Assembly of productive T cell receptor delta variable region genes exhibits allelic inclusion.

The generation of a productive "in-frame" T cell receptor beta (TCR beta), immunoglobulin (Ig) heavy (H) or Ig light (L) chain variable region gene can result in the cessation of rearrangement of the alternate allele, a process referred to as allelic exclusion. This process ensures that most alphabeta T cells express a single TCR beta chain and most B cells express single IgH and IgL chains. Assembly of TCR alpha and TCR gamma chain variable region genes exhibit allelic inclusion and alphabeta and gammadelta T cells can express two TCR alpha or TCR gamma chains, respectively. However, it was not known whether assembly of TCR delta variable regions genes is regulated in the context of allelic exclusion. To address this issue, we have analyzed TCR delta rearrangements in a panel of mouse splenic gammadelta T cell hybridomas. We find that, similar to TCR alpha and gamma variable region genes, assembly of TCR delta variable region genes exhibits properties of allelic inclusion. These findings are discussed in the context of gammadelta T cell development and regulation of rearrangement of TCR delta genes.

Antigen-independent appearance of recombination activating gene (RAG)-positive bone marrow B cells in the spleens of immunized mice.

Splenic B lineage cells expressing recombination activation genes (RAG(+)) in mice immunized with 4-hydroxy-3-nitrophenyl-acetyl coupled to chicken gamma-globulin (NP-CGG) and the adjuvant aluminum-hydroxide (alum) have been proposed to be mature B cells that reexpress RAG after an antigen encounter in the germinal center (GC), a notion supported by findings of RAG expression in peripheral B lymphocyte populations activated in vitro. However, recent studies indicate that these cells might be immature B cells that have not yet extinguished RAG expression. Here, we employ RAG2-green fluorescent protein (GFP) fusion gene knock-in mice to show that RAG(+) B lineage cells do appear in the spleen after the administration of alum alone, and that their appearance is independent of T cell interactions via the CD40 pathway. Moreover, splenic RAG(+) B lineage cells were detectable in immunized RAG2-deficient mice adoptively transferred with bone marrow (BM) cells, but not with spleen cells from RAG(+) mice. Although splenic RAG(+) B cells express surface markers associated with GC B cells, we also find the same basic markers on progenitor/precursor BM B cells. Finally, we did not detect RAG gene expression after the in vitro stimulation of splenic RAG(-) mature B cells with mitogens (lipopolysaccharide and anti-CD40) and cytokines (interleukin [IL]-4 and IL-7). Together, our studies indicate that RAG(+) B lineage cells from BM accumulate in the spleen after immunization, and that this accumulation is not the result of an antigen-specific response.

Substandard medicines in resource-poor settings: a problem that can no longer be ignored.

The circulation of substandard medicines in the developing world is a serious clinical and public health concern. Problems include under or over concentration of ingredients, contamination, poor quality ingredients, poor stability and inadequate packaging. There are multiple causes. Drugs manufactured for export are not regulated to the same standard as those for domestic use, while regulatory agencies in the less-developed world are poorly equipped to assess and address the problem. A number of recent initiatives have been established to address the problem, most notably the WHO pre-qualification programme. However, much more action is required. Donors should encourage their partners to include more explicit quality requirements in their tender mechanisms, while purchasers should insist that producers and distributors supply drugs that comply with international quality standards. Governments in rich countries should not tolerate the export of substandard pharmaceutical products to poor countries, while developing country governments should improve their ability to detect substandard medicines.

Left ventricular performance and coronary flow after coronary embolization with plastic microspheres.

