Transgenic mice overproducing human thioredoxin-1, an antioxidative and anti-apoptotic protein, prevents diabetic embryopathy.

AIMS/HYPOTHESIS: Experimental studies have suggested that apoptosis is involved in diabetic embryopathy through oxidative stress. However, the precise mechanism of diabetic embryopathy is not yet clear. Thioredoxin (TRX) is a small, ubiquitous, multifunctional protein, which has recently been shown to protect cells from oxidative stress and apoptosis. Using transgenic mice that overproduce human TRX-1 (TRX-Tg mice), we examined whether oxidative stress is involved in fetal dysmorphogenesis in diabetic pregnancies. METHODS: Non-diabetic and streptozotocin-induced diabetic (DM) female mice were mated with male TRX-Tg mice. Pregnant mice were killed either at day 10 or day 17 of gestation, and viable fetuses and their placentas were recovered, weighed and assessed for gross and histological morphology, biochemical markers and gene expression. RESULTS: In both wild-type (WT) and transgenic (Tg) groups, fetal and placental weights in the diabetic group were significantly decreased compared with the non-diabetic group. The incidence of malformation was higher in the diabetic group, and was significantly decreased in the TRX-Tg group (DM-WT vs DM-Tg; 28.6% vs 10.4%). Oxidative stress markers such as thiobarbituric acid reactive substances and 8-hydroxy-2'-deoxyguanosine were increased in DM-WT group fetuses but were decreased in fetuses from the DM-Tg group. Furthermore, immunohistochemically assayed apoptosis and cleaved caspase-3 production in embryonic neuroepithelial cells was significantly increased in the DM-WT group, and was significantly decreased in the DM-Tg group. CONCLUSIONS/INTERPRETATION: These results indicate that oxidative stress is involved in diabetic embryopathy, and that the antioxidative protein TRX at least partially prevents diabetic embryopathy via suppression of apoptosis.

Fruit and vegetable intake and risk of amyotrophic lateral sclerosis in Japan.

BACKGROUND: There has been little interest in the role of nutrition in the prevention of amyotrophic lateral sclerosis (ALS). We investigated the relationship between dietary intake of vegetables, fruit, and antioxidants and the risk of ALS in Japan. METHODS: Between 2000 and 2004, we recruited 153 ALS patients aged 18-81 years with disease duration of 3 years within the study period in accordance with El Escorial World Federation of Neurology criteria. Three hundred and six gender- and age-matched controls were randomly selected from the general population. Information on dietary factors was collected using a validated self-administered diet history questionnaire. RESULTS: A higher consumption of all fruits and vegetables and fruit alone in the highest quartiles was associated with a statistically significantly reduced risk of ALS. Although not statistically significant, a beneficial association between intake of all vegetables, green and yellow vegetables and other vegetables and ALS was found. No statistically significant dose-response relationship was observed between intake of beta-carotene, vitamin C and vitamin E and the risk of ALS. CONCLUSION: Our findings suggest that higher intake of food rich in antioxidants such as fruit and vegetables confer protection against the development of ALS.

Kii ALS dementia.

Epidemiological surveys in the foci of ALS of the Kii Peninsula of Japan started in the early 1960s. Continuous surveys conducted for decades revealed that there have been two foci in the Kii Peninsula: one in Kozagawa in the southern part, and the other in Hobara in the south-east. Clinically, ALS patients of the Kii foci occasionally showed parkinsonian features or dementia that have not been reported in the sporadic form of ALS. Neuropathologically, numerous NFT that are identical to those of Alzheimer's disease were observed in the cerebral cortex and in the brainstem nuclei. To elucidate the etiopathogenesis of this unique form of ALS, an analysis was conducted of the environment in the focus areas and of the specimens from the patients with ALS. It was hypothesized that the long exposure of these environments to low calcium and magnesium, and an excess of aluminum and manganese in the drinking water and the soil, might lead to the deposition of some trace elements in the CNS, eventually causing neuronal degeneration and death.

