Absolute depth of myometrial invasion in endometrial cancer is superior to the currently used cut-off value of 50%.

OBJECTIVE: In endometrial carcinoma, myometrial invasion is a well known predictor of recurrence, and important in the decision making for adjuvant treatment. According to the FIGO staging system, myometrial invasion is expressed as invasion of <50%> of the myometrium (50%MI). It has been suggested to use the absolute depth of invasion (DOI), or the tumor free distance to the serosa (TFD). The aim of this study was to compare DOI, 50%MI, and TFD. METHODS: All patients diagnosed with endometrioid endometrial carcinoma at the RUNMC, and the CWH from 1999 to 2009 were included. Histologic slides were reviewed for histologic type and grade, DOI, 50%MI, and TFD. After review, 335 patients were identified. DOI, 50%MI, and TFD were evaluated for their prediction of clinicopathologic characteristics. RESULTS: The prediction of recurrence was best performed by DOI when compared to TFD, with an area under the ROC curve of 0.726, and 0.638 respectively. The optimal cut-off value for DOI was 4mm. DOI independently correlated with recurrence of disease, and death of disease. TFD was associated with advanced age and large tumor diameter. DOI was the best predictor of progression-free and disease-specific survival next to 50%MI and TFD (HR 3.15, 95%CI 1.16-8.56) and (HR 10.35, 95%CI 1.23-86.93). CONCLUSIONS: DOI showed better predictive performance than TFD, and was more strongly correlated with clinicopathologic parameters than TFD and 50%MI. Possibly, DOI should substitute 50%MI as measure to express myometrial invasion in daily clinical practice. External validation is mandatory to confirm the proposed cut-off value of 4mm.

Does the method of dissecting in anterior colporraphy lead to a difference in thickness of removed vaginal tissue?

OBJECTIVE: To evaluate the difference in thickness of the anterior vaginal wall removed after different surgical dissecting techniques of anterior colporrhaphy. STUDY DESIGN: In patients undergoing primary anterior colporrhaphy, trimmed vaginal tissue was taken following different surgical techniques of vaginal wall dissection. Tissues were preserved in formalin and stained with hematoxylin-eosin and elastica-van Giesen stains. The examiner was an experienced pathologist blinded to the surgical technique. The specimens were examined for the epithelial thickness (ET), lamina propria thickness (LPT), muscular layer thickness (MT) and total thickness (TT). RESULTS: Tissue was analysed in 93 women who underwent anterior compartment pelvic organ prolapse surgery. There was no difference between the different surgical techniques in thickness measured in the three histological layers and for the total thickness. The use of hydrodissection was the only independent factor leading to thicker removed vaginal tissue. CONCLUSIONS: Dissecting the vaginal wall as thin as possible does not result in a thinner vaginal layer than dissecting in the most optimal surgical plane. The use of hydrodissection provides a thicker trimmed tissue.

Evidence for multiple distinct major histocompatibility complex class I lineages in teleostean fish.

In the context of studies on the expression of MhcCyca-Z sequences of the common carp, PCR amplifications of exon 4 were performed on cDNA obtained from pooled thymi of 20 carp F1 individuals. Five recombinant clones (Cyca-TC3, -TC13, -TC15, -TC17 and -TC18) were found to be 96% similar to the exon 4 region of Cyca-ZA1. Each of the five sequences was unique, and differed in a few positions in both the nucleotide and the derived amino acid sequences from any of the Cyca-Z sequences known to date. These data suggest that multiple Z genes per locus are present in the carp, which are transcribed in the thymus. In the course of analysing the amplified Cyca-Z sequences, serendipity yielded a clone, Cyca-TC16, containing a class I-like sequence substantially different from any other carp class I sequence. The predicted amino acid sequence of Cyca-TC16 was most similar to the class I genes (Lach-U) from the coelacanth (42-46% amino acid identity). Cyca-TC16 contains three conserved beta 2-microglobulin contact residues, and the secondary structure was predicted by computer algorithms to be similar to that of the alpha 3 domain of HLA-A2. Phylogenetic analysis shows that carp class I sequences reside in four distinct clusters: (i) Cyca-Z, Cyca-TC3, -TC13, -TC15, -TC17 and -TC18 together with Caau-Z from ginbuna crucian carp, (ii) Cyca-U with Bree-U (zebrafish) and Sasa-p30 (Atlantic salmon), (iii) Cyca-TC16 with Lach-U (coelacanth), and (iv) Cyca-C4.

