Structural and mechanistic basis of Parl activity and regulation.

The mitochondrial rhomboid protease Parl governs apoptosis, morphology, metabolism and might be implicated in Parkinson's disease, but the structural basis of its activity and complex regulation remain unknown. We report the discovery of γ-cleavage, a proteolytic event on the loop connecting the first transmembrane helix (TMH) of Parl to the 6-TMH catalytic rhomboid domain of the protease. This cleavage disrupts the '1+6' structure that defines every mitochondrial rhomboid and generates a new form of Parl, PROD (Parl-rhomboid-domain). Structure-function analysis of Parl suggests that γ-cleavage could be implicated in eliminating Parl proteolytic activity, and structural modeling of PROD reveals structural conservation with the bacterial rhomboid GlpG. However, unlike bacterial rhomboids, which employ a diad-based mechanism of catalysis, Parl appears to use a conserved mitochondrial rhomboid-specific Asp residue on TMH-5 in a triad-based mechanism of catalysis. This work provides unexpected insights into the structural determinants regulating Parl stability and activity in vivo, and reveals a complex cascade of proteolytic events controlling the function of the protease in the mitochondrion.

Loss of the Parkinson's disease-linked gene DJ-1 perturbs mitochondrial dynamics.

Growing evidence highlights a role for mitochondrial dysfunction and oxidative stress as underlying contributors to Parkinson's disease (PD) pathogenesis. DJ-1 (PARK7) is a recently identified recessive familial PD gene. Its loss leads to increased susceptibility of neurons to oxidative stress and death. However, its mechanism of action is not fully understood. Presently, we report that DJ-1 deficiency in cell lines, cultured neurons, mouse brain and lymphoblast cells derived from DJ-1 patients display aberrant mitochondrial morphology. We also show that these DJ-1-dependent mitochondrial defects contribute to oxidative stress-induced sensitivity to cell death since reversal of this fragmented mitochondrial phenotype abrogates neuronal cell death. Reactive oxygen species (ROS) appear to play a critical role in the observed defects, as ROS scavengers rescue the phenotype and mitochondria isolated from DJ-1 deficient animals produce more ROS compared with control. Importantly, the aberrant mitochondrial phenotype can be rescued by the expression of Pink1 and Parkin, two PD-linked genes involved in regulating mitochondrial dynamics and quality control. Finally, we show that DJ-1 deficiency leads to altered autophagy in murine and human cells. Our findings define a mechanism by which the DJ-1-dependent mitochondrial defects contribute to the increased sensitivity to oxidative stress-induced cell death that has been previously reported.

Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13.

SNAREs and Rab GTPases cooperate in vesicle transport through a mechanism yet poorly understood. We now demonstrate that the Rab5 effectors EEA1 and Rabaptin-5/Rabex-5 exist on the membrane in high molecular weight oligomers, which also contain NSF. Oligomeric assembly is modulated by the ATPase activity of NSF. Syntaxin 13, the t-SNARE required for endosome fusion, is transiently incorporated into the large oligomers via direct interactions with EEA1. This interaction is required to drive fusion, since both dominant-negative EEA1 and synthetic peptides encoding the FYVE Zn2+ finger hinder the interaction and block fusion. We propose a novel mechanism whereby oligomeric EEA1 and NSF mediate the local activation of syntaxin 13 upon membrane tethering and, by analogy with viral fusion proteins, coordinate the assembly of a fusion pore.

The importance of antibody-specificity in determining successful radioimmunotherapy of B-cell lymphoma.

We report the radioimmunotherapy of mouse B-cell lymphoma, BCL1, using a panel of anti-B-cell monoclonal antibodies (MoAb) (anti-CD19, anti-CD22, anti-major histocompatibility complex (MHC) II, and anti-idiotype (Id) radiolabeled with 131-iodine. When administered early in disease (day 4), the 131I-anti-MHCII MoAb cured tumors as a result of targeted irradiation alone, the unlabeled MoAb being nontherapeutic. In contrast, 131I-anti-Id, despite targeting irradiation and having therapeutic activity as an unconjugated antibody, protected mice for only 30 days; 131I-anti-CD19 and anti-CD22 were therapeutically inactive. Binding and biodistribution studies showed that the anti-Id, unlike anti-MHCII, MoAb was cleared from target cells in vivo and delivered 4 times less irradiation to splenic tumor. Treating later in the disease (day 14) increased tumor load and produced the expected reduction in therapeutic activity with the anti-MHCII, but surprisingly, allowed 131I-anti-Id to cure most mice. This unexpected potency of 131I-anti-Id late in the disease appeared to result from the direct cytotoxicity of the anti-Id MoAb, which was more active in established disease, in combination with targeted irradiation. We believe the ability of targeted irradiation and certain cytotoxic MoAb to work cooperatively against tumor in this way has important implications for the selection of reagents in radioimmunotherapy of B-cell lymphoma.

