Extra Arginine Research Articles

Outstanding Superoxide Dismutase Catalytic Activity Of Simple Peptide‐Based Nickel(II) Complexes

AbstractWe present here the most active synthetic Ni superoxide dismutase (NiSOD) mimic reported to date. Reactive oxygen species are aggressive compounds, which concentrations are tightly regulated in vivo. Among them, the superoxide anion, O2⋅−, is controlled by superoxide dismutases. Capitalizing on the versatility of the Amino‐Terminal CuII‐ and NiII‐binding (ATCUN) peptide motif, we introduced positive charges around the NiII center to favor the interaction with the superoxide radical anion. At physiological pH, the pentapeptide H−Cys−His−Cys−Arg−Arg−NH2 coordinates NiII after the deprotonation of one thiol, two amides, and either the second thiol or the N‐terminal ammonium, leading to an equilibrium between the two N3S1 and N2S2 coordination modes. Under catalytic conditions, a kcat value of 8.6(4)×106 L.mol−1.s−1 was measured. Within the first second, the catalyst remained undegraded with quantitative consumption of O2⋅− (completed up to 37 catalytic cycles). An extra arginine (Arg) was introduced at the peptide C‐terminus to increase the global charge of the NiII complex from +1 to +2. This had no effect on the catalytic performance, highlighting the critical role of charge distribution in space as a determining factor influencing the reactivity.

Outstanding Superoxide Dismutase Catalytic Activity Of Simple Peptide-Based Nickel(II) Complexes.

We present here the most active synthetic Ni superoxide dismutase (NiSOD) mimic reported to date. Reactive oxygen species are aggressive compounds, which concentrations are tightly regulated in vivo. Among them, the superoxide anion, O2⋅-, is controlled by superoxide dismutases. Capitalizing on the versatility of the Amino-Terminal CuII- and NiII-binding (ATCUN) peptide motif, we introduced positive charges around the NiII center to favor the interaction with the superoxide radical anion. At physiological pH, the pentapeptide H-Cys-His-Cys-Arg-Arg-NH2 coordinates NiII after the deprotonation of one thiol, two amides, and either the second thiol or the N-terminal ammonium, leading to an equilibrium between the two N3S1 and N2S2 coordination modes. Under catalytic conditions, a kcat value of 8.6(4)×106 L.mol-1.s-1 was measured. Within the first second, the catalyst remained undegraded with quantitative consumption of O2⋅- (completed up to 37 catalytic cycles). An extra arginine (Arg) was introduced at the peptide C-terminus to increase the global charge of the NiII complex from +1 to +2. This had no effect on the catalytic performance, highlighting the critical role of charge distribution in space as a determining factor influencing the reactivity.

Extra arginine supplementation during the suckling period alleviates weaning stress through the regulation of dendritic cells and Notch2 signaling in piglets.

This study aims to study the effects of extra arginine (Arg) supplementation during the suckling period on the weaning stress and intestinal barrier function of breastfed piglets. Forty 7-day-old breastfed piglets divided into the control group (CON) and Arg group (Arg) were fed with extra saline or Arg (250 mg per kg per d body weight), respectively. All piglets were weaned when they were 21 days old. Eight piglets from each group were sacrificed before weaning and on the 3rd-day after weaning, respectively. The results showed that Arg improved the average daily weight gain of piglets before weaning (P < 0.01) and decreased the average daily weight loss after weaning (P < 0.05). Weaning decreased the ratio of the villus length versus crypt depth (V/C) in the SI (P < 0.001), while Arg increased the V/C of the jejunum (P < 0.05). Arg increased the levels of immunoglobulins in the serum and SI (P < 0.05), decreased pro-inflammatory cytokines and increased anti-inflammatory cytokines in the SI (P < 0.05). In addition, Arg supplementation increased the numbers of SWC3a+CD40+ (P < 0.01) and SWC3a+SLAII+ DCs (P < 0.05), down-regulated Notch2 expression and up-regulated Jagged1 expression in the ilea of weaning piglets (P < 0.05). In conclusion, Arg supplementation during the suckling period decreased the LDH leakage in the SI, improved the intestinal morphology, down-regulated the contents of pro-inflammatory cytokines, accelerated the accumulation of DC precursors before weaning and increased the number of mature DCs after weaning, and thus improved the growth performance and reduced the weaning stress of piglets, and this might be associated with the regulation of Notch2 signaling.

