Cloning of TC-1 (C8orf4), a novel gene found to be overexpressed in thyroid cancer.

A novel gene highly expressed in thyroid cancer, designated TC-1 (thyroid cancer-1), was cloned from suppression subtractive hybridization between papillary thyroid carcinoma and its surrounding normal thyroid tissue. Overexpression of TC-1 in thyroid cancer was confirmed in 15/16 paired samples by RT-PCR and Northern analysis. Ubiquitously expressed in human tissues, the TC-1 sequence showed no homology to any known gene, but matched a cluster of ESTs. After alignment of our sequence with the ESTs, the missing transcription start site was obtained by 5'-RACE and verified by primer extension analysis. The full-length mRNA sequence of 1327 bp has an open reading frame of 321 bp, which encodes a highly conserved protein. Three regulatory motifs were identified at the expected positions within 1 kb of the 5' flanking sequence obtained by genome walking. Using fluorescence in situ hybridization, TC-1 was localized to chromosome 8p11.2. The overexpression of TC-1 in papillary carcinoma suggests that it may have an important role in thyroid carcinogenesis.

Prevalence and distribution of ret/ptc 1, 2, and 3 in papillary thyroid carcinoma in New Caledonia and Australia.

The world's highest incidence of thyroid cancer has been reported among females in New Caledonia, a French overseas territory in the Pacific located between Australia and Fiji. To date, no molecular genetic studies in this population are available. Over the past few years, the oncogenic rearrangement of the ret protooncogene (ret/ptc) has been studied in papillary carcinomas in different populations. In this study, we investigated the prevalence and distribution of ret/ptc1, 2, and 3 in papillary thyroid carcinoma from the New Caledonian population and compared the pattern with that of an Australian population. Fresh-frozen and paraffin-embedded papillary carcinomas from 27 New Caledonian and 20 Australian patients were examined for ret rearrangements by means of RT-PCR with primers flanking the chimeric region, followed by hybridization with radioactive probes. ret/ptc was present in 70% of the New Caledonian and in 85% of the Australian samples. Multiple rearrangements were detected and confirmed by sequencing in 19 cases, 4 of which had 3 types of rearrangements in the same tumor. This study demonstrates a high prevalence of ret/ptc in New Caledonian and Australian papillary carcinoma. The findings of multiple ret/ptc in the same tumor suggest that some thyroid neoplasms may indeed be polyclonal.

The economic burden of insulin resistance.

Diabetes mellitus has reached epidemic proportions worldwide as we enter the new millennium. The World Health Organization (WHO) has commented there is 'an apparent epidemic of diabetes which is strongly related to lifestyle and economic change'. Over the next decade the projected number will exceed 200 million, possibly reaching 250 million persons. Most will have type 2 diabetes and all are at risk of the development of complications. Better education, improved nutrition, more exercise, early diagnosis and prompt treatment are imperative. Diabetes is a serious disease, subject to the development of many complications affecting large vessels (heart, cerebral and peripheral), small vessels (kidney and retina), nerves and other organs. In type 2 diabetes these complications may precede diagnosis of the disease by many years. The process continues inexorably, with premature mortality and morbidity mainly from the development of vascular disease. Data from the WHO confirm the principal role of non-communicable disease on mortality in developed countries, while mortality in developing countries is rising rapidly, now often exceeding communicable disease. The non-communicable diseases are divided into cancer and degenerative diseases. In the developed world, degenerative diseases are grouped to include ischaemic heart disease, stroke, renal failure, hypertension and other macro- and microvascular diseases. The major complications of diabetes encountered most frequently and with the greatest impact are: 1. Neuropathy, both peripheral and autonomic, with principal manifestations in the lower limbs 2. Microvascular disease, mainly affecting the retina and kidney, resulting in blindness and renal failure 3. Macrovascular disease, presenting with atherosclerosis in the coronary arteries causing ischaemic heart disease, cerebrovascular disease causing stroke and peripheral vascular disease contributing to diabetic gangrene.

Competitive RT-PCR for measuring metalloproteinase gene expression in human mesangial cells exposed to a hyperglycemic environment.

