked by a long α helix (α1) between the first and second strand, and a short α helix (α2) between the fourth and fifth strand. This β sheet resembles a β barrel halved longitudinally and curled around α1 A. The α2subtilis  can be ssubtilis A. Conseqsubtilis  and Ub-ssubtilis .The two AfJAMM subunits in the asymmetric unit are connected through a parallel β sheet formed at the dimer interface B. The suɛ2 of H67 and H69 on β4, the carboxylate of D80 on α2, and a water molecule. The latter hydrogen-bonds to the sidechain of E22 on β2. Thus, the crystal structure confirms previous predictions that the histidine and aspartic acid residues in the JAMM motif are ligands for a metal , and neurolysin, a mammalian metalloprotease  into the pCRT7 vectors . During cloning, the alternate start codon, GTG, was replaced with the canonical start codon, ATG. The construct was expressed in BL21(DE3)pLysS cells (Novagen, Madison, Wisconsin, United States). The cells were grown to midlog phase in terrific broth media and induced with 0.5 mM IPTG. The cells were lysed by sonication and the protein was isolated by immobilized metal ion chromatography using a Ni-NTA resin . The protein was further purified by gel filtration on a Sephacryl S100 column  and concentrated. The amino-terminal tag was removed by limited digestion with trypsin. Mass spectrometry analysis revealed that trypsin only cut AfJAMM in the amino-terminal tag region, and only a single band was evident on a Coomassie-stained polyacrylamide gel. The tag and uncut protein were removed with Ni-NTA resin followed by anion-exchange chromatography with SOURCE 30Q resin (Amersham Pharmacia Biotech). The processed protein was then concentrated to approximately 30 mg/ml by ultrafiltration. The selenomethionine protein was produced as described elsewhere (The gene for dus JAMM , open relsewhere  and puri4H2PO4, 200 mM sodium citrate (pH 5) using vapor diffusion with sitting drops and hanging drops. Crystals were incubated for approximately 1 min in a cryo-solution of equal volumes of reservoir solution and 35% meso-erythritol for the selenomethionine crystals and supplemented with 5 mM ZnCl2 for the native crystals. The crystals belonged to the space group P65, with cell dimensions of a = b = 76 Å, c = 94 Å and two subunits per asymmetric unit. Data for the selenomethionine crystals were collected on Beamline 9.2 at the Stanford Synchrotron Radiation Laboratory (SSRL)  and data for the native crystals were collected on Beamline 8.2.1 at the Advanced Light Source (ALS)   see .Phases were obtained by the MAD technique using data collected from selenomethionine-substituted crystals see . Three S2 (0.54πl). As a result, reflections with l-indices such that cos2 (0.54πl) < ½ are systematically weak, leading to an R-factor higher than would be expected for a nonpseudocentered crystal structure. However, when only the reflections with cos2 (0.54πl) > ½  are used for the R-factor calculation, reasonable values for R are obtained.Since two monomers in the unit cell are related by a fractional translation along c of approximately 0.54, the intensities of the diffraction pattern are modulated by a factor of cosS. pombe were performed as previously described by The geometry of the final model was analyzed with PROCHECK . The RamThe accession numbers for the proteins discussed in this paper are 20S proteasomes (PDB ID 1RYP), AfJAMM , AMSH (Entrez Protein ID NP_006454), AtCSN5/AJH1 (Entrez Protein ID NP_173705), AtRpn11 (Entrez Protein ID NP_197745), C6.1A (Entrez Protein ID NP_077308), CeCSN5 (Entrez Protein ID NP_500841), CeRpn11 (Entrez Protein ID NP_494712), Csn5 (Entrez Protein ID NP_593131), Cul1 (Entrez Protein ID NP_594259), cytidine deaminase (PDB ID 1JTK), DmCsn5/CH5 (Entrez Protein ID NP_477442), DmRpn11/p37b (Entrez Protein ID AAF08394), EcRadC (Entrez Protein ID NP_418095), HsAMSH (Entrez Protein ID NP_006454), HsC6.1A (Entrez Protein ID NP_077308), HsCsn5 (Entrez Protein ID NP_006828), HsRpn11/POH1 (Entrez Protein ID NP_005796), JAB1 (Entrez Protein ID AAC17179), lambdaK (Entrez Protein ID AAA96551), Mov34 (Entrez Protein ID NP_034947), Mpr1p (Entrez Protein ID AAN77865), Nedd8 (Swiss-Prot ID Q15843), neurolysin (PDB ID 1I1I), Pad1p (Entrez Protein ID NP_594014), phage λ tail assembly protein K (Entrez Protein ID AAA96551), RadC (Entrez Protein ID NP_418095), Rpn11 (Entrez Protein ID AAN77865), SCF (PDB ID 1LDK), ScNP (PDB ID 1C7K), ScRpn11 (Entrez Protein ID AAN77865), Sic1 (Entrez Protein ID 1360441), SpCsn5 (Entrez Protein ID NP_593131), SpRpn11/Pad1 (Entrez Protein ID NP_594014), thermolysin (PDB ID 1FJQ), ubiquitin (Swiss-Prot ID P04838), UBP (PDB ID 1NB8), and UCH (PDB ID 1UCH).http://www.ncbi.nlm.nih.gov/entrez/ (Entrez Protein), http://www.rcsb.org/pdb/ (Protein Data Bank [PDB]), and http://us.expasy.org/sprot/ (Swiss-Prot).These databases may be found at


