Large Lung Volumes Delay the Onset of the Physiological Breaking Point During Simulated Diving.

During breath holding after face immersion there develops an urge to breathe. The point that would initiate the termination of the breath hold, the "physiological breaking point," is thought to be primarily due to changes in blood gases. However, we theorized that other factors, such as lung volume, also contributes significantly to terminating breath holds during face immersion. Accordingly, nine naïve subjects (controls) and seven underwater hockey players (divers) voluntarily initiated face immersions in room temperature water at Total Lung Capacity (TLC) and Functional Residual Capacity (FRC) after pre-breathing air, 100% O2, 15% O2 / 85% N2, or 5% CO2 / 95% O2. Heart rate (HR), arterial blood pressure (BP), end-tidal CO2 (etCO2), and breath hold durations (BHD) were monitored during all face immersions. The decrease in HR and increase in BP were not significantly different at the two lung volumes, although the increase in BP was usually greater at FRC. BHD was significantly longer at TLC (54 ± 2 s) than at FRC (30 ± 2 s). Also, with each pre-breathed gas BHD was always longer at TLC. We found no consistent etCO2 at which the breath holding terminated. BDHs were significantly longer in divers than in controls. We suggest that during breath holding with face immersion high lung volume acts directly within the brainstem to actively delay the attainment of the physiological breaking point, rather than acting indirectly as a sink to produce a slower build-up of PCO2.

The abortive infection of Syrian hamster cells with human adenovirus type 12.

Human adenovirus type 12 (Ad12) induces undifferentiated tumors in newborn Syrian hamsters, and this tumor model has been investigated in detail in our laboratory. One of the characteristics of the Ad12-hamster cell system is a strictly abortive infection cycle. In this chapter, we summarize previous and more recent results of studies on the interaction of Ad12 with the nonpermissive BHK21 hamster cell line. The block of Ad12 replication lies before viral DNA replication and late gene transcription which cannot be detected with the most sensitive techniques. Ad12 adsorption, cellular uptake and transport of the viral DNA to the nucleus are less efficient in the nonpermissive hamster cells than in permissive human cells. However, most of the early functions of the Ad12 genome are expressed in BHK21 cells, though at a low level. In the downstream region, the first exon, of the major late promoter (MLP) of Ad12 DNA, a mitigator element of 33 nucleotide pairs in length has been identified which contributes to the inactivity of the MLP in hamster cells and its markedly decreased activity in human cells. The E1 functions of Ad2 or Ad5 are capable of partly complementing the Ad12 deficiencies in hamster cells in that Ad12 viral DNA replication and late gene transcription can proceed, e.g. in a BHK hamster cell line, BHK297-C131,which carries in an integrated form and constitutively expresses the E1 region of Ad5 DNA. Nevertheless, the late Ad12 mRNAs, which are synthesized in this system with the authentic nucleotide sequence, fail to be translated to structural viral proteins. Hence, infectious virions are not produced in the partly complementing system. Probably there is also a translational block for late Ad12 mRNAs in hamster cells. We have recently shown that the overexpression of the Ad12 preterminal protein (pTP) gene or of the E1A gene facilitates the synthesis of full-length, authentic Ad12 DNA in BHK21 cells infected with Ad12. Apparently the pTP has a hitherto unknown function in eliciting full cycles of Ad12 DNA replication even in nonpermissive BHK21 cells when sufficient levels of Ad12 pTP are produced. We pursue the possibility that the completely abortive infection cycle of Ad12 in hamster cells ensures the survival of Ad12-induced hamster tumor cells which all carry, integrated in their genomes, multiple copies of Ad12 DNA. In this way, the viral genomes are immortalized and expanded in a huge number of tumor cells.

Cellular and early viral factors in the interaction of adenovirus type 12 with hamster cells: the abortive response.

