Hyperuricemia and Risk of Cardiovascular Outcomes: The Experience of the URRAH (Uric Acid Right for Heart Health) Project.

The latest European Guidelines of Arterial Hypertension have officially introduced uric acid evaluation among the cardiovascular risk factors that should be evaluated in order to stratify patient's risk. In fact, it has been extensively evaluated and demonstrated to be an independent predictor not only of all-cause and cardiovascular mortality, but also of myocardial infraction, stroke and heart failure. Despite the large number of studies on this topic, an important open question that still need to be answered is the identification of a cardiovascular uric acid cut-off value. The actual hyperuricemia cut-off (> 6 mg/dL in women and 7 mg/dL in men) is principally based on the saturation point of uric acid but previous evidence suggests that the negative impact of cardiovascular system could occur also at lower levels. In this context, the Working Group on uric acid and CV risk of the Italian Society of Hypertension has designed the Uric acid Right for heArt Health project. The primary objective of this project is to define the level of uricemia above which the independent risk of CV disease may increase in a significantly manner. In this review we will summarize the first results obtained and describe the further planned analysis.

Visceral obesity, but not metabolic syndrome, is associated with the presence of post-thrombotic syndrome.

INTRODUCTION: The relationship between metabolic syndrome (MetS), and the development of post-thrombotic syndrome (PTS) is currently unknown. MATERIALS AND METHODS: We enrolled 120 patients with a previous episode of deep venous thrombosis (DVT) diagnosed more than 2years apart from the enrollment. Presence of MetS was identified according to NCEP ATP III criteria and Villalta Score (VS) was used to establish the presence of PTS (VS≥5). RESULTS: We identified 49 (40.8%) subjects with clinical diagnosed of PTS. Patients with or without PTS showed comparable age and temporal distance from DVT event. We observed higher BMI (p=0.005) and waist circumference (p=0.006) among subjects with VS≥5 as compared to patients without PTS. No differences between the two groups were found in terms of lipid profile, blood pressure, diabetes, hs-CRP level and ongoing medications. The prevalence of MetS was equally distributed among patients with or without PTS (20% vs 26% respectively, p=0.64). Among the individual components of MetS only the prevalence of visceral adiposity was significantly increased in subjects affected by PTS (OR 2.81, p=0.008). Moreover, a significant linear correlation was found between VS and waist circumference in the entire cohort (r=0,354, p<0.0001). CONCLUSION: There is no evidence of association between MetS and PTS. However, the degree of visceral adiposity is strongly correlated with the presence and severity of post-thrombotic disease.

Predictors of vascular remodelling in hypertensive subjects with well-controlled blood pressure levels.

We evaluated the structural/functional characteristics of the arterial wall in a cohort of hypertensives with well-controlled blood pressure (BP) levels. We studied 40 hypertensives with well-controlled BP. We assessed by B-mode ultrasound the mean intima-media thickness (mean-IMT) and maximum-IMT (M-MAX) of carotid artery (common, bulb, internal) bilaterally. Endothelial function was evaluated by post-occlusion flow-mediated dilation (FMD) of the brachial artery. Along with traditional risk factors, we studied the impact of serum high-sensitivity C-reactive protein (hs-CRP) and osteoprotegerin (OPG). Forty normotensive subjects served as controls. In the hypertensives, the BP levels were well controlled (office BP: 129/79 mm Hg, ambulatory BP monitoring: 121/75 mm Hg). Compared with controls, higher BP levels and body mass index were present in hypertensives, whereas age and metabolic parameters were similar. In hypertensives, the IMT (mean-IMT 0.68 mm, M-MAX 0.81 mm) was significantly higher than in controls (mean-IMT 0.60 mm, M-MAX 0.71 mm). FMD was impaired in hypertensives (5.9%) compared with controls (9.2%). In multivariate analyses, it turned out that in hypertensives IMT parameters were related to age, hs-CRP and OPG. Low-density lipoprotein (LDL) cholesterol was the only factor related to FMD. IMT and FMD had no relationship with BP levels. In conclusion, in hypertensives with well-controlled BP, the pro-atherogenic remodelling (IMT) is mainly dependent on age and the inflammatory cytokines, OPG in particular. The functional impairment of the arterial wall (FMD) was related to the levels of LDL cholesterol. Under these conditions, when the impact of BP is minimized, the role of inflammatory cytokines and lipids on structural/functional remodelling becomes predominant.