Antitumor activity of tumor necrosis factor-alpha conjugated with polyvinylpyrrolidone on solid tumors in mice.

We attempted the development of a novel polymer conjugation to further improve the therapeutic potency of antitumor cytokines compared with PEGylation for clinical application. Compared with native tumor necrosis factor (TNF)-alpha in vitro, specific bioactivities of polyvinyl-pyrrolidone (PVP)-modified TNF-alphas (PVP-TNF-alphas) were decreased by increasing the degree of PVP attachment. PVP-TNF-alpha fraction 3, Mr 101,000, had the most effective antitumor activity of the various PVP-TNF-alphas in vivo. PVP-TNF-alpha fraction 3 had >200-fold higher antitumor effect than native TNF-alpha, and the antitumor activity of PVP-TNF-alpha fraction 3 was >2-fold higher than that of MPEG-TNF-alpha (Mr 108,000), which had the highest antitumor activity among the polyethylene glycol (PEG)-conjugated TNF-alphas. Additionally, a high dose of native TNF-alpha induced toxic side effects such as body weight reduction, piloerection. and tissue inflammation, whereas no side effects were observed after i.v. administration of PVP-TNF-alpha fraction 3. The plasma half-life of PVP-TNF-alpha fraction 3 (360 min) was about 80- and 3-fold longer than those of native TNF-alpha (4.6 mm) and MPEG-TNF-alpha (122 min), respectively. The mechanism of increased antitumor effect in vivo caused the prolongation of plasma half-life and increase in stability. These results suggested that PVP is a useful polymeric modifier for bioconjugation of TNF-alpha to increase its antitumor potency, and multifunctionally bioconjugated TNF-alpha may be a potentiated antitumor agent for clinical use.

Mechanical compression of the lumbar nerve root alters pain-related behaviors induced by the nucleus pulposus in the rat.

The purpose of this study was to refine a method of nerve-root injury in the rat to produce hyperalgesia, a pain-related behavior, and to determine if there were any relationships between the histological extent of nerve-root injury and the magnitude of hyperalgesia. Three methods were used to produce hyperalgesia: irritation of a nerve root by ectopic nucleus pulposus, silk loop alone, or both silk loop and ectopic nucleus pulposus. Autologous nucleus pulposus obtained from coccygeal intervertebral discs was relocated on the lumbar nerve roots after laminectomy. Two loops of 4-0 silk were placed around the exposed nerve roots. Hyperalgesia was measured preoperatively and postoperatively. The distribution of myelinated axons in the dorsal nerve roots was evaluated histologically. Mechanical hyperalgesia was detected in rats in which autologous nucleus pulposus was applied to the nerve root but not in those in which silk loops were used. Silk loops around the nerve root resulted in thermal hyperalgesia only in rats in which autologous nucleus pulposus was applied to the nerve root. Fewer large myelinated fibers were seen in the rats in which silk loops were used. Although a silk loop around the nerve root was not sufficient to produce hyperalgesia, supplemental application of autologous nucleus pulposus to the nerve root produced thermal hyperalgesia. It is possible that mechanical constriction of the nerve root alters the pain-related behavior elicited by chemical factors from the nucleus pulposus.

In vitro remodeling of tumor vascular endothelial cells using conditioned medium from various tumor cells and their sensitivity to TNF-alpha.

Prevention of tumor-associated blood vessel formation (angiogenesis) is a potentially powerful strategy to treat cancer. We found that tumor vascular endothelial cells were rearranged in vitro with conditioned culture medium derived from tumor cells and compared the sensitivity to the effects of TNF-alpha between normal and tumor endothelial cells. Incubation with tumor (Meth-A, Colon26)-derived conditioned medium showed that no effect was observed on cell growth. Tumor cells (Meth-A, Colon26, and B16BL6) only showed no sensitivity to TNF-alpha. Normal and control endothelial cells in culture showed little cytotoxicity in response to TNF-alpha treatment, but marked cytotoxicity of TNF-alpha was observed in endothelial cells cultured with tumor-derived conditioned medium. Sensitivity to TNF-alpha was different depending on the type of tumor from which the conditioned medium was derived. This difference in sensitivity was assumed to be due to the in vivo sensitivity to TNF-alpha. The results of this study suggested that the sensitivity of tumors to TNF-alpha is controlled by the sensitivity of tumor vasculature.