The interaction of human adenovirus type 12 (Ad12) with Syrian hamster cells is remarkable in that there is a block of viral DNA replication and late gene transcription. We have screened several cellular factors known to play a role in adenovirus replication for their possible contributions to the interactions of Ad12 in the abortive BHK21 hamster cell system. (1) Western blot analyses of total protein extracts from Ad12- or Ad2-infected BHK21 cells do not reveal a significant difference in the accumulation of NFIII protein at different times after infection. Transcriptional levels of the NFIII gene in BHK21 cells are not altered upon the abortive infection with Ad12 or the productive infection with Ad2. The amount of NFIII protein is markedly reduced in nuclear extracts from BHK21 cells as compared with extracts from C131 hamster cells or human HeLa cells. A presumptive defect in NFIII transport to the nuclei rather than overall reduced NFIII gene transcription might explain the low abundance of NFIII in the nuclei of uninfected or Ad12-infected BHK21 cells. The productive infection of BHK21 or C131 cells with Ad2 leads to an increase in the NFIII concentration in the nuclei of infected cells, late after infection to a decrease; (2) NFI levels in the nuclei of mock-infected or Ad2- or Ad12-infected BHK21 cells are comparable with those in HeLa or in C131 cells. Thus, deficiencies in NFI may not play a role in the abortive system; (3) The absence of morphological alterations in PML protein domains from globular to track-like structures in the nuclei of Ad12-infected hamster cells correlates with the inability of Ad12 DNA to replicate in BHK21 cells. In BHK21 cells, the E4-ORF3 of Ad12 DNA is only weakly transcribed and only small amounts of the gene product are synthesized. In Ad12-infected C131 cells, which allow the replication of Ad12 DNA, the E4-ORF3 of Ad12 DNA is expressed, and track-like PML protein structures are observed. Transfection of the 12-E4-ORF3-EGFP construct leads to the expression of both the green fluorescent protein (GFP) and of the 12-E4-ORF3 gene product in 20-30% of the transfected BHK21 cells and elicits the morphological reorganization of the PML protein structures in the successfully transfected BHK21 cells. Similar results are obtained upon transfection of the 2-E4-ORF3 construct. Untransfected cells or cells transfected with the empty pIRES2-pEGFP vector carry the globular PML protein phenotype; (4) The expression of the 12-E4-ORF3-EGFP and/or of the NFIII-EGFP constructs upon transfection following Ad12-infection of BHK21 cells fails to promote Ad12 DNA replication. Hence, the formation of track-like PML protein structures in BHK21 cells by itself is not a sufficient precondition for Ad12 DNA replication in this abortive system. The data demonstrate that the expression of NFI, NFIII, and/or the conversion of the PML domains do not suffice to elicit Ad12 DNA replication in the abortive hamster cell system.

Overexpression of the adenovirus type 12 (Ad12) pTP or E1A gene facilitates Ad12 DNA replication in nonpermissive BHK21 hamster cells.

In the adenovirus type 12 (Ad12) hamster cell system, abortive virus infection is one of the factors associated with the highly efficient oncogenesis in newborn Syrian hamsters. We have shown earlier that the replication and efficient late transcription of the Ad12 genome are blocked in Syrian hamster cells. Some of the early Ad12 functions are transcribed in these cells, although at a minimal rate. In the present study, we demonstrate that low expression levels of the E1A and precursor to terminal protein (pTP) genes of Ad12 seem to be responsible for the lack of Ad12 DNA replication in hamster cells. The Ad12 genes for the E1A functions or for pTP were tethered to the strong early promoter of the human cytomegalovirus and transfected into BHK21 cells. Subsequently, these cells were infected with Ad12 virions. In Ad12-infected BHK21 cells, which overexpressed pTP or E1A, full-length Ad12 DNA was de novo synthesized, as documented by metabolic labeling with [3H]thymidine and by zone velocity sedimentation in alkaline sucrose gradients followed by gel electrophoresis of the 3H-labeled DNA and Southern blot hybridization to 32P-labeled Ad12 DNA. Transfection of the cloned E1A region of Ad2 yielded similar results. The newly synthesized Ad12 DNA was covalently linked to pTP. The Ad12 DNA binding protein (DBP) and DNA polymerase (pol) genes were transcribed at levels similar to those in merely Ad12-infected cells. In pTP or E1A gene-transfected and Ad12-infected BHK21 cells, marginal levels of late Ad12 mRNA were detectable. Late Ad12 proteins were, however, not synthesized. Apparently, Ad12 DNA replication in hamster cells is rendered impossible due to insufficient threshold levels of the viral E1A and/or pTP.