Coronary flow, left ventricular circumference, and left ventricular pressure were observed in the isovolumically contracting, isolated canine heart supported with arterial blood from a donor. Systolic pressure, heart rate, and coronary perfusion pressure were held constant while the coronary bed was progressively embolized with either large (average 865 mu) or small (average 10 mu) polystyrene microspheres. During embolization with large microspheres, coronary flow diminished progressively. After sufficient embolization, decreased ventricular performance was indicated by a rise in end-diastolic pressure. During embolization with small microspheres, coronary flow initially increased, which suggests the effective release of a vasodilator substance. Return of coronary flow to control levels occurred only after the end-diastolic pressure rose, on the average, to above 30 mm Hg. After embolization with both sizes of microspheres, ventricular diastolic pressure-volume relationships showed decreased ventricular compliance. This was attributed, in part, to edema of the ventricular wall and, in part, to focal shortening of the sarcomeres where the circulation was compromised. Embolization with both sizes of microspheres ultimately caused a decrease in ventricular performance, although when the systolic pressure was increased the usual relationship between peak developed wall stress, and end-diastolic pressure showed less of a descending limb than that found in the nonembolized, isolated heart. It is felt that the data summarized above have bearing on ventricular performance and coronary flow in clinical situations where hearts are perfused through pump oxygenator systems and are thereby subject to embolization from aggregated clumps of platelets and fibrin.

The Anrep effect reconsidered.

Evidence is presented supporting the hypothesis that the positive inotropic effect after an abrupt increase in systolic pressure (Anrep effect) is the recovery from subendocardial ischemia induced by the increase and subsequently corrected by vascular autoregulation of the coronary bed. Major evidence consists of data obtained from an isolated heart preparation showing that the Anrep effect can be abolished with coronary vasodilation, and that with an abrupt increase in systolic pressure there is a significant reduction in the distribution of coronary flow to subendocardial layers of the ventricle. Furthermore, the intracardiac electrocardiogram shows S-T segment and T wave changes after an abrupt increase in ventricular pressure similar to that noted after coronary constriction. Major implications are that normally there may be ischemia of the subendocardial layers tending to reduce myocardial contractility which may account, in part, for the positive inotropic effect of various coronary vasodilators; that with an abrupt increase in ventricular pressure the subendocardium is rendered temporarily ischemic, placing the heart in jeopardy from arrhythmias until this is corrected; and that end-diastolic pressure and the intracardiac electrocardiogram may provide a means of evaluating the adequacy of circulation to subendocardial layers in diseased ventricles when systolic pressure is abruptly increased.

Identification of novel M phase phosphoproteins by expression cloning.

Using an expression cloning technique, we isolated cDNAs for eight M phase phosphoproteins (MPPs 4-11). We then used affinity-purified antibodies to fusion proteins to characterize the intracellular localization and some biochemical properties of these proteins and two others that we identified previously (MPPs 1-2). Each antibody immunoprecipitated one or two protein species of a characteristic size ranging from 17,000 to 220,000 Da. Each MPP, when immunoprecipitated from lysates of M phase cells, was reactive with MPM2, a monoclonal antibody that recognizes a group of related M phase phosphorylation sites, including F-phosphoT-P-L-Q. This reactivity indicated that all the MPPS encoded genuine M phase phosphoproteins. When antibodies to the MPPS were used for immunofluorescence microscopy, each anti-MPP gave a characteristic pattern of localization. In interphase, several of the MPPs were nuclear proteins, whereas others were cytoplasmic or distributed throughout the cell. Three MPPS were strikingly localized to interphase structures: MPP7 to centers of DNA replication, MPP9 to the Golgi complex, and MPP10 to nucleoli. In mitosis, most of the MPPs were distributed throughout the cells. Further studies of the 10 MPPs, most of which are previously undescribed, are expected to provide new understandings of the process of cell division.

The major form of the murine asialoglycoprotein receptor: cDNA sequence and expression in liver, testis and epididymis.