Molecular design of conjugated tumor necrosis factor-alpha: synthesis and characteristics of polyvinyl pyrrolidone modified tumor necrosis factor-alpha.

We conjugated tumor necrosis factor-alpha (TNF-alpha) with the synthetic polymeric modifier polyvinyl pyrrolidone (PVP) to facilitate its clinical use for anti-tumor therapy. TNF-alpha was chemically conjugated with the terminal carboxyl-bearing PVP at one end of its main chain, which was radically polymerized via the formation of an amide bond between the lysine amino groups of TNF-alpha and carboxyl group of PVP. In vitro specific bioactivity of PVP-conjugated TNF-alpha (PVP-TNF-alpha) relative to that of native TNF-alpha gradually decreased with increases in the degree of PVP attachment. In contrast, PVP-TNF-alpha in which 40% of TNF-alpha lysine residues were coupled with PVP (MPVP-TNF-alpha) exhibited the highest anti-tumor activity among the conjugated derivatives examined. MPVP-TNF-alpha had more than 200-fold higher anti-tumor efficacy than native TNF-alpha, and the anti-tumor activity of MPVP- TNF-alpha was more than 5-fold stronger than that MPEG- TNF-alpha which had the highest anti-tumor activity among PEG-conjugated TNF-alphas examined. Additionally, a high dose of native TNF-alpha induced toxic side-effects such as body weight reduction, piloerection and tissue inflammation, while no side effects were observed following i.v. administration of MPVP-TNF-alpha. The plasma half-life of MPVP-TNF-alpha (360 min) was about 80 and 3-fold longer than those of native TNF-alpha (4.6 min) and MPEG-TNF-alpha (122 min), respectively. These results suggested that PVP is a useful polymeric modifier for increasing the anti-tumor activity of PVP.

Epidemiology of motor neuron disease in the Kii Peninsula of Japan, 1989-1993: active or disappearing focus?

During the period 1989-1993, the incidence and migration patterns of patients with motor neuron diseases (MND) in Wakayama Prefecture, including one of the high-incidence Kii Peninsula foci ('Kozagawa focus'), were surveyed to determine whether the focus had truly disappeared or not. Overall, the crude average annual incidence was 1.43 per 100000 population; when age-adjusted to the 1990 Japanese population, it was 1.25 (1.85 for males and 0.61 for females). The average annual age- and sex-specific incidence steadily increased to a peak between 60 and 69 years and dropped after 70. Geographically, the rates varied in the five regions of Wakayama Prefecture from 0.38 to 2.48. The areas with high incidence were distributed in the central and southernmost regions; the highest was in the Kozagawa focus with 9.54 (two ALS cases within five years; 4193 base population, 1990). During the study period, four emigrants from Kozagawa had developed MND one to four decades after leaving the focus. Although the remarkable clustering of MND was thought to have disappeared, the southern Kii Peninsula remains a high-risk area for MND, especially if one interprets the data so as to include the emigrants. In general, the age at onset has increased in the past 20 years from 56.5 to 61.7; male predominance is observed.

Involvement of Onuf's nucleus in amyotrophic lateral sclerosis.