Dynamic nuclear localization of the baculovirus proteins IE2 and PE38 during the infection cycle: the promyelocytic leukemia protein colocalizes with IE2.

The early gene products IE2 and PE38 of Autographa californica multicapsid nuclear polyhedrosis virus localize to distinct nuclear domains after transient expression. Here, the nuclear localization pattern and the putative association with cellular proteins have been determined during virus infection to shed light on the functional significance of the nuclear domains. IE2 was always localized to distinct nuclear structures while PE38 was partly present in nuclear dots. Confocal imaging indicated colocalization of PE38 and IE2 to common domains, prominently at 2 h p.i. The nuclear dot localization of PE38 in infected cells was different from that in transfected cells. Hence, we have performed cotransfection experiments that suggested that a viral factor influences the nuclear distribution. Since the promyelocytic leukemia protein (PML) that localizes to distinct nuclear multiprotein complexes termed ND10/PODs in mammalian cells functions as a target for some immediate early viral proteins, we have investigated whether baculovirus proteins act similarly. Transiently expressed IE2 and PE38 were found to be associated with endogenous PML in the mammalian cell line BHK21. Infection with a recombinant virus that expresses the human pml gene in insect cells reveals IE2 and PML to be colocalized during the early phase of infection followed by a redistribution of both proteins. Taken together our results provide first evidence that the early baculovirus protein IE2 associates at least with one component of mammalian PODs during virus infection, suggesting that POD-like structures can be formed in insect cells.

[Self-mutilation in men--injury pattern and motivation]. / Selbst beigebrachte Verletzungen bei Männern--Verletzungsmuster und Motivation.

6 cases of self-inflicted injuries in male individuals are reported. The age of the affected men was between 15 and 46 years whereas the younger age predominated. Alleged incidents were robberies in 3 cases, rape in one case, violation in custody in one case and an assault originating in personal motives in one case. In 4 cases, the typical injury pattern of self-infliction was present showing parallel course and superficiality of the wounds in areas accessable by the persons's own hands. In 2 cases, atypical injuries (i.e. deep cuts and massive signs of strangulation respectively) were found. However, in most cases, the underlying motive was to gain affection. An autoerotic accident was tried to be disguised in one case. Profit was the leading motive in another case.

The human 20-kDa 5'-(CGG)(n)-3'-binding protein is targeted to the nucleus and affects the activity of the FMR1 promoter.

Previous reports have described the human DNA CGG repeat-binding protein (CGGBP1 or p20), which binds specifically to nonmethylated, but not to methylated, 5'-(CGG)(n)-3' repeats in the promoter of the fragile X mental retardation 1 (FMR1) gene. The results of transfection experiments into human HeLa cells using a p20-green fluorescent protein fusion construct indicate that the p20 protein is targeted to the nucleus. By deletion analyses, a nuclear localization signal has been found between amino acids 80 and 84. Deletions between amino acids 69 and 71 and between 95 and 167 interfere with 5'-(CGG)(n)-3' binding. The results of electrophoretic mobility shift assays using DNA with 5'-(CGG)(n)-3' repeats of different lengths render it likely that oligomers of the p20 protein bind to the repeat. In cotransfection experiments, the activity of the FMR1 promoter is reduced by the presence of p20. Upon transfection of the p20 cDNA construct into HeLa cells, transcription of the endogenous FMR1 gene is decreased. The green fluorescent protein-p20 fusion protein associates preferentially with the telomeres of the short arms of human chromosomes 13, 14, 15, 21, and 22. Their telomeres carry the genes for the 28 S rRNA, which contain 5'-(CGG)(n)-3' repeats. The translated region of the p20 gene from three healthy, five fragile X syndrome, and five premutation-carrying individuals has been sequenced, but mutations have not been detected.

Adenovirus type 12 DNA firmly associates with mammalian chromosomes early after virus infection or after DNA transfer by the addition of DNA to the cell culture medium.

