Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over.

Mice that are deficient in either the Pms2 or Msh2 DNA mismatch repair genes have microsatellite instability and a predisposition to tumours. Interestingly, Pms2-deficient males display sterility associated with abnormal chromosome pairing in meiosis. Here mice deficient in another mismatch repair gene, Mlh1, possess not only microsatellite instability but are also infertile (both males and females). Mlh1-deficient spermatocytes exhibit high levels of prematurely separated chromosomes and arrest in first division meiosis. We also show that Mlh1 appears to localize to sites of crossing over on meiotic chromosomes. Together these findings suggest that Mlh1 is involved in DNA mismatch repair and meiotic crossing over.

Microinjection of anti-p21 antibodies induces senescent Hs68 human fibroblasts to synthesize DNA but not to divide.

Replicative senescence is characterized by irreversible growth arrest and has been defined by four genetic complementation groups. One of these groups is associated with the predominance of underphosphorylated, growth-suppressive retinoblastoma tumor suppressor protein (pRb). Although certain members of the cyclin-dependent kinase (cdk)/cyclin family, some of which phosphorylate pRb, are underexpressed in senescent cells, others are expressed but inactive. This lack of cdk activity and arrest in the G1 phase of the cell cycle is likely attributable to the induction upon senescence of the G1-S cdk/cyclin inhibitors p21 (WAF1/CIP1/Sdi) and p16INK4. In fact, in early presenescent normal diploid fibroblasts in which p21 is inactivated, senescence is bypassed or postponed. Moreover, in senescent cells in which p53 function was inhibited, DNA synthesis was reinitiated, an effect likely attributable, in part, to the dependence of p21 expression on p53. We report here that the apparent inactivation of p21 in senescent human fibroblasts through the introduction of inhibitory alpha-p21 antibodies causes these cells to reenter the S-phase of the cell cycle. The disruption of p21 activity affects the p21-Rb-E2F pathway in that the expression of genes transcriptionally regulated by E2F, such as cyclin A and cdc2, were found to be up-regulated in injected cells. No evidence of cell division was observed. This suggests that p21 plays an important role in the maintenance of senescence and in the inhibition of S-phase progression, but inhibition of p21 activity is insufficient to permit cells to complete the cell cycle.

Reversal of quinone-induced chlorophyll fluorescence quenching.

We have used two methods to investigate the reversibility of the interaction of substituted quinones with the thylakoid membrane of plant chloroplasts. Treatment of chloroplasts with added quinones lowers the room-temperature Photosystem II chlorophyll fluorescence intensity by variable amounts depending on the identity and concentration of the quinone. The extent of restoration of the chlorophyll fluorescence level is used as a measure of the effectiveness of the reversal technique. One reversal method involves the addition of thiols to quinone-treated chloroplasts to alter the quinone in a chemical way via a nucleophilic 1,4-Michael addition. In general, the modified quinones exhibit a lower affinity for the thylakoid membrane, as evidenced by an accompanying increase in chlorophyll fluorescence. The thiol concentrations necessary for quenching reversal are found to be in the order [dithiothreitol] less than [2-mercaptoethanol] less than [glutathione]. The second reversal method examines the extent to which added quinones can be removed from thylakoid membranes using a concentration gradient established by resuspension of quinone-treated chloroplasts in quinone-free media. The results further support the reversible nature of the quinone inhibition and indicate that the extent of recovery is dependent upon the degree of fluorescence inhibition originally induced by the added quinone.

Repair of sequelae from artificial fiber hair implants with hair-bearing punch grafting.

Several thousand persons had artificial hairs of fiber implanted in their scalps 5 years ago with disastrous results. These persons may still require late repair to remove retained fiber fragments from the skin. The hair-bearing punch graft is a useful method of repair in some patients.

Treatment of radiation-induced alopecia.

Radiotherapy is frequently employed in the management of head and neck neoplasia, either as an adjunct to surgery or as the sole treatment modality. Consequently, radiation alopecia--a well-known complication of high-dosage radiotherapy--is seen often. Longer patient survival, especially with earlier discovery of the malignancy and more refined treatment regimens, will provide the surgeon with the opportunity to treat radiation alopecia by means of the punch graft technique of hair transplantation. The technique is substantially similar to that employed in treating male pattern baldness, although the approach to the recipient and donor areas must be modified. A successful case report is documented and a modified approach is highlighted.

Structural and functional similarities between synthetic HIV gp41 peptides and defensins.

