Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors.

Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

Intradialytic changes of plasma amino acid levels: effect of hemodiafiltration with endogenous reinfusion versus acetate-free biofiltration.

During hemodialysis, amino acids (AA) are lost in the ultrafiltrate with consequent modification of their plasma profile. The aim of this cross-sectional study was to evaluate intradialytic changes of plasma AA levels during a single session of hemodiafiltration with endogenous reinfusion (HFR) versus acetate-free biofiltration (AFB). 48 patients chronically treated with HFR or AFB were matched 1:1 for age, gender, Kt/V and diabetes. Blood samples were collected at the beginning and the end of dialysis. Baseline plasma levels (µmol/l) of total AA (3,176 ± 722), essential AA (889 ± 221), and branched chain AA (459 ± 140) levels in HFR were similar to those in AFB (3,399 ± 621, 938 ± 277, and 463 ± 71, respectively). Plasma intradialytic AA levels did not change in HFR, while in AFB there was a reduction by about 25%. In conclusion, as compared with AFB, HFR has a sparing effect on AA loss due to the lack of adsorption by cartridge and to their complete reinfusion in blood.

[Hyponatremia secondary to inappropriate antidiuretic hormone secretion]. / Iposodiemia da inappropriata secrezione di ADH.

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a disorder of sodium and water balance characterized by hypotonic hyponatremia and impaired water excretion in the absence of renal insufficiency , adrenal insufficiency or any recognized stimulus for the antidiuretic hormone (ADH). An inappropriate increase in ADH release of any cause produces hyponatremia by interfering with urinary dilution, thereby preventing the excretion of ingested water. Despite being the most common cause of hyponatremia in hospitalized patients, SIADH remains a diagnosis of exclusion. SIADH should be suspected in any patient with hyponatremia, hyposmolarity, urine osmolality above 100 mosmol/hgH2O, urine sodium concentration usually above 40 mEq/L, and clinical euvolemia. a number of modalities can be used to correct hyponatremia in SIADH, with water restriction and salt administration being the most important. The rate of correction is dependent upon the degree of hyponatremia and the presence or absence of symptoms. Patients with severe neurological symptoms require prompt correction; however, excessively rapid correction should be avoided because it can lead to the late onset of neurological complications from osmotic demyelination.

Mesangial hypercellularity predicts antiproteinuric response to dual blockade of RAS in primary glomerulonephritis.

The greater antiproteinuric efficacy of converting enzyme inhibitor and angiotensin II receptor blocker combination (CEI+ARB), versus monotherapy with either drug, is not a consistent finding. We evaluated the clinicopathologic predictors of response to CEI+ARB in 43 patients with primary glomerulonephritis (GN), never treated with immunosuppressive drugs, and with persistent proteinuria after CEI alone. Main histological lesions were analyzed by obtaining on 557 glomeruli and 165 arteries formal score of mesangial cellularity, glomerulosclerosis, tubulointerstitial damage, mononuclear cell infiltration, arteriosclerosis, and arteriolar hyalinosis. Duration of CEI and CEI+ARB therapy was similar (4.7+/-2.4 and 5.0+/-1.5 months). Proteinuria (g/day) decreased from 3.5+/-2.9 to 2.4+/-2.3 after CEI, and to 1.5+/-1.3 after CEI+ARB (P<0.0001). Reduction of proteinuria after CEI+ARB was greater in proliferative versus non-proliferative GN (-63.3+/-23.4 versus 42.4+/-23.7%, respectively; P=0.006). When patients were categorized in responders and non-responders to CEI+ARB, no difference between the two groups was detected in any demographic or clinical variable, whereas histology showed in responders a greater prevalence of proliferative GN (71.4 versus 31.8%, P=0.009) and higher score of mesangial cellularity (1.76+/-0.53 versus 1.20+/-0.22, P<0.0001). At multiple regression analysis (r(2)=0.476, P=0.001), response to CEI+ARB resulted independently related only to mesangial cellularity (P<0.0001). In conclusion, the best independent predictor of antiproteinuric efficacy of CEI+ARB in patients with primary GN is the degree of mesangial cellularity. This finding supports the experimental evidence that high angiotensin II contributes to proliferation of mesangial cells.

