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The synthesis and pharmacological properties of imidazobenzodiazepines with both high affinity and selectivity for alpha 5-containing GABAA receptors are described. Four of these compounds (5, 6, 8, and 9) inhibited [3H]flunitrazepam binding to recombinant alpha 5 beta 2 gamma 2 GABAA receptors with IC50 values between approximately 0.4 and 5 nM. These compounds were > or = 24-75-fold more selective for recombinant receptors containing alpha 5 subunits compared to other, "diazepam-sensitive" GABAA receptors containing either alpha 1, alpha 2, or alpha 3 subunits. Imidazobenzodiazepine 9 (used as the prototypical alpha 5 selective ligand) inhibited [3H]flunitrazepam binding to hippocampal membranes with high- and low-affinity components (IC50 0.6 +/- 0.2 and 85.6 +/- 13.1 nM, respectively), representing approximately 16% and approximately 84% of the receptor pool. Inhibition of [3H]flunitrazepam binding to cerebellar membranes with imidazobenzodiazepine 9 was best fitted to a single population of sites with an IC50 of 79.8 +/- 18.3 nM. These imidazobenzodiazepines behaved as GABA negative ligands in recombinant GABAA receptors expressed in Xenopus oocytes and were convulsant in mice after parenteral administration. The relative potencies of flumazenil and zolpidem in blocking convulsions induced by 9 and DMCM, respectively, indicated that occupation of alpha 5-containing GABAA receptors substantially contributed to the convulsant properties of acetylene analog 9. These 8-substituted imidazobenzodiazepines (5, 6, 8 and 9) should prove useful in examining the physiological roles of GABAA receptors bearing an alpha 5 subunit and may also lead to the development of novel, subtype selective therapeutic agents.

Spectrin is a major structural protein associated with the cytoplasmic surface of plasma membranes of many types of cells. To study the functions of spectrin, we transfected Caco-2 intestinal epithelial cells with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represented only a small fraction of neomycin-resistant cells, but they could be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibited a progressive decrease in amounts of endogenous beta G-spectrin, although alpha-spectrin was still present. Beta G-spectrin-deficient cells lost epithelial cell morphology, became multinucleated, and eventually disappeared after 10-14 d in culture. Spectrin-associated membrane proteins, ankyrin and adducin, as well as the Na+,K(+)-ATPase, which binds to ankyrin, exhibited altered distributions in cells transfected with beta G-spectrin domains. E-cadherin and F-actin, in contrast to ankyrin, adducin, and the Na+,K(+)-ATPase, were expressed, and they exhibited unaltered distribution in beta G-spectrin-deficient cells. Cells transfected with the same plasmid encoding beta-galactosidase alone survived in culture as the major population of neomycin-resistant cells, and they exhibited no change in morphology or in the distribution of spectrin-associated membrane proteins. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture.

The complete primary structure of the general form of human beta-spectrin (beta G) has been deduced from cDNAs isolated from human brain. beta G-Spectrin is encoded by a gene located on human chromosome 2. beta G-Spectrin and erythrocyte beta-spectrin (beta R) share identical domain organization, with sequence identity of 60% and sequence similarity of 77%. beta-Spectrins have closely related N-terminal domains implicated in binding to actin, and 17 copies of a 106-residue repeat motif with consensus residues that are highly conserved between beta-spectrins as well as alpha-spectrins. C-terminal domains of beta G and the 270-kDa beta R-spectrins are candidate regions to associate with alpha-spectrin, and exhibit 75% similarity. beta G- and beta R-spectrins exhibit different patterns of expression in tissues and follow different developmental programs in those tissues where they are co-expressed. beta G-Spectrin is present in all tissues examined except for erythrocytes, while beta R-spectrin could be detected only in erythrocytes, brain, and heart. beta G- and beta R-Spectrins are both expressed in brain, but beta R appeared later in post-natal development and was highly enriched in cerebellum in contrast to the broad regional distribution of beta G-spectrin. beta-Spectrins are likely to perform related but distinct functions, with beta G in a general, constitutive role and beta R-spectrin involved in more specialized activities of differentiated cells.

This report demonstrates that specific proteolysis of brain spectrin by a calcium-dependent protease, calpain I, abolishes association of brain spectrin with the ankyrin-independent binding site(s) in brain membranes. Calpain I cleaves the beta subunit of spectrin at the N-terminal end leaving a 218-kDa fragment and cleaves the alpha subunit in the midregion to produce 150- and 130-kDa fragments. Calpain-proteolyzed spectrin almost completely loses the capacity to displace binding of intact spectrin to membranes. Spectrin digested by calpain I under conditions that almost completely destroyed membrane-binding remained associated as a tetramer and retained about 60% of the ability to associate with actin filaments. Cleavage of spectrin occurred at sites distinct from the membrane-binding site which is located on the beta subunit since the isolated 218-kDa fragment of the beta subunit as well as a reconstituted complex of alpha and 218-kDa beta subunit fragment partially regained binding activity. Moreover, cleavage of the alpha subunit alone reduced the affinity of spectrin for membranes by 2-fold. A consequence of distinct sites for calpain I cleavage and membrane-binding is that calpain I can digest spectrin while spectrin is complexed with other proteins and therefore has the potential to mediate disassembly of a spectrin-actin network from membranes.

Involvement of adult Ixodes persulcatus ticks in the transmission of Lyme disease in Hailin County, Heilongjiang Province, China, is reported. In 1986 from April through August adult I. persulcatus was the dominant tick in this endemic area with an infection rate of 43% for the Lyme disease spirochaete, Borrelia burgdorferi. The incidence of Lyme disease cases presenting the symptom of erythema chronicum migrans (ECM) within this area was correlated with the seasonal abundance of adult I. persulcatus and the number of people bitten by ticks. The frequency of ECM formation in all age groups varied and was associated with the frequency of tick bites. In addition, a strain of B. burgdorferi was isolated from a pool of six female I. persulcatus collected from this area. We demonstrate that the seasonal abundance of adult I. persulcatus and its frequent attachment to humans result in the spring and summer transmission of Lyme disease in this endemic area. The role of immature I. persulcatus in Lyme disease transmission is apparently minimal.

Pathology and prognosis of 378 cases of IML collected from 28 colleges or hospitals, are reported. The ratio of male and female was 2:1. Ages ranged from 3 to 86 years with a peak age of 21-50. Main symptoms were abdominal pain, mass, intestinal obstruction, diarrhea and intestinal bleeding. The tumor occurred, in order of incidence, in small intestine (42%), ileocecal region (30%), colon (13%), cecum (8%), rectum (3%), duodenum (2%) and anorectum (1%). Multifoci were found in 43 cases (11%). Grossly, the tumor was divided into three types: exophytic, ulcerative and diffuse infiltrative. In histology, follicular type comprised 4% (16 cases) and diffuse type, 96% (362 cases). In the latter, the majority was cleaved, non-cleaved or mixed cell type originating from the follicular center cells. There were a few T cell lymphomas and true histiocytic types but no Hodgkin's lymphoma. Statistically, the prognosis was significantly related to the depth of invasion, gross type, tumor size and stage. By histology, lympho-plasmacytoid type and small lymphocytic type had better prognosis compared with the other diffuse types. In this series, 83 patients were treated by surgery (including operation plus chemotherapy). The survival rate of less than 1 year was 51%, nearly equal to the survival rate of more than 1 year (49%). 10 patients have survived more than 5 years and 6, more than 10 years, 2 of them, more than 18 years. It indicates that the operation plays an important role for prolonging survival. Early detection, early diagnosis and early operation are necessary.