Identification and characterization of a new major histocompatibility complex class I gene in carp (Cyprinus carpio L.).

In this study we report the finding of three representatives of a new group of major histocompatibility complex class I sequences in carp: Cyca-12 (Cyca-UA1*01), a full-length cDNA; Cyca-SP1 (Cyca-UAW1), a polymerase chain reaction (PCR) fragment from cDNA; and Cyca-G11 (Cyca-UA1*02), a partial genomic clone. Comparison of the amino acid sequences of Cyca-12, Cyca-SP1, and Cyca-G11 with classical and non-classical class I sequences from other species shows considerable conservation in regions that have been shown to be involved in maintaining the structure and function of class I molecules. The genomic organization of Cyca-12 has been elucidated by analysis of a partial genomic clone (Cyca-G11, in combination with PCR amplifications on genomic DNA of a homozygous individual. Although the genomic organization is similar to that found in class I genes from other species, the 3' untranslated region contains an intron which is unprecedented in class I genes, and intron 2 is exceptionally large (+/-14 kilobases). Southern blot analysis indicates the presence of multiple related sequences. In phylogenetic analyses, the Cyca-UA sequences cluster with class I genes from zebrafish and Atlantic salmon, indicating that the ancestral gene arose before the salmonid/cyprinid split, approximately 120-150 million years ago. The previously reported class I Cyca-Z genes from carp and Caau-Z genes from goldfish cluster as a completely separate lineage. A polyclonal antiserum (anti-Cyca12) was raised against a recombinant fusion protein containing most of the extracellular domains of Cyca-12. The antibodies showed substantial reactivity to the recombinant protein and an Mr 45000 protein in membrane lysates of spleen and muscle, as well as to determinants present on leucocytes in fluorescence-activated cell sorter analyses. Erythrocytes and thrombocytes were found to be negative.

Characterization of class II A and B genes in a gynogenetic carp clone.

A prerequisite for carrying out functional studies on major histocompatibility complex (Mhc) molecules of fish is the availability of genetically well-defined homozygous strains. Previously we have applied gynogenetic reproduction to generate isogenic carp, denoted clone A410. This clone has recently been demonstrated to express a single class I gene, Cyca-UA1(*)01, and in the present study two class II B and two class II A transcripts were obtained. The two class II B transcripts, Cyca-D(CB3)B and Cyca-D(CB4)B, as well as the class II A transcripts, Cyca-D(10A)A and Cyca-D(15A)A, appear to be bona fide class II transcripts, based on the presence of conserved protein characteristics of the inferred class II molecules. With the isolation of class II A sequences, representatives of all major classes of Mhc genes have been identified in the carp. To assess the relationship between the different class II genes, segregation studies, comparison of cDNA and intron 1 sequence data, and phylogenetic analyses were undertaken. These showed that the class II B transcripts, Cyca-D(CB3)B and Cyca-D(CB4)B, are derived from related, closely linked loci. In addition, these studies indicated that the previously described Cyca-DAB*01 and Cyca-DAB*02 are also closely linked, but that this linked pair segregates independently from the Cyca-D(CB3)B and Cyca-D(CB4)B loci. The class II A transcripts are most likely derived from separate loci and do not represent alleles, as they were found not to segregate in the individuals of the clone which was generated by meiogenetic gynogenesis.

Fish major histocompatibility complex genes: an expansion.