Synthetic peptides derived from the putative immunosuppressive region of HIV-1 gp41 were examined to identify the amino acids required for suppressive activity. The active sequence was similar to the pseudosubstrate regulatory peptide of protein kinase C and to a sequence from the cyclic immune peptides known as defensins. The activity of the gp41 peptides appeared to be dependent upon a structural conformation, possibly a looped motif like that found in defensins. This similarity to defensins, which can insert themselves into lipid bilayers, was further strengthened by demonstrating that gp41 peptides have the ability to associate with liposomes and lymphocyte membranes. These results suggest that the immunosuppressive region of gp41 may participate in the membrane interactions required for viral fusion and infectivity.

Inhibition of lymphoproliferation by a synthetic peptide with sequence identity to gp41 of human immunodeficiency virus type 1.

Peptides were synthesized that contained sequences from two regions (env amino acids [aa] 581 to 597 and 655 to 671) of the transmembrane protein gp41 and one region of the external envelope glycoprotein gp120 (aa 457 to 464) of human immunodeficiency virus type 1. Selection of these sequences was based on their homology to the highly conserved and immunosuppressive sequence contained within the transmembrane proteins p15E and gp21 of animal and human retroviruses, respectively. Peptide aa581-597 was found to specifically inhibit human and murine lymphoproliferation, whereas peptides aa655-671 and aa457-464 had no activity. These results suggest a mechanism by which human immunodeficiency virus type 1 gp41 exerts a direct immunosuppressive effect in vivo, analogous to that postulated for p15E and gp21, which could contribute to the immune dysfunction observed in patients suffering from acquired immunodeficiency syndrome. It is of particular interest that the sequence aa 584 to 609, shown to contain B- and T-helper-cell epitopes, overlaps with the sequence aa 581 to 597 that is shown here to inhibit lymphoproliferation. The potential implications of this overlap of immunologic activities are discussed.

Identification, using synthetic peptides, of the minimum amino acid sequence from the retroviral transmembrane protein p15E required for inhibition of lymphoproliferation and its similarity to gp21 of human T-lymphotropic virus types I and II.

Synthetic peptides containing portions of a highly conserved region of retroviral transmembrane proteins of human and animal retroviruses were tested for their ability to inhibit lymphoproliferation to determine the minimum amino acid sequence required. The previously reported immunosuppression mediated by the peptide CKS-17 was confirmed and further localized to a sequence of eight residues essentially identical to the sequence present in the transmembrane protein gp21 of human T-lymphotropic virus types I and II (HTLV-I and -II). To substantiate the physiological relevance of the inhibition of lymphoproliferation observed with the synthetic peptides and to relate this activity to the intact protein, we purified the Rauscher murine leukemia virus transmembrane protein p15E by immunoaffinity chromatography and report that this purified component presented in the form of protein micelles inhibited the interleukin-2-dependent proliferation of the murine T-cell line CTLL-2 in a dose-dependent manner, with a half-maximal inhibitory dose (ID50) of approximately 16 nM. In comparison, the ID50 concentration of a recombinant form of p15E required to inhibit lymphoproliferation was approximately 2.2 microM. The results reported here support the hypothesis that the transmembrane protein gp21 of HTLV-I and -II participates in the mechanism of immunosuppression previously reported for the transmembrane proteins of feline leukemia virus and other animal retroviruses. Thus, the transmembrane protein of HTLV-I, the etiological agent of adult T-cell leukemia-lymphoma, may be partially responsible for the immunocompromised clinical course of this disease that results in fatal opportunistic infections in a majority of cases.

Assessment of the antibody response to the immunosuppressive/immunodominant region of HIV gp41 in a 5-year longitudinal study.

The antibody response of HIV-infected individuals to the 581-609 amino acid (aa) region of HIV-1 gp41 containing the putative immunosuppressive and immunodominant sequences was examined. Sera collected every 5 to 6 months over a period of 5 years from 50 HIV-1-infected homosexual and bisexual men, 25 of whom progressed to AIDS during the collection period, were monitored for changes in ELISA reactivity against synthetic peptides encompassing aa581-609 of gp41. The data obtained in this blinded, historical prospective study were analyzed with respect to changes in mean ELISA absorbance over time and differences in absorbance between patient groups (those who progressed to AIDS and those who did not). No correlation was found between time or disease state and the presence of antibodies to the aa581-597 immunosuppressive sequence. In contrast, ELISA absorbance against the aa598-609 immunodominant sequence continued to increase over time in both the AIDS and non-AIDS groups. The rate of increase in absorbance was similar for both groups; however, the AIDS group had a significantly higher mean absorbance level at the outset of the study and maintained this difference throughout the study. This region of gp41 has been proposed to play a role in complement-mediated antibody-dependent enhancement of infection.

