The LIM homeobox gene Lhx9 is essential for mouse gonad formation.

During mammalian embryonic development, the ovaries and testes develop from somatic cells of the urogenital ridges as indifferent gonads, harbouring primordial germ cells that have migrated there. After sex determination of the gonads, the testes produce testosterone and anti-Mullerian hormone which mediate male sexual differentiation, and the female developmental pathway ensues in their absence. Here we show that transcripts of the LIM homeobox gene Lhx9 are present in urogenital ridges of mice at embryonic day 9.5; later they localize to the interstitial region as morphological differentiation occurs. In mice lacking Lhx9 function, germ cells migrate normally, but somatic cells of the genital ridge fail to proliferate and a discrete gonad fails to form. In the absence of testosterone and anti-Mullerian hormone, genetically male mice are phenotypically female. The expression of steroidogenic factor 1 (Sf1), a nuclear receptor essential for gonadogenesis, is reduced to minimal levels in the Lhx9-deficient genital ridge, indicating that Lhx9 may lie upstream of Sf1 in a developmental cascade. Unlike mice lacking other genes that mediate early stages of gonadogenesis, Lhx9 mutants do not exhibit additional major developmental defects. Thus, LHX9 mutations may underlie certain forms of isolated gonadal agenesis in humans.

Diazepam binding inhibitor (DBI) in the plasma of pediatric and adult epileptic patients.

The polypeptide diazepam binding inhibitor (DBI) displays epileptogenic activity by binding to benzodiazepine receptors. We analyzed DBI concentrations in the plasma of pediatric and adult epileptic patients, as a possible peripheral marker in epilepsy. DBI plasma concentrations are significantly higher (+ 62%, P < 0.001) in adult patients and slightly but significantly higher (+15%, P < 0.01) in pediatric patients, compared to age-related controls. Strikingly, plasma DBI is much higher (+81%, P < 0.001) in generalized epilepsy in adults and in drug-resistant pediatric and adult patients. Based on these findings, plasma DBI may be considered as a peripheral biological marker of epilepsy and, in association with lymphocyte benzodiazepine receptor density, of anticonvulsant drug responsiveness.

Decreased density of lymphocyte benzodiazepine receptors in drug-resistant epileptic patients.

Peripheral benzodiazepine receptors (PBR) may have a role in epilepsy and in mediating antiepileptic drug effects. Since PBR in blood mononuclear cells may be acted on by anticonvulsant drugs, we investigated possible modifications of these receptors in newly diagnosed patients, before and after antiepileptic treatment and in drug-resistant epileptic patients. A significantly lower receptor density, with no difference in affinity, was observed in drug-resistant patients, compared to newly diagnosed patients and to normal age-related controls. We suggest the possible use of PBR as a peripheral marker of drug response.

Identification of in vivo target RNA sequences bound by thymidylate synthase.

We developed an immunoprecipitation-RNA-random PCR (rPCR) method to isolate cellular RNA sequences that bind to the folate-dependent enzyme thymidylate synthase (TS). Using this approach, nine different cellular RNAs that formed a ribonucleoprotein (RNP) complex with thymidylate synthase (TS) in human colon cancer cells were identified. RNA binding experiments revealed that seven of these RNAs bound TS with relatively high affinity (IC50 values ranging from 1.5 to 6 nM). One of the RNAs was shown to encode the interferon (IFN)-induced 15 kDa protein. Western immunoblot analyses demonstrated that the level of IFN-induced 15 kDa protein was significantly decreased in human colon cancer H630-R10 cells compared with parent H630 cells. While the level of IFN-induced 15 kDa mRNA expression was the same in parent and TS-overexpressing cell lines, the level of IFN-induced 15 kDa RNA bound to TS in the form of a RNP complex was markedly higher in H630-R10 cells relative to parent H630 cells. These studies begin to define a number of cellular target RNA sequences with which TS interacts and suggest that these TS protein-cellular RNA interactions may have a biological role.

Basic mutant Max reverses a c-Myc block to differentiation.

