Hybrid T cell receptor genes formed by interlocus recombination in normal and ataxia-telangiectasis lymphocytes.

In this paper, using polymerase chain reaction (PCR), we demonstrated the occurrence of hybrid genes formed by interlocus recombination between T cell receptor gamma (TCR-gamma) variable (V) regions and TCR-beta joining (J) regions in the peripheral blood lymphocytes (PBL) from normal individuals and patients with ataxia-telangiectasia (AT). Sequence analysis of the PCR-derived hybrid genes confirmed that site-specific V gamma-J beta recombination had occurred and showed that 10 of 23 genomic hybrid genes maintained a correct open reading frame. By dilution analysis, the frequency of these hybrid genes was 8 +/- 1/10(5) cells in normal PBL and 587 +/- 195/10(5) cells in AT PBL. These frequencies and the approximately 70-fold difference between the normal and AT samples are consistent with previous cytogenetic data examining the occurrence of an inversion of chromosome 7 in normal and AT PBL. We also demonstrated expression of these hybrid genes by PCR analysis of first-strand cDNA prepared from both normal and AT PBL. Sequence analysis of the PCR-amplified transcripts showed that, in contrast to the genomic hybrid genes, 19 of 22 expressed genes maintained a correct open reading frame at the V-J junction and correctly spliced the hybrid V-J exon to a TCR-beta constant region, thus allowing translation into a potentially functional hybrid TCR protein. Another type of hybrid TCR transcript was found in a which a rearranged TCR-gamma V-J exon was correctly spliced to a TCR-beta constant region. This form of hybrid gene may be formed by trans-splicing. These hybrid TCR genes may serve to increase the repertoire of the immune response. In addition, studies of their mechanism of formation and its misregulation in AT may provide insight into the nature of the chromosomal instability syndrome associated with AT. The mechanism underlying hybrid gene formation may be analogous to the mechanism underlying rearrangements between putative growth-affecting genes and the antigen receptor loci, which are associated with AT lymphocyte clones and lymphoid malignancies.

First-in-human phase 0 study of 111In-CHX-A"-DTPA trastuzumab for HER2 tumor imaging.

INTRODUCTION: Tumors over-expressing the human epithelial receptor 2 (HER2) or exhibiting amplification or mutation of its proto-oncogene have a poorer prognosis. Using trastuzumab and/or other HER2 targeted therapies can increase overall survival in patients with HER2(+) tumors making it critical to accurately identify patients who may benefit. We report on a Phase 0 study of the imaging agent, 111In-CHX-A"-DTPA trastuzumab, in patients with known HER2 status to evaluate its safety and biodistribution and to obtain preliminary data regarding its ability to provide an accurate, whole-body, non-invasive means to determine HER2 status. METHODS: 111In-CHX-A"-DTPA trastuzumab was radiolabeled on-site and slowly infused into 11 patients who underwent single (n=5) or multiple (n=6) ɣ-camera (n=6) and/or SPECT (n=8) imaging sessions. RESULTS: No safety issues were identified. Visual and semi-quantitative imaging data were concordant with tissue HER2 expression profiling in all but 1 patient. The biodistribution showed intense peak liver activity at the initial imaging timepoint (3.3h) and a single-phase clearance fit of the average time-activity curve (TAC) estimated t1/2=46.9h (R2=0.97; 95%CI 41.8 to 53h). This was followed by high gastrointestinal (GI) tract activity peaking by 52h. Linear regression predicted GI clearance by 201.2h (R2 =0.96; 95%CI 188.5 to 216.9h). Blood pool had lower activity with its maximum on the initial images. Non-linear regression fit projected a t1/2=34.2h (R2 =0.96; 95%CI 25.3 to 46.3h). Assuming linear whole-body clearance, linear regression projected complete elimination (x-intercept) at 256.5hr (R2=0.96; 95%CI 186.1 to 489.2h). CONCLUSION: 111In-CHX-A"-DTPA trastuzumab can be safely imaged in humans. The biodistribution allowed for visual and semiquantitative analysis with results concordant with tissue expression profiling in 10 of 11 patients. Advances in Knowledge and Implications for Patient Care Using readily available components and on-site radiolabeling 111In-CHX-A"-DTPA trastuzumab SPECT imaging may provide an economical, non-invasive means to detect HER2 over-expression.

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins.