Effect of Extra Arginine Supplementation on Growth Performance, Carcass Characteristics and Immune Response in Broilers

Effect of Extra Arginine Supplementation on Growth Performance, Carcass Characteristics and Immune Response in Broilers

Posttranslational Arginylation Enzyme Arginyltransferase1 Shows Genetic Interactions With Specific Cellular Pathways in vivo.

Arginyltransferase1 (ATE1) is a conserved enzyme in eukaryotes mediating posttranslational arginylation, the addition of an extra arginine to an existing protein. In mammals, the dysregulations of the ATE1 gene (ate1) is shown to be involved in cardiovascular abnormalities, cancer, and aging-related diseases. Although biochemical evidence suggested that arginylation may be involved in stress response and/or protein degradation, the physiological role of ATE1 in vivo has never been systematically determined. This gap of knowledge leads to difficulties for interpreting the involvements of ATE1 in diseases pathogenesis. Since ate1 is highly conserved between human and the unicellular organism Schizosaccharomyces pombe (S. pombe), we take advantage of the gene-knockout library of S. pombe, to investigate the genetic interactions between ate1 and other genes in a systematic and unbiased manner. By this approach, we found that ate1 has a surprisingly small and focused impact size. Among the 3659 tested genes, which covers nearly 75% of the genome of S. pombe, less than 5% of them displayed significant genetic interactions with ate1. Furthermore, these ate1-interacting partners can be grouped into a few discrete clustered categories based on their functions or their physical interactions. These categories include translation/transcription regulation, biosynthesis/metabolism of biomolecules (including histidine), cell morphology and cellular dynamics, response to oxidative or metabolic stress, ribosomal structure and function, and mitochondrial function. Unexpectedly, inconsistent to popular belief, very few genes in the global ubiquitination or degradation pathways showed interactions with ate1. Our results suggested that ATE1 specifically regulates a handful of cellular processes in vivo, which will provide critical mechanistic leads for studying the involvements of ATE1 in normal physiologies as well as in diseased conditions.

Supplemental arginine improves intestinal morphology, but not expression of enzymes related with synthesis of polyamines in heat stress pigs

Animals exposed to high ambient temperature suffer heat stress (HS); this condition reduces feed intake and damages their intestinal epithelia, affecting nutrient digestion and absorption. Polyamines are involved in the restoration of intestinal epithelia by stimulating the proliferation of enterocytes. Because arginine is a precursor for the synthesis of polyamines, we examined the effect of supplying extra arginine in the diet on the expression of enzymes involved in polyamine synthesis and the intestinal morphology of HS pigs. This experiment was conducted when natural ambient temperature (AT) fluctuated daily from 28.2 to 38.7 °C. Twenty pigs (25.3±4.4 kg BW) were assigned to one of two treatments: control wheat‐soybean meal diet, and the control diet added with 0.20% free arginine. Pigs were fed at 0700 and 1900‐h, same amount (900 g) each time. At the end of a 21‐d trial, six pigs per treatment were sacrificed to collect intestinal mucosa samples and 5‐cm fragments from duodenum, jejunum, and ileum to analyze the intestinal morphology, and mRNA expression of spermidine/spermine N1‐acetyltransferase (SSAT), ornithine decarboxylase (ODC), and argininosuccinate synthase (ASS). Extra dietary arginine increased the villi height in duodenum, jejunum, and ileum, and the villi height:crypt depth ratio in duodenum and jejunum (P&lt;0.01). However, no effect of extra arginine was observed on the expression of polyamine‐related enzymes (P&gt;0.10). These results indicate that supplementing extra free arginine restores the morphology of the small intestine, but there is no clear explanation for the lack of effect of extra arginine on expression of enzymes involved in the polyamine synthesis.Support or Funding InformationCONACYT, México, CB‐2016‐01‐287052. Evonik Nutrition &amp; Care GmbH.