Murine erythroleukemia (MEL) cells overexpressing a transfected c-myc gene are blocked in their ability to undergo inducer-mediated differentiation, whereas overexpression of a transfected max gene mutated within the basic region (bm-max) accelerates differentiation. Based on these findings, we cotransfected MEL cells with plasmids which express human c-Myc constitutively and bm-Max in a zinc-inducible manner. Competition of endogenous proteins for binding to bm-Max can be considered negligible in cells expressing such high constitutive levels of c-Myc. Thus, this system provides a cell culture model for studying Myc:Max complex formation and its effect on erythroid differentiation. Clones expressing high levels of c-Myc and low levels of bm-Max are blocked in their ability to undergo N,N'-hexamethylene bisacetamide (HMBA)-mediated differentiation, presumably due to a preponderance of growth-promoting Myc:Max complexes. However, increased expression of bm-Max, in these clones, allows differentiation to occur by decreasing the levels of functional Myc:Max complexes. Although the exogenously expressed c-Myc and bm-Max associate in vivo, the basic region mutation in bm-Max abolishes the binding of Myc:bm-Max complexes to the specific E-box consensus sequence. We demonstrate that this sequestering of c-Myc by bm-Max reverses the c-Myc block to differentiation.

Regulation of murine Max (Myn) parallels the regulation of c-Myc in differentiating murine erythroleukemia cells.

Max is a basic region-helix-loop-helix-leucine zipper protein that consists of two major isoforms, p22 (long form, Max-L) and p21 (short form, Max-S). These proteins are encoded by two [the 1.9- and the predominant 2.3-kilobase (kb) forms] of the five alternatively spliced max mRNA species. We now demonstrate that N,N'-hexamethylene bisacetamide-mediated differentiation of murine erythroleukemia cells leads to a pattern of biphasic down-regulation of the 1.9- and the 2.3-kb myn (murine max) mRNAs that closely parallels that which occurs for myc mRNA. In contrast, the p22/Myn-L and p21/Myn-S protein isoforms down-regulate in monophasic fashion. Unlike the short-lived myc mRNA, the myn message is quite stable. However, its half-life of 3-6 h is still consistent with the biphasic down-regulation that accompanies differentiation. Furthermore, unlike myc, the overexpression of which prevents differentiation, elevated max levels merely delay differentiation. Coincident with this is a delay in the second decline of c-myc mRNA. In N,N'-hexamethylene bisacetamide-induced cells blocked from differentiating by overexpression of c-, N- or L-myc, myn mRNA expression is constitutive. These findings suggest that myn may also be involved in differentiation.

Differential-effects of max proteins (long and short) on growth and differentiation of murine erythroleukemia-cells.

The myc gene product belongs to a family of basic, Helix-Loop-Helix, Leucine Zipper (b-HLH-LZ) proteins involved in cell growth, differentiation and tumorigenesis. The Max protein dimerizes with Myc to form a complex which binds to DNA and promotes transcription of target genes. Max exists in two major forms, Max-9 (long) and Max (short), which differ by 9 amino acids just amino to the basic region. We compared the in vivo behavior of the two forms in stable transfectants of long and short wild type (Lwt, Swt) and basic region mutants (Lbm, Sbm). While both Lwt-max and Swt-max clones exhibit delays in cell growth and differentiation, these delays are far more pronounced in the Lwt-max clones. In contrast, no difference is noted between Lbm-Max and Sbm-Max in their observed ability to delay growth, to accelerate HMBA-induced differentiation and to induce spontaneous differentiation. We suggest that a difference in affinity for DNA underlies the differential activities of Lwt- and Swt-Max.

Transfected wild-type and mutant max regulate cell growth and differentiation of murine erythroleukemia cells.