Competitive reverse transcription (RT)-PCR is now considered a reliable technique for determining the relative or absolute copy number of target mRNAs in tissue or cells. The method relies on a synthetic RNA competitor molecule with identical sequences to the PCR primers that is added to the RT step allowing reproducible quantitation between samples. In this report, we demonstrate a strategy for developing competitive RT-PCR assays using a multi-template RNA competitor molecule that is synthesized using solely PCR-based techniques. The approach we describe is both cost- and time-effective and importantly, the approach saves RNA because it allows the simultaneous quantitation of three target mRNAs from as little as one microgram of total RNA. The procedure for preparing the RNA competitor molecule, the RT-PCR protocol and the use of the competitor molecule in our studies of diabetic nephropathy are described in detail.

The role of the mesangial cell and its matrix in the pathogenesis of diabetic nephropathy.

Mesangial cells are pericyte-like cells which are found the glomeruli of the kidney. It is well known that they have important contractile and synthetic properties regulating the function of the glomerulus. During diabetes the synthesis of various extracellular matrix (ECM) components by mesangial cells are increased. In recent years it has been recognized that degradation of ECM may also be decreased in diabetes, contributing to the process of mesangium accumulation. The major enzymes responsible for ECM degradation are a large group of enzymes collectively known as matrix metalloproteinases (MMPs). The physiology of MMPs is complex and their activity is tightly regulated at many levels. The MMPs are synthesized as proenzymes and require activation via catalytic cleavage to become fully active. In this regard it is of importance that the mesangial cell and its pericellular matrix have a very active plasminogen cascade that can liberate plasmin locally to mediate matrix degradation both directly and indirectly, by activating the MMPs. In addition, the MMPs are regulated by transforming growth factor beta (TGF-beta). There is evidence that each of these pathways regulating the matrix degradation is affected by the diabetic environment and this will be the subject of this contribution.

Effect of glucose on matrix metalloproteinase activity in mesangial cells.

Mesangial cells are known to secrete matrix metalloproteinases (MMPs). These enzymes play a major role in the degradation and remodelling of extracellular matrix, and alterations in their activity may contribute to the mesangium enlargement of diabetic nephropathy. MMPs are secreted as latent forms which are cleaved in the pericellular environment to form active enzymes. In this study, we used a biosynthetically labelled matrix as substrate and conditioned medium obtained from mesangial cells, as a source of enzymes to investigate the effect of a high glucose concentration on degradative capacity. Inhibitor studies showed that MMPs were responsible for 72.2% of the degradation. A high glucose concentration caused a significant reduction in matrix degradation (low glucose 33.5 +/- 5.6%, high glucose 24.2 +/- 4.8%). Addition of aminophenyl mercuric acetate to activate latent MMPs increased matrix degradation by 2.3-fold in both low- and high-glucose media, but the decreased degradation caused by a high glucose concentration was still apparent. Activation with plasmin also increased matrix degradation and abolished the effect of the high glucose concentration. Gelatin zymography showed that mesangial cells grown at a low glucose concentration secreted both 72- and 92-kD gelatinases; however, at high glucose concentrations the 92-kD gelatinase was no longer apparent. These results suggest that a high glucose concentration causes a reduction in the amount of MMPs secreted by the mesangial cells. This reduction may contribute to the mesangium enlargement of diabetic nephropathy.

High glucose inhibits effect of ascorbic acid on [35S] sulphate incorporation in mesangial cell and matrix proteoglycan.

Expansion of the glomerular mesangium is a consistent finding of diabetic nephropathy. Negatively charged proteoglycans are an integral part of the mesangium and their synthesis and degradation is disturbed in many forms of glomerulosclerosis. The metabolism of ascorbic acid (AA), which plays an important role in extracellular matrix regulation, is known to be abnormal in diabetes. The action of AA has also been shown to be inhibited by high glucose (HG) concentration. In this study we investigated the effect of AA and HG on proteoglycan (PG) synthesis by examining the incorporation of [35S] sulphate into PG in the cellular, matrix and media components of rat mesangial cell (MC) cultures. MC were grown in 9 or 25 mM glucose for 8 days, with and without the addition of AA. Sulphation of PG was measured by adding 50 microCi of [35S] sulphuric acid to the culture medium and precipitating 35S-labelled PG with cetylpyridinium chloride. In this study AA was shown to have a stimulatory effect on the overall incorporation of [35S] sulphate into cell and matrix PG and this was inhibited by 25 mM glucose. Correcting for protein synthesis and specific activity of [35S] sulphate showed that HG inhibits AA stimulation by decreasing sulphation of the individual PG molecules. These findings may be of particular importance in the pathophysiology of nephropathy in diabetes, a condition where AA concentration is already compromised.