The Onufrowicz (Onuf's) nuclei from eight amyotrophic lateral sclerosis (ALS) cases and nine neurological control cases were studied histologically and morphometrically. To clarify the factors relating to the involvement of the Onuf's nucleus in ALS, we correlated the relationships among the age at death, clinical duration, morphometric findings for Onuf's neurons, and neuronal numbers in the posteroposterolateral (PPL) nuclei in the ALS cases with those in neurological controls. Intracytoplasmic inclusions such as Bunina bodies, ubiquitin-reactive inclusions, and conglomerate inclusion were found in the Onuf's neurons in ALS, but not in the controls. The total number of Onuf's neurons in the ALS cases was not decreased, but that of normal-appearing neurons was decreased while that of atrophic neurons was increased. Significantly decreased perikaryal, nuclear and nucleolar areas and decreased perikaryal (P)/nuclear and P/nucleolar area ratios of Onuf's neurons were found in ALS, not only in the atrophic neurons but also in the normal-appearing neurons, compared with the controls. The shrinkage in Onuf's neurons of ALS was different from that seen in the ageing process or in the axonal reactions of controls with atonic bladder. In ALS, the morphometric findings for the Onuf's neurons showed no correlation with age at death, clinical duration, or number of PPL neurons. Our results indicate that in ALS Onuf's nucleus is principally vulnerable to the ALS process, although the degree of degeneration differs from that seen in other motor neurons. The involvement of Onuf's nucleus might be slowed due to factors specific to this nucleus, including the biochemical and autonomic properties of the nucleus; nevertheless, it is histologically classified as part of the somatic cell column.

PEGylation of interleukin-6 effectively increases its thrombopoietic potency.

The in vivo thrombopoietic activity of polyethylene glycol-modified interleukin-6 (MPEG-IL-6), in which 54% of the 14 lysine amino groups of IL-6 were coupled with PEG, was compared to that of native IL-6. Native IL-6 and MPEG-IL-6, which showed about 51% of the specific bioactivity of native IL-6, were administered subcutaneously to mice every 2 days for 7 days. Native IL-6 increased not only the peripheral platelet count, but also the plasma-IgG1 level in a dose-dependent manner. MPEG-IL-6 showed about 500 times higher thrombopoietic potency than native IL-6. Further, in comparison to native IL-6, MPEG-IL-6 did not enhance IgG1 production as much as it enhanced platelet production. MPEG-IL-6 significantly stimulated platelet recovery in mice treated with 5-fluorouracil, whereas the administration of native IL-6 had a negligible effect. The plasma half-life of MPEG-IL-6 was about 100-fold longer than that of native IL-6. The decrease in the plasma clearance of MPEG-IL-6 was thought to be due, in part, to the shielding of the proteolytic sites in the IL-6 molecule by the PEG chain. The uptake of IL-6 by the reticuloendothelial system, such as the liver and spleen, was markedly limited by PEGylation. The PEGylation of IL-6 markedly enhanced the blood-residency of IL-6, resulting in effective augmentation of its thrombopoietic activity and a marked decrease in its side-effects. These findings suggest that MPEG-IL-6 may be a potential candidate for thrombopoietic agent.

Molecular design of hybrid tumour necrosis factor-alpha. II: The molecular size of polyethylene glycol-modified tumour necrosis factor-alpha affects its anti-tumour potency.

To design hybrid tumour necrosis factor-alpha (TNF-alpha) applicable to systemic anti-tumour therapeutic use, we assessed the relationships among the molecular size of hybrid TNF-alpha, in vitro bioactivity and in vivo anti-tumour potency. Natural human TNF-alpha was covalently modified with polyethylene glycol (PEG) of various number-average molecular weights (Mn = 2000, 5000, 12,000). The in vitro bioactivity of PEG-modified TNF-alpha s decreased with an increase in the degree of PEG modification, irrespective of the molecular weight of PEG. This decrease in the specific bioactivity markedly increased with an increase in the molecular weight of the attached PEG. The in vivo anti-tumour effects of the hybrid TNF-alpha s with a molecular size from 100 to 110 kDa, which had more than 50% of specific bioactivity of native TNF-alpha, were significantly superior to other PEG-TNF-alpha s. These hybrid TNF-alpha s showed over ten times greater anti-tumour effects than native TNF-alpha. Thus, the molecular size, which was determined by the degree of PEG modification and PEG molecular weight, influences the specific activity and anti-tumour effects of hybrid TNF-alpha.