The Rab5 effector EEA1 is a core component of endosome docking.

Intracellular membrane docking and fusion requires the interplay between soluble factors and SNAREs. The SNARE hypothesis postulates that pairing between a vesicular v-SNARE and a target membrane z-SNARE is the primary molecular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system. However, several observations demonstrate that SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting. Moreover, other proteins have been shown to be important in membrane docking or tethering. Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting, docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is regulated by the small GTPase Rab5. Here we show that Rab5-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the Rab5 effector EEA1 is the only factor necessary to confer minimal fusion activity. Rab5 and other associated proteins seem to act upstream of EEA1, implying that Rab5 effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. We further show that EEA1 mediates endosome docking and, together with SNAREs, leads to membrane fusion.

Monoclonal antibody therapy of B cell lymphoma: signaling activity on tumor cells appears more important than recruitment of effectors.

Despite the recent success of mAb in the treatment of certain malignancies, there is still considerable uncertainty about the mechanism of action of anti-cancer Abs. Here, a panel of rat anti-mouse B cell mAb, including Ab directed at surface IgM Id, CD19, CD22, CD40, CD74, and MHC class II, has been investigated in the treatment of two syngeneic mouse B cell lymphomas, BCL1 and A31. Only three mAb were therapeutically active in vivo, anti-Id, anti-CD19, and anti-CD40. mAb to the other Ags showed little or no therapeutic activity in either model despite giving good levels of surface binding and activity in Ag-dependent cellular cytotoxicity and complement assays, and in some cases inhibiting cell growth in vitro. We conclude that the activity of mAb in vitro does not predict therapeutic performance in vivo. Furthermore, in vivo tracking experiments using fluorescently tagged cells showed that anti-Id and anti-CD40 mAb probably operate via different mechanisms: the anti-Id mAb cause growth arrest that is almost immediate and does not eliminate cells over a period of 5 or 6 days, and the anti-CD40 mAb have a delayed effect that allows tumor to grow normally for 3 days, but then abruptly eradicates lymphoma cells. This work supports the belief that mAb specificity is critical to therapeutic success in lymphoma and that, in addition to any effector-recruiting activity they may possess, in vivo mAb operate via mechanisms that involve cross-linking and signaling of key cellular receptors.

A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function.

The small GTPase Rab5 plays an essential role in endocytic traffic. Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5. Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5. Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion. Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic. Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane. This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.

The human mitochondrial import receptor, hTom20p, prevents a cryptic matrix targeting sequence from gaining access to the protein translocation machinery.

Yeast Mas70p and NADH cytochrome b5 reductase are bitopic integral proteins of the mitochondrial outer membrane and are inserted into the lipid-bilayer in an Nin-Ccyto orientation via an NH2-terminal signal-anchor sequence. The signal anchor of both proteins is comprised of a short, positively charged domain followed by the predicted transmembrane segment. The positively charged domain is capable of functioning independently as a matrix-targeting signal in yeast mitochondria in vitro but does not support import into mammalian mitochondria (rat or human). Rather, this domain represents a cryptic signal that can direct import into mammalian mitochondria only if proximal components of the outer membrane import machinery are removed. This can be accomplished either by treating the surface of the intact mitochondria with trypsin or by generating mitoplasts. The import receptor Tom20p (Mas20p/MOM19) is responsible for excluding the cryptic matrix-targeting signal from mammalian mitochondria since replacement of yeast Tom20p with the human receptor confers this property to the yeast organelle while at the same time maintaining import of other proteins. In addition to contributing to positive recognition of precursor proteins, therefore, the results suggest that hTom20p may also have the ability to screen potential matrix-targeting sequences and exclude certain proteins that would otherwise be recognized and imported by distal components of the outer and inner membrane protein-translocation machinery. These findings also indicate, however, that cryptic signals, if they exist within otherwise native precursor proteins, may remain topogenically silent until the precursor successfully clears hTom20p, at which time the activity of the cryptic signal is manifested and can contribute to subsequent translocation and sorting of the polypeptide.