[Daily nutrient intake in hemodialysis]. / Variabilità dell'introito giornaliero di nutrienti nei pazienti emodializzati.

BACKGROUND: Although there is a higher nutrient requirement, food intake in haemodialysis patients is often inadequate. Protein nitrogen appearance (PNA) indirectly estimates the mean protein intake during the short interdialysis period, but it does not measure the daily nutrient intake, which is generally unknown. We carried out a longitudinal study aimed at estimating the daily nutrient intake and its relationship with the nutritional status of haemodialysis patients. METHODS: We selected 28 haemodialysis patients with adequate nutritional status and no evidence of risk-factor for malnutrition. Patients were treated with biocompatible membranes, low-flux and high bicarbonate dialysis, Kt/V > 1.2, PNA > 1.1 g/kg/day and erythropoietin. We measured every four months daily PNA, protein and calorie intake (DPI, DCI) as well as weight gain (WG) during an entire week for one-year. The nutritional status was assessed by biochemical and BIA markers. RESULTS: Twenty seven subjects (8 F, 19 M; age 57.1 +- 2.7 yeas; dialysis age 105 +- 13 months) completed the trial. The mean interdialytic PNA did not change in both long- and short-interdialysis periods, resulting in the "normal" range (> 1.1 g/kg/day); however, daily levels of protein and calorie intake were significantly reduced on the third day during the long interdialysis interval. Eight patients showed time-averaged values of DPI and DCI lower than 0.8 g/kg/day and 25 Kcal/kg/day, respectively, on the third day (LOW group), values that were associated with similar changes in WG. Such a highly reduced nutrient intake during the third interdialysis day was associated with a normal PNA value (1.23 +- 0.05 g/kg/day vs 1.30 +- 0.06 in CON, NS) when measured during the short interdialysis period (S), just as it is in clinical practice; in contrast, when the PNA value was measured during the long interdialysis period it was found to be significantly reduced (1.07 +- 0.08 g/kg/day vs 1.37 +- 0.06 in CON, p < 0.05 and vs S, p < 0.05). During the study, the body weight progressively decreased from 68.0 +- 5.5 to 65.8 +- 5.9 kg (p < 0.05) in the LOW group, due to the decrease in lean body mass, as suggested by the reduction in serum creatinine (9.2 +- 1.1 vs 8.1 +- 0.7 mg/dL, p < 0.05), creatinine generation (835 +- 155 vs 723 +- 106 mg/die, p < 0.05) and serum albumin (3.96 +- 0.07 vs 3.66 +- 0.06 g/dL, p < 0.05). Moreover, reactance and phase angle declined in the LOW group (from 54 +- 4 to 44 +- 3 ohms, p < 0.05 and 5.5 +- 0.3 to 4.5 +- 0.3 degrees, p < 0.05, respectively). At the end of the study the nutritional status in the LOW group was reduced as compared to the CON group. CONCLUSIONS: In stable, well-nourished haemodialysis patients, in absence of known risk factors for malnutrition, the daily nutrient intake is variable and progressively reduce during the interdialytic interval. The measurement of interdialytic PNA, as is done in clinical practice, does not enable the discovery of such abnormal eating behaviour; the low daily nutrient intake, on the contrary, can be evidenced by the daily measurement of either PNA or weight gain, and it can also be inferred by the reduced PNA during the long interdialytic period. Finally, the persistent reduction in nutrient intake below the threshold of 0.8 g/kg/day of proteins and 25 Kcal/kg/day one day a week, is capable of inducing body protein wasting and moderate impairment of the nutritional status.

Storage in the yolk platelets of low MW DNA produced by the regressing follicle cells.