The overexpression or mutation of tyrosine kinases (TKs), such as the epidermal growth factor receptor (EGFR), can lead to the development of cancer. The most common mutation of the EGFR in glioblastomas is the deletion of exons 2-7 known as the EGFRvIII. This mutant receptor cannot bind EGF but, instead, is constitutively active. The Cbl family of ubiquitin ligases (Cbl, Cbl-b, and Cbl-c) targets the activated EGFR for degradation. As the EGFRvIII is transforming, we investigated whether it could be downregulated by the Cbl proteins. The overexpression of all three Cbl proteins resulted in the ubiquitination and degradation of the EGFRvIII. As with the wild-type EGFR, the TK-binding domain and the RING finger of Cbl-b are sufficient for the downregulation of the EGFRvIII. Also, we found that Cbl-b is recruited to the EGFRvIII and inhibits the transformation of NIH 3T3 cells by the EGFRvIII. Mutation of the Cbl-binding site (Y1045F) in the EGFRvIII inhibits its ubiquitination and downregulation by Cbl-b and enhances its ability to transform. Furthermore, the EGFR TK inhibitor, AG 1478, prevents the downregulation of the EGFRvIII by the Cbl proteins and antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor. In conclusion, the EGFRvIII does not transform by escaping regulation by Cbl proteins and this activation-induced downregulation of the EGFRvIII has an important role in mediating the toxicity of anti-EGFRvIII immunotoxins.

The death domain kinase RIP is essential for TRAIL (Apo2L)-induced activation of IkappaB kinase and c-Jun N-terminal kinase.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand [Apo2L]) is a member of the TNF superfamily and has been shown to have selective antitumor activity. Although it is known that TRAIL (Apo2L) induces apoptosis and activates NF-kappaB and Jun N-terminal kinase (JNK) through receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the components of its signaling cascade have not been well defined. In this report, we demonstrated that the death domain kinase RIP is essential for TRAIL-induced IkappaB kinase (IKK) and JNK activation. We found that ectopic expression of the dominant negative mutant RIP, RIP(559-671), blocks TRAIL-induced IKK and JNK activation. In the RIP null fibroblasts, TRAIL failed to activate IKK and only partially activated JNK. The endogenous RIP protein was detected by immunoprecipitation in the TRAIL-R1 complex after TRAIL treatment. More importantly, we found that RIP is not involved in TRAIL-induced apoptosis. In addition, we also demonstrated that the TNF receptor-associated factor 2 (TRAF2) plays little role in TRAIL-induced IKK activation although it is required for TRAIL-mediated JNK activation. These results indicated that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in TRAIL-induced IKK and JNK activation.

A measure of genomic instability and its relevance to lymphomagenesis.

A recent pilot study that we performed on 12 individuals who are involved in the cultivation and processing of grains and legumes suggests to us that we may have in hand a relatively quick, inexpensive, and highly sensitive assay that identifies individuals at increased risk for the development of lymphoid malignancy. The generation of this assay evolved from our interest in the causes and consequences of lymphocyte-specific chromosomal aberration.

Fas expression and function in normal and malignant breast cell lines.

Expression and function of the Fas apoptotic pathway was investigated in normal and malignant human breast epithelial cells. Nontransformed mammary epithelial cell lines all expressed high levels of Fas mRNA and protein, but only one of seven breast cancer cell lines (T47D) expressed high levels of Fas. Apoptosis was induced in the nontransformed lines when they were incubated with the anti-Fas antibody. However, all of the breast cancer cell lines tested, except T47D, were resistant to Fas-mediated apoptosis. Four of five Fas-resistant breast cancer cell lines became sensitive to Fas-mediated apoptosis upon treatment with IFN-gamma. Fas mRNA increased slightly in both cell lines that became sensitive and in the cell line that remained resistant to Fas-mediated apoptosis upon IFN-gamma treatment. However, the cell surface expression of Fas showed little or no increase in any of the cell lines tested upon IFN-gamma treatment. In contrast to Fas expression, interleukin-1beta-converting enzyme (ICE) expression increased only in the cell lines that became Fas sensitive after IFN-gamma treatment. The importance of ICE and/or ICE-like proteases in Fas-mediated apoptosis in these cells was confirmed by inhibition of Fas-mediated apoptosis by a specific ICE inhibitor, YVAD-cmk. Fas sensitivity was reconstituted in the IFN-gamma-resistant cell line by transfection of ICE into that cell line. Together, these data suggest that down-regulation of Fas and its pathway may be a step in tumor progression and that modulation of Fas expression may provide an approach to inducing apoptosis in breast cancer cells.