Max protein forms specific DNA-binding dimeric complexes with itself and with proteins of the c-myc gene family. A large volume of data has accumulated on the role of the c-myc proto-oncogene in cell proliferation, differentiation and tumorigenesis. To elucidate the role of max in regulating c-myc functions and the effect of both proteins on cell proliferation and differentiation, we transfected murine erythroleukemia (MEL) cells with a full-length wild-type (wt) human max gene and a mutant containing a double point mutation in the basic region (bm), which abolishes specific DNA binding. All clones expressing wt-max grow slowly, and the process of inducer-mediated differentiation is delayed. Furthermore, cells transfected with the mutated max exhibit growth retardation, accumulation in the G0/G1 phase of the cell cycle and spontaneous differentiation. Our findings are consistent with a model in which a large excess of wt-Max in the cells enhances the formation of Max-Max growth-suppressor complexes, while elevated bm-Max deprives the cell of growth-promoting Myc-Max heterodimers in a dominant-negative manner, presumably by inactivating endogenous Myc and Max.

Correlation between cytogenetic data and ganglioside pattern in human meningiomas.

Partial or total loss of chromosome 22 is often associated with tumors of the central nervous system and in particular with meningiomas. As in the case of other tumors, the ganglioside pattern is modified in transformed tissues. Cytogenetic analysis of 30 human meningiomas has been performed and the results compared to biochemical analysis of ganglioside distribution on the membrane surface. The meningiomas were divided into 2 groups on the basis of the presence or absence of chromosome 22. Thirteen tumors exhibited partial or total monosomy of the chromosome, whereas 17 were normal or showed other chromosomal anomalies. The GM3 and GD3 content of the meningiomas belonging to the 2 groups revealed a significant correlation between amount and reciprocal ratio of these 2 gangliosides and cytogenetic data. Tumors with monosomy 22 had a higher content of ganglioside GD3 than samples without monosomy 22, where the main ganglioside was GM3. Other gangliosides such as GM1, GD1a, GD1b and GT were present in various amounts in the 2 groups. Considering the biosynthetic pathway of gangliosides, we hypothesize the involvement of a gene located on chromosome 22 in the regulation of the enzymes which catalyze either GD3 synthesis (sialyltransferase 2, SAT-2) or its degradation to GM3 (neuraminidase).

Preparation and biological characterization of conjugates consisting of ricin and a tumor-specific non-internalizing MAb.

MOv18, a non-internalizing monoclonal antibody (MAb) with restricted tumor specificity, was conjugated to ricin toxin (RT). According to their ability to bind to galactose residues of Sepharose 6B, the immunoconjugates were fractionated into Bound and Unbound MOv18-RT. The two conjugates could be distinguished by SDS-PAGE, in vivo toxicity and agglutination capability. When the binding activity of both fractions was compared by solid-phase RIA to that of native MAb, it proved to be similar on the relevant target cells but significantly increased on the non relevant cells. On the latter, galactose totally cancelled the binding of the Unbound immunoconjugate, whereas it could only partially reverse that of the Bound MOv18-RT. By in vitro cytotoxic activity, either in the presence or absence of galactose, only a slight selectivity for relevant versus non-relevant target cells was observed for both conjugates. It seems that in the presence of a MAb which is incapable of internalization, the conjugate cytotoxicity could only be attributed to RT, with a loss of the MAb's specificity.

Fractionation of the ribosome inactivating protein preparations with triazine dyes.

Aspergillins are ribosome-inactivating proteins (RIPs), isolated from several strains of Aspergillus. The interaction between Cibacron Blue F3GA and two members of this family, alpha sarcin and mitogillin, and other RIPs of type I, was studied. Alpha sarcin retention depended on pH and ionic strength. By chromatography on Affi-Gel Blue in mild experimental conditions, mitogillin and PAP-I did not interact with the dye, whereas 40% of alpha sarcin and 70-90% of briodin, RTA and gelonin were recovered in the bound fraction. In all cases, the major fraction showed a higher toxicity level in protein synthesis inhibition assays. The unbound alpha sarcin, conjugated with the anti-ovarian carcinoma monoclonal antibody MOv17, showed on OVCA 432 a cytotoxicity which was 900 times higher than that exerted by the alpha sarcin alone.