Human adenovirus type 12 (Ad12) infects human cells productively and leads to viral replication, whereas infection of hamster cells remains abortive, with total blocks in viral DNA replication and late viral gene transcription. The intranuclear fate of Ad12 DNA in productively infected human cells and in abortively infected hamster cells was monitored by using the fluorescent in situ hybridization (FISH) technique. Human HeLa cells, primary human umbilical cord fibroblasts, hamster BHK21 cells, primary embryonal hamster cells, and the Ad12-transformed T637 hamster cell line were studied. As early as 2 h after infection, extensive association of Ad12 DNA with metaphase chromosomes was demonstrated by FISH in all of these cells. Chromosomal association continued until late (24 to 28 h) after infection, when about 100% of the human cell nuclei and 70 to 80% of the hamster cell nuclei showed distinct FISH signals. This chromosomal association of Ad12 DNA in infected cells seemed to be rather firm, since it proved to be resistant to mechanically stretching the chromosomes and to different types of chemical treatment. Moreover, laser scan microscopy of mechanically stretched chromosomes from Ad12-infected HeLa cells and from the Ad12-transformed T637 cell line, with about 20 copies of Ad12 DNA provably integrated, revealed identical FISH patterns. Therefore, it was likely that even in infected cells the chromosomal association of Ad12 DNA was very similar to the integrated state. Late in productively infected cells, large nuclear areas were taken over by viral DNA replication, as visualized by FISH in interphase nuclei. Chromosomal association at many sites was frequently limited to one chromatid, but signals in adjacent positions on both chromatids were also seen. Upon the long-term cultivation and passage of abortively infected BHK21 cells for 96 h after infection, a gradual decrease of viral DNA association with chromosomes was observed. Integration of Ad12 DNA in hamster cells early after infection was previously documented, and recombination between viral and cellular DNAs in human cells was also shown. The FISH data on extensive chromosomal association of Ad12 DNA suggest a means to study the pathway of Ad12 DNA from early steps in viral infection via chromosomal interactions to integration events. In a different approach, Ad12 DNA, Ad12 DNA with the terminal protein covalently linked to its ends (Ad12 DNA-TP), or Ad2 DNA was simply added to the culture medium of HeLa or BHK21 cells. Precipitation or selection procedures were avoided. Depending on the experimental conditions, up to 25 to 30% of the interphase nuclei of HeLa cells and 9 to 19% of the interphase nuclei of BHK21 cells showed positive FISH signals at 24 h after the addition of DNA. Viral DNA also became associated in some cases with both chromatids. The uptake of Ad12 DNA-TP appeared to be 10 to 20 times more efficient than that of Ad12 DNA completely freed of proteins. Control bacteriophage lambda, M13, or plasmid DNA could not be detected in the nuclei under these conditions.

The structure of adenovirus type 12 DNA integration sites in the hamster cell genome.

Foreign DNA can integrate into the genomes of mammalian cells, and this process plays major roles in viral oncogenesis and in the generation of transgenic organisms and will be important in evolving regimens for human somatic gene therapy. In the present study, the insertion sites of adenovirus type 12 (Ad12) DNA genomes have been analyzed in detail in the Ad12-transformed hamster cell line T637, its revertants, which have lost most of the >20 Ad12 genome equivalents integrated chromosomally in cell line T637, and in the Ad12-induced tumor T191. Some of these junction sites have been molecularly cloned, and the nucleotide sequences at the sites of transition between viral and cellular DNAs have been determined. The sites of linkage between the hamster cellular and the foreign (viral) DNA are characterized by the frequent occurrence of patch homologies between the recombination partners. The cellular junction sites investigated here are not transcriptionally active. One of the cellular DNA sequences abutting the right Ad12 DNA terminus in cell line T637 (os2) is represented only once in the hamster genome and has a strikingly low abundance of 5'-CG-3' dinucleotide sequences. One 5'-GCGC-3' sequence close to the Ad12 DNA integration site is heavily methylated in normal cells, Ad12-transformed cells, and Ad12-induced tumor cells. The second such sequence is more remote from the junction site, is partly methylated in BHK21 hamster cells, and shows differences in methylation in different Ad12-transformed cell lines. This site is unmethylated in liver DNA. The cellular DNA sequence at the site of Ad12 linkage in the tumor T191 exhibits homologies to highly repetitive sequences of the Alu family and to an origin of hamster DNA replication containing an Alu element. A number of junction sites between Ad12 DNA and hamster or mouse DNA in Ad12-transformed cell lines or Ad12-induced tumor cell lines, investigated here and previously, are characterized by stem-loop structures encompassing the junction sites.