The protein tyrosine phosphatase DEP-1 is induced during differentiation and inhibits growth of breast cancer cells.

Sodium butyrate-induced differentiation of breast cancer cell lines was used to identify protein tyrosine phosphatases (PTPs) involved in differentiation and growth inhibition of breast cancer cells. Of 42 PTPs analyzed, 31 were expressed in the ZR75-1 breast cancer cell line. Expression of four PTPs (DEP-1, SAP, PTP gamma, and PAC) was regulated in ZR75-1 cells undergoing differentiation. Expression of two of these PTPs (DEP-1 and SAP) was also regulated in the SKBr-3 cell line undergoing differentiation. In view of its marked induction with differentiation in an estrogen receptor (ER)-positive and an ER-negative breast cancer cell line, DEP-1 was investigated for a role in growth inhibition or induction of differentiation in breast cancer cells. A DEP-1 cDNA construct under control of a constitutively active cytomegalovirus promoter was transfected into the ZR75-1, SKBR-3, and MCF-7 breast cancer cell lines, and resistant colonies were selected with G418. DEP-1 expression inhibited the development of resistant colonies by 3-5-fold in all three lines compared to transfection with vector alone. Three stable MCF-7 cell lines expressing DEP-1 under control of an inducible metallothionein promoter were then established. In these lines, induction of DEP-1 expression inhibited breast cancer cell growth by 5-10-fold. These data describe PTPs expressed and regulated in breast cancer cell lines during differentiation and identify one PTP, DEP-1, that inhibits the growth of breast cancer cells in vitro.

Chemotherapy augments TRAIL-induced apoptosis in breast cell lines.

Expression and function of the TRAIL apoptotic pathway was investigated in normal and malignant breast epithelial cells. Glutathione-S-transferase (GST)-TRAIL extracellular domain fusion proteins were produced to analyze TRAIL-induced apoptosis. Only GST-TRAIL constructs containing regions homologous to the Fas self-association and ligand binding domains could induce apoptosis. GST-TRAIL induced significant (>90%) apoptosis in just one of eight normal and one of eight malignant breast cell lines. All other lines were relatively resistant to TRAIL-induced apoptosis. Activating TRAIL receptors DR4 and DR5 were expressed in all normal and malignant breast cell lines. The inhibitory receptor TRID was highly expressed in one of four normal and two of seven malignant breast cell lines. DR4, DR5, or TRID expression did not correlate with sensitivity to TRAIL-induced apoptosis. Incubation of cell lines with doxorubicin or 5-fluorouracil significantly augmented TRAIL-induced apoptosis in most breast cell lines. By fractional inhibition analysis, the toxicity of the combination of TRAIL and doxorubicin or 5-fluorouracil was synergistic compared with either agent alone. In contrast, melphalan and paclitaxel augmented TRAIL-induced apoptosis in few cell lines, and methotrexate did not augment it in any cell line. Augmentation of TRAIL-induced apoptosis by doxorubicin or 5-fluorouracil was mediated through caspase activation. This was evidenced by the fact that chemotherapy agents that synergized with TRAIL (e.g., doxorubicin) themselves caused cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and their toxicity was blocked by the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2 (ZVAD-fmk). The combination of TRAIL and doxorubicin caused significantly greater caspase-3 and PARP cleavage, and the combined toxicity also was inhibited by ZVAD-fmk. In contrast, chemotherapy agents that did not augment TRAIL-induced apoptosis (e.g., methotrexate) caused minimal caspase-3 and PARP cleavage by themselves, and their toxicity was not inhibited by ZVAD-fmk. These drugs also did not increase caspase-3 or PARP cleavage when combined with TRAIL. In summary, few breast cell lines are sensitive to TRAIL-induced apoptosis, and no difference in sensitivity is found between normal and malignant cell lines. Treatment with chemotherapy provides an approach to sensitize breast cancer cells to TRAIL-induced apoptosis.

Down-regulation of the erbB-2 receptor by trastuzumab (herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2.

