Membrane attachment of recombinant G-protein alpha-subunits in excess of beta gamma subunits in a eukaryotic expression system

Recombinant cDNAs encoding the alpha-subunits of Gi1, Gi2, Gi3, Go and Gs were transfected into COS cells with the pCD-PS mammalian expression vector. Expression of each G alpha was verified using subtype-specific peptide antisera on immunoblots. Quantitative immunoblotting of alpha and beta subunits indicated: i) that there was no change in expression of endogenous beta subunits, and ii) overexpression of alpha subunits could achieve a ratio of alpha:beta greater than 25:1. Despite the excess of alpha over beta, the G alpha subunits were found predominantly in the membrane fraction. The results demonstrate that G alpha subunits can attach to the membrane independently of beta gamma subunits.     

Caldesmon structure and function: sequence analysis of a 35 kilodalton actin- and calmodulin-binding fragment from the C-terminus of the turkey gizzard protein

We have determined the amino acid sequence of a 35 kDa proteolytic fragment ("CaD35") derived from the C-terminus of turkey gizzard caldesmon. This 239-residue peptide contains binding sites for actin and calmodulin. Residues 1-96 of CaD35 comprise "CaD15", an actin-binding subfragment which we previously showed to resemble the tropomyosin-binding segment of troponin T. The remainder of the CaD35 sequence shows no significant similarity to other proteins. Residues 111-128 may form a basic, amphipathic helix which interacts with calmodulin.     

Immunoliposomes with different acid sensitivities as probes for the cellular endocytic pathway

By combining dioleoylphosphatidylethanolamine (DOPE) with oleic acid (OA), palmitoylhomocysteine (PHC) or dipalmitoylsuccinylglycerol (DPSG) we have prepared pH-sensitive liposomes with different acid sensitivities. DOPE/OA liposomes are the most acid sensitive, while DOPE/DPSG liposomes are the least acid sensitive. Incubation of DOPE/OA liposomes with mouse L929 cells reduces the pH-sensitivity of these liposomes by altering the lipid composition. Using diphtheria toxin fragment A as a marker for cytoplasmic delivery, we find that the delivery kinetics of pH-sensitive immunoliposomes closely correlates with the modified acid sensitivities of the liposomes. Immunoliposomes encounter pH 6-6.2 with a t1/2 of 5-15 min after internalization. By contrast, acidification of the endosomes to pH 5.0 takes longer (t1/2 approximately 25 min). We also used a whole cell null point technique (Yamishiro and Maxfield (1987) J. Cell Biol. 105, 2713-2721) to directly determine the average pH encountered by the endocytosed immunoliposomes. We find that acidification determined by the null point method proceeds less rapidly than that estimated from DTA delivery data. This is likely due to the fact that the measured DTA delivery is done by those liposomes which first arrive at the endosomes with sufficient acidity. Our data suggests that DOPE/PHC immunoliposomes deliver at the early endosome while DOPE/DPSG immunoliposomes deliver at the late endosomes. The DOPE/OA immunoliposomes, with the altered composition and acid sensitivity, deliver with a kinetics intermediate between the other two immunoliposomes. Thus, pH-sensitive liposomes represent useful probes for studying the kinetics of endosome acidification.     

Common expression of a tumor necrosis factor resistance mechanism among gynecological malignancies

Summary The efficacy of tumor necrosis factor (TNF) as an anticancer agent is limited. This limitation might be related to the expression of a protein-synthesis-dependent resistance mechanism that prevents the lysis of tumor cells by TNF. To test this possibility eight randomly selected human cell lines, three derived from ovarian carcinomas and five derived from cervical carcinomas, were tested for their in vitro sensitivity to TNF-mediated lysis. The results of this analysis showed that all eight cell lines are normally resistant to lysis by TNF. However, in the presence of inhibitors of protein synthesis, seven of them showed a significant increase in TNF-mediated lysis. Measurement of protein synthesis showed that there is a linear correlation between the level of inhibition of protein synthesis and the level of TNF-mediated lysis. The fact that seven of eight randomly selected cell lines are resistant to TNF because they express a protein-synthesis-dependent resistance mechanism suggests that this mechanism of resistance may be common among gynecological cancers. The results also suggest that a therapy involving TNF and inhibitors of protein synthesis might be useful for the treatment of gynecological malignancies.     

Localization of 27 DNA markers to the region of human chromosome 22q11-pter deleted in patients with the DiGeorge syndrome and duplicated in the der22 syndrome

DiGeorge syndrome is a human developmental field defect with the pathological features of an abnormality of embryogenesis at 4 to 6 weeks of gestation. Cytogenetic analyses of patients have revealed a number of instances of monosomy 22q11-pter in this condition. We have analyzed 52 DNA markers that map to 22q11-pter and have found 27 that are deleted in DiGeorge syndrome patients with known monosomy for part of this region and that are duplicated in patients with the der22 syndrome. The set of clones mapping to the DiGeorge region was further assigned to a proximal or a distal location within the deletion.     

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44/66 (67%) of these clones cross-hybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested (n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of greater than 370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3-4 Mbp.     

Caseload or workload? Scoring complexity of operative procedures as a means of analysing workload.

OBJECTIVE--To present a more realistic assessment of surgical workload than that provided by a case count. DESIGN--Prospective study of all the operative procedures performed in one year, classified according to the British United Provident Association''s schedule of procedures and scored by the "intermediate equivalent" value (taking the recommended fee value of an intermediate operation as 1.0) compared with the number of operations performed. SETTING--General surgical unit of Taunton and Somerset Hospital, comparing four consultant surgeons and their teams. PATIENTS--Inpatients and day patients admitted under the care of general surgeons during 1989. MAIN OUTCOME MEASURE--Difference between the apparent workload represented by simple case counting (caseload) and the actual workload represented by calculation of the total "intermediate equivalent" value. RESULTS--The workload assessed in terms of intermediate equivalent values was greater than that suggested by case counting for complex operations (12% v 4%), operations at the district hospital (82% v 74%), and operations performed by consultants (53% v 35%) and was lower for minor operations (20% v 42%), operations at the community hospitals (18% v 26%), and operations performed by surgeons in training grades and clinical assistants (47% v 66%). CONCLUSIONS--The use of the intermediate equivalent values as an indicator of complexity allows a more realistic assessment of the operative workload than a simple case count of the number of different operations and is recommended for comparing workload in different hospitals and departments.     

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins.