A case of GNE myopathy mimicking hereditary motor neuropathy.

A 36-year-old woman who presented with upper limb distal weakness since the age of 15 years, with gradual progression to the lower limbs, is reported. Hereditary motor neuropathy was initially suspected based on distal weakness and hyporeflexia; however, whole exome sequencing accidentally revealed a compound heterozygous variant in the GNE gene, and ultrasound revealed increased homogeneous echogenicity in the involved muscles, which is characteristic of myopathic changes. Muscle magnetic resonance imaging revealed fatty infiltration in all limb muscles, sparing the triceps brachii, vastus lateralis and vastus medialis. Muscle biopsy revealed intracytoplasmic rimmed vacuole, supporting the diagnosis of GNE myopathy.

Increased risk of osteoporotic vertebral fracture in rheumatoid arthritis patients with new-onset cardiovascular diseases: a retrospective nationwide cohort study in Taiwan.

INTRODUCTION: Both cardiovascular diseases (CVD) and osteoporosis are common comorbidities in rheumatoid arthritis (RA) patients. Although accumulating evidence indicates a link between CVD and osteoporotic fracture, whether CVD contributes to osteoporotic fracture risk in RA has yet to be explored. We examined the incidence rate and risk factors of osteoporotic vertebral fracture in RA patients with new-onset CVD (RA-CVD) and evaluated the effects of medications on such fracture risk. METHODS: A retrospective study was conducted using a nationwide database from 2000 to 2010: 1267 RA-CVD and 1267 non-CVD patients were enrolled from 30,507 patients with newly diagnosed RA. The main outcome was the development of osteoporotic vertebral fracture. After being adjusted for age, gender, and comorbidities, the Cox proportional hazard model was used to identify independent factors contributing to osteoporotic vertebral fracture. RESULTS: The adjusted hazard ratio (aHR) of developing osteoporotic vertebral fracture was 1.47-fold greater in RA-CVD group than in non-CVD group (95% confidence interval 1.19-1.81, p < 0.001). Both the age above 40 years and female gender were significant risk factors for developing osteoporotic vertebral fracture in RA-CVD patients. Using patients not taking medication as a reference group, the aHR of osteoporotic vertebral fracture was significantly lower in those receiving statins (0.50), low-dose corticosteroids (0.57), or hydroxychloroquine (0.12). CONCLUSIONS: The risk of osteoporotic vertebral fracture was significantly increased in RA-CVD patients, particularly women above 40 years of age, and could be reduced by statin therapy. However, the protective effect of low-dose corticosteroids or hydroxychloroquine on osteoporotic vertebral fracture risk needs further validation.

Phosphoproteomics in translational research: a sarcoma perspective.

Phosphoproteomics has been extensively used as a preclinical research tool to characterize the phosphorylated components of the cancer proteome. Advances in the field have yielded insights into new drug targets, mechanisms of disease progression and drug resistance, and biomarker discovery. However, application of this technology to clinical research has been challenging because of practical issues relating to specimen integrity and tumour heterogeneity. Beyond these limitations, phosphoproteomics has the potential to play a pivotal role in translational studies and contribute to advances in different tumour groups, including rare disease sites like sarcoma. In this review, we propose that deploying phosphoproteomic technologies in translational research may facilitate the identification of better defined predictive biomarkers for patient stratification, inform drug selection in umbrella trials and identify new combinations to overcome drug resistance. We provide an overview of current phosphoproteomic technologies, such as affinity-based assays and mass spectrometry-based approaches, and discuss their advantages and limitations. We use sarcoma as an example to illustrate the current challenges in evaluating targeted kinase therapies in clinical trials. We then highlight useful lessons from preclinical studies in sarcoma biology to demonstrate how phosphoproteomics may address some of these challenges. Finally, we conclude by offering a perspective and list the key measures required to translate and benchmark a largely preclinical technology into a useful tool for translational research.

Mitochondrial DNA variants as genetic risk factors for Parkinson disease.

