Jacob Hochman

A disulfide conjugate between anti‐tetanus antibodies and HIV (37–72)Tat neutralizes tetanus toxin inside chromaffin cells

Conjugates between anti‐tetanus F(ab′)2 fragments and the (37–72) fragment of the HIV Tat protein were taken up by chromaffin cells, NG108‐15 neurohybridoma cells and Rev‐2‐T‐6 lymphoma cells. The uptake could not be inhibited by competition with (37–72)Tat, but was reduced in the presence of metabolic inhibitors or at low temperature. The disulfide as well as the thioether conjugate were translocated to the cytoplasmic space, but only the disulfide conjugate moderately restored the stimulated transmitter release inhibited by tetanus toxin. Therefore, disulfide conjugates are more promising than thioethers for the neutralization of intracellular antigens. These conjugates provide new tools to study neuroprotection against bacterial neurotoxins.

Cytotoxic salan-titanium(IV) complexes: high activity toward a range of sensitive and drug-resistant cell lines, and mechanistic insights.

The cytotoxicities of highly efficient salan–TiIV complexes toward a range of cell lines, including drug‐resistant cells, are reported along with preliminary mechanistic insights. Five salan–TiIV complexes were investigated toward eight different human and murine cancer‐derived cell lines, including colon, ovarian, lung, cervical, pancreatic, leukemic, skin, and breast. The salan complexes are more active toward the cells analyzed than cisplatin and the known titanium compound (bzac)2Ti(OiPr)2, and no cell line resistant to the salan complexes was identified. Moreover, the salan–TiIV complexes are highly active toward both cisplatin‐sensitive (A2780) and cisplatin‐resistant (A2780CisR) human ovarian cancer cell lines. Similarly, the salan complexes are cytotoxic toward multi‐drug‐resistant (ABCB1‐expressing) mouse lymphoma cell lines HU‐1 and HU‐2. Importantly, minimal or no activity was observed toward primary murine cells (bone marrow, heart, liver, kidney, spleen, and lung), supporting selectivity for cancer cells. Additionally, the salan complexes maintain high cytotoxicity for up to 24 h following exposure to cell culture medium, whereas reference complexes (bzac)2Ti(OiPr)2 and Cp2TiCl2 rapidly lose much of their activity upon exposure to medium, within ∼1 h. The upregulation of p53 followed by cell‐cycle arrest in G1 phase is likely one mechanism of action of the salan complexes. Taken together, the results indicate that these compounds are selectively toxic to cancer cells and are able to circumvent two independent mechanisms of drug resistance, thus expanding the scope of their potential medicinal utility.

International Central Nervous System and Ocular Lymphoma Workshop: Recommendations for the Future

Purpose: To bring together multidisciplinary experts to discuss primary central nervous system lymphoma (PCNSL) and primary intraocular lymphoma (PIOL). Methods: NIH campus workshop discussion focusing on future work in both clinical and basic lymphoma research. Results: The discussion lead to recommendations on elucidating disease pathobiology, improving diagnostic accuracy and sensitivity, and novel therapeutic strategies. Conclusions: Approaches which have been successfully applied to other neoplasms, such as microarray, may be applied to improve diagnostic accuracy and sensitivity of PCNSL and PIOL and should be systematically incorporated into clinical trials of both. Development of animal models of PCNSL and PIOL may be useful in understanding the unique ocular and CNS milieu. Disease detection by radiological, nuclear medicine, molecular and flow cytometric approaches should be systematically studied to improve early diagnosis, accurate staging, and response evaluation. Improved therapy remains the ultimate goal. Efforts in these arenas should be coordinated on a national and international level.

Ultrastructural immunohistochemical localization of vasopressin in the hypothalamic-neurohypophysial system of three murids.

SummaryVasopressin was immunohistochemically localized at the electron microscopic (EM) level in the hypothalamic-neurohypophysial system (HNS) of three murids. Antiserum to vasopressin was produced in rabbits injected with lysine vasopressin (LVP) conjugated to egg albumin (EA), anti-EA being precipitated prior to staining. Sternbergers unlabeled antibody peroxidase technique was employed, immunoreactivity being designated by peroxidase anti-peroxidase (PAP) molecules and electron opacity. Immunoreactive neurosecretory granules (NSG) were found in the perikarya of the supraoptic nucleus (SON) in all three murids investigated, although far more profusely in the two wild strains. Immunoreactive axonal NSG were observed in the inner and outer zones of the median eminence (ME), and within most of the axons and terminals in the neurohypophysis. The concentration of primary serum effective for staining the SON (1∶10–1∶50) was far higher than that required for the ME and the neurohypophysis (1:500–1:1,200). AntiLVP also induced electron opacity of granules in cells of the pars intermedia (PI). Discussion centers on the significance of immunoreactive NSG in the neurosecretory (NS) perikarya, on the possibility of an extragranular pool of hormone, and on speculation about the electron opacity of the PI granules.