Impact of hypertension on vascular remodeling in patients with psoriatic arthritis.

We studied the impact of hypertension along with traditional and new cardiovascular risk factors on the structural and functional properties of arteries in psoriatic arthritis (PsA) patients. We examined 42 PsA subjects (aged 51±9 years) stratified according to hypertensive status (19 normotensive, PsA-NT and 23 hypertensives, PsA-HT). Thirty-eight normotensive subjects (C-NT) and 23 hypertensives (C-HT) comparable by age and sex served as controls. Mean carotid intima-media thickness (mean-IMT) and mean of the maximum IMT (M-Max) were evaluated by ultrasound in carotid artery segment bilaterally. Post-occlusion flow-mediated dilation (FMD) of the brachial artery was evaluated by ultrasonography. These parameters were correlated with risk factors, markers of inflammation and disease activity. Values of mean-IMT were higher in both groups of PsA patients compared with C-NT (0.68 mm in PsA-NT and 0.75 mm in PsA-HT versus 0.61 mm in C-NT). PsA-HT displayed higher M-Max (0.95 mm) versus both C-HT (0.71 mm) and PsA-NT (0.79 mm). FMD was impaired in PsA subjects compared with C-NT (5.7% in PsA-NT and 6.0% PsA-HT versus 9.3% in C-NT), whereas there was no difference among PsA-HT, PsA-NT, and C-HT groups. Values of carotid IMT were directly related to tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), blood pressure and lipid profile levels. FMD showed an inverse relationship with TNF-α and blood pressure, but no correlation with lipids. In conclusion, PsA per se implies a pro-atherogenic remodeling, which is enhanced by the hypertensive status. TNF-α and OPG may have an independent role in the development of such vascular damage.

Innate immunity and atherogenesis.

Atherosclerosis is recognized as the pathological basis of cardiovascular disease (CVD) and recent advances in basic science have shown that it should be considered as a chronic inflammatory process. Both elements of the innate and the adaptive immunity appear to be actively involved in atherogenesis. In fact, the potential role played by pattern-recognition receptors (Toll-like receptors and scavenger receptors), cytokines (such as IL-1, IL-6, TNFalpha), chemokines and pentraxines (such as CRP and PTX3) represents an emerging field of investigation in atherogenesis. In the near future we expect a better definition of the real biological and clinical impact on CVD of these mediators. On one side, they could become useful to complement traditional risk factors, in order to identify new categories of subjects prone to CVD development. On the other, they could become an additional potential target for therapeutic strategies.

In vivo noninvasive identification of cell composition of intimal lesions: a combined approach with ultrasonography and immunocytochemistry.

PURPOSE: We investigated whether differences in cellular composition of the shoulder region of carotid plaque, a cell-rich, debris-free area, can be revealed with computer-driven analysis of ultrasound scans. METHODS: In 26 patients referred for carotid endarterectomy, the shoulder region of plaque eligible for surgical removal was identified with ultrasound scanning. Digital images were obtained and evaluated with a specially developed computer-driven system (Medical Image Processing [MIP]). The gray level distribution of the region of interest (ROI), along with some statistical parameters exploring the spatial distribution of pixels, such as entropy and second angular moment, were analyzed. In the specimen retrieved at surgery, the area corresponding to the ROI was selected. Cryosections were tested at immunocytochemistry with monoclonal antibodies specific to smooth muscle cells (SMCs), macrophages), and lymphocytes. Computerized image analysis was performed to quantify each cellular component of the lesion. RESULTS: Mean gray levels were related positively to the content of SMCs (r = 0.576, P =.002) and negatively to the content of macrophages (r = -0.555, P =.003). Lymphocytes did not show any correlation. Prevalence of SMCs, expressed as the ratio SMC/(SMC + macrophages), was related positively with entropy (r = 0.517, P =.007) and negatively with the second angular moment (r = -0.422, P =.032). The quartiles of gray level were useful for detecting significant differences in terms of cellular composition. CONCLUSIONS: Some cellular features of the shoulder region of plaque are associated with specific videodensitometric patterns evaluated with MIP. This approach enables in vivo noninvasive prediction and monitoring of cell composition of the shoulder region, and could be extended to study of the thickened intima.