The human steroidogenic acute regulatory (StAR) gene is expressed in the urogenital system and encodes a mitochondrial polypeptide.

The first enzymatic step in the biosynthesis of steroid hormones occurs in the mitochondrial inner membrane and is dependent on the mobilization of cholesterol from cellular stores. We report on the isolation of a human cDNA which encodes a mitochondrial protein called steroidogenic acute regulatory (StAR) protein, implicated in transport of cholesterol into mitochondria. Nucleotide and predicted amino acid sequence analyses indicate that the human and murine polypeptides are highly conserved, sharing 87% identity with an overall homology of 92%. Analysis of the distribution of StAR mRNA transcripts in human tissues by Northern blotting reveals several mRNA species, the most abundant of which is a 1.8 kb mRNA transcript present in testes, ovaries and kidneys. Using in vitro translated protein, we demonstrate that the StAR gene product can be efficiently imported into exogenously added mitochondria.

Insertion of an uncharged polypeptide into the mitochondrial inner membrane does not require a trans-bilayer electrochemical potential: effects of positive charges.

Mitochondria with a ruptured outer membrane exhibited impaired import into this membrane of an outer membrane fusion protein containing the signal-anchor sequence of Mas70p. However, the Mas70p signal-anchor efficiently targeted and inserted the protein directly into exposed regions of the inner membrane. Import into the inner membrane was dependent on delta psi and this dependence was due to the presence of the positively-charged amino acids located at positions 2, 7, and 9 of the signal-anchor. In contrast to wild-type signal-anchor, mutants lacking the positively-charged residues mediated import into the inner membrane in both the presence and absence of delta psi. The results suggest two conclusions: (1) delta psi-dependent import of the signal-anchor sequence was due exclusively to an effect of delta psi on the positively-charged domain of the signal-anchor, rather than to an effect of delta psi on a property of the inner membrane import machinery; (2) in the absence of delta psi, the positively-charged domain of the signal-anchor prevented the otherwise import-competent signal-anchor from inserting into the membrane. This suggests that the positively-charged domain leads import across the inner membrane, and that delta psi is required to vectorially clear this domain in order to allow the distal region of the signal-anchor to enter the translocation pathway. The implications of these findings on the mechanism of import into the mitochondrial inner membrane and matrix are discussed.

Import and insertion of proteins into the mitochondrial outer membrane.

Nuclear-encoded proteins destined for insertion into the mitochondrial outer membrane, follow the same general pathway for import as proteins that are translocated to interior compartments within the organelle. This observation is true both for beta-barrel-type proteins and for proteins that contain hydrophobic alpha-helical transmembrane segments. In this review, we describe what is known about the various steps leading to protein insertion into the outer membrane, and discuss the energetics that favor vectorial translocation into and across this membrane. The selection of the outer membrane during import may involve a lateral release of the translocating polypeptide from the import machinery so that the appropriate domains of the protein become embedded in the lipid bilayer. One type of topogenic domain that can guarantee such selection of the outer membrane is a signal-anchor sequence of the type characterized for the bitopic protein Mas70p. It is suggested that a signal-anchor sequence selective for the mitochondrial outer membrane causes abrogation of polypeptide translocation and triggers the release of the transmembrane segment into the surrounding lipid bilayer, prior to any possibility for the commitment of translocation to the interior of the organelle. Specific structural features of the signal-anchor sequence specify its orientation in the membrane, and can confer on this sequence the ability to form homo-oligomers and hetero-oligomers. Strategies other than a signal-anchor sequence may be employed by other classes of proteins for selection of the outer-membrane. Of note is the ability of the outer-membrane import machinery to catalyze integration of the correct set of proteins into the outer-membrane bilayer, while allowing proteins that are destined for integration into the bilayer of the inner membrane to pass through unimpeded. Again, however, different proteins may employ different strategies. One model proposes that this can be accomplished by a combination of a matrix-targeting signal and a distal stop-transfer sequence. In this model, the formation of contact sites, which is triggered when the matrix-targeting signal engages the import machinery of the inner membrane, may prevent the outer-membrane translocon from recognizing and responding to the downstream stop-transfer domain. This allows the transmembrane segment to pass across the outer-membrane, and subsequently integrate into the inner membrane.