We investigated whether combined treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and trastuzumab could enhance the specific killing of cells that overexpress the erbB-2 receptor. The combination resulted in an enhancement of TRAIL-mediated apoptosis in all cell lines overexpressing erbB-2 receptor compared with either reagent alone. In contrast, there was no effect in cell lines with low levels of the erb-B2 receptor. Trastuzumab treatment resulted in down-regulation of the erbB-2 receptor in all erbB-2-overexpressing cell lines. Similar enhancement of TRAIL toxicity was observed when the erbB-2 receptor was down-regulated using antisense oligodeoxynucleotides. Down-regulation of the erbB-2 receptor protein by trastuzumab or antisense oligodeoxynucleotides decreased Akt kinase activation but not mitogen-activated protein kinase activation. Down-regulation of Akt kinase activity by a phosphatidylinositol 3'-kinase inhibitor (LY294002) also resulted in enhancement of TRAIL-mediated apoptosis. Expression of a constitutively active form of Akt kinase in an erbB-2-overexpressing cell line completely abrogated the increase in TRAIL-mediated apoptosis by trastuzumab and significantly reduced the biological effect of either reagent alone. Therefore, down-regulation of the erbB-2 receptor by trastuzumab enhances TRAIL-mediated apoptosis by inhibiting Akt kinase activity. These data suggest that the combination of trastuzumab and TRAIL may allow enhanced therapeutic efficacy and specificity in the treatment of erbB-2-overexpressing tumors.

Cloning and characterization of cbl-b: a SH3 binding protein with homology to the c-cbl proto-oncogene.

We have cloned a new gene, cbl-b, with homology to the c-cbl proto-oncogene. A large protein is predicted (approx. MW 108,000) that has a proline rich domain, a nuclear localization signal, a C3HC4 zinc finger and a putative leucine zipper. There is striking nucleotide and amino acid homology to the c-cbl proto-oncogene most notably in the structural motifs described above. Cbl-b is expressed in normal and malignant mammary epithelial cells, in a variety of normal tissues, and in hematopoietic tissue and cell lines. Cbl-b expressions is up-regulated with macrophage/monocyte differentiation of the HL60 and U937 cell lines. There is direct association of the cbl-b protein with the Src Homology 3 domains of several proteins including signaling, cytoskeletal and adaptor proteins. Our data suggest that cbl-b encodes a protein which can interact with signal transduction proteins to regulate their function or to be regulated by them. Together, cbl-b and c-cbl are members of a novel family of proto-oncogenes involved in signal transduction.

cbl-3: a new mammalian cbl family protein.

We have cloned a new human gene, cbl-3, which encodes a protein with marked homology to the cbl family of proteins. The predicted protein encoded by this gene retains the conserved phosphotyrosine binding domain (PTB) in the N-terminal and the zinc finger but is significantly shorter (MW 52.5 kDa) than the other mammalian cbl proteins. The protein lacks the extensive proline rich domain and leucine zipper seen in c-cbl and cbl-b and structurally most resembles the C. elegans and Drosophila cbl proteins. The gene is ubiquitously expressed with highest expression in the aerodigestive tract, prostate, adrenal gland, and salivary gland. The protein is phosphorylated and recruited to the EGFR upon EGF stimulation and inhibits EGF stimulated MAP kinase activation. In comparison to the other mammalian cbl proteins (e.g. cbl-b), cbl-3 interacts with a restricted range of proteins containing Src Homology 3 regions. An alternatively spliced form of the cbl-3 protein was also identified which deletes a critical region of the PTB domain and which does not interact with the EGFR nor inhibit EGF stimulated MAP kinase activation. These data demonstrate that cbl-3, a novel mammalian cbl protein, is a regulator of EGFR mediated signal transduction.

Tyrosine phosphorylation and complex formation of Cbl-b upon T cell receptor stimulation.

Cbl-b, a mammalian homolog of Cbl, consists of an N-terminal region (Cbl-b-N) highly homologous to oncogenic v-Cbl, a Ring finger, and a C-terminal region containing multiple proline-rich stretches and potential tyrosine phosphorylation sites. In the present study, we demonstrate that upon engagement of the T cell receptor (TCR), endogenous Cbl-b becomes rapidly tyrosine-phosphorylated. In heterogeneous COS-1 cells, Cbl-b was phosphorylated on tyrosine residues by both Syk- (Syk/Zap-70) and Src- (Fyn/Lck) family kinases, with Syk kinase inducing the most prominent effect. Syk associates and phosphorylates Cbl-b in Jurkat T cells. A Tyr-316 Cbl-binding site in Syk was required for the association with and for the maximal tyrosine phosphorylation of Cbl-b. Mutation at a loss-of-function site (Gly-298) in Cbl-b-N disrupts its interaction with Syk. Cbl-b constitutively binds Grb2 and becomes associated with Crk-L upon TCR stimulation. The Grb2- and the Crk-L-binding regions were mapped to the C-terminus of Cbl-b. The Crk-L-binding sites were further determined to be Y655DVP and Y709KIP, with the latter being the primary binding site. Taken together, these results implicate that Cbl-b is involved in TCR-mediated intracellular signaling pathways.