A mannose receptor mediates mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle cells.

The putative mannose receptor (MR), previously implicated in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle (ASM) cells, was studied to determine its properties. Specific binding of the mitogenic neoglycoprotein, mannosylated bovine serum albumin (Man-BSA) to ASM cells was saturable, with an apparent Kd = 5.0 x 10(-8) M. Cell-bound ManBSA-colloidal gold conjugate was localized by electron microscopy to clathrin-coated pits on the cell surface, and was found to undergo internalization to endosomes; this was inhibitable by weak bases and swainsonine, that also inhibited ligand-induced mitogenesis. The ASM-MR, isolated by mannose-affinity chromatography, had the same apparent molecular mass as the macrophage (Mø) MR (M(r) = 175 kD), and was immunoprecipitated by an anti-MøMR immune serum. This antiserum blocked 125I-labeled-ManBSA binding to intact ASM cells, stimulated mitogenesis, and immunolocalized the ASM-MR in cytoplasmic vesicles compatible with endosomes. A monoclonal antibody directed against the MøMR also reacted with the ASM-MR; like the polyclonal antibodies, it stimulated mitogenesis as effectively as beta-hexosaminidases. These data indicate that the ASM-MR shares a number of functional and structural properties with the MøMR and suggest that similar receptors may have different main functions in different cells.

Molecular cloning, reconstruction and expression of the gene encoding the alpha-chain of the bovine CD8--definition of three peptide regions conserved across species.

We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8 alpha). A bovine thymus cDNA library was hybridized at low stringency with a human CD8 alpha cDNA clone. The first round of screening of 5 x 10(4) independent colonies yielded 12 clones containing incomplete BoCD8 alpha genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8 alpha clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8 alpha clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8 alpha. Sequence analysis confirmed that the clone under study encoded the BoCD8 alpha. The overall similarity of the BoCD8 alpha coding region with the human CD8 alpha coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8 alpha. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among all species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8 alpha to the alpha 3 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8 alpha clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8 alpha gene, we constructed a chimeric gene containing the coding sequence of the BoCD8 alpha clone and synthetic sequences corresponding to the first two amino acids of the human CD8 alpha leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8 alpha construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8 alpha. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8 alpha gene, as previously suggested on serological grounds.

Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin.

Development of a strategy for efficient delivery of exogenous enzyme to neuronal lysosomes is essential to achieve enzyme replacement in neurodegenerative lysosomal storage diseases. We tested whether effective lysosomal targeting of the human enzyme beta-N-acetylhexosaminidase A (Hex A; beta-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase, EC 3.2.1.52) can be obtained by coupling it via disulfide linkage to the atoxic fragment C of tetanus toxin (TTC) that is bound avidly by neuronal membrane. TTC-Hex A conjugation resulted in neuronal surface binding and enhanced endocytosis of enzyme as observed in immunofluorescence studies with rat brain cultures. In immunoelectrophoretic quantitative uptake studies, rat neuronal cell cultures contained 16- and 40-fold greater amounts of enzyme after incubation with TTC-Hex A than with nonderivatized Hex A. In cerebral cortex cell cultures from a feline model of human GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases), binding and uptake patterns of the enzymes were similar to those in the rat brain cell cultures. After exposure to extracellular concentrations of enzyme attainable in vivo, lysosomal storage of immunodetectable GM2 ganglioside was virtually eliminated in neurons exposed to TTC-Hex A, whereas a minimal effect was observed with Hex A. These findings demonstrate the usefulness of TTC adducts for effective neuronal lysosomal enzyme replacement.

Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture.