High glucose reduces generation of plasmin activity by mesangial cells.

Mesangium enlargement is a central feature of diabetic nephropathy and almost certainly plays a pathogenic role in this condition. Previous studies have shown that mesangium degradation is reduced in a high glucose mileau. Plasmin has been shown to play an important role in extracellular matrix degradation, both directly and through its ability to activate the matrix metalloproteinases. We therefore investigated how high glucose concentration may affect the various components of the plasminogen cascade on mesangial cells and whether it impairs the ability of the mesangial cell to generate plasmin activity. Result showed decreased binding of plasminogen and the urokinase type plasminogen activator to the mesangial cell surface while the tissue type plasminogen activator and the plasminogen activator-1 associated with mesangial cells were increased. The net effect of these changes was a reduced capacity of mesangial cell layers to generate plasmin activity in a high glucose environment. We postulate that this may be of importance in the reduced mesangium degradation which occurs in diabetes.

Why does ethnicity affect prevalence of gestational diabetes? The underwater volcano theory.

To study why gestational diabetes (GDM) is more common in some ethnic groups than others, we tested the hypothesis that GDM is more common in people who are temporally closer to developing non-insulin-dependent (Type 2) diabetes mellitus (NIDDM). The prevalence of GDM and the mean age of affected women in each major ethnic group were determined. From our database of NIDDM 6052 patients, the mean age of onset in each ethnic gorup was calculated and the mean difference between age of developing GDM and age of developing NIDDM derived (NIDDM-GDM age gap). This age gap was used to adjust for the susceptibility to GDM of each group. The overall prevalence of GDM was 6.7% (CI 6.0%-7.4%). In Anglo-Celtic women it was 3.0% (CI 2.3%-3.7%). For the other ethnic groups the prevalence and odds ratio (OR) were: Chinese (15.0% CI 11.8%-18.2% OR 5.6), Vietnamese (9.6% CI 6.6%-12.5% OR 3.6), Indian (16.7% CI 9.8%-23.5% OR 6.4), Arabic (7.3% CI 4.6%-10.1% OR 2.5) and Aborigines (10.1% CI 3.8%-16.4% OR 3.7). The OR for susceptibility to GDM did not change after adjustment for BMI and maternal age and it correlated significantly with the NIDDM-GDM age gap (r = -0.85; p = 0.03). However, it fell substantially after adjustment for NIDDM-GDM age gap. For women of different ethnic origins there is a difference in the time gap between their pregnancies and the time at which they would on average be expected to develop diabetes. This difference may be an important factor underlying the higher prevalence of GDM in some ethnic populations.

Urinary glycosaminoglycan excretion in NIDDM subjects: its relationship to albuminura.

Nephropathy is a serious microvascular complication of diabetes mellitus which is preceded by a period of microalbuminura. Increased loss of proteoglycan (PG) from glomerular basement (GBM) has been postulated to alter glomerular charge selectivity which contributes to urinary loss of albumin. In this study we measured the excretion of urinary glycosaminoglycans (GAG), the degradation products of PG, in 82 non-insulin-dependent (NIDDM) (Type 2) diabetic and 34 non-diabetic subjects. We found that diabetic subjects had a significantly higher GAG urinary excretion rate compared to non-diabetic subjects (12.54 +/- 5.67 vs 8.80 +/- 3.99 micrograms glucuronic acid min-1, p = 0.0001). Categorizing for albuminuric status shows that the diabetic normo-, micro- and macroalbuminuric groups have a higher GAG excretion rate than non-diabetic subjects. Heparan sulphate (HS) GAG urinary excretion was measured in 25 samples from diabetic subjects and 18 non-diabetic subjects. Diabetic subjects excreted more HS GAG than controls both as a rate or as a percentage of total GAG (3.70 +/- 1.94 vs 2.38 +/- 1.48 micrograms glucosamine min-1, p = 0.02; 31.6% +/- 12.5 vs 23.1% +/- 10.4, p = 0.02). Categorizing for albuminuric status shows that micro- and macro-albuminuric groups have a significantly higher HS GAG excretion rate than non-diabetic subjects. We conclude that, as in IDDM, excretion of GAG and HS GAG is higher in NIDDM and may precede the development of microalbuminuria.