Posttranslational arginylation enzyme Ate1 regulates cell death through a mitochondrial‐dependent pathway

Arginyltransferase1 (Ate1) is an evolutionarily conserved enzyme that mediates posttranslational addition of one extra arginine to a protein substrate in a process called arginylation. Recent evidence suggests that Ate1 is essential for normal stress response. The level of Ate1 and total arginylation activity appear to increase follow acute stress and leads to cell death. Vice versa, the removal or reduction of this enzyme minimized cellular sensitivity to high doses of common cellular stressors such as oxidants, radiations, heavy metal, and high temperature. All these evidence suggest that Ate1 is a master regulator of cell death/growth. However, how does Ate1 accomplish this role remain unknown. In this study, we found that Ate1 regulates cell death in a mitochondrial‐dependent manner. By using S.cerevisiae, which contains a conserved Ate1 gene, we found that during acute stress, Ate1 is highly recruited to mitochondria. By using recombinant Ate1 that is bound to cell membrane, we found that the ability of locating to mitochondria is essential for Ate1‐mediated cell death. By screening of dozens of other players with known roles in cell death, we discovered that Ate1‐mediated cell death appears to be independent of the pan‐caspase in yeast, the mitochondrial membrane potential, or the ubiquitin‐proteasome system in general. Instead, the Ate1‐mediated cell death is dependent on the integrity of the mitochondrial pore complex and the existence of apoptosis‐inducing factor (AIF), a mitochondrial protein that is often released into cytosol during mitochondrial stress. Consistently, the Ate1‐mediated cell death can be at least partially blocked by the over‐expression of mammalian pro‐survival protein bcl2, which is known to block the formation of mitochondrial permeability transition pore. Finally, in mammalian cells, where the intrinsic and extrinsic cell‐death pathways are well‐separated, we found that the absence of Ate1 only has impacts on intrinsic pathway that is mainly mediated by mitochondria, but has little effects on cell‐death triggered by death receptors such as TNF‐R.In summary, our study has discovered a novel cell death mechanism that is regulated by a posttranslational modification enzyme.Support or Funding InformationNIGNMS R01 GM107333DoD (CDMRP), Idea Award, PC140622This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Posttranslational arginylation enzyme Ate1 is a mitochondrial‐derived master regulator that coordinates glycolysis and respiration in the Warburg effect