Studies were conducted to assess the mitogenic effect of lysosomal hydrolases, enzymes known to have an association with allergen- or ozone-induced airway hyperreactivity, on bovine tracheal myocytes in culture. Addition of purified human placental beta-hexosaminidase and partially purified bovine liver beta-glucuronidase resulted in the doubling of cell count after 4 d of incubation in medium M199 with 0.4% FBS. Unstimulated cells remained quiescent without a significant increase of cell count. Lysosomal hydrolases also selectively enhanced 3H-thymidine incorporation four to seven times more than that in vehicle-treated cells or cells treated with endotoxin, a common contaminant of purified enzymes. Ovalbumin (glycoprotein control), pronase, and lysozyme caused a modest but statistically insignificant increase (up to twofold) in 3H-thymidine incorporation. Elastase, collagenase and dialyzed E. coli beta-glucuronidase had no effect. The mitogenic effect of hydrolases was equally seen in quiescent, serum-depleted cells as well as in those maintained in medium with 10% FBS, suggesting that it was independent of serum factors. The effect of lysosomal hydrolases was inhibited by exposure to yeast mannan, and mannosylated human serum albumin had a mitogenic effect, suggesting the involvement of a mannose receptor. We conclude that lysosomal hydrolases may play a role in the development of the hyperplasia/hypertrophy of respiratory smooth muscle.

Neuroaxonal dystrophy in neuronal storage disorders: evidence for major GABAergic neuron involvement.

The formation of focal granular enlargements within axons (axonal spheroids or "torpedoes"; neuroaxonal dystrophy) is a well known phenomenon occurring in a variety of neurological diseases. The relative susceptibility of different types of neurons to this kind of axonal pathology, however, is largely unknown. An immunocytochemical study directed at localizing glutamic acid decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), in various CNS regions in feline models of lysosomal storage disorders has revealed vast numbers of axonal spheroids containing this enzyme. In some storage diseases (GM1 and GM2 gangliosidosis), GAD-immunoreactive spheroids were a common occurrence in many brain regions, whereas in other disorders these structures were more limited in distribution (alpha-mannosidosis), or were absent (mucopolysaccharidosis type I). Axonal spheroids unreactive for GAD were encountered in large numbers in subcortical white matter in GM2 gangliosidosis, but were infrequently observed in the other diseases. The incidence and distribution of GAD-immunoreactive spheroids in the various diseases under study were found to correlate closely with the type and degree of neurological deficits exhibited by affected animals. This study indicates that the neuroaxonal dystrophy occurring in some types of storage disorders commonly involves axons of GABAergic neurons and suggests that a resulting defect in neurotransmission in inhibitory circuits may be an important factor underlying brain dysfunction in this family of diseases.

Distribution of ectopic neurite growth and other geometrical distortions of CNS neurons in feline GM2 gangliosidosis.

Golgi and combined Golgi-electron microscopic (EM) studies were carried out on cats in the terminal stages of GM2 ganglioside storage disease and the resulting data were compared with those from similar studies of other neuronal storage diseases in cats, including GM1 gangliosidosis. The results support the view that only limited types of neurons affected by the lysosomal hydrolase deficiency and subsequent intracellular storage have the capacity to sprout new dendritic-like growth processes from their axon hillocks, and that these neurons are essentially the same in all of these diseases studied to date. Golgi studies of CNS tissues from GM2 gangliosidosis cats revealed ectopic neurite growth on pyramidal neurons of cerebral cortex and multipolar cells of amygdala and claustrum, whereas other types of neurons responded to the metabolic defect with aspiny meganeurite formation or somatic enlargement, or appeared normal in terms of soma-dendritic morphology. Combined Golgi-EM studies of cortical pyramidal neurons revealed that ectopic, axon hillock neurites commonly possessed asymmetrical synapses which were similar to those observed in other storage disorders.

Initiation and growth of ectopic neurites and meganeurites during postnatal cortical development in ganglioside storage disease.