The present work was carried out to clarify the nature and origin of the yolk DNA present in vitellogenic oocytes of the lizard Podarcis sicula. Morphological and biochemical evidences indicate that it has an intrafollicular origin, from the apoptotic bodies resulting from follicle cells regression at the end of previtellogenesis. This conclusion is reinforced by the observation that the oocyte membrane, in in vitro experiments, is unpermeable to exogenous DNA. Biochemical evidences reveal that the yolk DNA has a low (200bp) molecular weight and this suggests that it is produced by the endonucleases typically involved in apoptotic DNA laddering. Indeed, immunocytochemical analyses demonstrate that follicle cells contain significant amounts of DNAse I. In immunoblots, carried out during different periods of the ovarian cycle, the enzyme shows a MW of about 33, 66 or 100 kDa thus indicating that its activity in the follicle of Podarcis is modulated by dimerization and/or binding to regulatory factors. Mol. Reprod. Dev. 59: 422-430, 2001.

Abnormalities of bioimpedance measures in overweight and obese hemodialyzed patients.

BACKGROUND: The body composition in overweight and obese hemodialyzed patients (HD) remains ill-defined. This study evaluates in HD patients the influence of body size, as indicated by body mass index (BMI, kg/m(2)), on body composition by measuring bioimpedance analysis (BIA)-derived variables (phase angle (PA), fat-free mass (FFM) and body cell mass (BCM). METHODS: We studied 50 Caucasian patients (mean age 62.8+/-9.2 y) on standard bicarbonate hemodialysis for at least 12 months who regularly achieved dry weight in post-HD, received similar dialysis doses and were free from inflammation/infection. Thirty-eight gender- and age-matched healthy subjects were included as controls (CON). Both HD and CON were divided into three groups on the basis of their BMI(kg/m2) 18.5-24.9, normal-weight (NW); 25-29.9, overweight (OW); and > or =30, obese (OB). In HD patients, BIA was performed 30 min after the end of dialysis. RESULTS: Seven patients were obese (12%) while 16 were overweight (32%); in CON, 12 were obese (31%) and 12 overweight (31%). BIA-measured extracellular water was comparable in all groups. PA, which was similar in normal-weight HD and CON (6.2+/-0.9 degrees and 6.3+/-0.8 degrees ), decreased in OW- and OB-HD patients (5.3+/-1.0 degrees and 5.2+/-0.6 degrees, respectively; P<0.05 vs NW-HD) while it was unchanged in OW- and OB-CON (6.1+/-0.8 degrees and 5.9+/-0.5 degrees, P<0.05 vs respective HD groups). In OW and OB patients, the lower PA values were coupled with a major reduction of BIA-derived percentage BCM and FFM (P<0.05 vs NW-HD, and vs OW- and OB-CON). In patients, PA and BCM correlated with anthropometry-measured FFM. Of note, serum albumin and protein catabolic rate were significantly reduced in OB patients. CONCLUSION: In overweight and obese HD patients, BIA-derived FFM, BCM and PA are significantly lower with respect to normal-weight patients and BMI-matched controls. These abnormalities of body composition are coupled with reduction of anthropometric measures of lean mass and a decrease of protein intake that, however, becomes significant only in the obese. We therefore suggest that overweight and obese HD patients are at risk of protein malnutrition in spite of excessive energy intake. BIA may be considered as a useful diagnostic tool to detect such a condition early.

Control of oocyte recruitment: regulative role of follicle cells through the release of a diffusible factor.

To determine whether oogonial proliferation and oocyte recruitment are under control of hypophyseal and/or ovarian factors, we carried out a series of investigations using Podarcis sicula, a lizard inhabiting the temperate lowlands of Europe in which oocyte recruitment occurs throughout the year, as animal model. Germinal beds containing oogonia and oocytes in prefollicular stages were cocultured with different ovarian compartments in presence/absence of FSH, and the effects of different treatments were evaluated by counting the number of prelepto-leptotene oocytes. Results revealed that oocyte recruitment from the pool of oogonia is under the control of a factor released by follicle cells while FSH has an indirect effect on modulating oogonial proliferation. SDS-PAGE analyses carried out on media conditioned by follicles suggest that the factor involved in the control of oocyte recruitment may be a small protein (about 21 kDa) and that its release is dependent on the period of the ovarian cycle but apparently not on the circulating levels of FSH.

Triterpene saponins and iridoid glucosides from galium rivale.