In the Warburg effect observed in vast majority of cancer cells, glycolysis is preferred over mitochondrial respiration. However, the role of mitochondria and its relationship with glycolysis in cancer metabolism is poorly understood. Arginyltransferase 1(Ate1) is an evolutionarily conserved enzyme that exist in most eukaryotes and a large subset of bacteria. In eukaryotes, this enzyme mediates arginylation, the posttranslational addition of one extra arginine to a protein. Recently, Ate1 was identified to be down‐regulated in many types of cancer but the significance of such observation remained unclear. Here, by examining classic glycolytic markers such glucose intake rate, sensitivity to glucose starvation, lactate generation, and the effects of glycolysis inhibitors including 2‐DG and 2‐FDG, we found that a deletion of Ate1 gene is sufficient to causes the Warburg effect on the cell level. This effect was reproduced by shRNA‐mediated knockdown of Ate1 and can be reversed by the expression of a recombinant Ate1 in the KO cells. In a screening of typical players involved in glycolysis, we found that the deletion of Ate1 increases of the protein level of hypoxia‐inducible factor 1a (HIF1a), a protein with known role in glycolysis. We found that the knockdown of HIF1a in Ate1‐KO cells was sufficient to reverse the glycolytic phenoytype in these cells, suggesting Ate1 regulates glycolysis through HIF1a. Furthermore, we found that Ate1 regulates the protein level of HIF1a through both conventional and unconventional pathways. In particularly, HIF1a appears to be a previous unknown substrate of Ate1‐mediated arginylation, which promotes the degradation of HIF1a through the N‐end rule. In addition, the deletion of Ate1 generate prominent effects on the morphology and functions of mitochondria, with significant defect on the assembly of mitochondrial complex II and a consequential accumulation of succinate, which is expected to stabilize the HIF1a protein by an inhibition of the PHD proteins. Finally, we found that eukaryotic Ate1 has significant homology to its counterpart in alpha‐proteobacteria, the ancestor of mitochondria. Also, although the majority of Ate1 is located in the cytosol, a detectable portion of Ate1 is located inside mitochondria. These evidence suggest that the Ate1 enzyme was derived from a gene transfer during the domestication of mitochondria, which further explains the previous unknown role of Ate1 as a master regulator of the balance between glycolysis and mitochondrial function.In summary, our study discovered a novel regulator of cell metabolism with a evolution root concomitant with mitochondria and our data suggest this regulator coordinates the balance between glycolysis and mitochondrial respiration in the cancer Warburg effect. Our data also reveal the existence of a non‐canonical degradation pathway of HIF1a.Support or Funding InformationNIGMS R01 GM107333This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enteral Glutamine Administration in Critically Ill Nonseptic Patients Does Not Trigger Arginine Synthesis.

Glutamine supplementation in specific groups of critically ill patients results in favourable clinical outcome. Enhancement of citrulline and arginine synthesis by glutamine could serve as a potential mechanism. However, while receiving optimal enteral nutrition, uptake and enteral metabolism of glutamine in critically ill patients remain unknown. Therefore we investigated the effect of a therapeutically relevant dose of L-glutamine on synthesis of L-citrulline and subsequent L-arginine in this group. Ten versus ten critically ill patients receiving full enteral nutrition, or isocaloric isonitrogenous enteral nutrition including 0.5 g/kg L-alanyl-L-glutamine, were studied using stable isotopes. A cross-over design using intravenous and enteral tracers enabled splanchnic extraction (SE) calculations. Endogenous rate of appearance and SE of glutamine citrulline and arginine was not different (SE controls versus alanyl-glutamine: glutamine 48 and 48%, citrulline 33 versus 45%, and arginine 45 versus 42%). Turnover from glutamine to citrulline and arginine was not higher in glutamine-administered patients. In critically ill nonseptic patients receiving adequate nutrition and a relevant dose of glutamine there was no extra citrulline or arginine synthesis and glutamine SE was not increased. This suggests that for arginine synthesis enhancement there is no need for an additional dose of glutamine when this population is adequately fed. This trial is registered with NTR2285.

Arginyltransferase (Ate1) is a Pro‐apoptotic Enzyme Essential for Stress Response

Arginylation consists of addition of an extra arginine to an existing peptide chain, thus changing the de facto primary sequence as well as the surface change. This process is evolutionarily conserved and mainly mediated by arginyltransferase (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in stress response. By using multiple types of eukaryotic cells as test models, we found that Ate1 is a pro‐apoptotic factor. Knockout of Ate1 in cells leads to a retardation of stress response and the diminish of apoptosis and cell death during exposure of high doses of various stressing factors including heat shock, oxidative and osmotic stress, and heavy metal. Oppositely, over‐expression of Ate1 leads to cell death in an Aif1‐dependent and caspase‐independent manner. Our data further suggest that localization of Ate1 to mitochondria and its enzymatic activity is essential for apoptosis during stress. Intriguingly, we also found that the activity of arginylation is essential for the wellbeing of the cells during mitochondria‐dependent metabolism as well as nutrient‐deprivation. Our data suggests that arginylation is a global regulator for metabolism and stress response, and provides an explanation for the observed role of Ate1 in carcinogenesis and injury.

