Arginine-427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane.

To investigate the role of charged intramembrane residues in the function of the rabbit Na+/glucose cotransporter (rbSGLT1) we substituted arginine-427 (R427) by alanine in the putative domain M9 SGLT1. This residue is conserved in all the members of the SGLT1 family. The mutant protein (R427A) was expressed in Xenopus oocytes and, although Western blot analysis revealed that it was produced in amounts comparable to wild-type, no function was measured. Freeze-fracture analysis showed that R427A SGLT1 was not in the plasma membrane while immunocytochemical experiments localized the transporter to just beneath it. These results indicate that arginine-427 plays a critical role in SGLT1 trafficking to the plasma membrane.

An examination of intraspinal sprouting in dorsal root axons with the tracer horseradish peroxidase.

Postdeafferentation reorganization in the central terminal fields of spared dorsal root axons was evaluated by examining the intraspinal distribution of horseradish peroxidase-labeled sciatic nerve afferent fibers at various intervals following the removal of several lumbar dorsal root ganglia. The sciatic projection to the spinal cord, as determined by the pattern and density of intraspinal reaction product, was remarkably stable following the ganglionectomies. For as long as 3 months later, there was no evidence that sciatic afferent fibers had formed anomalous connections either with new spinal segments or in denervated areas within normal segments of entry. These findings cast doubt upon the existence of anatomic reorganization within the spinal cord following its partial deafferentation and suggest that physiological processes other than new axonal growth underlie observations such as postdenervation alterations in the response properties of dorsal horn neurons and the recovery of behavioral function.

Mechanism of antitumor activity of a single-chain interleukin-12 IgG3 antibody fusion protein (mscIL-12.her2.IgG3).

We have constructed an antibody interleukin-12 (IL-12) fusion protein (mscIL-12.her2.IgG3) that demonstrates significant antitumor activity against the murine carcinoma CT26-expressing human HER2/neu. We now report that this antitumor activity is dose dependent and comparable to or better than recombinant murine IL-12 (rMuIL-12) using subcutaneous and metastatic models of disease. The antitumor activity of mscIL-12.her2.IgG3 is reduced in Rag2 knockout mice, suggesting that T cells play a role in tumor rejection. In SCID-beige mice, the antitumor activity is further reduced, suggesting that natural killer (NK) cells or macrophages or both are also important. The isotype of the antibody response to HER2/neu is consistent with a switch from a Th2 to a Th1 immune response and the infiltration of mononuclear cell in tumors from mice treated with mscIL-12.her2.IgG3. Immunohistochemistry reveals that mscIL-12.her2.IgG3 is antiangiogenic. Thus, the mechanism of the antitumor activity exhibited by mscIL-12.her2.IgG3 is highly complex and involves a combination of T and NK cell activity, a switch to a Th1 immune response, and antiantiogenic activity. This is the first study comparing the in vivo antitumor activity of an antibody-IL-12 fusion protein and free IL-12. Our results suggest that antibody-IL-12 fusion proteins may be useful for the treatment of human cancer.

Thin-fiber cutaneous innervation and its intraepidermal contribution studied by labeling methods and neurotoxin treatment in rats.

Sensory nerves innervating rat distal limb skin were labeled by axonal transport of an enzyme-lectin conjugate injected into lumbar dorsal root ganglia (DRG), with emphasis on tracing the course of the thin axons. Selective neonatal neurotoxin destruction of most unmyelinated sensory or sympathetic axons was achieved by treatment with capsaicin (CAP) and 6-hydroxydopamine (6-OHDA), respectively. The relationship of substance P-immunoreactive (SPI) axons to the patterns of axonal transport-labeled thin axons was compared in normal and neurotoxin-treated animals. SPI is restricted to a limited population of unmyelinated axons, and electron-microscopic observation reveals its total absence in myelinated axons. SPI fibers of sensory origin, as determined by CAP susceptibility, can be traced into the epidermal stratum spinosum, in relation to guard hair follicles, mast cells, and a specific class of small blood vessels. These morphological features are not eliminated by neurotoxin sympathectomy, and some are inferred to contribute to the initial events associated with the neurogenic vasodilation and plasma extravasation associated with the inflammatory response. A re-evaluation of the concept of "free nerve endings" is suggested in the context of the variety of afferent and efferent patterns displayed by sensory peptidergic unmyelinated axons, their putative nociceptive role, and the functional diversity of sensory C fibers.

In vivo properties of three human HER2/neu-expressing murine cell lines in immunocompetent mice.

BACKGROUND AND PURPOSE: Expression of the HER2/neu proto-oncogene, a receptor-like transmembrane protein expressed at low levels on some normal cells, is markedly increased in a subset of human breast, colon, lung, and ovarian cancers. A humanized HER2/neu antibody has been tested as a therapeutic agent in several clinical trials, with promising results. We have developed a family of anti-HER2/neu fusion proteins. To evaluate the immunologic efficacy of these proteins, it is critical that tumors expressing the target antigen can grow in immunologically intact mice. METHOD: To produce murine tumors expressing human HER2/neu on the surface, CT26, MC38, and EL4 murine cell lines were transduced by use of a retroviral construct containing the cDNA encoding the human HER2/neu gene. RESULTS: Histologic features and kinetics of tumor growth in subcutaneous space of the human HER2/neu-expressing cells were similar to those of the respective parental cell lines. Intravenous inoculation with these cells induced disseminated malignant disease. Flow cytometric and immmunohistochemical analyses of freshly isolated tumors revealed in vivo expression of human HER2/neu. Secretion of antigen was not detected by use of an ELISA. CONCLUSION: Although an antibody response against the human HER2/neu antigen was observed, this response does not affect the growth rate of the HER2/neu-expressing cells. These murine models may be useful tools for evaluation of anti-cancer therapeutic approaches that target human HER2/neu.