BACKGROUND AND PURPOSE: Investigation of the relationship between mitochondrial DNA (mtDNA) variants and Parkinson disease (PD) remains an issue awaiting more supportive evidence. Moreover, an affirming cellular model study is also lacking. METHODS: The index mtDNA variants and their defining mitochondrial haplogroup were determined in 725 PD patients and 744 non-PD controls. Full-length mtDNA sequences were also conducted in 110 cases harboring various haplogroups. Cybrid cellular models, composed by fusion of mitochondria-depleted rho-zero cells and donor mitochondria, were used for a rotenone-induced PD simulation study. RESULTS: Multivariate logistic regression analysis revealed that subjects harboring the mitochondrial haplogroup B5 have resistance against PD (odds ratio 0.50, 95% confidence interval 0.32-0.78; P = 0.002). Furthermore, a composite mtDNA variant group consisting of A10398G and G8584A at the coding region was found to have resistance against PD (odds ratio 0.50, 95% confidence interval 0.33-0.78; P = 0.001). In cellular studies, B4 and B5 cybrids were selected according to their higher resistance to rotenone, in comparison with cybrids harboring other haplogroups. The B5 cybrid, containing G8584A/A10398G variants, showed more resistance to rotenone than the B4 cybrid not harboring these variants. This is supported by findings of low reactive oxygen species generation and a low apoptosis rate in the B5 cybrid, whereas a higher expression of autophagy was observed in the B4 cybrid particularly under medium dosage and longer treatment time with rotenone. CONCLUSIONS: Our studies, offering positive results from clinical investigations and cybrid experiments, provide data supporting the role of variant mtDNA in the risk of PD.

Systemic treatment of advanced clear cell sarcoma: results from a retrospective international series from the World Sarcoma Network.

BACKGROUND: Clear cell sarcoma (CCS) is a translocated aggressive malignancy with a high incidence of metastases and poor prognosis. There are few studies describing the activity of systemic therapy in CCS. We report a multi-institutional retrospective study of the outcomes of patients with advanced CCS treated with systemic therapy within the World Sarcoma Network (WSN). MATERIALS AND METHODS: Patients with molecularly confirmed locally advanced or metastatic CCS treated with systemic therapy from June 1985 to May 2021 were included. Baseline demographic and treatment information, including response by Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, was retrospectively collected by local investigators. Descriptive statistics were carried out. RESULTS: Fifty-five patients from 10 institutions were included. At diagnosis, the median age was 30 (15-73) years and 24% (n = 13/55) had metastatic disease. The median age at diagnosis was 30 (15-73) years. Most primary tumours were at aponeurosis (n = 9/55, 16%) or non-aponeurosis limb sites (n = 17/55, 31%). The most common fusion was EWSR1-ATF1 (n = 24/55, 44%). The median number of systemic therapies was 1 (range 1-7). The best response rate was seen for patients treated with sunitinib (30%, n = 3/10), with a median progression-free survival of 4 [95% confidence interval (CI) 1-7] months. The median overall survival for patients with advanced/metastatic disease was 15 months (95% CI 3-27 months). CONCLUSIONS: Soft tissue sarcoma-type systemic therapies have limited benefit in advanced CCS and response rate was poor. International, multicentre prospective translational studies are required to identify new treatments for this ultra-rare subtype, and access to early clinical trial enrolment remains key for patients with CCS.

Intravenous tissue plasminogen activator for acute ischemic stroke in patients with renal dysfunction.

OBJECTIVE: This study used the Taiwan Stroke Registry data to evaluate the efficacy and safety of intravenous tissue plasminogen activator (tPA) in treating acute ischemic stroke in patients with renal dysfunction. DESIGN: We identified 3525 ischemic stroke patients and classified them into two groups according to the estimated glomerular filtration rate (eGFR) at the emergency department: ≥60, and <60 ml/min/1.73 m2 or on dialysis and by the propensity score from August 2006 to May 2015. The odds ratio of poor functional outcome (modified Rankin Scale ≥2) was calculated for patients with tPA treatment (N = 705), compared to those without tPA treatment (N = 2820), by eGFR levels, at 1, 3 and 6 months after ischemic stroke. We also evaluated the risks of intracerebral hemorrhage, upper gastrointestinal bleeding, mortality, between the two groups by eGFR levels. RESULTS: Among patients with eGFR levels of <60 ml/min/1.73 m2, tPA therapy reduced the odds ratio of poor functional outcome to 0.60 (95% confidence interval = 0.42-0.87) at 6 months after ischemic stroke. The tPA therapy was not associated with increased overall risk of upper gastrointestinal bleeding, but with increased risk of intracerebral hemorrhage. The low eGFR was not a significant risk factor of intracerebral hemorrhage among ischemic stroke patients receiving tPA treatment. CONCLUSIONS: tPA for acute ischemic stroke could improve functional outcomes without increasing the risks of upper gastrointestinal bleeding for patients with or without renal dysfunction. The low eGFR was not a significant risk factor for intracerebral hemorrhage among patients receiving tPA treatment.