A signal-anchor sequence selective for the mitochondrial outer membrane.

pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase. The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29). The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro. The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9. Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro. Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import. We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.

Monoclonal antibodies raised against the idiotype of the murine B cell lymphoma, BCL1 act primarily with heavy chain determinants.

Anti-idiotypic antibodies can be used as probes to distinguish neoplastic cells from their normal counterparts. In addition they have been used in the passive therapy of B cell tumors. In this report we describe a panel of 7 rat monoclonal antibodies raised against idiotypic determinants carried by the IgM molecule of the BCL1 lymphoma. The majority (6/7) of these antibodies recognize private idiotypic determinants that are carried on the isolated mu heavy chain of the molecule, and do not require the lambda chain for reactivity. This is unusual for antibodies raised against the idiotype of the whole immunoglobulin molecule, which normally require both chains for reactivity. The antibodies do not, however, bind peptides corresponding to the complementarity determining regions of the mu heavy chain of BCL1. The antibodies perform well in complement mediated cytotoxicity, and, in at least one case, are effective in the passive immunotherapy of BCL1 lymphoma.

Production and characterization of monoclonal antibodies specific for human mast cell tryptase.

Human mast cell tryptase was purified from lung tissue by high salt extraction, ammonium sulphate precipitation, octyl Sepharose and heparin-agarose chromatography. The tryptase isolated was a tetramer with a molecular weight of 132 kD on gel filtration, and on SDS-polyacrylamide gel electrophoresis was reduced to a single diffuse band with a mean molecular weight of 32.5 kD. Purified tryptase catalysed the cleavage of the tryptic substrates tosyl L-arginine methyl ester and benzoyl DL-arginine p-nitroanilide; enzymatic activity was enhanced in the presence of heparin but markedly decreased in the presence of 2 M sodium chloride. Rabbit antisera and three new monoclonal antibodies (AA1, AA3 and AA5) were produced which were specific for tryptase in indirect ELISAs, immunoenzymatic overlay in crossed immunoelectrophoresis and by Western blotting. Additive and competitive ELISA experiments suggested that the three monoclonal antibodies all recognized epitopes within a single highly immunogenic area of the tryptase molecule, and enzyme assays indicated that this site was distant from the active site. Binding of monoclonal antibodies to tryptase was not affected by the presence of heparin, or by periodate treatment of the antigen suggesting that carbohydrate epitopes were not recognized. Western blotting indicated that some heterogeneity in molecular weight for monomeric tryptase was not reflected in antigenic differences. An immunofluorescence procedure with cytocentrifuge preparations of enzymatically dispersed lung, colon and skin revealed highly specific localization of tryptase to the granules of all mast cells, but there was no binding to other cells in these preparations, to cultured keratinocytes, to basophils or to any other blood leucocyte.

Bispecific F(ab' gamma)2 antibody for the delivery of saporin in the treatment of lymphoma.