The incidence of cortical pyramidal neurons displaying meganeurites or enlarged axon hillocks with ectopic spines and neurites was evaluated developmentally using feline models of GM1 and GM2 gangliosidosis. Results of these studies demonstrated that the onset of ectopic neurite growth occurred after the elaboration of dendrites on cortical pyramidal neurons, and that the time of onset of this renewed dendritogenesis was similar in the two diseases. Initiation and growth of ectopic neurites also correlated in a general way with onset and progression of clinical deterioration in both diseases. In GM1 gangliosidosis there was a greater tendency toward formation of meganeurites, whereas in cats with GM2 gangliosidosis the growth of ectopic axon hillock neurites without meganeurites predominated. At end-stage disease in GM2 gangliosidosis, nearly 90% of pyramidal cells displayed some degree of axon hillock neurite growth as opposed to less than half this number for GM1 gangliosidosis cats at the same age. These data are consistent with the hypothesis that there are two separate driving forces behind these somadendritic abnormalities of pyramidal neurons in the gangliosidoses. Excessive intraneuronal accumulation of storage vacuoles accounts for the formation of meganeurites, whereas some type of intrinsic metabolic defect results in axon hillock neurite growth which in turn offers new surface area for synaptic input. Currently available data indicate that GM2 or GM3 ganglioside, or a closely related metabolic product other than GM1 ganglioside, may be primarily associated with the growth of ectopic dendritic processes on morphologically mature neurons in storage diseases.

Sphingomyelin lipidosis in a cat.

A 7-month-old Balinese cat with progressive neurological dysfunction had histopathological lesions of brain, liver, kidney, spleen, and lung consistent with a lysosomal storage disease. Ultrastructural examination revealed lysosomal hypertrophy with membranous inclusions. Hepatic sphingomyelin and cholesterol were elevated 10 times normal, and total phospholipids were increased 3.6 fold. Sphingomyelinase activity measured with 14C labeled sphingomyelin at pH 5.0 was virtually absent in brain and liver. Other lysosomal hydrolase activities were normal or elevated. Clinical, morphological, and biochemical findings suggest that this cat had sphingomyelin lipidosis similar to human Niemann-Pick disease type A, and that feline sphingomyelin lipidosis provides another model of human lysosomal storage disease.

Modified beta-D-N-acetylhexosaminidase isozymes for enzyme replacement in GM2 gangliosidosis.

The therapeutic potential of enzyme replacement in lysosomal storage disorders has remained largely unfulfilled, perhaps because of negative reactions to the initial disappointing results. Despite the existence of several animal models that can be utilized to explore solutions to the problems of exogenous enzyme targeting, the interest in ERT prevalent during the 1970's seems to have subsided to be replaced by active interest in bone marrow transplantation (BMT, Krivit and Paul [1986]). This is a logical approach to enzyme replacement in storage disorders of the RE system, and indeed some encouraging results have been obtained. However, in addition to having high morbidity and mortality, in the ultimate analysis BMT presents the same targeting problems as conventional ERT. In our opinion, these problems can be solved more easily in the case of ERT by exploiting the existing cellular uptake mechanisms and infusing enzymes whose structure has been suitably modified by simple biochemical manipulations. Accordingly, we have explored a methodology that takes advantage of negative charges on the cell surface to obtain nonspecific but effective membrane binding of beta-hex coupled to the highly positively charged PLL, followed by internalization and routing to the lysosomes. This system increases uptake of exogenous enzyme by some neurons in vitro and possibly in vivo, but its efficiency depends on the cells' endocytic activity that, in the case of neuronal soma, apparently is low. Thus, we have chosen as recognition marker for specific neuronal uptake a nontoxic fragment of TTx that is efficiently taken up by these cells. The initial results are encouraging; they support our contention that effective enzyme replacement methodologies can be devised, and encourage us to continue our work in this direction. Finally, recombinant DNA techniques are now being applied to a number of LSD, and the genes for several of the pertinent enzymes have been or are being isolated. In addition to representing a first step towards gene replacement therapy, the results of this work will permit the generation of large amounts of human enzymes from bacteria by recombinant DNA methods, thus obviating the problem of enzyme supply for ERT. Since human lysosomal enzymes obtained from bacteria will be nonglycosylated, to obtain cell uptake it will be necessary to resort to the type of modifications that we are trying to develop at this time, i.e., covalent linkage to moieties that allow non-glycosyl-mediated cellular uptake. Thus, our work on beta-hex may provide a model for biochemical manipulations of bacterially produced enzymes applicable to several LSD.