Three new glycosides of the oleanene-type triterpenes, rivalosides C-E (1-3), along with three known triterpene saponins, momordin IIb (4) and rivalosides A-B (5-6), and five known iridoid glucosides: monotropein, scandoside, deacetylasperulosidic acid, geniposidic acid and asperulosidic acid, were isolated from aerial parts of Galium rivale. The structures of the new compounds were elucidated by spectral methods and chemical means as 2alpha-acetoxy-3alpha, 19alpha-dihydroxy-olean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl-(1--> 6)-beta-D-glucopyranoside, 2alpha,3alpha, 19alpha-trihydroxy-olean- 12-en-28-oic acid 28-O-beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranoside and 3-O-beta-D-glucuronopyranosyl-24-hydroxy-olean-12-en-28-oic acid 28-O-beta-D-glucopyranoside, for rivalosides C-E, respectively. The taxonomic significance of the rivalosides in G. rivale was discussed.

Comparison of vector and conventional bioelectrical impedance analysis in the optimal dry weight prescription in hemodialysis.

BACKGROUND: Dry weight prescription is commonly based on symptoms induced by inappropriate fluid removal by hemodialysis (HD). Aim of this study was to compare the assessment of volume status by conventional bioelectrical impedance analysis (BIA) and the resistance-reactance (RXc) graph method in HD patients achieving their target dry weight determined on clinical criteria. METHODS: We studied 39 HD patients (23 males and 16 females, mean age 52 +/- 17 years, dialytic age 41.2 +/- 37 months). Dry weight, prescribed according to the standard clinical criteria, was constantly achieved in the last 3 months. Patients symptom-free over the last 3 months were defined as asymptomatic. Patients with either muscular cramps or hypotensive episodes were defined as symptomatic. Thirty-three healthy volunteers (11 males, 22 females, mean age 50 +/- 11 years) constituted the control group. Standard, single frequency (50 kHz), tetrapolar, BIA measurements were obtained in controls, and in patients before, every 60 min, and 30 min after one HD session. Total body water (TBW), and extracellular water (ECW) were calculated using conventional BIA regression equations. In both groups, tissue hydration was also assessed by the RXc graph method. RESULTS: On the basis of 95% tolerance interval (mean +/- 2 SD) for the ECW (%) calculated in healthy subjects (ECW = 35-44%), HD patients were divided into 3 groups according to their post-HD ECW: 72% normohydrated with ECW 35-44%, 10% overhydrated with ECW >44%, and 18% underhydrated with ECW <35%. Patients were also classified into 3 categories according to the RXc graph method: 38% normohydrated with vectors within the reference 75% tolerance ellipse, 0% overhydrated with short vectors below the lower pole of the 75% tolerance ellipse, and 62% underhydrated with long vectors above the upper pole of the 75% tolerance ellipse. The progressive removal of body fluid during HD treatment was associated with a progressive increase in both impedance vector components, R and Xc. Eleven of thirty-nine patients (28%) were symptomatic during HD treatment in the last 3 months. The majority of these (73%) were classified as normohydrated according to ECW estimates, while 9 and 18% were classified as over- and underhydrated, respectively. This frequency distribution was significantly different from that obtained with the RXc graph method (chi(2) = 6.9, p = 0.03) where the majority (73%) were classified as underhydrated, while 0 and 27% were classified as over- and normohydrated, respectively. The frequency distribution of the 28 asymptomatic patients also significantly differed between conventional BIA and RXc graph hydration categories (chi(2) = 10.8, p = 0.005), since 11, 71 and 18% vs. 0, 43 and 57% of patients were classified as over-, normo-, and underhydrated, respectively. CONCLUSIONS: The classification of volume status based on conventional BIA was insensitive to either clinical situation (presence or absence of symptoms). In contrast, the classification based on the RXc graph was consistent with the clinical course in symptomatic patients (73% dehydrated, and 27% normohydrated), while it did not reflect the clinical course in asymptomatic patients, 57% of whom were classified as (already) underhydrated. A longitudinal study will establish the clinical usefulness of RXc graph indications in asymptomatic patients.

Current indications for renal biopsy: a questionnaire-based survey.