We sought to determine how quickly carbon monoxide would accumulate in the passenger compartment of a snow-obstructed vehicle.A 1992 sedan was buried in snow to the level of the undercarriage, the ignition was then engaged and carbon monoxide levels recorded at 2.5-minute intervals. The primary outcome was the time at which a lethal carbon monoxide level was detected. Six trials were conducted: windows closed; windows open one inch; windows open 6 inches; windows closed and tailpipe swept clear of snow; windows closed and one cubic foot of snow removed around tailpipe; windows closed and tailpipe completely cleared of snow to ground level in a path 12 inches wide.Lethal levels of carbon monoxide occurred within 2.5 minutes in the vehicle when the windows were closed, within 5 minutes when the widows were opened one inch, and within 7.5 minutes when the widows were opened six inches. Dangerously high levels of carbon monoxide were detected within the vehicle when the tailpipe had been swept clear of snow and when a one cubic foot area had been cleared around the tailpipe. When the tailpipe was completely unobstructed the carbon monoxide level was zero.Lethal levels of carbon monoxide occurred within minutes in this snow-obstructed vehicle. Carbon monoxide poisoning is the number one cause of toxin related death in the United States . It has One of the manners in which people die from carbon monoxide poisoning is hypoxia secondary to entrapment within a snow-obstructed vehicle. Recommendations for prevention of these poisonings has been well documented but not thoroughly studied. The Centers for Disease Control (CDC) recommends that "following heavy snowfall, the public should be reminded to inspect vehicles to ensure that exhaust pipes are cleared of snow before engines are started" . Brian HIn reviewing case reports regarding snow emergencies, it appears that there have been situations where venting carbon monoxide through an open window may not have been sufficient to prevent dangerously high levels of carbon monoxide from accumulating within the passenger compartment . This raWe performed a pilot study with a single vehicle to simulate a snow emergency whereby a person stranded in a vehicle during a snowstorm would perform a safety maneuver. Our hypothesis was that opening a window a few inches for ventilation or clearing a tailpipe of snow would be sufficient to keep carbon monoxide levels in a non-lethal range.In 1995, a 1992 four-door sedan whose exhaust system had been inspected by the State of Massachusetts and found to be functioning normally was placed in a driveway and snow was shoveled around its base on all four sides until the car was obstructed by snow to the level of the bumpers. A Nighthawk, battery powered, digital readout, 80 mAmp carbon monoxide detector was attached to the front of the driver's seat headrest. The instrument had the ability to detect carbon monoxide levels in the range of zero to nine hundred ninety nine parts per million (0 – 999 ppm) [Six trials were performed. Between each trial all doors and windows were opened, the carbon monoxide was allowed to exhaust from the vehicle until the detector registered zero parts per million.Dangerously high carbon monoxide concentrations were recorded in the passenger compartment within three minutes when the windows were closed, within five minutes when the front windows were open one inch and within 7.5 minutes when the windows were opened six inches Table . With thCarbon monoxide is an odorless, tasteless by-product of fossil fuel combustion . When itIn this study, only opening a window was not enough to prevent accumulation of carbon monoxide within the passenger compartment of the vehicle. As the guidelines suggest -6, snow The authors declare that they have no competing interests.JG gathered data and prepared the manuscript. SP conceived of the project and gathered data. BO gathered data and presented the abstract at the Society for Academic Emergency Medicine. HS oversaw the project and provided statistical support.The pre-publication history for this paper can be accessed here:


Endometrial cancer represents a tumor entity with a great variation in its incidence throughout the world (range 1 to 25). This suggests enormous possibilities of cancer prevention due to the fact that the incidence is very much endocrine-related, chiefly with obesity, and thus most frequent in the developed world. As far as treatment is concerned, it is generally accepted that surgery represents the first choice of treatment. However, several recommendations seem reasonable especially with lymphadenectomy, even though they are not based on evidence. All high-risk cases are generally recommended for radiotherapy.A literature search of the Medline was carried out for all articles on endometrial carcinoma related to diagnosis and treatment. The articles were systematically reviewed and were categorized into incidence, etiology, precancerosis, early diagnosis, classification, staging, prevention, and treatment. The article is organized into several similar subheadings.In spite of the overall good prognosis during the early stages of the disease, the