Antimicrobial Peptide Piscidin Permeabilizes Bacterial Membranes and Binds Intracellular Targets at Sub-Lethal Concentrations

Piscidins 1 (P1) and 3 (P3) are cationic α-helical membrane active antimicrobial peptides originally isolated from the hybrid striped seabass. The peptides, which possess broad-spectrum activity against bacteria, fungi, and viruses, bind to anionic membranes and lyse them once a sufficient concentration has been reached. In this study, we used confocal microscopy and FITC-labeled piscidins to show that at sub-inhibitory concentrations (0.75 μM), P1 and P3 are able to translocate across the membrane of Gram-positive and -negative bacteria. The FITC signal of the peptides was co-localized with the DAPI signal of the nucleoid region of the bacteria. CD using a 15 base pair piece of DNA and a gel retardation assay using a 1782 base pair piece of DNA confirmed that P1 and P3 could bind to DNA in vitro. It appears that P3 binding was more disruptive to the structure of DNA, and this may be due to the extra arginine residue in P3's sequence. However, these results did not indicate whether the peptides translocated through the membrane or entered the cells via pores formed in the membrane. To probe this question, we used the bacterial strain E. coli ML35 in a permeabilization assay to determine at which peptide concentration the membrane became permeabilized. Our data indicate that at the lowest concentration tested (0.1 μM) the membrane of ML35 cells became permeabilized by P1 and P3. Our results agree with prior findings that permeabilization of bacterial cell membranes can occur prior to lysis and cell death. The implications of piscidin's ability to translocate into bacterial cells and bind to intracellular targets at sub-lethal concentrations will be discussed in terms of its mechanism of action, which was previously believed to be based on its membrane activity.

Posttranslational arginylation is essential for stress response (555.15)

Arginylation consists of addition of an extra arginine to an existing peptide chain, thus changing the de facto primary sequence as well as the surface change. This process is evolutionarily conserved and mainly mediated by arginyltransferase1 (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in stress response. By using multiple types of eukaryotic cells as test models, we found that Ate1 is a pro‐apoptotic factor. Knockout of Ate1 in cells leads to a retardation of stress response and the diminish of apoptosis and cell death during exposure of high doses of various stressing factors including heat shock, oxidative and osmotic stress, and heavy metal. Oppositely, over‐expression of Ate1 leads to cell death. Intriguingly, we also found that the activity of arginylation is essential for the wellbeing of the cells during mitochondria‐dependent metabolism as well as nutrient‐deprivation. Our data suggests that arginylation is a global regulator for metabolism and stress response, and provides an explanation for the observed role of Ate1 in carcinogenesis and injury.Grant Funding Source: NIGMS

Isolation and characterization of NAI-802, a new lantibiotic produced by two different Actinoplanes strains

Lantibiotics are biologically active peptides produced by Gram-positive bacteria. Starting from fermentation broth extracts preselected from a high-throughput screening program for discovering cell-wall inhibitors, we successfully isolated a new lantibiotic produced by Actinoplanes sp., designated as NAI-802. MS and NMR analysis together with explorative chemistry established that NAI-802 consists of 21 amino acids, 19 of which are identical to those present in the class II lantibiotic actagardine. Interestingly, NAI-802 carries one extra alanine and one extra arginine at the N- and C-termini, respectively. As expected from the overall higher positive charge, NAI-802 was slightly more active than actagardine against staphylococci and streptococci. Further improvement of its antibacterial activity was achieved by adding one additional positive charge through conversion of the C-terminal carboxylate into the corresponding basic amide. NAI-802 thus represents a novel promising candidate for treating Gram-positive infections caused by multidrug-resistant pathogens.