Apoptosis is associated with cleavage of a 5 kDa fragment from RB which mimics dephosphorylation and modulates E2F binding.

Dephosphorylation of the RB protein has been reported to be associated with apoptosis. In contrast, we show that treatment of HL60 cells with etoposide or cytosine arabinoside or treatment of breast epithelial cells with alpha-FAS is associated with the cleavage of a 5 kDa fragment from the C-terminus of RB, resulting in a truncated product that we have designated as p100cl. This cleavage event coincides with the activation of cysteine proteases at the onset of apoptosis, is blocked by the addition of iodoacetamide to cells prior to the onset of apoptosis, and results in the expression of faster migrating protein species which can mimic dephosphorylated RB. The free 5 kDa fragment is detected only during apoptosis, predicts a cleavage site that we have mapped to a unique CPP32-like recognition sequence which is present at the C-terminus of all reported RB homologues, and results in a truncated RB protein with enhanced E2F binding affinity. While the causality for this cleavage event in the apoptotic process is still under investigation, our findings suggest distinct post-translational pathways for the RB product between cells examined during growth arrest (p105 hypophosphorylated RB) or apoptosis (p100cl).

cbl-b inhibits epidermal growth factor receptor signaling.

The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this finding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling.

N-(4-hydroxyphenyl) retinamide (4HPR) enhances TRAIL-mediated apoptosis through enhancement of a mitochondrial-dependent amplification loop in ovarian cancer cell lines.

The majority of ovarian cancer cells are resistant to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Subtoxic concentrations of the semisynthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) enhanced TRAIL-mediated apoptosis in ovarian cancer cell lines but not in immortalized nontumorigenic ovarian epithelial cells. The enhancement of TRAIL-mediated apoptosis by 4HPR was not due to changes in the levels of proteins known to modulate TRAIL sensitivity. The combination of 4HPR and TRAIL enhanced cleavage of multiple caspases in the death receptor pathway (including the two initiator caspases, caspase-8 and caspase-9). The 4HPR and TRAIL combination leads to mitochondrial permeability transition, significant increase in cytochrome c release, and increased caspase-9 activation. Caspase-9 may further activate caspase-8, generating an amplification loop. Stable overexpression of Bcl-xL abrogates the interaction between 4HPR and TRAIL at the mitochondrial level by blocking cytochrome c release. As a consequence, a decrease in activation of caspase-9, caspase-8, and TRAIL-mediated apoptosis occurs. These results indicate that the enhancement in TRAIL-mediated apoptosis induced by 4HPR is due to the increase in activation of multiple caspases involving an amplification loop via the mitochondrial-death pathway. These findings offer a promising and novel strategy for the treatment of ovarian cancer.

SETA is a multifunctional adapter protein with three SH3 domains that binds Grb2, Cbl, and the novel SB1 proteins.

Expression of the src homology 3 (SH3)-encoding, expressed in tumorigenic astrocytes (SETA) gene is associated with astrocyte transformation in culture and tumors in the adult brain. SETA binds to the apoptosis regulator apoptosis-linked gene 2 (ALG-2) interacting protein 1 (AIP1), and modulates apoptosis in astrocytes. The predicted protein structure of SETA revealed two SH3 domains, while related proteins were reported to have three. Here we report the identification of an additional SH3 domain N-terminal to the previously identified SETA sequence. Yeast two-hybrid screening of a p53(-/-) astrocyte cDNA library with this SH3 domain identified a novel gene, SETA binding protein 1 (SB1), with 55% amino acid identity to the renal tumor antigen, NY-REN-45. In vitro confrontation and co-immunoprecipitation experiments confirmed the binding of SB1 to SETA. Evidence that SETA binds to the CD2 protein, the proto-oncogene c-Cbl, and the signal transduction molecule Grb2, and can dimerize via its C-terminal coiled coil (CC) domain is also presented.

Interlocus V-J recombination measures genomic instability in agriculture workers at risk for lymphoid malignancies.