survival is poor in advanced stages or recurrences. Diagnostic measures are very well able to detect asymptomatic recurrences. These only seem justified if patients' chances are likely to improve, otherwise such measures increases costs as well as decrease the patients' quality of life. To date neither current nor improved concepts of endocrine treatment or chemotherapy have been able to substantially increase patients' chances of survival. Therefore, newer concepts into the use of antibodies e.g. trastuzumab in HER2-overexpressing tumors and the newer endocrine compounds will need to be investigated. Furthermore, it would seem highly desirable if future studies were to identify valid criteria for an individualized management, thereby maximizing the benefits and minimizing the risks. Nearly 170,000 new cases of endometrial carcinomas were estimated worldwide in 1997 .Endometrial carcinomas occur in advanced age . The age-related incidence for Germany is shown in Figure The etiology of the endometrial carcinoma is not fully understood. Most cases appear sporadically whereas about 10% are hereditary. Chief among the latter is the autosomal dominantly inherited hereditary non-polyposis colorectal cancer (HNPCC) -5. The rWithin the current concept of multi-step progression of normal cells to malignancy, recent molecular work has identified several gene alterations important for tumor development. In summary, mutations and amplifications of oncogenes K-ras and HER2/neu, mutations or deletions of tumor suppressor genes p53, p21, p16, and pTEN/MMAC1 as well as impaired DNA repair functions through mutations of hMLH1, hMSH2, and hMSH6 have been connected with the development of endometrial carcinomas .Environmental, dietary and hormonal factors as well as an aging female population have been attributed to an observed increase of endometrial carcinomas over the past few years. Epidemiologic studies have observed correlations between the incidence of endometrial cancer and the usage of estrogens, especially when applied to alleviate perimenopausal and postmenopausal symptoms. Therefore, it appears that estrogen plays a key role in the development and progression of endometrial carcinomas.There is also convincing evidence that high body mass increases the risk of endometrial carcinomas. Current estimations figure that about 40% of these relate to excess body weight . A plausPrevious irradiation of the pelvis, estrogen replacement therapy (HRT)  and tamoxifen therapy represent exogenous risk factors. Prolonged and exclusive intake of synthetic estrogens is associated with an up to 15-fold higher incidence of endometrial carcinomas ,17. TamoRecently a case-control study identified antipsychotic drugs as being a great risk factor for endometrial cancer . These fIn some cases endometrial cancer develops from atypical endometrial hyperplasia. The likelihood of this happening correlates with the degree of hyperplasia Table  20]. Ac. Ac20]. Endometrial hyperplasia is regarded as a preliminary stage of endometrioid carcinomas. Serous and clear cell carcinomas are on the other hand frequently associated with an atrophic endometrium . A precuTo prevent an outbreak of the disease in menopausal and postmenopausal women long-term estrogen replacement therapy  should be supplemented by intermittent application of gestagens. A recent study suggested the use of intrauterine devices (IUD) also possibly reducing the risk for endometrial cancer due to improved elimination or decrease of hyperplastic endometrial cells . Numerou® intrauterine system maintains that irregular menstrual bleeding should not be treated simply with this system without prior diagnostic [To date there is no procedure that seems appropriate as a screening method for early detection of endometrial carcinomas. Current guidelines of the American Cancer Society suggest informing patients of risks and symptoms involved with endometrial cancer and furthermore firmly emphasize the importance of reporting unexpected bleeding or spots to their physician . The facagnostic .The necessity of finding a screening method is discussed controversially in this context. So far, most epidemiological studies have failed to show significant effects of screening on mortality.In some cases the Pap smear may lead to the diagnosis of endometrial cancer. However, in many cases cells from inside the uterus are not assessed by the sampling procedure. Positive cervical cytology was found to correlate with nodal spread in 91% of cases, whereas the risk of lymph node spread in patients with normal cervical cytology was estimated at around 2% . It woulTransvaginal ultrasound has also been suggested as a potential means of early detection of endometrial carcinomas. A recent meta-analysis involving 9,031 patients and covering 57 separate studies on the diagnostic accuracy and positive predictive power of endometrial thickness measurement by pelvic ultrasound in patients with postmenopausal bleeding concluded that these measurements cannot