A general assay for antibody catalysis using acridone as a fluorescent tag.

A simple and highly sensitive catalysis assay is demonstrated based on analyzing reactions with acridonetagged compounds by thin-layer chromatography. As little as 1 pmol of product is readily visualized by its blue fluorescence under UV illumination and identified by its retention factor (Rf). Each assay requires only 10 microliters of solution. The method is reliable, inexpensive, versatile, and immediately applicable in repetitive format for screening catalytic antibody libraries. Three examples are presented: (i) the epoxidation of acridone labeled (S)-citronellol. The pair of stereoisomeric epoxides formed is resolved on the plate, which provides a direct selection method for enantioselective epoxidation catalysts. (ii) Oxidation of acridone-labeled 1-hexanol to 1-hexanal. The activity of horse liver alcohol dehydrogenase is detected. (iii) Indirect product labeling of released aldehyde groups by hydrazone formation with an acridone-labeled hydrazide. Activity of catalytic antibodies for hydrolysis of enol ethers is detected.

Effect of chronic autoimmune nerve growth factor deprivation on sympathetic neuroaxonal dystrophy in rats.

Nerve growth factor (NGF) deficiency has been proposed as a possible pathogenetic mechanism underlying the sympathetic autonomic neuropathy which develops in clinical and experimental diabetes and aging. To determine if long-term NGF deficiency alone would reproduce the distinctive sympathetic neuropathology of streptozocin-induced diabetes or aging in rats, nondiabetic animals were deprived of NGF for 12 months using an autoimmune paradigm. Neuroaxonal dystrophy (NAD), the neuropathologic hallmark of experimental sympathetic diabetic neuropathy and aging, was not increased in frequency in prevertebral superior mesenteric or paravertebral superior cervical ganglia in comparison to age-matched controls. Residual neurons in chronically NGF deprived sympathetic ganglia did not show significant atrophy, chromatolysis, active neuronal degeneration or intraganglionic debris. Postganglionic noradrenergic axons in ileal mesenteric nerves also failed to develop NAD in chronic autoimmune NGF-deprived rats as they would have in animals diabetic for the same duration. These results suggest that simple, isolated NGF deficiency maintained for long periods of time in nondiabetic animals is not sufficient to produce NAD in the pattern of experimental rat diabetes and aging.

On the insertion of foreign DNA into mammalian genomes: mechanism and consequences.

We have studied the integration of adenovirus type 12 (Ad12) DNA in transformed and hamster tumor cells over many years. Upon infection of hamster cells with Ad12, viral DNA has been found in association with hamster chromosomes, possibly in part integrated into the host genome. Ad12 DNA integration is not sequence specific. Transcriptionally active sites of the host genome show a preponderance for foreign DNA insertion. We are pursuing the mechanism of Ad12 DNA integrative recombination in a cell-free system prepared from hamster cell nuclear extracts. In a number of Ad12-transformed hamster cell lines or in cell lines carrying foreign DNA, we have located the inserted Ad12 DNA copies on hamster chromosomes by fluorescent in situ hybridization (FISH). Among the consequences of Ad12 DNA integration, we have studied the de novo methylation of the integrated foreign (Ad12) DNA and increases in DNA methylation in several cellular genes and DNA segments in Ad12-transformed and hamster tumor cells. Several lines of evidence argue for the notion that parameters in addition to nucleotide sequence, in particular site of integration and/or the chromatin configuration of the integrated DNA, are important in generating de novo methylation patterns. The de novo methylation of integrated foreign DNA can be interpreted as an old cellular defense mechanism against the activity of foreign genes in an established genome. Pursuing this concept, we have asked for the most likely portal of entry of foreign DNA, supposedly the gastrointestinal tract in most animals. This hypothesis has been tested by feeding mice linearized or circular, double-stranded bacteriophage M13mp18 DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Fine structure of presynaptic axonal terminals in sympathetic autonomic ganglia of aging and diabetic human subjects.