An experimental model for infiltration of malignant lymphoma to the eye and brain

Abstract Currently there is no adequate experimental model available whereby the lethal infiltration of malignant lymphoma to the eye and CNS can be studied. Variant S49 mouse lymphoma cells that exhibit cell-cell adhesion properties (named Rev-2-T-6) were inoculated intraperitoneally into Balb/C mice at the ages of 6–60 days postnatal. Mice inoculated between days 6–11 postnatal developed signs of eye and CNS involvement with an apparent peak (58% of mice) at day 7. None of the mice inoculated beyond day 11 exhibited such signs. Histological analysis of these sites revealed tumorous infiltrates into a variety of structures in the orbit, intraocular tissues, along the optic nerve and in the brain. Additional analysis of the histopathological data, based on the structures demonstrating the highest frequency of lymphoma infiltration, suggests preferred routes of lymphoma entry to the brain and eye. Thus, entry to the brain can occur mainly through the choroid plexus and cranial nerves or cranial nerve ganglia. Entry to the eye may occur from the brain (along the optic nerve), and through hematogenous infiltration of orbital structures. No data were found that would support retrograde infiltration of the lymphoma from the eye to the brain. These findings present an experimental model for addressing the molecular mechanisms that govern homing of malignant lymphoma to the eye and brain, as well as the development of experimental therapeutic modalities for malignant lymphoma in these organs.

A Key role for cyclic AMP-responsive element binding protein in hypoxia-mediated activation of the angiogenesis factor CCN1 (CYR61) in Tumor cells.

Hypoxia is a prominent feature of solid tumors known to contribute to malignant progression and therapeutic resistance. Cancer cells adapt to hypoxia using various pathways, allowing tumors to thrive in a low oxygen state. Induction of new blood vessel formation via the secretion of proangiogenic factors is one of the main adaptive responses engaged by tumor cells under hypoxic conditions. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays a pivotal role in mediating such responses. In addition, several other transcription factors have also been implicated in hypoxic gene regulation, either independently or in cooperation with HIF-1. In this work, we show that the expression of the angiogenesis-related, immediate early gene CCN1 (formerly known as CYR61), considered to be involved in tumor growth and invasiveness, is enhanced upon hypoxia stress primarily in a protein kinase A and cyclic AMP-responsive element binding protein (CREB) and CRE–dependent manner in various cell lines. The hypoxia-mediated activation of the CCN1 promoter is independent of HIF-1 and HIF-2, as shown by small interfering RNA knockdown. We identify the cis element in the mouse CCN1 promoter responsible for CREB binding to be one of two partial CRE sites present in the promoter. Moreover, we report for the first time that CREB-mediated CCN1 transcription is enhanced in hypoxic regions of tumors in vivo. Identifying and characterizing the molecular mechanisms that govern the response of tumors to hypoxia may be instrumental to identify the tumors that will respond favorably to inhibition of angiogenesis and thus lead to the development of treatments that could complement hypoxia-inducing treatment modalities. (Mol Cancer Res 2008;6(9):1397–409)

Isolation, characterization and synthesis of a novel pardaxin isoform

We report the isolation of a novel pardaxin isoform from the toxic secretion of the Red Sea Moses sole (Pardachirus marmoratus). Mass spectrometrical analysis of the newly purified peptide revealed a different primary structure compared to the previously known pardaxin isoforms. Sequence analysis disclosed an aspartic acid residue instead of glycine at position 31 of the new isoform. According to the novel sequence, a synthetic Asp‐31‐peptide was compared with the native compound as well as with synthetic Gly‐31‐pardaxin. The isolated Asp‐31‐pardaxin isoform and its synthetic analog exhibited identical elution properties during reverse‐phase HPLC, as well as similar dose‐dependent lytic effects on human erythrocytes at a concentration of 10−6 to 10−5 M. The hemolytic activity of Asp‐31‐pardaxins was lower than that of Gly‐31‐pardaxin and no synergistic effect between these peptides was found. The additional negative charge introduced by Asp‐31 is likely to affect the selectivity of pardaxin pores towards a variety of ions.

Polyamines and protein kinase II. Effect of polyamines on cyclic AMP--dependent protein kinase from rat liver.

Abstract The effect of polyamines on the activity of cAMP-dependent protein kinase from rat liver was determined. It has been shown that polyamines inactivate the enzyme in the decreasing order of activity: spermine > spermidine > putrescine. This effect is due to inhibition of the catalytic subunit. On the other hand, binding of cAMP to the regulatory subunit of the enzyme is not affected by polyamines. It is suggested that the inhibition of protein kinase by polyamines is a general phenomenon.

Mouse Mammary Tumor Virus Env–Derived Peptide Associates with Nucleolar Targets in Lymphoma, Mammary Carcinoma, and Human Breast Cancer

We have previously shown that the leader peptide (p14) of the Env-precursor of mouse mammary tumor virus is translocated into the nucleoli of murine T cell lymphomas that harbor this virus. Using a polyclonal antibody against recombinant p14, we show here that p14 is also localized to the nucleoli of murine mammary carcinomas and some human breast cancer samples. Affinity purification studies define a number of proteins, mostly nucleolar, that bind p14. Taken together, these findings point towards a more general involvement of p14 in lymphomagenesis and mammary carcinogenesis.

The leader peptide of MMTV Env precursor localizes to the nucleoli in MMTV-derived T cell lymphomas and interacts with nucleolar protein B23

We have previously described two nucleolar proteins, named p14 and p21, in MMTV-induced T cell lymphomas. These proteins were identified by a monoclonal antibody (M-66) generated from a nontumorigenic, immunogenic variant of S49 T cell lymphoma. While p14 was common to several MMTV-derived T cell lymphomas, p21 was found only in highly tumorigenic variants of S49 cells. Here we report that p14 is the leader peptide of the MMTV env precursor. The epitope recognized by M-66 contains a putative nuclear localization signal. Actinomycin D was found to induce redistribution of p14/p21 from the nucleolus to the nucleoplasm. p14 coimmunoprecipitated and colocalized with the cellular protein, B23. Association with B23 has been previously reported for other auxiliary nucleolar retroviral proteins, such as Rev (HIV) and Rex (HTLV).