In vivo anti-tumor efficacy of polyethylene glycol-modified tumor necrosis factor-alpha against tumor necrosis factor-resistant tumors.

We previously reported that the optimally PEGylated tumor necrosis factor-alpha (MPEG-TNF-alpha), in which 56% of the TNF-alpha-lysine amino groups were coupled with polyethylene glycol (PEG), had about 100-fold greater anti-tumor effect than native TNF-alpha. Here, we assessed the usefulness of MPEG-TNF-alpha as a systemic anti-tumor therapeutic drug, using B16-BL6 melanoma and colon-26 adenocarcinoma, which have been reported to be resistant to TNF-alpha in vivo, as compared with Meth-A fibrosarcoma. MPEG-TNF-alpha markedly inhibited the growth of both tumors without causing any TNF-alpha-mediated side-effects, whereas native TNF-alpha had no anti-tumor effects and caused adverse side-effects. In addition, MPEG-TNF-alpha drastically inhibited the metastatic colony formation of B16-BL6 melanoma. MPEG-TNF-alpha may, thus, be a potential systemic anti-tumor therapeutic agent.

Molecular design of hybrid tumor necrosis factor-alpha III: polyethylene glycol-modified tumor necrosis factor-alpha has markedly enhanced antitumor potency due to longer plasma half-life and higher tumor accumulation.

We have reported that chemical modification of tumor necrosis factor-alpha (TNF-alpha) with polyethylene glycol (PEG) markedly increases its antitumor potency without any adverse side effects. MPEG-TNF-alpha, especially, in which 56% of the lysine amino groups of TNF-alpha are coupled with PEG, exhibits 100-fold more antitumor activity in vivo than native TNF-alpha in the Meth-A murine sarcoma model. In this study, we investigated the pharmacokinetics of PEG-modified TNF-alpha with various molecular sizes to clarify the mechanisms of the enhanced antitumor potency of MPEG-TNF-alpha. The plasma half-lives of modified TNF-alpha increased with increasing molecular size. The decreased plasma clearance of modified TNF-alpha was partially caused by the shielding effect of the proteolytic sites in TNF-alpha by the attached PEG and the decreased transport from blood to various tissues. Almost all native TNF-alpha was uniformly distributed to the kidney and reticuloendothelial system within 1 hr of an intravenous administration, and rapidly disappeared from these tissues at 3 hr. However, very little native TNF-alpha was transported into the tumor. The absolute distributed amount and distribution profile of modified TNF-alpha to tissues other than the tumor were the same as those of native TNF-alpha, whereas the plasma levels of the modified TNF-alpha were higher than plasma levels of the native TNF-alpha. The tumor distribution of modified TNF-alpha was markedly enhanced compared with native TNF-alpha and gradually increased over time. About 9-fold more MPEG-TNF-alpha was distributed to the tumor than native TNF-alpha. Thus, we found that the marked increase in the antitumor potency of PEG-modified TNF-alpha resulted from the enhanced blood residency and tumor accumulation. The antitumor effect of MPEG-TNF-alpha against sarcoma-180 other than Meth-A fibrosarcoma was also about 100 times greater than that of native TNF-alpha when systemically administered. The optimal PEGylation of TNF-alpha facilitated its antitumor potency and MPEG-TNF-alpha may be useful systemic antitumor therapeutic drug.

An intergenerational contraction of the CTG repeat in Japanese myotonic dystrophy.