The neuropathologic changes that may underlie autonomic nervous system dysfunction in nondiabetic elderly human subjects or as a complication of diabetes have been systematically examined in sympathetic ganglia of a series of autopsied human subjects. As in animal models of aging and diabetes, enormously swollen terminal axons were found closely apposed to the perikarya of principal sympathetic neurons in prevertebral superior mesenteric sympathetic ganglia of aged and diabetic human subjects. Dystrophic axons consisted of two stereotyped forms: the first was composed of large numbers of misaligned aggregates of neurofilaments surrounded by variable numbers of small dense core vesicles; the second was characterized by large numbers of mitochondria, vacuoles, and dense and multivesicular bodies. The fine structural characteristics of neuroaxonal dystrophy, its predilection for prevertebral rather than paravertebral sympathetic ganglia, and the tendency for multiple dystrophic axons to cluster preferentially around selected neurons were identical in aged and diabetic human ganglia and were similar to changes seen in animal models of aging and diabetes. Neither diabetic nor aging ganglia demonstrated evidence of neuronal degeneration. Such structural changes may represent a degenerative influence of diabetes and aging on the normal remodeling of nerve terminals in autonomic ganglia, i.e., the continually ongoing process of turnover and replacement of axonal terminals. Similarity of lesions in human diabetes and aging suggests the possibility of pathogenetic mechanisms that are common to diabetes and the aging process. The substantial parallels between humans and animal models provide support for the validity of testing some proposed pathogenetic mechanisms directly in animal models.

Ad spending: growing market share.

If I yell louder but you yell even louder, the audience will hear you. So I shouldn't expect to be heard if I also start yelling louder, unless you become quieter. That, in essence, is the key to the relative share of voice effect in advertising. In most markets, consumer goods markets are in a state of equilibrium, where advertising expenditures are relatively stable and changes in market share are small. To gain ground in market share, a competitor has to launch a huge ad campaign for a sustained period that outspends the biggest rival by at least double.

Insulin bound to the insulin receptor of IM-9 lymphocytes is more accessible to antiinsulin antibodies after treatment with dithiothreitol: bound insulin is deeply buried in the receptor-binding site.

Binding of insulin to its receptor followed by covalent cross-linking with disuccinimidyl suberate (DSS) dramatically impairs the ability of antiinsulin antibodies (both polyclonal and monoclonal) to bind to the insulin moiety. We have used dithiotheitol, which has major effects on the oligomeric structure of the insulin receptor, to determine if this decreased antibody recognition is due to alteration in the conformation of insulin itself or to steric factors. Treatment of the covalently cross-linked insulin-receptor complex with dithiothreitol (DTT) increased the ability of the polyclonal and two monoclonal antiinsulin antibodies to immunoprecipitate the insulin-receptor complex. This treatment decreased immunoprecipitation by antireceptor antibodies. The effect of DTT may have been due to a reversal of either a binding-induced conformational change in the insulin moiety or an alteration in the conformation of the insulin-receptor complex, thereby decreasing steric hindrance. In an effort to choose between these two possible explanations, we prepared a biotinylated derivative of insulin which was cross-linked to the receptor. Since the biotin moiety is relatively rigid, it seemed improbable that binding to the receptor would alter the conformation of the epitope recognized by antibiotin antibodies and that the change would be reversed by DTT. Treatment of the cross-linked biotin-insulin-receptor complex with DTT did increase the ability of both antiinsulin and antibiotin antibodies to immunoprecipitate the cross-linked receptor complex. Identification of the cross-linked receptor on reduced sodium dodecyl sulfate-polyacrylamide gels confirms that the DTT treatment alters the distribution of the oligomeric forms of the receptor. These studies favor the hypothesis that when bound to its receptor, most of the insulin molecule is sequestered within the receptor-binding site such that there is steric hindrance to the approach of antiinsulin antibodies. Moreover, DTT alters the conformation of the cross-linked insulin-receptor complex so as to decrease this steric hindrance.

Binding of insulin to its receptor impairs recognition by monoclonal anti-insulin antibodies.