Indications for renal biopsy are still ill defined. We recently sent a detailed questionnaire to 360 nephrologists in different areas of the world with the aim of providing information on this critical issue by evaluating the replies. The questionnaire was organized in four sections that included questions on renal biopsy indications in patients with normal renal function, renal insufficiency, and a transplanted kidney. In addition, the questions included methods applied to each renal biopsy procedure and to specimen processing. We received 166 replies; North Europe (50 replies), South Europe (47 replies), North America (31 replies), Australia and New Zealand (24 replies), and other countries (14 replies). In patients with normal renal function, primary indications for renal biopsy were microhematuria associated with proteinuria, particularly greater than 1 g/d of protein. In chronic renal insufficiency, kidney dimension was the major parameter considered before renal biopsy, whereas the presence of diabetes or serological abnormalities was not considered critical. In the course of acute renal failure (ARF) of unknown origin, 20% of the respondents would perform renal biopsy in the early stages, 26% after 1 week of nonrecovery, and 40% after 4 weeks. In a transplanted kidney, the majority of nephrologists would perform a renal biopsy in the case of graft failure after surgery, ARF after initial good function, slow progressive deterioration of renal function, and onset of nephrotic proteinuria. The last section provided comprehensive information on the technical aspects of renal biopsy. This survey represents the first attempt to provide a reliable consensus that can be used in developing guidelines on the use of kidney biopsy.

Endothelin-1 released by vascular smooth muscle cells enhances vascular responsiveness of rat mesenteric arterial bed exposed to high perfusion flow.

Vasodilation of resistance vessels ensues in response to increased perfusion flow to maintain tissue perfusion. The flow-induced vasodilation is mainly dependent on nitric oxide (NO), which also regulates vascular responsiveness to vasoconstrictors. Besides NO, however; high flow increases endothelin-1 (ET-1) production from endothelial cells. It is likely, therefore, that the interaction between NO and ET-1 may play a critical role in the control of arterial vascular tone under high perfusion flow. In this study, the vascular responsiveness (VR) to high flow rate and the role of ET-1 released by vascular smooth muscle cells (VSMC) were evaluated in isolated and in vitro-perfused mesenteric arteries (MA). MA were perfused at constant (3.5 mL/min; CPF) and increased flow rate (4.5, 5.5, 6.5 mL/min; IPF). VR was evaluated by infusing norepinephrine (NE; 5 micromol/L) and potassium chloride (KCl; 80 mmol/L). Mesenteric vascular resistance (MVR), ET-1, and cGMP release were measured under different flow rates. The role of endothelium-derived ET-1 was evaluated by perfusing MA with phosphoramidon (endothelin converting enzyme inhibitor), whereas the role of other endothelium-derived vasoactive substances was excluded by measuring VR in MA without endothelium. Finally, ETA and ETB receptor antagonists were perfused in disendothelized MA. In the IPF group of intact MA, MVR dropped (P<.05) and both ET-1 and cGMP increased in the perfusate (P<.05). VR was enhanced by high flow after NE (101+/-9 v. 56+/-12 mm Hg in CPF, P<.005) and KCl (119+/-12 v. 51+/-10 mm Hg in CPF, P<.005) and it was unaffected by either phosphoramidon or endothelium removal. On the contrary, BQ-610 abolished the flow-dependent increase in VR. No further additive effect was achieved with BQ-788. In conclusion, in MA, high flow reduces MVR and concurrently enhances VR, likely through VSMC-derived ET-1.

A new cacospongionolide derivative from the sponge Fasciospongia cavernosa.

Cacospongionolide F (4a), a new bioactive cacospongionolide-related sesterterpene, has been isolated from the Northern Adriatic sponge Fasciospongia cavernosa. The structure was proposed on the basis of spectroscopic data and chemical transformations. The absolute configuration was established using the modified Mosher's method. A molecular mechanics study of the dehydrodecalin ring explained the observed differences in dynamic behavior between cacospongionolide F and mamanuthaquinone, a related compound. Antimicrobial activity, brine shrimp and fish lethalities of this new compound are reported.

How the ovarian follicle of Podarcis sicula recycles the DNA of its nurse, regressing follicle cells.