Upregulated ankyrin repeat-rich membrane spanning protein contributes to tumour progression in cutaneous melanoma.

BACKGROUND: We have previously demonstrated that overexpression of ankyrin repeat-rich membrane spanning (ARMS) protein facilitates melanoma formation via conferring apoptotic resistance. This study aims to investigate whether ARMS contributes to melanoma progression. METHOD: Using immunohistochemistry, we graded the expression level of ARMS in 54 cases of primary melanoma and 46 cases of metastatic melanoma. The immunointensity of ARMS was statistically correlated with individual clinicopathological characteristics. By RNA interference, stable melanoma cell clones with ARMS-knockdown were constructed, and were used for in vitro scratch wound, transwell invasion assays, and in vivo lung metastasis experiment. RESULTS: Stronger immunointensity of ARMS was observed mostly in melanomas with Breslow tumour thickness >1.0 mm (Fisher's exact test, P=0.002) or with nodal metastasis (Fisher's exact test, P=0.026), and was correlated with a worse overall survival in melanoma patients (log-rank test, P=0.04). Depletion of ARMS inhibited migration, invasion, and metastatic potential of melanoma cells in vitro and in vivo. Moreover, ARMS mediated melanoma cell migration and invasion through activation of the extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signalling pathway. CONCLUSION: Ankyrin repeat-rich membrane spanning expression, conjunctly with tumour thickness or ulceration, may serve as a prognostic factor in patients with cutaneous melanoma.

Using mass spectrometry to detect buffalo salivary odorant-binding protein and its post-translational modifications.

A large number of mammalian odorant-binding proteins, which are lipocalins, have been studied. These proteins participate in peri-receptor events by selecting and carrying odorant molecules. The present study aimed at identifying the buffalo salivary odorant-binding protein (sOBP), and to determine its post-translational modification using mass spectrometry. The buffalo salivary 21 kDa protein was initially separated adopting sodium dodecyl sulfate-polyacrylamide gel electrophoresis and it was identified as sOBP with high statistical reliability using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and SEQUEST, for the first time. Further, the post-translationally modified peptides were screened adopting MS/MS. A total of four post-translational modifications, namely glycation at lysine-(59), hydroxylation at lysine-(134), ubiquitination at lysine-(121), and dihydroxylation in lysine-(108), were recorded. Moreover, these modifications have not been identified in buffalo salivary odorant-binding protein.

Correction: Role of integrin-linked kinase in regulating the protein stability of the MUC1-C oncoprotein in pancreatic cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of novel vesicles in the cytosol to vacuole protein degradation pathway.

The key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose. FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated. In some vid mutants, FBPase is found in punctate structures in the cytoplasm. When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants. In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells. We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity. Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway. Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles. Under EM, these vesicles are 30-40 nm in diam. Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles. We propose that FBPase is imported into these vesicles before entering the vacuole.

The adequacy of tissue microarrays in the assessment of inter- and intra-tumoural heterogeneity of infiltrating lymphocyte burden in leiomyosarcoma.