The interaction between insulin and its receptor was investigated using both monoclonal and polyclonal anti-insulin antibodies. After covalent cross-linking of 125I-insulin to the insulin receptor on cultured human lymphocytes (IM-9 cells) using disuccinimidyl suberate, we inquired whether the insulin-receptor complex could be immunoprecipitated with anti-insulin antibodies. While a polyclonal guinea pig anti-insulin antiserum succeeded in immunoprecipitating receptor-bound 125I-insulin, binding to the receptor decreased the avidity of the antiserum for the insulin moiety by a factor of approximately 1000-fold. Sixteen distinct monoclonal murine anti-insulin antibodies were employed to immunoprecipitate receptor-bound 125I-insulin. Of these 16 monoclonal antibodies, only one (antibody 5.9F4) could be shown to recognize receptor-bound 125I-insulin. Moreover, even with antibody 5.9F4, binding of 125I-insulin to its receptor reduced the affinity of the antibody by a factor of 10- to 100-fold. These data strongly suggest that, when insulin binds to its receptor, the majority of the insulin molecule is unavailable for binding by anti-insulin antibodies. It seems likely that the hormone binding site on the receptor may be very large, thereby allowing for sequestration of the majority of the insulin molecule with relatively little of the hormone remaining exposed.

Fine specificity of cloned insulin-specific T cell hybridomas: evidence supporting a role for tertiary conformation.

Two groups of cloned insulin-specific T cell hybridomas were derived from the fusion of (BALB/c X A/J)F1 T cells with the BW5147 tumor line. Group I responded only to the immunogen pork insulin and failed to respond to rat insulin, which differs only at amino acids A4 and B3. The second group of T cell hybridomas exhibited a broader pattern of reactivity to heterologous insulins because they could be stimulated with the B chain-identical pork, beef, sheep, and human insulins, as well as by isolated oxidized B chain. These hybridomas, however, displayed a much greater reactivity to pork insulin than to beef insulin or to the oxidized B chain. Comparison of the reactivity profiles of these two groups of hybridomas with the three-dimensional structure of the insulin molecule strongly suggested that group I cells were recognizing an immunogenic moiety composed of residues A4 and/or B3 in association with A chain loop determinants and that group II hybrids recognized a moiety composed of the amino acid sequence of the B chain and the A chain loop. Both groups of hybrids were restricted to the high-responder I-Ad allele but could be distinguished by their pattern of alloreactivity. We were unable to show any effect of a panel of monoclonal anti-insulin antibodies on the induction of interleukin 2 secretion by these hybridomas or to demonstrate the presence of idiotypic determinants on these T hybrids by using a panel of anti-idiotypic reagents raised against the monoclonal anti-insulin antibodies.

Idiotypes on monoclonal antibodies to bovine insulin. I. Two public idiotypes on anti-bovine insulin hybridomas define idiotypically distinct families of hybridomas.

We used isoelectric focusing (IEF) to show that individual mice responding to bovine insulin (BI) present complex spectrotypes that are not conserved between individuals. Competition of 125I-BI-reactive bands with cold BI or cold pork insulin (PI), showed that the majority of the antibody response is sensitive to the two amino acid difference between BI and PI. This difference resides in the three amino acid intrachain disulfide-bonded A-loop. To begin dissection of the antibody response to BI, we prepared a panel of 30 monoclonal antibodies against insulin. Anti-idiotypic sera were produced in guinea pigs against seven of these hybridoma proteins (HP), and two, anti-5. 10C6 and anti-5.2B8, were found to define public idiotypes. The level of the 5.10C6 idiotype is controlled by genes linked to the Igh locus. Both the G.P. anti-5.10C6 and G.P. anti-5.2B8 were found to define distinct groups of idiotypically related HP. The 5.10C6 idiotype group contains three closely related members, 5.10C6, 5.5F10, and 5.4C5, which are shown to bear the same L chain by IEF. The 5.2B8 idiotype group has six members that are less closely related, defined by inhibition solid phase RIA. The two most closely related members of this family, 5.2B8 and 5.3C6, possess co-focusing L chains. Within each idiotype group, however, members were found that bind to distinct determinants on insulin. Four of the members of the 5.2B8 idiotype group appear to bind overlapping determinants on insulin that define a topographic region that includes the three amino acid A-loop. Two other HP that have been shown to bind the A-loop do not bear determinants that cross-react with the G.P. anti-5.2B8 antiserum.