Methyl inosine monophosphate: a potential immunotherapeutic for early human immunodeficiency virus (HIV) infection.

MIMP is a new thymomimetic purine under development for immunorestorative therapy. Lymphocytes were obtained from eight patients with acquired immunodeficiency disease (AIDS), eight with symptomatic pre-AIDS (ARC), and 22 normal controls and were stimulated in vitro with phytohemagglutinin (PHA). AIDS patients (mean CD4 counts of 40) showed PHA responses less than 10% of control while ARC patients (mean CD4 counts of 544) showed responses approximately 50% of the control responses. MIMP (0.1, 1, 10 and 100 micrograms/ml) progressively augmented the PHA responses in all these groups. The augmentation of the responses of the leukocytes of AIDS patients while statistically significant was minimal. The augmentation of the responses of ARC patients was significant and their maximal responses approached control levels. The effect of 1 micrograms/ml MIMP was comparable with that observed with indomethacin (10(-6) M) and interleukin-2 (IL2 - 4 units/ml) and was additive with each of these stimulants. In a parallel manner, MIMP restored the suppression of control lymphocytes induced by the immunosuppressive 17 amino acid fragment of the P41 peptide of HIV. In vivo experiments showed that MIMP significantly delayed death in a murine FLV AIDS model at a dose of 1 mg/kg by the oral or parenteral route. MIMP is under preclinical development for early HIV disease to forestall progression to AIDS by attenuating virus-induced immunosuppression.

Monoclonal antibodies specific to the acute lymphoblastic leukemia t(1;19)-associated E2A/pbx1 chimeric protein: characterization and diagnostic utility.

Nonrandom chromosomal abnormalities are found in most human malignancies, particularly leukemias and lymphomas. A characteristic t(1;19) (q23;p13.3) chromosomal translocation is detected in 5% of childhood acute lymphoblastic leukemia (ALL) cases. This translocation results in the formation of a fusion gene, which leads to the expression of an oncogenic E2A/pbx1 protein. Breakpoints in the E2A gene almost invariably occur within a single intron, and the identical portion of PBX1 is joined consistently to exon 13 of E2A in fusion mRNA. In this article, we report the development of monoclonal antibodies against E2A/pbx1 fusion protein using a specific peptide that corresponds to the junction region of the protein. The obtained antibodies recognize specifically the chimeric E2A/pbx1 fusion protein and lack cross-reactivities with E2A and pbx1. Immunohistochemical staining and flow cytometric studies show that these antibodies can distinguish t(1;19)-positive from t(1;19)-negative leukemic cells. These results indicate that the obtained E2A/pbx1-specific monoclonal antibodies might prove to be valuable diagnostic reagents and important tools for elucidating the mechanisms involved in oncogenesis and progression of t(1;19)-positive childhood ALL.

Characterization of a conserved T cell epitope in HIV-1 gp41 recognized by vaccine-induced human cytolytic T cells.

A human CTL epitope located in a region of the HIV-1 envelope protein gp41 that is highly conserved among various HIV-1 strains was identified. This epitope was recognized by CD4+ CTL clones that were induced in seronegative humans by immunization with recombinant gp160. Fusion proteins carrying portions of the HIV-1 env gene and synthetic peptides were used to localize this epitope to amino acids 584-595 of the HIV-1 BRU env sequence. Only two positions within this epitope showed variation among North American HIV-1 isolates, and the substitutions were conservative in nature. The Lys to Arg substitution at position 593 abolished recognition, probably by interfering with the peptide-MHC interactions. This epitope was recognized in association with at least one subtype of the widely distributed human class II MHC specificity DPw4, namely DPw4.2. The relatively high frequency of this allele (27.2% among Caucasians) makes it likely that a larger fraction of the population would generate a response directed at this epitope than would be the case for epitopes recognized in the context of gene products of most other class II and class I loci. Interestingly, the closely related DP beta-chain allele types 4.1 and 2.1, which differ from 4.2 by 3 and 1 amino acids, respectively, were unable to present this gp41 peptide to DPw4.2-restricted clones. Comparison of the structure of this epitope with that of other peptides recognized in the context of DPw4.2 led to the identification of a consensus sequence for DPw4.2 binding peptides. Because the gp41 CTL epitope 584-595 identified here is highly conserved and is recognized in the context of a common DP allele, it may represent an important target region for vaccine development. Our results indicate that vaccines containing this epitope may induce in a significant fraction of those immunized CTL active against at least half of all HIV-1 strains.