The characterisation and clinical relevance of tumour-infiltrating lymphocytes (TILs) in leiomyosarcoma (LMS), a subtype of soft tissue sarcoma that exhibits histological heterogeneity, is not established. The use of tissue microarrays (TMA) in studies that profile TIL burden is attractive but given the potential for intra-tumoural heterogeneity to introduce sampling errors, the adequacy of this approach is undetermined. In this study, we assessed the histological inter- and intra-tumoural heterogeneity in TIL burden within a retrospective cohort of primary LMS specimens. Using a virtual TMA approach, we also analysed the optimal number of TMA cores required to provide an accurate representation of TIL burden in a full tissue section. We establish that LMS have generally low and spatially homogenous TIL burdens, although a small proportion exhibit higher levels and more heterogeneous distribution of TILs. We show that a conventional and practical number (e.g. ≤3) of TMA cores is adequate for correct ordinal categorisation of tumours with high or low TIL burden, but that many more cores (≥11) are required to accurately estimate absolute TIL numbers. Our findings provide a benchmark for the design of future studies aiming to define the clinical relevance of the immune microenvironments of LMS and other sarcoma subtypes.

Clinical effectiveness and safety experience with alefacept in the treatment of patients with moderate-to-severe chronic plaque psoriasis in Taiwan: results of an open-label, single-arm, multicentre pilot study.

BACKGROUND: The effectiveness and safety of alefacept for the treatment of moderate-to-severe chronic plaque psoriasis has been established in several clinical trials conducted in the United States and Europe. No clinical trial of alefacept has been conducted in Asia. OBJECTIVE: To determine the effectiveness and safety of alefacept in the treatment of psoriasis in Chinese population. DESIGN AND METHODS: This was an open-label, single-arm, multicentre pilot study conducted at three centres. Patients with a body surface area > or = 10% and psoriasis area and severity Index (PASI) > or = 12 were given 15 mg alefacept intramuscularly once a week for 12 weeks and were then followed up for a further 12 weeks. RESULTS: A total of 46 patients was enrolled. Only one subject (2%) achieved a > or = 75% improvement in PASI at week 14. The median improvement in PASI at week 14 after the 12-week treatment was 39%. At any time during the 6-month course, 3 subjects (7%) achieved a Physician Global Assessment (PGA) of 'almost clear', and a > or = 50% and > or = 75% improvement in PASI was seen in 46% and 9%, respectively. There is a trend for decreased counts of CD4(+) and CD8(+) cells after alefacept treatment, but subjects who achieved PASI50 showed a lesser degree of decrease in CD4(+) and CD8(+) counts compared with those in patients who did not achieve PASI50. CONCLUSIONS: This small pilot study indicated that intramuscular alefacept was effective and safe in psoriasis in Chinese patients over 12 weeks of treatment. Further studies are needed to clarify the reason for low PASI 75 effectiveness and the paradoxical lesser decline of CD4(+) and CD8(+) T cells in those who responded.

Role of integrin-linked kinase in regulating the protein stability of the MUC1-C oncoprotein in pancreatic cancer cells.

MUC1-C overexpression has been associated with the progression of pancreatic tumors by promoting the aggressive and metastatic phenotypes. As MUC1 is a STAT3 target gene, STAT3 plays a major role in regulating MUC1-C expression. In this study, we report an alternative mechanism by which integrin-linked kinase (ILK) post-transcriptionally modulates the expression of MUC1-C by maintaining its protein stability in pancreatic cancer cells. We found that ILK acts in concert with STAT3 to facilitate IL-6-mediated upregulation of MUC1-C; ILK depletion was equally effective as STAT3 depletion in abolishing IL-6-induced MUC1-C overexpression without disturbing the phosphorylation or cellular distribution of STAT3. Conversely, ectopic expression of constitutively active ILK increased MUC1-C expression, though this increase was not noted with kinase-dead ILK. This finding suggests the requirement of the kinase activity of ILK in regulating MUC1-C stability, which was confirmed by using the ILK kinase inhibitor T315. Furthermore, our data suggest the involvement of protein kinase C (PKC)δ in mediating the suppressive effect of ILK inhibition on MUC1-C repression. For example, co-immunoprecipitation analysis indicated that ILK depletion-mediated MUC1-C phosphorylation was accompanied by increased phosphorylation of PKCδ at the activation loop Thr-507 and increased binding of PKCδ to MUC1-C. Conversely, ILK overexpression resulted in decreased PKCδ phosphorylation. From a mechanistic perspective, the present finding, together with our recent report that ILK controls the expression of oncogenic KRAS through a regulatory loop, underscores the pivotal role of ILK in promoting pancreatic cancer progression.