solely be used to accurately rule out endometrial pathology. Measurement of both endometrial layers of ≤ 5 mm coincides with endometrial pathology in only 2.5% . Other sIt seems that biopsy remains the only accurate way of diagnosing endometrial cancer . Under oNewer methods, like magnetic resonance imaging (MRI), positron emission tomography (PET), intraoperative ultrasound or three dimensional sonography are not likely to gain importance with respect to the diagnosis or early diagnosis of the disease -56. It iThe term endometrial carcinoma describes a variety of different tumor types originating from the inner ling of the uterus. Many authors differentiate between two basic types, which may be divided into estrogen-dependent and estrogen-independent types or tumors with favorable or unfavorable prognosis . Althoug1. It comprises of estrogen related tumors occurring in younger, perimenopausal women. These tumors are often said to be highly differentiated, mainly adenocarcinomas with positive steroid hormone receptor status (ER, PR), the known risk factors (estrogen etc.) originating from atypical endometrial hyperplasia. The patients in this group have a longer history, lower grade tumors, less myometrial invasion and low potential for lymphatic spread. They may be associated with concomitant carcinomas of the ovary, breast and colon and respond to progestin therapy. The overall prognosis is generally favorable.2. This category is comprised of tumors of patients with shorter history, higher grade tumors, deeper myometrial invasion and high risk of lymphatic spread. The tumors will not respond to progestin therapy and there are no associated tumors. Histologically these tumors are identified as serous carcinomas or clear cell adenocarcinomas. The prognosis of patients with this type of tumor is poor.3. Findings of carcinomas in atrophic endometrium being associated with an intermediate prognosis lead to the suggestion of a third category. In these cases the endometrioid carcinomas are not likely to be estrogen-related .4. It was furthermore suggested to incorporate endometrial neoplasia originating from an inherited predisposition into a fourth category. These types of tumors tend to develop about 15 years earlier and are associated with a favorable prognosis. Most patients in this category have a hereditary non-polyposis colorectal carcinoma syndrome (HNPCC).The following section will briefly describe the most important characteristics of endometrial carcinomas. Detailed descriptions may be found in specialized pathological textbooks e.g. Anderson et al. .Endometrioid adenocarcinoma: The endometrioid adenocarcinoma, whose glands resemble those of the normal endometrium, is the most common type (60 – 80%). This type is considered part of the 1st category• Endometrioid adenocarcinoma with squamous cell differentiation (adenoacanthoma, adenosquamous carcinoma): Approximately 25% of endometrioid adenocarcinomas display partial squamous differentiation. The squamous elements are interpreted as terminally differentiated indicating that the tumor is incapable of independent growth. Prognosis depends on the glandular components of the lesion. Highly differentiated tumors (adenoacanthomata) have a favorable prognosis, whereas poorly differentiated tumors (adenosquamous carcinomas) have an unfavorable course.• Serous adenocarcinoma: This tumor type is similar to the serous ovarian carcinoma. It is characterized by aggressive growth and poor prognosis. Lymphogenic and hematogenic metastases are usually already present at the time of diagnosis. Nearly all tumors are poorly differentiated. Serous adenocarcinomas belong to the 2nd category of endometrial cancer.• Clear cell adenocarcinoma: This type comprises 3 to 6% of all endometrial carcinomas. Like the serous adenocarcinoma, clear cell adenocarcinomas tend to progress rapidly. They share the 2nd category.• Mucinous adenocarcinoma: Diagnosis is based on the presence of mucus within the tumor cells. Purely mucinous carcinomas are rare, although a mucinous component within endometrioid carcinomas is more common. The tumors are usually highly differentiated and have a good prognosis. It is important to exclude a primary mucinous adenocarcinoma originating from the endocervix, spreading into the uterine body. Mucinous adenocarcinomas are part of the 1st category.• Squamous cell carcinoma (2nd category): A very rare entity associated with very poor survival. Primary squamous cell carcinomas of the uterine cervix should be ruled out. Adjuvant platinum-based radiochemotherapy may result in improved survival.• Mixed carcinoma: A carcinoma composed of two or more different non-squamocellular components with each component occupying at least 10% of the tumor. Prognosis varies, e.g. in case of a serous component, the prognosis is poor.• Undifferentiated carcinoma: A rare carcinoma without glandular, squamous or sarcomatous differentiation. Prognosis is unfavorable.