Beta-hexosaminidase isozymes and replacement therapy in Gm2 gangliosidosis.

The problem of cell targeting of lysosomal enzymes is a critical one in the development of strategies for therapeutic enzyme replacement in lysosomal storage diseases. In principle, posttranscriptional isozymes with different carbohydrate-chain composition may be helpful in exploiting existing glycosyl-specific receptors on target cells, if the receptor specificities are known and match the glycosyl composition of available isozymes. In practice, however, the choice is limited to isozymes that can be obtained from tissues available in abundance, such as placenta or blood plasma. Our early experiments show that one can interfere with the interaction between hepatic (RES) receptor and enzyme glycosyl chain, to obtain extrahepatic targeting of beta-hexosaminidase, with catabolic effects. This approach, of course, does not have an immediate therapeutic application, as it involves injection of large amounts of foreign material in order to inhibit hepatic uptake. Modification of the glycosyl chain may be the method of choice in selected instances [Furbish et al. 1981], but is applicability again depends on the knowledge of receptor specificity on target cells and on composition of the glycosyl chain of the enzyme in question. Our recent experiments are a first step toward obtaining enzyme forms that can be endocytosed efficiently by mechanisms that are independent of glycosyl-specific receptors. Charge-mediated, absorptive endocytosis can be obtained by covalent coupling of cationic PLL to beta-hexosaminidase. Given the abundance of negative surface charges on most cell types [Weiss, 1969], this approach may be applicable to different target cells and organs, and possibly also to lysosomal enzymes other than beta-hexosaminidase. The existence of glycosyl recognition signals on beta-hexosaminidase can be obviated by simple chemical manipulations, such as Na-metaperiodate oxidation, which effectively prevents hepatic RES uptake [Rattazzi et al, 1982]. In combination with PLL conjugation, this may ultimately result in an enzyme form that escapes the undesired, preferential RES uptake and is efficiently endocytosed by most cells. It will remain to be seen if this artificially created isozyme (for which we propose the name "ersatzyme") is catabolically effective. This can easily be verified in our animal model, along the lines followed to demonstrate the catabolic effects of native Hex A. Finally, the recent developments in molecular genetics, which allows production of human proteins in bacterial systems by recombinant DNA techniques, make it very likely that abundant beta-hexosaminidase may be similarly obtained for therapeutic applications.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiation of human mitochondria DNA types analyzed by restriction endonuclease cleavage patterns.

Human mitochondrial DNA (mtDNA) restriction endonuclease fragment patterns were analyzed using total blood cell DNA isolated from 200 individuals representing five different populations. Thirty-two fragment patterns (morphs) were observed with the enzymes Hpa I, Bam HI, Hae II, Msp I and Ava II yielding thirty-five different combinations of fragment patterns (mt DNA types). The major ethnic groups exhibit quantitative as well as qualitative differences in their mtDNA types, all of which are related to each other by a tree in which the closely related mtDNA types cluster according to geographic origin. Three mtDNA types are postulated to be 'central' to ethnic radiations due to their high frequencies, their appearance in more than one ethnic group, or their presence in other primate species. Genetic distances among populations were computed and employed in construction of an average linkage tree. If one of the three central mtDNA types is the root of the tree, differences in evolutionary rates among the branches become apparent. In particular, the Bushmen appear to have a higher evolutionary rate for mtDNA than the other four populations. Comparisons with nuclear gene frequencies suggest that this higher evolutionary rate may be the product of an elevated mutation rate or fixation of mutations in mtDNA.

The Florence sextuplets. Report of a case. Part two: progression of pregnancy, the onset of labor and delivery.

The paper presents a successful case of sextuplet pregnancy after HMG-HCG induced ovulation. The pregnancy was brought to 34, 5 weeks of gestation with all six babies in good conditions and high birth weight (1200-1750 gr.). The effectiveness of early diagnosis, intensive follow-up, and elective caesarean section is discussed. Suggestions are made for the prenatal, intrapartum, and post natal problems connected with mulitple gestation of high fetal number.