Nitric oxide and asthma severity: towards a better understanding of asthma phenotypes

Nitric oxide and asthma severity: towards a better understanding of asthma phenotypes

Meal Amino Acids with Varied Levels of Arginine do Not Affect Postprandial Vascular Endothelial Function in Healthy Young Men

Postprandial endothelial dysfunction is a key event mediating the effects of diet on early atherogenesis. The potential effects of protein intake have been overlooked in the past, although amino acids are precursors for homocysteine and nitric oxide (NO). Our objective was to study the effect of amino acids on postprandial vascular function, in relation to the utilization of meal arginine for NO production. In a crossover design, 9 men ingested 50 g of a complete amino acid mixture, trace-labeled with 13C-glycine and 15N2-arginine, without (meal A) or with (meal B) 3 g extra arginine. The postprandial utilization of meal arginine for NO production was determined from urinary 15NO3. We monitored endothelial function of the brachial artery, the stiffness of the common carotid artery, aortic pulse wave velocity and soluble markers related to endothelial function for 8 h. Meal A did not significantly increase plasma homocysteine and did not alter endothelial function markers. The amount of NO synthesized from meal arginine doubled after meal B (107.1 ± 16.5% increase vs. meal A, P < 0.01) but was very low (271 ± 84 ppm vs. 332 ± 73 ppm, P < 0.05, respectively). After meal B, flow-mediated and nitroglycerine-induced dilation decreased but common carotid artery compliance, pulse wave velocity, plasma soluble intercellular adhesion molecule-1, and von Willebrand factor, and urinary cGMP did not differ when compared with meal A. Together, the data indicate that, in healthy men, meal amino acids do not adversely affect endothelial function, and meal arginine only slightly enters the NO pathway. Unexpectedly, arginine in physiological amounts may acutely lessen smooth muscle cell reactivity to a high dynamic NO release.

The lung in the balance: arginine, methylated arginines, and nitric oxide

the lung is a major source of nitric oxide (NO), be it from nitric oxide synthase (NOS) III in the endothelium of the vast pulmonary circulation, NOS II in the epithelium of the large surface area of the airways, or NOS I in the nonadrenergic noncholinergic nerves ([6][1]). NO produced in the lung

Haplotype analysis and age estimation of the 113insR CDKN2A founder mutation in Swedish melanoma families.

Germline mutations in the CDKN2A tumor suppressor gene located on 9p21 have been linked to development of melanomas in some families. A germline 3-bp insertion in exon 2 of CDKN2A, leading to an extra arginine at codon 113 (113insR), has been identified in 17 Swedish melanoma families. Analysis of 10 microsatellite markers, spanning approximately 1 Mbp in the 9p21 region, showed that all families share a common allele for at least one of the markers closest to the CDKN2A gene, suggesting that the 113insR mutation is an ancestral founder mutation. Differences in the segregating haplotypes, due to meiotic recombinations and/or mutations in the short-tandem-repeat markers, were analyzed further to estimate the age of the mutation. Statistical analysis using a maximum likelihood approach indicated that the mutation arose 98 generations (90% confidence interval: 52-167 generations), or approximately 2,000 years, ago. Thus, 113insR would be expected to have a more widespread geographic distribution in European and North American regions with ancestral connections to Sweden. Alternatively, CDKN2A may lie in a recombination hot spot region, as suggested by the many meiotic recombinations in this narrow approximately 1-cM region on 9p21.

Processing, disulfide pattern, and biological activity of a sugar beet defensin, AX2, expressed in Pichia pastoris.