• • Rare types of endometrial carcinomas: small cell carcinomas, microglandular adenocarcinomas, signet-ring cell carcinomas, transitional cell carcinomas, glassy cell carcinomas, mucinous adenocarcinomas of the intestinal type, lymphoepithelioma-like carcinomas, and endometrial adenocarcinomas with trophoblastic differentiation have been reported.The tumor grading is a highly significant prognostic parameter although it is subjective with a considerable inter-observer and intra-observer variability. It is determined by the percentage of non-squamocellular, solid portions as follows: G1: 5% and less, G2: 5–50%, G3: more than 50%. A significant nuclear atypia increases the grade of differentiation by one grade.After histological confirmation of an endometrial carcinoma clinical palpation and vaginal sonography should be performed. Several additional examinations have been suggested: Rectoscopy, cystoscopy, computed tomography and/or MRI. They may be omitted in clearly diagnosed early cases but are strongly recommended in advanced stages. Recent studies indicate some value of tumor marker diagnostics. At a cut-off level of 40 U/ml elevated CA125 serum levels indicate nodal metastases with a sensitivity of 77.8% and a specificity of 81.1% ,61.In all stages of endometrial carcinomas, surgery is the primary treatment of choice. Preoperative intracavitary radiation treatment, often recommended in earlier times, is not considered advisable any more since the information about the depth of myometrial invasion and thus information on an important prognostic factors is lost . A studyThere is some controversy on the value of radical hysterectomy in stage II carcinomas. While several earlier studies advocated this procedure -70 a recComplications after pelvic lymphadenectomy may be reduced by omentoplasty and omentopexy . In advaVaginal hysterectomy as primary treatment of endometrial cancer has also been investigated especially in medically compromised women ,73. SuchThe aforementioned basic goals of surgery  should be reached especially in medically fit patients, because treatment along the recognized guidelines has been found to be prognostically favorable. Some studies, however, have reported the standards mentioned in many cases as not having been realized -80. As sThe problem mentioned may partly be due to the fact that the current guidelines are insufficiently supported by randomized surgical trials. Interestingly the COSA-NZ-UK Endometrial Cancer Study Group trial showed that lymphadenectomy showed no advantage for endometrial cancer if primary surgery was followed by adjuvant radiotherapy . TherefoEndometrial carcinomas are staged surgically. Procedures previously used for determination of stages, such as fractional dilatation and curettage to differentiate between stage I and II, are no longer applicable unless the patient is to be treated by primary radiation therapy. The old (1971) and new (1988) staging system of the International Federation of Gynecology and Obstetrics (FIGO) are shown in table Tumor stage, patient age, histologic type and grade, hormone receptors and DNA ploidy represent the traditional prognostic factors. Respective of the response to progestin therapy steroid hormone receptors may also be regarded a predictive factors in recurrent and advanced disease . The strThere is a divergence of opinion concerning the value of a positive peritoneal cytology as an independent prognostic factor . In stagIn medically compromised women, primary irradiation may be suitable. Analyses from Germany show that approximately 20% of all patients undergo primary radiotherapy ,64. TherOnly few clinical trials on primary radiotherapy have been performed. The results suggest that dividing doses into smaller fractions allows better tumor control and has less side effects . The oldMost studies indicate that advance age, grade 3 histology or deep myometrial invasion relate to a higher risk of disease recurrence. Therefore adjuvant radiotherapy seems important in this subset. External beam radiation should also be considered in cases of multiple infiltrated lymph nodes (> 5). But many questions remain unanswered and the value of postoperative irradiation is still under debate. Survival rates of patients with early stage disease are excellent, no matter whether they underwent extended-surgical staging with more restricted use of the adjuvant therapy or simple hysterectomy bilateral salpingoophorectomy with more frequent use of adjuvant radiotherapy . ProspecThe American Brachytherapy Society has now issued recommendations for brachytherapy for carcinomas of the endometrium . AccordiThe data on adjuvant medical treatment is not conclusive. Most studies have their limitations and therefore there is still no final answer to the question, who should receive what type of adjuvant treatment. Merely in cases of uterine papillary serous carcinomas, which affect 1% to 10% of patients, there is consensus that patients should receive chemotherapy (with or without adjuvant radiotherapy) with a platinum-based regimen, combined with