This work describes the successful use of bispecific F(ab' gamma)2 antibody (Ab) in combination with a ribosome-inactivating protein (RIP), saporin, for the treatment of neoplastic disease in vivo. A total of three thioether-linked F(ab' gamma)2 heterodimers were prepared, each having dual specificities for saporin and the guinea pig lymphoblastic leukemia, L2C. In all three cases specificity for the L2C cells was provided by a high affinity mouse anti-idiotype (anti-Id) mAb, whereas the antisaporin activity came from either one of two mouse mAb or an affinity-purified rabbit polyclonal Ab. In vitro studies, measuring protein synthesis, showed that all three derivatives were extremely efficient at delivering saporin to L2C cells, to the extent that addition of the rabbit Fab' gamma-containing bispecific Ab to cell culture at 1 microgram/ml increased the toxicity of saporin (50% inhibiting concentration) by close to 90,000-fold. Similarly, in leukemic guinea pigs, a small dose of saporin (10 micrograms) which by itself showed no therapeutic effect, was able to completely eradicate Id-positive tumor when given in combination with an excess of bispecific Ab. Although tumors did eventually emerge in most of these animals, immunofluorescence analysis showed that in almost all instances the escaping cells were Id- variants of the L2C. Experiments to define the optimal treatment regimen in this model showed that, although the administration of saporin and bispecific Ab at separate sites could be therapeutically effective, mixing the Ab and saporin to form immune complexes before injection did generally enhance their performance. A molar surplus of bispecific Ab in these mixtures both extended the metabolic survival of the saporin and enhanced the therapeutic performance, with molar ratios above 3:1 generally being required for optimum treatment when using saporin at 10 micrograms. Derivatives containing polyclonal antisaporin were more efficient than those containing mAb, yielding optimum therapeutic results with a molar ratio of 1.5:1 when combined with 10 micrograms saporin. These findings have shown that bispecific F(ab' gamma)2 Ab, as well as being straightforward to prepare, can also function as an extremely efficient vector for delivering cytotoxic agents such as ribosome-inactivating protein to unwanted cells in vivo.

Preparation and performance of bispecific F(ab' gamma)2 antibody containing thioether-linked Fab' gamma fragments.

A simple and efficient method is described for the production of pure bispecific F(ab' gamma)2 heterodimers, in which the individual antibody Fab' gamma fragments are joined via a stable thioether linkage. Hybrid molecules were constructed from both mouse monoclonal and rabbit polyclonal antibodies with equal efficiency, in the combinations mouse-rabbit and mouse-mouse. Peptic F(ab' gamma)2 fragments from the two chosen antibodies were first reduced to provide Fab' gamma SH. The SH groups on one of the Fab' gamma SH partners were then fully alkylated with o-phenylenedi-maleimide to provide free maleimide groups. Finally the two preparations, Fab' gamma mal and Fab' gamma SH, combined under conditions which allowed cross-linking of the maleimide and SH groups and avoided reoxidation of SH groups. The major product isolated from the reaction mixture after chromatography was always the F(ab' gamma)2 heterodimer (50 to 70%), other products being unreacted Fab' gamma and trace amounts of putative F(ab' gamma)3. Immunochemical analysis revealed that the thioether-linked F(ab' gamma)2 molecules were essentially all heterodimers, most of which had been joined via their Fd chains. The dual specificity of F(ab' gamma)2 heterodimers was tested functionally in three systems: 1) the combination (anti-idiotype + anti-phycoerythrin) linked L2C cells to the fluorochrome phycoerythrin, allowing fluorescence analysis; 2) the combination (anti-idiotype + anti-saporin) linked L2C cells to the ribosome-inactivating protein saporin, and transformed a subtoxic dose of saporin into a highly toxic mixture which prevented further protein synthesis by L2C cells; and 3) the combination of anti-idiotype with 3G8 (antibody to the Fc gamma receptor CD16) subjected L2C cells to cytotoxic attack by human mononuclear effectors.

Emergence of immunoglobulin variants following treatment of a B cell leukemia with an immunotoxin composed of antiidiotypic antibody and saporin.

The potency and specificity of immunotoxins consisting of monoclonal antiidiotype conjugated to the ribosome-inactivating protein, saporin, have been evaluated in the treatment of guinea pig L2C B lymphocytic leukemia. The immunotoxins were therapeutically much more effective than their parent antibodies. Their specificity reflected that of their antiidiotype component. Although the leukemia emerged eventually in most animals treated with these conjugates, most of the cells showed altered Ig expression, which rendered them resistant to the therapy. Commonly, the emerging cells had lost mu heavy chain production, leaving them negative for intracellular, surface, and secreted IgM, but still positive for lambda light chain production. In addition, a minor group of L2C variants was identified in a protocol designed to detect mutants at very low frequency: here the cells were exposed in vitro to immunotoxin and, while still viable as judged by dye-exclusion, inoculated in large numbers into animals. In tumor that emerged under these circumstances, the majority of cells were again immunoglobulin-negative; however a minority exhibited IgM with an altered idiotype (Idiotope-loss variants), rendering them unreactive with immunotoxin. Immunotherapy with unmodified anti-Id antibody alone does not reveal these variants, and we suggest it is the increased selective force exerted by the highly potent immunotoxins that allow these minor nonreactive populations to emerge.