In Podarcis sicula specialized follicle cells send reserve materials to the previtellogenic oocyte via intercellular bridges. Immediately before the onset of vitellogenesis this transferring becomes particularly massive so that the cell volume significantly reduces, meanwhile in the nucleus the morphological alterations typical of apoptosis appear. To clarify why these follicle cells are not simply fully resorbed by the oocyte and to determine whether their DNA is discarded or recycled, we carried out a series of morphological and biochemical investigations. The finding that large macromolecular scaffolds are formed and that these are able to retain the DNA until it is extensively cut by two different endonucleases suggests that regression of the follicle cells is programmed and that the fate of their DNA is strictly controlled. Following its genetical neutralization via fragmentation, the DNA is apparently recycled by being transferred into the oocyte via the intercellular bridges, that, in fact, remain open until the very late stages of cell regression. The small DNA fragments reaching the oocyte cytoplasm would not interfere with meiosis completion but could significantly contribute to the stock of reserve materials to the advantage of the growing oocyte and/or developing embryo.

A new cacospongionolide inhibitor of human secretory phospholipase A2 from the Tyrrhenian sponge Fasciospongia cavernosa and absolute configuration of cacospongionolides.

A new inhibitor of human secretory phospholipase A2 (PLA2), cacospongionolide E (4a), has been isolated from the Tyrrhenian sponge Fasciospongia cavernosa. The structure was proposed on the basis of spectroscopic data and by chemical transformations. The absolute configuration of cacospongionolides 2a-4a was established using the modified Mosher's method. Cacospongionolide E was the most potent inhibitor toward human synovial PLA2, showing higher potency than the reference compound manoalide and exerting no signs of toxicity on human neutrophils. It showed high activity in the Artemia salina bioassay and moderate toxicity in the fish (Gambusia affinis) lethality assay.

Cacospongionolide B, a new sesterterpene from the sponge Fasciospongia cavernosa.

Cacospongionolide B [2a], a new cacospongionolide-related sesterterpene, has been isolated from the Adriatic sponge Fasciospongia cavernosa. The structure was elucidated by spectral and chemical means. The antimicrobial activity and brine shrimp and fish lethalities of 2a are reported.

Biological effects of prenylated hydroquinones: structure-activity relationship studies in antimicrobial, brine shrimp, and fish lethality assays.

Twenty-three hydroquinone and quinone derivatives were assayed for antimicrobial effects and brine shrimp and fish lethalities, to establish relevant structure-activity relationships (SAR). Linear 2-prenyl-1,4-hydroquinones used for bioassay were obtained either by isolation from the sponge Ircinia spinosula or by synthesis. Corresponding quinones, as well as hydroquinones possessing saturated side-chains composed of one to eight isopentane units, were also synthesized and biologically evaluated. Terpenoid 1,4-benzoquinones displayed moderate antimicrobial activity against three microorganisms, SAR studies indicate the optimum length of the side-chain is in the range of five to fifteen carbon atoms.

Spinocerebellar ataxia (SCA1) in two large Italian kindreds: evidence in favour of a locus position distal to GLO1 and the HLA cluster.

Two large Italian pedigrees with HLA-linked spinocerebellar ataxia (SCA1) were typed for HLA-A, -B and -DR as well as for markers either distal (F13A, D6S8) or proximal (D6S29, GLO1) to HLA. Pairwise linkage analyses of SCA1 vs. HLA-A, -B, and -DR showed peak lodscores of 5.3, 5.6 and 3.3 respectively at 7% recombination. Negative lodscores significantly excluded linkage with F13A at less than 5% and with GLO1 at less than 10%. The lodscores with D6S8 and D6S29 had only low peaks. Recombination events in the two pedigrees and the estimated genetic distances of SCA1 from GLO1 and HLA favour the hypothesis of a SCA1 location distal to both of them. An order cen-GLO1-HLA-SCA1-tel appears therefore most likely with present data. These results are discussed in relation to previous reports placing SCA1 distal to HLA in two families and

The human Y chromosome shows a low level of DNA polymorphism.

Six new Y-specific probes have been isolated and are reported. Along with another six already described they have been used in a systemic search for male specific RFLPs. An overall number of 46515 nucleotides have been screened with 12 enzymes and no polymorphic pattern observed. Our data reveal a greatly reduced level of polymorphism compared with other chromosomes.