Reduction of leucocyte proteolytic enzyme activity in diabetic patients with microalbuminuria and proteinuria: its possible role in diabetic nephropathy.

Mesangium enlargement and glomerular basement membrane thickening are cardinal features of diabetic nephropathy. The reasons for these changes are uncertain but decreased degradation of extracellular matrix may play a role. Mesangium degradation can be modulated by factors intrinsic to the kidney or by factors in the circulation. In this study the capacity of leucocyte proteolytic enzymes to degrade mesangium matrix materials was investigated. Leucocytes were obtained from 57 patients with NIDDM (age 58.3 +/- 8.8 years, duration 9.4 +/- 7.3 years, body mass index (BMI) 30 +/- 6 kg m-2, HbA1c 7.7 +/- 2.0%) and 21 control subjects (age 55.1 +/- 14.6 years, BMI 25 +/- 4 kg m-2). Leucocyte lysates from control and NIDDM subjects with normal AER degraded matrix to the same extent (40.6 +/- 8.2% vs 42.9 +/- 13.5%) while lysates from patients with microalbuminuria and proteinuria were less able to degrade matrix (33.0 +/- 14.2% and 26.1 +/- 12.7%, respectively). There was a significant inverse correlation between matrix degradation and AER (r = -0.49) and multiple regression analysis showed that AER was the most important factor determining degradation rate (R2 = 0.24). Degree of metabolic control, age, and blood pressure were not significant factors. The major enzyme(s) responsible for the matrix degradation was identified as metalloproteinase(s). We conclude that leucocytes from diabetic patients with abnormal albumin excretion have a decreased proteolytic capacity to degrade extracellular matrix. This may play a role in the glomerular basement membrane thickening and mesangium expansion which occurs in diabetic nephropathy.

Cell-associated proteoglycans of retinal pericytes and endothelial cells: modulation by glucose and ascorbic acid.

Abnormalities of retinal pericytes and endothelial cells are prominent features of diabetic retinopathy. In this study, we used cultures of bovine retinal cells to examine the regulation of cell-associated proteoglycans, a class of highly sulfated macromolecules important in the regulation of cell growth. Bovine retinal pericytes and endothelial cells were radiolabeled with 35SO4 and cell-associated proteoglycans were removed from the cell surface, quantified, and characterized. The effects of high glucose concentration (25 mM), phorbol 12,13-dibutyrate (PDBu, 0.1 microM), and ascorbic acid (0.1 mM) on cell-associated proteoglycans and growth of these cells were studied. Our results showed that both the ionically bound and the membrane-intercalated forms of cell-associated proteoglycans are present on retinal cells. The predominant cell-associated proteoglycan of pericytes is chondroitin sulfate and for endothelial cells it is heparan sulfate. High glucose concentration and ascorbic acid increased the cell-associated proteoglycans on pericytes but reduced them on endothelial cells. In contrast to this divergent trend, high glucose concentration and ascorbic acid inhibited the growth of both pericytes and endothelial cells. The effects of high glucose on retinal cell-associated proteoglycans were mimicked by PDBu added in a manner to stimulate protein kinase C activity. We conclude that cell-associated proteoglycans are present on retinal pericytes and endothelial cells. High glucose concentration and ascorbic acid affect cell-associated proteoglycans of these two cell types in opposite directions, whereas both suppress the growth of the two cell types. Therefore, it is not likely that high glucose concentration and ascorbic acid change the rate of retinal cell growth directly by affecting cell-associated proteoglycan levels.

High glucose concentration causes a decrease in mesangium degradation. A factor in the pathogenesis of diabetic nephropathy.