Analysis of the interaction of antibodies with immunoglobulin idiotypes on neoplastic B lymphocytes: implications for immunotherapy.

We have examined the reactions of a panel of nine monoclonal anti-idiotype antibodies with the surface immunoglobulin in situ on guinea pig L2C leukemic lymphocytes. Equilibrium binding constants were shown to range between 10(7) and 10(8) M-1 for univalent Fab' gamma fragments and between 10(8) and 10(9) M-1 for intact IgG. Saturation of the cell surface binding sites was achieved with 2.9 X 10(5) Fab' gamma molecules/cell and 1.2 X 10(5) IgG molecules/cell for each antibody, a result that is consistent with a bivalent mode of interaction for the IgG. Despite these overall similarities in binding characteristics antibodies showed striking differences in their ability to clear Ig from the cell surface by antigenic modulation in vitro. This suggested differences in the readiness with which the antibodies cross-linked neighboring surface Ig molecules. Such an interpretation was supported by differences in the times required to achieve bivalent binding at 0 degree C, and in the rates at which labeled antibody dissociated from the cell surface in the presence or absence of an excess of unlabeled antibody. The data are consistent with there being two functionally distinct types of anti-idiotype antibody: those that form predominantly intra-Ig bridges, with each antibody Fab being linked to an Fab on one target molecule ("monogamous" binding) and not favoring modulation; and those that form predominantly inter-Ig bridges ("bigamous" binding) and favor modulation. The nature of interaction is presumably dictated by the orientation of the particular idiotope concerned. This distinction could be of great importance in the therapeutic use of anti-idiotype to ablate B cell neoplasms.

Characterisation of a new murine B cell lymphoma.

The characterisation of a new murine B cell lymphoma, A31, is described. Histopathological examination of passaged tumour indicates that initial infiltration occurs in the spleen, lymph nodes, Peyer's patches and liver, while in the terminal phase the bone marrow, gonads and occasionally the central nervous system become involved. The terminal spread is coincidental with the leukaemic phase in the tumour. The tumour cells show typical B cell characteristics in vitro. These include surface immunoglobulin (Ig) of mu, kappa isotype, surface Ia, Thy-1 negativity and an increased uptake of tritiated thymidine following incubation with lipopolysaccharide. A31 cells secrete low levels of IgM into the tissue culture fluid. Short-term culture produced only 100 ng IgM per 10(7) cells over 8 h and no tumour-associated monoclonal band could be detected in the serum of tumour-bearing mice. Chromosomal karyotypes of A31 cells gave model numbers 2n=40 normal, and 2n=41, with partial trisomy of chromosome 2, and trisomy of 17. There was loss of a chromosome 6 and the Y chromosome, together with the translocation of part of an 11 to one of the two unidentified marker chromosomes. The responses of lymphoma-bearing mice to therapeutic levels of cyclophosphamide and vincristine sulphate and also to whole body X-radiation are illustrated. This tumour may help in unravelling the complex biology of B cell lymphoma and because of its low level of Ig secretion, be of particular value in experimental immunotherapy.

Development of core competencies in clinical nutrition.

A nationwide survey of core competencies of nutrition knowledge and skills that primary care physicians should know and/or be able to do has been completed. Nutrition competencies were synthesized from a review of clinical nutrition practices as reported in the medical literature and by medical school faculty group discussions. A nutrition competency questionnaire was sent to 445 practicing physicians and to 752 department chairpersons in every US medical school in the disciplines of family practice, medicine, obstetrics and gynecology, pediatrics, psychiatry, and surgery. The overall response rate for practitioners and faculty combined was 46.03%. Of the 55 nutrition competencies, 28 items received very strong support (greater than 90% of respondents marked agree or completely agree). Twenty-two competencies received strong support (80 to 89% of respondents marked agree or completely agree), and five items received mixed support (40 to 79% of respondents marked agree or completely agree). Statistical comparisons of the item responses between practitioners and faculty were significantly different (p less than 0.05) on 19 (35%) of the items. It is anticipated that the 50 nutrition competencies that have received strong or very strong agreement among the 551 physicians representing primary care disciplines across the US will serve as guidelines for continued development of medical school curriculum and continuing medical education in clinical nutrition.