Northern blot analysis of poly(A)+RNAs isolated from mouse liver or mouse testis (Te)/epididymis (Ep) reveals that both tissues express 1.5- and 7.5-kb transcripts which have extensive homology to the major form of the rat asialoglyco-protein receptor (ASGP-R). In situ hybridization studies have localized the expression of this ASGP-R-like transcript to late-stage sperm from Te and Ep of several different strains of mice. Swiss Webster mice express this ASGP-R-like transcript in late-stage spermatids at the time of release into the seminiferous tubule and in Ep sperm, while Balb/C, NIH Swiss and C57Bl/6 mice express this ASGP-R-like transcript predominantly in Ep sperm. cDNAs containing the entire coding region for this ASGP-R-like transcript have been cloned from mouse liver and mouse Te/Ep. These cDNAs are 100% identical in the coding region and 3'-untranslated region (UTR), but differ in the 5'-UTR. The gene encoding these cDNAs is called MHL-1, designating the major form of the mouse ASGP-R. The deduced amino acid (aa) sequence of MHL-1 shares 88% homology to the rat hepatic (He) lectin form 1 (RHL-1) and 78% homology to the human asialoglycoprotein receptor form 1 (H1). The three sites for N-linked glycosylation in the RHL-1 sequence are all conserved in the deduced MHL-1 sequence. Taken collectively, these data describe the cloning and sequencing of the MHL-1 cDNA and illustrate its deduced aa homology to RHL-1 and H1.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the "hepatic" asialoglycoprotein receptor in rat late-stage spermatids and epididymal sperm.

Northern blot analysis of rat testicular (Te) poly(A)+RNA reveals that a transcript homologous to the major form of the asialoglycoprotein receptor (ASGP-R), designated RHL-1, is expressed as early as one week postnatally and that steady-state levels are approx. 8-times higher in the Te of an 8-week-old rat (sexually mature) as compared to an 84-week-old rat (aged). Partial cDNAs encoding RHL-1 and the minor form of the ASGP-R, designated RHL-2/3, have been cloned from two rat Te/epididymal (Ep) cDNA libraries and rat Te poly(A)+RNA. Sequence analysis of the Te/Ep RHL-1 cDNA and the Te/Ep RHL-2/3 cDNA indicates that these cDNAs are identical to the forms expressed in rat liver. Western blot analysis demonstrates the presence of a 49-kDa Te/Ep RHL-1-related protein band and a 54-kDa Te/Ep RHL-2/3-related protein band in both rat Te membrane fractions (MF) and rat Ep sperm MF. The RHL-1-related protein has been localized to late-stage Te spermatids at the time of release from the seminiferous tubules and to Ep sperm in the region of the sperm tail, referred to as the middle piece. Taken collectively, these data indicate that the authentic RHL-1 and RHL-2/3 genes of the ASGP-R are expressed in late-stage spermatids; however, the Te/Ep RHL-1-related protein differs in size from the hepatic RHL-1 polypeptide, possibly indicating a specific function of the RHL-1-related protein in spermatogenesis.

The cDNA sequence and characterization of the Ca2+/calmodulin-dependent protein kinase-Gr from human brain and thymus.

We have isolated and sequenced cDNAs encoding Ca2+/calmodulin-dependent protein kinase type Gr (CaM-K-Gr, also called CaM-K-IV) from human brain and thymus. The sequence of the protein coding region of the cDNA is identical in both brain and thymus, although Northern hybridization analysis shows variation of the mRNA transcripts in these tissues. The sequence predicts a protein of M(r) 51,897 that is 83.7% identical and shows 89.2% similarity with the rat homologue. The deduced human CaM-K-Gr is identical to the rat and mouse proteins in the portion of the enzyme involved in ATP binding, the catalytic domain and Ca2+/calmodulin-binding domain; however, the N terminus of the human kinase, which may comprise a second regulatory domain [McDonald et al., J. Biol. Chem. 268 (1993) 10054-10059], contains a 4-amino-acid (aa) insertion relative to the rodent enzymes. Additionally, the C-terminal association domain shows only 45.2 and 41.6% identity with the rat and mouse proteins, respectively, suggesting that this domain is not constrained by stringent structural and functional requirements. Based on the predicted aa sequence of the human kinase, we produced polyclonal antisera against a C-terminal peptide that recognizes two forms of CaM-K-Gr in human T-cell lymphoma and neuroblastoma cell lines. The human antiserum cross-reacts with the rat and mouse proteins and immunoprecipitates the active kinase.

Maintenance electroconvulsive therapy.

Maintenance electroconvulsive therapy remains an infrequently used and insufficiently researched treatment for the prevention of relapse and recurrence of affective illnesses. The foundation for this treatment is examined by reviewing the relevant scientific literature, with particular attention to identifying those patients who may benefit most from this form of therapy, as well as establishing standard techniques of application. A critical assessment of current research is made, and areas for future investigation are proposed.