TGFß promotes mesenchymal phenotype of pancreatic cancer cells, in part, through epigenetic activation of VAV1.

The highly homeostasis-resistant nature of cancer cells leads to their escape from treatment and to liver metastasis, which in turn makes pancreatic ductal adenocarcinoma (PDAC) difficult to treat, especially the squamous/epithelial-to-mesenchymal transition (EMT)-like subtype. As the molecular mechanisms underlying tumour heterogeneity remain elusive, we investigated whether epigenetic regulation might explain inter-individual differences in the progression of specific subtypes. DNA methylation profiling performed on cancer tissues prior to chemo/radiotherapy identified one hypermethylated CpG site (CpG6882469) in the VAV1 gene body that was correlated with demethylation of two promoter CpGs (CpG6772370/CpG6772811) in both PDAC and peripheral blood. Transforming growth factor ß treatment induced gene-body hypermethylation, dissociation of DNMT1 from the promoter, and VAV1 expression via SMAD4 and mutant KrasG12D. Pharmacological inhibition of TGFß-VAV1 signalling decreased the squamous/EMT-like cancer cells, promoted nuclear VAV1 localization, and enhanced the efficacy of gemcitabine in prolonging the survival of KPfl/flC mice. Together, the three VAV1 CpGs serve as biomarkers for prognosis and early detection, and the TGFß-VAV1 axis represents a therapeutic target.

Accuracy and Trending of Continuous Noninvasive Hemoglobin Monitoring in Patients Undergoing Liver Transplantation.

BACKGROUND: Shift in large fluid volumes and massive blood loss during liver transplantation frequently leads to rapid changes in hemoglobin (Hb) concentration; thus, to ensure adequate tissue oxygenation, accurate and rapid determination of Hb concentration is essential in transplant recipients. The Radical-7 Pulse CO-Oximeter provides a noninvasive and continuous way to monitor Hb concentration (SpHb) in real time and is an ideal candidate for use during liver transplantation. In this study, we assessed the relationship between SpHb and total Hb (tHb) obtained from arterial blood samples during surgery. METHODS: Forty patients undergoing liver transplantation were enrolled in this study. tHb and time-matched SpHb were measured at 5 different phases throughout surgery. Paired SpHb and tHb levels were assessed using linear regression, Bland-Altman analysis, and the Critchley polar plot method. RESULTS: A total of 161 paired measurements with sufficient signal quality were analyzed. The correlation between SpHb and tHb was 0.59 (P < .001). Bland-Altman analysis revealed that a bias between SpHb and tHb was 2.28 g/dL, and limits of agreement (LoA) were from -0.78 to 5.34 g/dL. Trending analysis showed that 87% of data were located within the acceptable trending area, indicating that the trending ability was not satisfied. CONCLUSIONS: The Radical-7 Pulse CO-Oximeter was not sufficient to monitor Hb levels and trends during liver transplantation surgery in our cohort. In particular, in critical patients and in those with low Hb levels, invasive Hb measurement should be used for assessment.

Cardiac Output Assessed by the Fourth-Generation Arterial Waveform Analysis System Is Unreliable in Liver Transplant Recipients.

BACKGROUND: Liver transplant recipients often have violent hemodynamic fluctuation during surgery that may be related to perioperative and postoperative morbidity. Because there are some considerations for the risk of the pulmonary arterial catheter (PAC), the conventional invasive device for cardiac output (CO) measurement, a reliable and minimally invasive alternative is required. We validated the reliability of CO measurements with the use of a minimally invasive FloTrac system with the latest fourth-generation algorithm in liver transplant recipients. METHODS: Forty liver transplant recipients without atrial fibrillation, valvular pathology, or intracardiac shunt were recruited in this prospective, observational study. CO values measured by use of PAC with continuous thermodilution method (COTh) and FloTrac devices (COFT) were collected simultaneously throughout the operation for reliability validation. RESULTS: Four hundred pairs of CO data points were collected in total. The linear regression analysis showed a high correlation coefficient (73%, P < .001). However, the percent error between COTh and COFT was 42.2%, which is worse than the established interchangeability criterion of 30%. The concordance rates were calculated at 89% and 59% by 4-quadrant plot and polar plot analysis, respectively. Neither met the preset validation criteria (>92% for the 4-quadrant plot and >90% for polar plot analyses). CONCLUSIONS: Our study demonstrates that the CO measurements in liver transplant recipients by the latest FloTrac system and the PAC do not meet the recognized interchangeability criterion. Although the result showed improvement in linear regression analysis, it failed to display a qualified trending ability.