V(D)J [variable-(diversity)-joining] rearrangements occur between, as well as within, immune receptor loci, resulting in the generation of hybrid antigen-receptor genes and the formation of a variety of lymphocyte-specific chromosomal aberrations. Such hybrid genes occur at a low frequency in the peripheral blood lymphocytes (PBL) of normal individuals but show a markedly increased incidence in the PBL of individuals with the autosomal recessive disease ataxia-telangiectasia. In this manuscript we demonstrate that the frequency of hybrid antigen-receptor genes is 10- to 20-fold increased in the PBL of an occupational group, agriculture workers, with related environmental exposures. Both ataxia-telangiectasia patients and this population of agriculture workers are at increased risk for lymphoid malignancy. This result suggests that the measurement of hybrid antigen receptor-genes in PBL may be a sensitive assay for a type of lymphocyte-specific genomic instability. As a corollary, this assay may identify populations at risk of developing common types of lymphoid malignancy.

Acetylcholine-receptor-mediated ion flux in electroplax membrane microsacs (vesicles): change in mechanism produced by asymmetrical distribution of sodium and potassium ions.

The kinetics of acetylcholine-receptor-mediated efflux of inorganic ions from electroplax microsacs of Electrophorus electricus in the presence of varying alkali metal ion concentrations on both sides of the membrane have been investigated. The efflux, a monophasic process when the ion distribution is symmetrical (the same concentrations and types of ions on both sides of the membrane), becomes a biphasic process, consisting of a very rapid initial release of ions followed by a slower first-order process, under conditions that resemble the physiological state of the neural membrane (potassium ions inside the microsacs and sodium ions on the outside). The initial phase of the efflux discriminates between calcium and sodium ions and is inhibited by potassium ions in the external solution. The rate constant associated with this phase is at least 40 times larger than the rate constant associated with the slower efflux. Both phases depend on the concentration of acetylcholine or carbamoylcholine, and are inhibited by receptor inhibitors (d-tubocurarine and alpha-bungarotoxin).A simple model is proposed which relates the kinetics of the flux to ligand-induced conformational changes in the receptor. We also indicate the relationship between the biphasic kinetics of the flux observed in microsacs to "desensitization," the phenomenon in which, on addition of acetylcholine, the transmembrane voltage of muscle and nerve cells first increases and then decreases to its resting value within a few seconds.

Inversion of chromosome 7 in ataxia telangiectasia is generated by a rearrangement between T-cell receptor beta and T-cell receptor gamma genes.

Specific and recurrent chromosomal rearrangements are often observed in the karyotypes of phytohemagglutinin-stimulated lymphocytes. The percentage of cells demonstrating these rearrangements is dramatically increased in the genetic disease ataxia telangiectasia. Inversion of chromosome 7 represents approximately half of the chromosomal rearrangements in this disease. Because the chromosomal locations of the inv(7) breakpoints coincide precisely with those of the T-cell antigen receptor (TCR) beta and gamma genes, it has been hypothesized that this rearrangement may occur by recombination between those two loci. Here, we present direct evidence that inversion of chromosome 7 in ataxia telangiectasia is generated by site-specific recombination between a TCR gamma variable segment and a TCR beta joining segment.

Two new Drosophila genes related to human hematopoietic and neurogenic transcription factors.

We have identified two new basic domain helix-loop-helix (bHLH) genes in Drosophila melanogaster, DroSCL and DroNHLH. DroSCL was identified because of its homology to the mammalian hematopoietic transcription factor SCL. DroNHLH was similarly identified by homology to NHLH1 and NHLH2, two bHLH genes expressed in the developing mammalian nervous system. A partial DroSCL complementary DNA clone was obtained from an early pupal (5.5-7.5-day) Drosophila library. DroSCL is 73% identical to SCL within the 55-amino acid region of the bHLH domain. A DroNHLH complementary DNA clone was obtained from an early instar (I and II) Drosophila library. Its coding region consists of 162 amino acids and encodes a predicted protein of 18,312 daltons. DroNHLH is 87% identical to NHLH1 and NHLH2 within the bHLH domain. DroSCL and DroNHLH are located on the X chromosome. A 1.7-kilobase DroSCL transcript and a 1.5-kilobase DroNHLH transcript were detected by Northern analysis of total Drosophila RNA. Examination of Drosophila embryos by tissue in situ hybridization reveals restricted expression of both genes in a subset of cells in the developing central nervous system.