AX2 is a 46-amino-acid cysteine-rich peptide isolated from sugar beet leaves infected with the fungus Cercospora beticola (Sacc.). AX2 strongly inhibits the growth of C. beticola and other filamentous fungi, but has little or no effect against bacteria. AX2 is produced in very low amounts in sugar beet leaves, and to study the protein in greater detail with respect to biological function and protein structural analysis, the methylotrophic yeast Pichia pastoris was used for large-scale production. The amino acid sequence, processing of the signal peptide, disulfide bridges, and biological activity of the recombinant protein were determined and compared with that of the authentic AX2. In P. pastoris, the protein was expressed with an additional N-terminal arginine. The disulfide bonding was found to be identical to that of the authentic AX2. However, when tested in in vitro bioassay, the biological activity of the recombinant protein was slightly lower than that measured for the authentic protein. Furthermore, the recombinant protein was significantly more sensitive to Ca(2+) than the authentic protein. This is most probably due to the extra arginine, since no other differences between the two proteins have been found.

185. Expression of human α-defensin HD-5 in Pichia pastoris

AX2 is a 46-amino-acid cysteine-rich peptide isolated from sugar beet leaves infected with the fungus Cercospora beticola (Sacc.). AX2 strongly inhibits the growth of C. beticola and other filamentous fungi, but has little or no effect against bacteria. AX2 is produced in very low amounts in sugar beet leaves, and to study the protein in greater detail with respect to biological function and protein structural analysis, the methylotrophic yeast Pichia pastoris was used for large-scale production. The amino acid sequence, processing of the signal peptide, disulfide bridges, and biological activity of the recombinant protein were determined and compared with that of the authentic AX2. In P. pastoris, the protein was expressed with an additional N-terminal arginine. The disulfide bonding was found to be identical to that of the authentic AX2. However, when tested in in vitro bioassay, the biological activity of the recombinant protein was slightly lower than that measured for the authentic protein. Furthermore, the recombinant protein was significantly more sensitive to Ca2+ than the authentic protein. This is most probably due to the extra arginine, since no other differences between the two proteins have been found.

Screening of germline mutations in the CDKN2A and CDKN2B genes in Swedish families with hereditary cutaneous melanoma.

Approximately 10% of human cutaneous melanomas occur in families in which several members are affected. The familial predisposition to this disease is often associated with dysplastic nevus syndrome, a condition in which afflicted family members have multiple dysplastic nevi (atypical moles). The chromosome region 9p21 and markers on chromosomes 1p and 6p have been linked to melanoma susceptibility. The tumor suppressor genes CDKN2A and CDKN2B have been mapped to the 9p21 region, and genetic analyses have revealed the presence of germline CDKN2A alterations in melanoma families. The reported frequencies of such alterations, however, vary among these families. The present investigation was carried out to determine the frequencies of CDKN2A and CDKN2B germline gene mutations among members in a population-based cohort of Swedish melanoma families (i.e., melanoma kindreds). DNA was prepared from blood samples obtained from 181 individuals belonging to 100 melanoma kindreds. The polymerase chain reaction (PCR) technique, followed by single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses, were used to identify the types and frequencies of mutations in exons 1, 1beta, 2, and 3 of the CDKN2A gene and in exons 1 and 2 of the CDKN2B gene. CDKN2A gene aberrations were independently identified by both SSCP and nucleotide-sequence analyses. Nucleotide-sequence analysis identified a single point mutation leading to a substitution of leucine for proline in codon 48 of exon 1 in a family with a history of melanoma and several other cancers. A second abnormality, leading to an insertion of an extra arginine residue at codon number 113 of exon 2, was seen in four separate families. The CDKN2A exon-3 coding region had the wild-type sequence in all samples. No germline mutations were found in the alternative exon 1beta of the CDKN2A gene or in exons 1 and 2 of the CDKN2B gene. The present investigation demonstrates that CDKN2A germline gene mutations were observed in 7.8% of the 64 Swedish melanoma kindreds that each included at least two first-degree relatives with melanoma and dysplastic nevus syndrome. No CDKN2A exon 1beta or CDKN2B mutations were identified. The critical genes responsible for the inheritance of a susceptibility to develop melanoma among family members in this population have yet to be identified.