The Distribution, Drug Resistance, and Clinical Characteristics of Acinetobacter baumannii Infections in Solid Organ Transplant Recipients.

OBJECTIVE: Acinetobacter baumannii has become a major problem among solid organ transplant (SOT) recipients. The aim of this study was to investigate the distribution, drug resistance, and clinical characteristics of A baumannii infections in SOT recipients. METHODS: We retrospectively identified 78/1,850 (4.2%) SOT recipients who developed A baumannii infections from January 1, 2003, to April 1, 2015, and evaluated the distribution, drug resistance, and clinical characteristics of these infections. RESULTS: Over the study period, 101 episodes of A baumannii infection occurred in 78 SOT recipients, with respiratory tract (37.6%) and blood (35.6%) as the most common sites of infection. Fifty-six episodes of A baumannii infection were accompanied with a serum creatinine level of >1.5 mg/dL. Multidrug resistance (MDR) or extensive drug resistance (XDR) occurred in 83.2%. Antibiotic resistance rate of all A baumannii to 8 of 9 antibiotics investigated was >50%. Seventy-eight percent of A baumannii were carbapenem-resistant. All but one A baumannii isolates tested against polymyxin B were susceptible. There were 40 (51.3%) overall in-hospital deaths and 31 (39.7%) infection-related deaths. CONCLUSIONS: A baumannii infections are associated with high morbidity and mortality in SOT recipients, and MDR or XDR is common. Prevention measures are essential, and combination therapy of antibiotics are needed to improve the outcomes of SOT recipients with A baumannii infections.

Mice deficient in hepsin, a serine protease, exhibit normal embryogenesis and unchanged hepatocyte regeneration ability.

Hepsin, a liver-enriched novel serine protease, has been implicated in participating with normal cell growth, embryogenesis, and blood coagulation pathway. To study its function in vivo, we have disrupted the mouse hepsin gene by homologous recombination. Targeted disruption of the hepsin gene and ablation of hepsin message were demonstrated by Southern blotting, Northern blotting and RT-PCR analysis. Homozygous hepsin -/- mice were viable, fertile, and exhibited no gross abnormalities, as judged by the size, weight and blood coagulation (PT) assays. However, the serum concentration of the bone form of alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase of the hepsin -/- mice was mildly elevated, in spite of no obvious pathological change of hepatocytes. To examine whether hepsin is involved in controlling cell growth in adult tissues, 70% hepatectomy was applied to the hepsin -/- mice. Liver regeneration proceeded normally in the hepsin -/- mice as judged by the liver mass restoration rate. These results suggest that loss of hepsin function causes no effect in cell growth and embryogenesis in vivo, which is in contradiction to the studies using in vitro cell culturing system. Moreover, gross mass regeneration of liver after damage proceeds normally in the absence of functional hepsin.

Short-term carcinogen-induced changes in composition of rat liver chromatin protein.

Changes in the composition of rat liver chromatin protein were detected by electrophoretic analysis 4 h after a single injection of 10-30 mg/kg body weight dimethylnitrosamine. Non-histone bands were reduced in staining intensity and increases in all F2 histone subfractions were observed. The staining intensity of F1 and F3 histone was unchanged throughout the experiment. By 24 h after administration of a non-necrotizing dose of dimethylnitrosamine, the proportion of chromatin proteins, when compared to controls, was marked by an increase in non-histones and decreased amounts of F2 histone subfractions. Slight differences between treated and control were evident at 48 h, suggesting that at least this interval is required for recovery from in vivo methylation.