We present the first report of a Japanese family with myotonic dystrophy (DM) that showed an intergenerational contraction of the CTG repeat. The size of the expanded CTG repeats was 3.2 kb for the father and 2.2 kb for the daughter, indicating that the expansion decreased during transmission from the father to the daughter. Despite the CTG repeat contraction, the daughter showed earlier age of onset than the father. However, she appeared to be less severely affected than the father. We discuss the correlation between the CTG repeat contraction and the clinical phenotype. The presence of the CTG repeat contraction in Japanese DM is important for genetic counseling of Japanese DM families.

Hemolytic anemia provoked by recombinant alpha-interferon.

A 30-year-old female with chronic hepatitis C treated with recombinant alpha-interferon developed serious hemolytic anemia after receiving 10 million units a day for 4 weeks. The results of Coombs' tests were negative before and after interferon therapy. The hemolytic anemia gradually improved after the withdrawal of recombinant alpha-interferon and the administration of methyl-prednisolone. The clinical course suggested that the recombinant alpha-interferon had provoked the hemolytic anemia.

Late onset hepatic failure due to hepatitis B virus with mutations in the pre-core region.

A 60-year-old man complained of severe general fatigue on October 11, 1992. Pertinent laboratory findings were: aspantate aminotransferase (AST) 1920 IU, alanine aminotransferase (ALT) 2050 IU, and total bilirubin (T. Bil) 124 micromol/l (normal range, 0-17 micromol/l). Virological assay revealed that hepatitis B surface antigen (HBsAg), anti-hepatitis B e (HBe), anti-HBc, and immunoglobulin M (IgM) anti-HBc were positive, and anti-HBs, HBeAg, and anti-delta antibody were negative. A diagnosis of acute hepatitis due to hepatitis B virus was made. Despite a decrease in transaminase, jaundice worsened and prothrombin time was prolonged. On the 60th day of hospitalization, massive ascites developed, but the patient's consciousness was not impaired. Although, albumin and diuretics were given, the ascites further increased. Paracentesis of 2000 ml of ascitic fluid was performed twice a week. On the 120th day of hospitalization, the patient passed black stools and he exhibited renal failure 3 weeks later. Although severe jaundice persisted, he was still alert. On the 150th day of hospitalization, massive gastrointestinal bleeding occurred, due to hemorrhagic gastritis. Despite receiving intensive care, the patient died. Determination of the HBV DNA sequence revealed two point mutations in the pre-core region; these have not been reported elsewhere.

Induction of IgA B cell differentiation of bone marrow-derived B cells by Peyer's patch autoreactive helper T cells.

The objective of this study was to demonstrate in vitro that bone marrow-derived pro/pre-B cells bearing mu mRNA can switch their Ig heavy-chain isotype to that of alpha mRNA-expressing B cells after contact with Peyer's patches-derived activated autoreactive CD4+ T cells. Bone marrow-derived pro/pre-B cells and activated autoreactive Peyer's patch, mesenteric lymph node, or spleen CD4+ T cells were co-cultured in the presence of recombinant (r) IL-2, rIL-7, and Con A for 3 days. The mixed cultured cells were isolated for preparation of total RNA. Dot/slot hybridization, using murine C mu (pu3741) and C alpha (P alpha J558) Ig heavy-chain cDNA probes, detected C mu and C alpha Ig heavy-chain mRNA transcripts. The magnitude of each mRNA expression was measured demsitometrically. In addition, the secreted class-specific Ig contents from the co-cultured supernatants were measured. The results indicate that activated autoreactive Peyer's patch and mesenteric lymph node CD4+ T cells provide a specific Ig heavy-chain switch from mu to alpha (Peyer's patch CD4+ T cells > mesenteric lymph node CD4+ T cells) in bone marrow-derived pro/pre-B cells and also assist to develop IgA-secreting plasma cells. The alpha heavy-chain switch and IgA production do not occur in the presence of activated autoreactive spleen CD4+ T cells. These results support the view that autoreactive gut Peyer's patch CD4+ T cells, at least, regulate IgA B cell heavy-chain switching and terminal differentiation during gut mucosal B cell development.