The advent of polymerase chain reaction technology has provoked a large amount of progress in the field of fish major histocompatibility complex (MHC) research. Many new teleost sequences have been reported in the last four years, including representatives of all classes of MHC genes. While the intron-exon structure of teleost MHC genes is now becoming clear, the organisation of the genes within the teleost MHC is still unclear. The sequences reported to date have been used for phylogenetic analysis and, due to their evolutionary position, are discussed in relation to hypotheses regarding the origin of the MHC. Teleost MHC gene sequences are also examined to see if conserved features of the both the nucleotide and amino acid sequences of higher vertebrate MHC genes are present. Differences in these features will reflect functional differences between teleost and mammalian MHC genes and may also have evolutionary implications.

Polymorphism and estimation of the number of MhcCyca class I and class II genes in laboratory strains of the common carp (Cyprinus carpio L.).

Restriction fragment length polymorphisms (RFLPs) have been identified in the Mhc of the carp (MhcCyca) using class I (Cyca-Z) and class II (Cyca-YB) specific probes. The K1-5 and K2-1 probes were obtained as PCR products after amplification of genomic DNA from a European carp using primers deduced from genomic sequences, and were shown to be 90% and 80% similar to Cyca-Z exon 3 and Cyca-YB exon 2 sequences, respectively. Six carp strains of different geographical origins and genomic status were studied. In homozygous gynogenetic carp strains the class I probe K1-5 hybridized to 9-12 fragments, whereas the class II probe K2-1 hybridized to 3-5 fragments. Thus, the Cyca consists of multiple class I and class II genes. The level of polymorphism of the Cyca genes of the strains studied was calculated as the percentage of polymorphic fragments among the total number of fragments observed, and was shown to be 70% for class I and 40-66% for class II genes. In addition, a possible correlation was investigated between a serologically defined locus K, which was demonstrated previously to incorporate class I-like characteristics, and molecular genotyping using the class I probe. Two gynogenetic families, which were serologically typed K1 and K2 homozygous, also differed in their RFLPs using a class I probe. This would suggest that the K locus is part of the Cyca complex.

Characterization of beta 2-microglobulin transcripts from two teleost species.

Using degenerate primers based on published beta 2-microglobulin sequences we were able to obtain an expected 111 base pairs (bp) polymerase chain reaction (PCR) fragment from tilapia genomic DNA. The sequence of this fragment showed a high degree of similarity to mouse beta 2-microglobulin at the protein level. We used these primers in an "anchored PCR" to obtain a 213 bp PCR fragment from a carp cDNA library. This was then used to clone a full-length beta 2-microglobulin cDNA from carp. The carp sequence showed the highest similarity to rabbit beta 2-microglobulin. Both sequences showed strong similarities to all previously published vertebrate beta 2-microglobulin sequences. The predicted protein secondary structure of both the carp and tilapia clones was almost identical to the corresponding regions of previously known vertebrate beta 2-microglobulin protein sequences. When either the carp or tilapia probes were used against corresponding northern blots, they hybridized to a message of approximately 800-1000 bases long, which corresponds to the previously published lengths of beta 2-microglobulin mRNAs. Southern blotting indicated that beta 2-microglobulin was encoded by a single copy gene in both cases. Phylogenetic analysis indicated that the sequences were related to the beta 2-microglobulins of higher vertebrates but grouped together in an ancestral position.

The effect of water content and nature of organic solvent on enzyme activity in low-water media. A quantitative description.

A simple theoretical model was suggested to describe quantitatively the effect of water content and nature of organic solvents on catalytic behavior of enzymes suspended in low-water media. The model was based on a generally accepted notion that the destruction of the protein hydration shell is one of the main reasons for protein denaturation by organic solvents. The validity of the model was confirmed by the example of catalytic behavior of immobilized laccase suspended in water/organic mixtures of different compositions. In addition, the results were used to demonstrate that the effect of organic solvents and/or water content on catalytic behavior of enzymes in low-water media can be adequately assessed only in terms of the full kinetic description based on properly determined Vm and Km values.