doxorubicin and cyclophosphamide. Newer regimens consider paclitaxel, with or without platinum .A multicenter, open, controlled, prospectively randomized trial on adjuvant endocrine treatment with medroxyprogesterone acetate (MPA) or tamoxifen in stage I and II endometrial carcinomas failed to detect differences in the disease-free and overall survival rates for a tamoxifen group compared with a control or a MPA group . HoweverThe generally good prognosis of endometrial carcinomas does not justify a general recommendation of chemotherapy, especially in the early stages. Even so, patients at high risk  seem to profit from adjuvant chemotherapy ,103. StuThe development of endometrial cancer in young patients is usually related to unopposed estrogen stimulation. In patients with continuing a desire to have children, approaches have been made to preserve fertility. Primary hormonal therapy with progestins was suggested as an alternative treatment for surgery hereby offering the preservation of fertility. These must be interpreted with caution because of low case numbers and a publication bias. Treatment with megestrol acetate at 160 mg/day for 3 months or medroxyprogesterone acetate (MPA) at 200–800 mg/day for 2–14 months resulted in disease regression in 60 to 75%, however, the percentage of patients who actually delivered healthy children was much lower, ranging from 20 to 25% -107. FurIn essence, the problems regarding the optimal selection of patients for conservative progestin therapy are unsolved. Only cases with good prognostic factors are to be selected: Well differentiated tumors and tumors with known favorable prognoses (e. g. endometrioid type tumors) with no or minimal myometrial invasion and early stage disease. The greatest problem lies in the difficulty of appropriate staging, resulting in a potential underestimation of the problem. Maybe positron emission tomography, magnetic resonance imaging or a combination of both may be useful in this respect in due course.Patients have to be carefully informed that this fertility preserving concept is still experimental. Moreover, these women must realize the low overall pregnancy rate which may partially be related to the origin of the disease (obesity, irregular menses, polycystic ovarian disease (PCOD) with chronic anovulation and infertility) . NowadayGenerally the prognosis for patients with recurrent disease is poor, therefore a thorough staging procedure should be performed. Analyses from other tumor entities, e.g. ovarian carcinomas, have demonstrated that a second surgical intervention may be useful in improving patients' overall survival ,110. ThuSome studies also suggest a potential benefit for luteinizing hormone-releasing hormone analogues (GnRH), although studies in endometrial carcinomas have not shown any convincing activity -118. To With respect to chemotherapy, several cyctostatic compounds have demonstrated efficiency. Among these are paclitaxel, carboplatinum, doxorubicin, cisplatin, etoposide, and 5 fluorouracil. Topotecan showed only limited activity -124. AllGenerally recommendation for clinical follow-up of patients advocate patients to be monitored at three-monthly intervals during the first 3 years, at 6-monthly intervals up to the 5th year and at yearly intervals thereafter. Apart from provision of general information on all aspects of the disease and its treatment including unconventional cancer therapies, the documentation of patients' history, a clinical gynecological examination including a pap smear, a vaginal sonography and even the determination of tumor markers (SCC, CA125) have been recommended. Up to 95% of all recurrences may be detected early this way . Again tHormone replacement therapy (HRT) with estrogen with or without progestins is frequently used to alleviate menopausal symptoms but also to reduce the risk of osteoporosis and cognitive dysfunctions. In endometrial carcinomas HRT may be believed to be critical due to fear of initiating growth of occult residual tumor cells, resulting in disease recurrence and shortened survival. As summarized by the American College of Obstetricians and Gynecologists (ACOG) there is not enough data to draw any final conclusion. Any decision on the subject should thus be individualized based on potential benefit and risk to the patient . Some st®) could perhaps improve the outcome in HER-2/neu overexpressing tumors [Endometrial carcinomas represent a very frequent tumor entity in industrialized countries. It is hard to believe how little evidence-based data exists on even the important aspects of the disease. This may be partially due to the overall good prognosis even if surgery is reduced to hysterectomy and adnectomy. However, the high incidence in the developed world and consequently many women suffering relapses, necessitates the research of new approaches for cancer recurrence. Recent research suggests that therapy with trastuzumab  have a substantially better overall and recurrence free survival Figure  139]. D. D139].