Hepatocyte regeneration in chronic hepatitis C and interferon treatment: analysis of immunohistological identification of proliferating cell nuclear antigen (PCNA).

To evaluate the usefulness of proliferating cell nuclear antigen (PCNA) immunostaining in the assessment of the efficacy of interferon (IFN) therapy in chronic hepatitis C, we investigated the proliferative activity of hepatocytes in 67 patients with chronic hepatitis C, using this immunostaining method. The percentage of PCNA-positive hepatocytes was 2.4% in patients with chronic persistent hepatitis, 2.5% in those with chronic aggressive hepatitis 2A, and 3.9% in those with chronic aggressive hepatitis 2B. The PCNA count increased with the progression of the liver disease. Patients were classified as complete, partial, and non-responders to IFN; the percentage of PCNA-positive hepatocytes before IFN therapy was 1.6% in the complete responders, 3.9% in the partial responders, and 4.9% in the non-responders. There was a significant negative correlation between the percentage of PCNA-positive hepatocytes and the response to IFN treatment. Thirty-two of 53 cases (60.4%) in which the PCNA labeling index (LI) was less than 5.0 were complete responders compared with 13 of 14 cases (92.9%) in which the PCNA LI was higher than 5.0, representing partial responders or non-responders (P < 0.001). Most complete responders had a low PCNA LI, irrespective of HCV genotype. Our findings indicate that PCNA immunostaining is a simple and reliable index of cell proliferation in liver regeneration, and may be a useful predictor of the response to IFN treatment in chronic hepatitis C.

Bunina body formation in amyotrophic lateral sclerosis: a morphometric-statistical and trace element study featuring aluminum.

Clinico-environmental and pathological variables were obtained from 10 patients with amyotrophic lateral sclerosis using particle-induced X-ray emission spectrometry (PIXE) and morphometric-statistical analysis. Statistical analysis identified a model that maximally predicts the Bb% (frequency of Bunina bodies) from a selected set, four variables: (1) nucleolar index, (2) magnesium (Mg) content, (3) aluminum (Al) content, and (4) duration of illness. Among them, only the Al content proved important. To determine their chemical nature, electron energy loss spectrometry (EELS) was applied at the ultrastructural level; it revealed that within the motor neuron, Al strongly binds to the Bunina body as well as rough endoplasmic reticulum (rER), and lesser strongly to mitochondria and lipofuscin granule. Thus, it is chemically similar to the rER, providing preferential binding sites to aluminum. The Bunina bodies may be an end-product of the nucleic acid dysmetabolism at rER caused by Al along with Mg depletion.

Molecular design of hybrid tumour necrosis factor alpha with polyethylene glycol increases its anti-tumour potency.

This study was conducted to increase the anti-tumour potency and reduce the toxic side-effects of tumour necrosis factor alpha (TNF-alpha). Natural human TNF-alpha was chemically conjugated with monomethoxy polyethylene glycol (PEG) using succinimidyl coupling of lysine amino groups of TNF-alpha. The number-average molecular weight of PEG-modified TNF-alpha (PEG-TNF-alpha) increased with an increase in the reaction time and the initial molar ratio of PEG relative to TNF-alpha. The resulting modified TNF-alpha was separated into fractions of various molecular weights. The specific activity of separated PEG-TNF-alpha s relative to that of native TNF-alpha gradually decreased with an increase in the degree of PEG modification, but the plasma half-life was drastically increased with the increase in molecular weight of modified TNF-alpha. PEG-TNF-alpha s, in which 29% and 56% of lysine residues were coupled to PEG, had anti-tumour activity approximately 4 and 100 times greater than unmodified TNF-alpha in the murine Meth-A fibrosarcoma model. Extensive PEG modification did not increase its in vivo activity. A high dose of unmodified TNF-alpha induced toxic side-effects, but these were not observed with the modified TNF-alpha s. Optimal PEG modification of TNF-alpha markedly increased its bioavailability and may facilitate its potential anti-tumour therapeutic use.