Mesangium enlargement is a constant feature of diabetic nephropathy and is likely to be important in the pathogenesis of this diabetic complication. Whether decreased degradation of mesangium plays any role in causing the enlargement is uncertain. We developed a system of preparing radioactively labeled mesangium matrix from mesangial cell cultures to be used as substrates for studies of mesangium degradation. Degradation is commenced by growing mesangial cells on the labeled matrix and monitored by the release of radioactivity into the culture medium. High glucose concentration (30 mM), whether present 1) when the matrix is being made or 2) when the degradation is taking place, reduces the rate of mesangium degradation. The second but not the first of these two phenomena was abolished by aminoguanidine. Phorbol 12-myristate 13-acetate, added in a manner to antagonize the action of protein kinase C, inhibited mesangium degradation and was not able to nullify the effect of high glucose. Thus it appears unlikely that a high glucose concentration inhibits mesangium degradation by increasing mesangial cell protein kinase C activity. We conclude that decreased degradation of mesangium as a result of hyperglycemia may play a role in causing the mesangium enlargement that occurs in diabetic nephropathy.

A single visit diabetes complication assessment service: a complement to diabetes management at the primary care level.

Modern diabetes management emphasizes the early detection and prompt treatment of diabetic complications. However it is difficult to organize comprehensive screening at the primary care level. To address this problem we established a complication assessment service whereby all the major diabetes-specific complications were assessed in a single 3 h visit. A report with results and recommendations was sent to the general practitioner (GP). Being philosophically a complication-specific service, no attempt was made to intervene with metabolic management. This paper describes our experience with the first 743 patients of whom 92% had been referred from GPs. Of the diabetes-specific complications, 22% of patients had one, 5% had two, and 1% had three major complications. Many of the patients were unaware of the presence of these complications. One hundred and three people had attended the service on more than one occasion with an average time between visits of 1.7 years. The results demonstrated that GPs were very good at following a recommendation to refer a patient for ophthalmic assessment (85% of cases) and improving hypertension but were less successful in treating hyperlipidaemia. This service has proven to be an excellent forum for the collection of data and the teaching of health professionals. It is a move away from the traditional format of hospital-based clinics providing comprehensive diabetes management.

Sharing the care of diabetic patients between hospital and general practitioners: does it work?

A randomized controlled trial was conducted to compare three forms of diabetes follow-up: (1) general practitioner care, (2) a system of care shared between the general practitioner (GP) and clinic and (3) conventional clinic care. Two hundred and six diabetic patients without significant diabetes-related or other medical complications were randomized to one of these follow-up systems. Metabolic control and blood pressure improved significantly and equally in all three groups (p < 0.05). The shared care group performed as well as or better than either of the other two groups in all other outcome measures. In particular, final attendance rates were 72% for shared care compared with only 35% for GP care and 53% for clinic care. Data collection rates for shared care were comparable with the clinic group for random blood glucose (88.9% vs 95.1%), weight (93.5% vs 98.3%), and blood pressure (94.8% vs 92.7%). Only in the case of glycosylated haemoglobin did shared care have poorer data collection (66.0% vs 98.4%). In all these parameters, except blood pressure, shared care out-performed the GP group. We conclude that with adequate support from and communication with hospital-based diabetes services, GPs are capable of providing care appropriate to the needs of uncomplicated diabetic patients.

Diabetes education: whose priorities are met?

Two hundred Type 2 diabetic patients newly referred to the diabetes centre at a large university teaching hospital were studied over an 8-month period. Patients completed a diabetes knowledge questionnaire, and specified their educational priorities by selecting six diabetes-related topics from a list of 14. After giving 1 h of individual education and using the same list, the educators selected six topics which they considered to be most important for that particular patient to know. Choice of educational priorities differed between the patients and the corresponding educator (p less than 0.001). In only 38% of cases did the educators' first three priorities coincide with those of the patients. The major discrepancies were in the selection of 'sick day management' and 'complications', especially favoured by patients, as against 'oral hypoglycaemic agents' and other therapy-related topics, especially favoured by educators. Diabetes knowledge was a determinant of educational priority for patients (p less than 0.001) but not educators. In contrast, only the educators' overall choices were affected by duration of diabetes (p less than 0.001). Diabetes treatment type influenced both patients' and educators' selection of priorities (p less than 0.001). We conclude that an educational strategy which relies on health professionals' perceptions to determine what diabetic patients need to know may be inadequate.